Cannabinoid NANO-micelles and methods of use thereof

Abstract

The present disclosure relates to a novel cannabinoid nano-micelle composition comprising a cannabinoid inclusion complex encapsulated within nano-micelles. The disclosed composition facilitates enhanced delivery of the cannabinoid to the brain, resulting in a rapid onset of action and elevated intracerebral cannabinoid concentrations. Administration of the composition was found to significantly improve cannabinoid bioavailability and to produce robust analgesic effects in models of neuropathic pain. These effects are achieved, at least in part, by suppressing aberrant neuronal hyperactivity within the somatosensory nervous system. The disclosed compositions and methods provide an effective therapeutic approach for the treatment of neuropathic pain and related conditions.

Description

[0001] CANNABINOID NANO-MICELLES AND METHODS OF USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 662,516, filed June 21, 2024. The foregoing application is incorporated by reference herein in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under AT010779 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] This invention relates generally to cannabinoid nano-micelles and compositions thereof, methods of preparation thereof, and methods of use thereof.

[0008] BACKGROUND OF THE INVENTION

[0009] Cannabinoids derived from Cannabis saliva, including delta-9-tetrahydrocannabinol (THC), a psychoactive component, and cannabidiol (CBD), a non-psychoactive component, have garnered increasing attention for their potential therapeutic applications, particularly in the management of pain. Neuropathic pain, a complex and chronic condition, is widely regarded as one of the most difficult types of pain to treat effectively. Existing pharmacological treatments for neuropathic pain, such as gabapentinoids, antidepressants, and opioids, frequently yield limited efficacy and are often associated with undesirable side effects.

[0010] Emerging clinical evidence suggests that combination therapies comprising THC and CBD may provide relief from neuropathic pain. However, such combinations have been linked to a heightened risk of adverse effects. Conversely, monotherapy with CBD demonstrates a more favorable safety profile, though its clinical efficacy in the treatment of neuropathic pain remains inconclusive. Limited clinical studies to date indicate that current CBD formulations often fail to produce significant therapeutic benefit compared to placebo controls. The underlying cause of this limited efficacy is not well understood and may result from suboptimal central nervous system (CNS) exposure or an inherent lack of analgesic potency. While basic research has identified numerous putative molecular targets for CBD that could potentially contribute to its analgesic effects, conclusive evidence of effective CNS delivery and analgesic efficacy in vivo remains lacking. For example, in murine models of chemotherapy- induced peripheral neuropathy, prophylactic administration of CBD has been shown to attenuate the development of mechanical hypersensitivity. However, in the same models, post-onset treatment with CBD failed to reverse established mechanical allodynia, even at doses exceeding those effective in a prophylactic context. These disparate outcomes may be attributable to limitations in CNS penetration and activity of CBD.

[0011] Neuropathic pain is known to involve a constellation of dysfunctions across diverse CNS pathways, including spinal nociceptive circuits and higher-order somatosensory centers within the cerebral cortex. Accordingly, a comprehensive investigation of CNS pharmacodynamics of CBD, including brain distribution and modulation of neuronal activity, may provide important insights into its analgesic potential. Notably, most prior studies have not evaluated CBD concentrations within the brain or characterized its effects on neural activity throughout the CNS in the context of neuropathic pain models.

[0012] CBD is classified as a Class II compound under the Biopharmaceutics Classification System (BCS), due to its high lipophilicity and poor aqueous solubility. These properties contribute to extensive first-pass hepatic metabolism and low oral bioavailability, which may partially explain the ineffectiveness of orally administered CBD oil in clinical trials for neuropathic pain. Preclinical studies have examined alternative delivery modalities, including intraperitoneal administration and solubilization in aqueous formulations using non-ionic surfactants such as ethoxylated castor oil (Cremophor EL). However, concerns regarding the toxicity of such excipients limit the dose of CBD that can be safely delivered to the CNS, thereby constraining its therapeutic utility.

[0013] Therefore, there is a need for improved cannabinoid compositions, for example, as a nonpsychoactive pharmacotherapy for neuropathic pain.

[0014] SUMMARY OF THE INVENTION

[0015] This disclosure addresses the need mentioned above in a number of aspects by providing a novel nano-micelle composition comprising a cannabinoid inclusion complex encapsulated in nano-micelles formed by a micelle-forming agent, wherein the cannabinoid inclusion complex comprises a cannabinoid encapsulated in cyclodextrin, and the cannabinoid inclusion complex slows down precipitation and / or crystallization of the cannabinoid during entrapment in the nanomicelles by the micelle-forming agent, wherein the micelle-forming agent is in an amount above a critical micelle concentration of the micelle-forming agent and in an amount of from about 0.2 to about 1.6 mole per mole of the cannabinoid, and wherein the cyclodextrin is in an amount of from about 1 to 2 mole per mole of the cannabinoid.

[0016] In some embodiments, a mean particle diameter of the nano-micelles is from about 16 nm to about 32 nm. In some embodiments, an overall size distribution of the nano-micelles is from about 5 nm to about 100 nm.

[0017] In some embodiments, the amount of the cyclodextrin is about 1 mole per mole of the cannabinoid. In some embodiments, the amount of the micelle-forming agent is about 0.81 mole per mole of the cannabinoid. In some embodiments, the cannabinoid, the cyclodextrin, and the micelle-forming agent have a molar ratio of 1 : 1 : 0.81.

[0018] In some embodiments, the cannabinoid is an amount of from about 0% to about 22.8% by weight of the nano-micelle composition. In some embodiments, the cyclodextrin is an amount of from about 0% to about 56.7% by weight of the nano-micelle composition. In some embodiments, the micelle-forming agent is an amount of from about 38.4% to about 100% by weight of the nanomicelle composition.

[0019] In some embodiments, the nano-micelle composition comprises: about 11.3% by weight of the cannabinoid; about 50.3% by weight of the cyclodextrin; and about 38.4% by weight of the micelle-forming agent.

[0020] In some embodiments, the cannabinoid is selected from the group consisting of A9- tetrahydrocannabinol (THC), A8-tetrahydrocannabinol, A9-tetrahydrocannabinol propyl analog (THCV), CBD, cannabidiol propyl analog (CBDV), cannabinol (CBN), cannabichromene, cannabichromene propyl analog, and cannabigerol or derivatives, and combinations thereof. In some embodiments, the cannabinoid is CBD.

[0021] In some embodiments, the cyclodextrin is selected from the group consisting of a- cyclodextrin, y-cyclodextrin, P-cyclodextrin, 2-hydroxypropyl-P-cyclodextrin, sulfobutylether P- cyclodextrin sodium salt, randomly methylated P-cyclodextrin, branched P-cyclodextrin, y- cyclodextrin, and derivatives thereof. Tn some embodiments, the cyclodextrin is 2-hydroxypropyl - P-cyclodextrin.

[0022] In some embodiments, the micelle-forming agent comprises stearoyl polyoxyl-32 glycerides, lauroyl polyoxyl-32 glycerides, polysorbate 80, polysorbate 60, polysorbate 20, or a combination thereof. In some embodiments, the micelle-forming agent comprises polysorbate 80.

[0023] In some embodiments, the cannabinoid inclusion complex is formed before entrapment in the nano-micelles by the micelle-forming agent.

[0024] In some embodiments, the nano-micelle composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the nano-micelle composition is a fluid pharmaceutical formulation for parenteral or oral use.

[0025] In some embodiments, the cannabinoid has a Cmax of about 6 pg / g in brain when the nanomicelle composition is administered to a mouse at 100 mg / kg intraperitoneal condition. In some embodiments, the cannabinoid has a Cmax of about 7.8 pg / ml in serum when the nano-micelle composition is administered to a mouse at 100 mg / kg intraperitoneal condition. In some embodiments, the cannabinoid has a half life of about 2 hours in brain after the nano-micelle composition is administered to a mouse.

[0026] In another aspect, this disclosure provides a kit comprising the nano-micelle composition described herein.

[0027] In another aspect, this disclosure also provides a method for preparing a nano-micelle composition described herein. In some embodiments, the method comprises: preparing a cannabinoid solution by dissolving the cannabinoid in a solvent; gradually adding the cyclodextrin to the cannabinoid solution with agitation to obtain a mixture, incubating the mixture at a predetermined temperature for a predetermined time period to form the cannabinoid inclusion complex in which the cannabinoid is encapsulated in the cyclodextrin; and adding the micelleforming agent to the mixture to form nano-micelles that encapsulate the cannabinoid inclusion complex.

[0028] In some embodiments, the solvent comprises ethanol. In some embodiments, the cannabinoid and the solvent have a molar ratio of about 1 :20.

[0029] In some embodiments, the predetermined temperature is 25-45° Celsius. In some embodiments, the predetermined time period is from about 1 hour to about 96 hours.

[0030] In another aspect, this disclosure further provides a method of treating a disease or disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the nano-micelle composition described herein.

[0031] In some embodiments, the disease or disorder comprises neuropathic pain.

[0032] In some embodiments, the nano-micelle composition suppresses neuropathic pain-related activation of neurons in somatosensory pathways. In some embodiments, the nano-micelle composition suppresses al lodynia-r elated hyperactivity of somatosensory corticospinal neurons in the subject. In some embodiments, the nano-micelle composition suppresses tactile allodynia or hyperalgesia in a subject having a spared nerve injury (SNI) but does not disturb tactile and nociceptive responses in a healthy subject. In some embodiments, the nano-micelle composition only suppresses hyperactive responses of somatosensory corticospinal neurons (CSNs) in a neuropathic pain state, but not in an intact healthy state.

[0033] In some embodiments, the nano-micelle composition exerts effects through spinal cord dorsal horn hyperactive circuits in the subject.

[0034] In some embodiments, the method comprises administering to the subject the nano-micelle composition in one or more doses to provide a dose of cannabinoid (e.g., CBD) equivalent to from about 0.2 mg / kg to about 8 mg / kg by body weight of the subject (e.g., human).

[0035] In some embodiments, the method comprises administering to the subject the nano-micelle composition via an oral, intravenous, intraperitoneal, ophthalmic, parenteral, topical, transdermal, subcutaneous, subdural, intravenous, intramuscular, intradermal, intrathecal, intraperitoneal, intracerebral, intraarterial, intralesional, pulmonary, nasal spray, sublingual, or mucosal route.

[0036] In another aspect, this disclosure provides use of the nano-micelle composition, as described herein, in the manufacture of a medicament for the treatment of neuropathic pain.

[0037] In yet another aspect, this disclosure additionally provides a nano-micelle composition, as described herein, for use in treating neuropathic pain.

[0038] The foregoing summary is not intended to define every aspect of the disclosure, and additional aspects are described in other sections, such as the following detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, or paragraph, or section of this document. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figures 1A, IB, 1C, ID, and IE show the development of an Inclusion-complex-enhanced Nano-micelle formulation for cannabidiol (CBD-IN). Figure 1A shows an illustration of the preparation of the CBD-IN formulation using pharmaceutical excipients including 2- hydroxypropyl-P-cyclodextrin (HPPCD), deionized water (dilLO), and polysorbate 80 (PS80). Figure IB shows the aqueous CBD concentrations in three different preparations: the CBD- HPPCD inclusion complex (CBD-I), the CBD-PS80 nano-micelle formulation (CBD-N), and the combined CBD-IN formulation, based on three independent batches per preparation, analyzed using one-way analysis of variance (ANOVA; F2,e = 509.0, P < 0.0001) followed by Bonferroni correction (CBD-I vs. CBD-IN, P < 0.0001; CBD-N vs. CBD-IN, P < 0.0001). Figure 1C shows Cryo-transmission electron microscopy (Cryo-TEM) images (upper panel) of micelles in PS80 vehicle, CBD-N, and CBD-IN solutions, with a scale bar of 50 nm, and micelle size distributions (lower panel) in the respective solutions. Figure ID shows cumulative micelle size distributions for PS80, CBD-N, and CBD-IN solutions based on ten images per preparation, analyzed using the Kolmogorov-Smirnov test (PS80 vs. CBD-IN, P < 0.0001; CBD-N vs. CBD-IN, P < 0.0001). In all panels, data are presented as mean ± standard error of the mean from independent samples, with significance indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and n.s., not significant. Figure IE shows aqueous CBD concentrations measured on Day 0 (DO), Day 14 (DI 4) and Day 28 (D28) after CBD-IN solution preparation (N=3 batches, one-way ANOVA, F2, 6=2.855, P=0.1345).

[0041] Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 21 show that CBD-IN improved brain delivery and analgesic efficacy. Figure 2A shows von Frey pain thresholds measured in spared nerve injury (SNI) mice 30 minutes after administration of various doses of CBD-IN (4, 20, and 100 mg / kg, intraperitoneally, N=10-12 mice), and compared to CBD-N at 100 mg / kg (intraperitoneally, N=8 mice). A one-way ANOVA (Fs, 37=20.37, P<0.0001) followed by Bonferroni correction revealed significant differences between CBD-IN 100 mg / kg and the lower doses (CBD-IN 20 mg / kg, P=0.0029; CBD-IN 4 mg / kg, P<0.0001) as well as between CBD-IN 100 mg / kg and CBD-N 100 mg / kg (P<0.0001). Vehicle control groups (VEH-IN and VEH-N) were tested to establish baseline pain thresholds. Figure 2B shows CBD brain concentrations measured at 0.5, 1, 1.5, and 2 hours following administration of CBD-IN or CBD-N at 100 mg / kg intraperitoneally (CBD-IN, N=6 mice; CBD-N, N=4 mice). A two-way ANOVA (Fi, 32=44.07, P<0.0001) with Sidak correction demonstrated significantly higher brain concentrations of CBD- IN at 0.5 hours (P<0.0001) and 1 hour (P=0.0051) compared to CBD-N. Figure 2C shows von Frey pain thresholds in SNI mice 30 minutes after oral administration of CBD-IN at 2, 10, or 50 mg / kg (N=10 mice per group), and compared to CBD-0 at 50 mg / kg (N=10 mice). A one-way ANOVA (F3, 36=20.41, P<0.0001) followed by Bonferroni correction showed that CBD-IN at 50 mg / kg was significantly more effective than CBD-IN at 10 mg / kg (P=0.0001), CBD-IN at 2 mg / kg (P<0.0001), and CBD-0 at 50 mg / kg (P<0.0001). Vehicle control groups (VEH-IN and VEH-O) were used to assess baseline thresholds. Figure 2D shows brain CBD concentrations measured at 0.5, 1, 2, and 4 hours following oral administration of CBD-IN or CBD-0 at 50 mg / kg (N=4 mice per group). A two-way ANOVA (Fi, 24=7.879, P=0.0098) with Sidak correction indicated significantly higher brain concentrations for CBD-IN compared to CBD-0 at 0.5 hours (P=0.0007) and 1 hour (P=0.0004). Figure 2E shows serum CBD concentrations measured at different time points (0.5, 1, 1.5, 2h) after CBD-IN and CBD-N (100 mg / kg i.p.) administration (each time point: CBD-IN, N=6; CBD-N, N=4. Two-way ANOVA, Fl, 32=0.432, P=0.5200). Figure 2F shows serum CBD concentrations measured at different time points (0.5, 1, 2, 4h) after CBD-IN and CBD-0 (50 mg / kg p.o.) administration (each time point: CBD-IN, N=4; CBD-O, N=4. Two-way ANOVA, Fl, 24=8.034, P=0.0092, followed by Sidak correction. CBD-IN vs. CBD-0 0.5h, P=0.0003; Ih, P=0.0003). Figure 2G shows liver CBD concentrations measured at different time points (0.5, 1, 2, 4h) after CBD-IN and CBD-0 (50 mg / kg p.o.) administration (each time point: CBD-IN, N=4; CBD-O, N=4. Two-way ANOVA, Fi, 24=4.627, P=0.0417, followed by Sidak correction. CBD-IN vs. CBD-0 Ih, P=0.0007). Figure 2H shows aqueous concentration of Cannabidiol (CBD-IN), Cannabinol (CBN-IN), and Cannabigerol (CBG-IN) in the Inclusion-complex- enhanced Nano-micelle formulation. The dotted line indicates the theoretical concentration of the cannabinoids in the aqueous solution if the cannabinoids used for the solubility test were fully dissolved (19 mM). Tire bottom panel shows the chemical structures of CBD, CBN, and CBG. Note that CBN and CBG are solubilized even more completely than CBD. Figure 21 shows von Frey pain threshold in SNI mice was measured 30 min after Cannabinol (CBN-IN) and Cannabigerol (CBG-IN) i.p. injection and compared with vehicle (VEH-IN). For CBN injection (100 mg / kg), a significant analgesic effect was observed. VEH-IN vs. CBN-IN, N=5, Mann Whitney test, P=0.0079, Cliff s delta=-l. For CBG injection (100 mg / kg), VEH-IN, CBG-IN, N=5, no analgesic effect was observed.

[0042] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31, 3J, 3K, 3L, and 3M show that CBD suppresses neuropathic pain while preserving normal somatosensory responses. Figure 3A shows the von Frey sensitivity spectrum in intact mice with no injection, CBD administered at 100 mg / kg intraperitoneally, and Buprenorphine (BUP) administered at 25 pg / kg intraperitoneally (N=6 mice per group, two-way repeated measures ANOVA, F2, 15=10.35, P=0.0015; Bonferroni correction: Intact vs. BUP at 1.4 g, P=0.043; Intact vs. CBD at 1.4 g, P=0.55). Figure 3B shows the von Frey spectrum in spared nerve injury (SNI) mice following vehicle (VEH) or CBD (100 mg / kg intraperitoneally) injection (VEH, N=10; CBD, N=ll; two-way RM ANOVA, Fl, 19= 175.2, P<0.0001 ). Figure 3C shows the von Frey threshold in intact mice treated with VEH or CBD (N=6; unpaired t-test, t=0.9159, df=10, P=0.3813, Cohen’s d=0.48796). Figure 3D shows the von Frey threshold in SNI mice treated with VEH (N=15) or CBD (N=15) (Mann-Whitney test, P<0.0001, Cliff’s delta= -0.99111). Figure 3E shows results of the dynamic light brush test in VEH-treated (N=ll) and CBD-treated (N=15) mice (unpaired t-test, t=2.406, df=24, P=0.0242, Cohen’s d=0.86157). Figure 3F shows the acetone test for cold allodynia in VEH and CBD-treated mice (N=12; Mann-Whitney test, P<0.0001, Cliff’s delta=0.93056). Figure 3G shows the results of the 56°C hot plate test in VEH and CBD-treated mice (N=15; Mann-Whitney test, P=0.1729, Cliff’s delta= -0.29333). Figure 3H shows the latency to fall, Figure 31 shows the distance traveled, and Figure 3J shows the maximum revolutions per minute (RPM) in the rotarod test in SNI mice 30 minutes post-injection with VEH or CBD (N=15; unpaired t-tests: Figure 3H, t=0.5258, df=28, P=0.6031, Cohen’s d=0.18681; Figure 31, t=0.5856, df=28, P=0.5629, Cohen’s d=0.20803; Figure 3J, t=0.5252, df=28, P=0.6036, Cohen’s d=0.18658). Figure 3K shows the total distance traveled and Figure 3L shows the velocity in the open field test in SNI mice 30 minutes after VEH or CBD treatment (N=15; unpaired t-tests: Figure 3K, t=1.414, df=28, P=0.1682, Cohen’s d=0.50251; Figure 3L, t=1.405, df=28, P=0.1709, Cohen’s d=0.49932). Figure 3M shows that repeated CBD- IN administration in SNI animals did not result in cumulative effects or drug tolerance. Von Frey threshold measurements in SNI animals after repeated CBD-IN administration. Baseline (N = 9) and VEH injection days: Day 0 (DO), Day 2 (D2), Day 4 (D4), Day 6 (D6), Day 8 (D8). One-way repeated measures (RM) ANOVA: F1.587.12.70 = 0.9612, P = 0.3886. CBD-IN injection (N = 9) on Day 1 (DI), Day 3 (D3), Day 5 (D5), Day 7 (D7), Day 9 (D9). One-way RM ANOVA: F3.240, 25.92 = 0.7395, P = 0.5477.

[0043] Figures 4A and 4B show that CBD reduces neuropathic pain-related neuronal activation across somatosensory pathways. Figure 4A shows a schematic illustrating the tactile stimulation- induced neuronal activation labeling method in FosTRAP2 Ail4 mice with spared nerve injury (SNI) Figure 4B shows representative images and statistical analysis of c-Fos expression patterns in different spinal cord and brain regions following VEH or CBD treatment in SNI animals subjected to tactile stimulation on the injured side (N = 7 mice per group). The laminae I-II and III-V of the dorsal horn at the L4 segment of the spinal cord (L4 DH Laminae I-II / III-V) show a significant reduction in c-Fos expression in laminae I-II with VEH versus CBD treatment (unpaired t-test, t = 11.32, df = 12, P < 0.0001, Cohen’s d = 5.6991), while no significant difference was observed in laminae III-V (t = 1.024, df = 12, P = 0.3260, Cohen’s d = 0.51232). Figure 4B also shows that the ventrobasal thalamus (VB) displayed reduced activation with CBD compared to VEH (Mann-Whitney test, P = 0.0023, Cliff’s delta = 0.91837). In the brain, reduced neuronal activation was also observed in the primary somatosensory cortex hindlimb area (HLS1) (t = 4.211, df = 12, P = 0.0012, Cohen’s d = 2.1067), and in the insular cortex (IC) (t = 3.610, df = 12, P = 0.0036, Cohen’s d = 1.8062). No significant difference was noted in the dentate gyrus (DG) (t = 0.9511, df = 12, P = 0.3603, Cohen’s d = 0.47583). Scale bar represents 200 pm.

[0044] Figure 5A, 5B, 5C, 5D, and 5E show that CBD attenuated neuronal hyperactivity in ex vivo spinal dorsal horn slices. Figure 5A shows a schematic illustration of the breeding strategy used to generate transgenic Vglut2:GCaMP6f mice. Figure 5B shows the experimental paradigm for calcium imaging performed in spinal cord slices. Figure 5C shows calcium event traces from excitatory neurons located in laminae I-II, averaged per slice, under 4-AP+VEH (gray) and 4- AP+CBD (green) conditions. Figure 5D shows the results of an area-under-the-curve (AUC) analysis of the slice-averaged calcium event traces measured between 360 and 540 seconds following drug incubation (4-AP+VEH: N=10 slices from 5 animals; 4-AP+CBD: N=9 slices from 5 animals), with statistical significance determined by a Mann-Whitney test (P=0.0172; Cliff’s del ta=O.6444).

[0045] Figures 6A, 6B, 6C, and 6D show that CBD suppresses allodynia-related hyperactivity of somatosensory corticospinal neurons in vivo. Figures 6A and 6C show an average CSN response (0-ls after stimulus onset) to von Frey stimuli after VEH or CBD injection in intact (Figure 6A) and SNI (Figure 6C) animals (Intact, VEH vs. CBD, N=5 mice, paired t-test, t=0.4369, df=4 P=0.6847. Cohen’s d=0.28672; SNT, VEH vs. CBD, N=6 mice, paired t-test, t=4.580, df=5, P=0.0059, Cohen’s d=2.0326). Figures 6B and 6D show a percentage of von Frey-responsive neurons after VEH or CBD injection in intact (Figure 6B) and SNI (Figure 6D) animals (intact, N=5 mice, paired t-test, VEH vs. CBD, t=0.6169, df=4, P=0.5707, Cohen’s d=-0.32177; SNI, N=6 mice, paired t-test, VEH vs. CBD. t=3.03, df=5, P=0.0291. Cohen’s d=1.2015).

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] Cannabis derivatives, including cannabidiol (CBD), have long been investigated for their therapeutic potential in the treatment of chronic pain. Although cannabidiol is considered nonpsychoactive and relatively safe, its low bioavailability, attributable to its highly hydrophobic lipid nature, poses a significant challenge for therapeutic applications. Accordingly, the present disclosure provides a novel cannabinoid nano-micelle composition comprising a cannabinoid inclusion complex encapsulated within nano-micelles. As demonstrated, the disclosed cannabinoid nano-micelle composition enables rapid delivery and results in elevated concentrations of the cannabinoid in the brain. Treatment with the cannabinoid nano-micelle composition significantly enhances cannabinoid bioavailability and produces robust analgesic effects against neuropathic pain by suppressing hyperactivity in the somatosensory nervous system.

[0048] Cannabinoid Nano-micelle Compositions

[0049] This disclosure addresses the need mentioned above in a number of aspects by providing a novel nano-micelle composition comprising a cannabinoid inclusion complex encapsulated in nano-micelles formed by a micelle-forming agent, wherein the cannabinoid inclusion complex comprises a cannabinoid encapsulated in cyclodextrin, and the cannabinoid inclusion complex slows down precipitation and / or crystallization of the cannabinoid during entrapment in the nanomicelles by the micelle-forming agent, wherein the micelle-forming agent is in an amount above a critical micelle concentration of the micelle-forming agent. In some embodiments, the micelle-forming agent is in an amount of from about 0.2 to about 1.6 mole per mole of the cannabinoid. In some embodiments, the micelle-forming agent is in an amount of from about 0.3 mole to about 1.5 mole, from about 0.4 mole to about 1.4 mole, from about 0.5 mole to about 1.3 mole, from about 0.6 mole to about 1.2 mole, from about 0.7 mole to about 1.1 mole, or from about 0.8 mole to about 1.1 mole, per mole of the cannabinoid.

[0050] In some embodiments, the micelle-forming agent is in an amount of from about 0.2 mole to about 0.4 mole, from about 0.4 mole to about 0.6 mole, from about 0.6 mole to about 0.8 mole, from about 0.8 mole to about 1.0 mole, from about 1.0 mole to about 1.2 mole, from about 1.2 mole to about 1.4 mole, or from 1.4 mole to about 1.6 mole, per mole of the cannabinoid.

[0051] In some embodiments, the micelle-forming agent is in an amount of about 0.2 mole, about 0.3 mole, about 0.4 mole, about 0.5 mole, about 0.6 mole, about 0.7 mole, about 0.8 mole, about 0.9 mole, about 1 mole, about 1.1 mole, about 1.2 mole, about 1.3 mole, about 1.4 mole, about 1.5 mole, or about 1.6 mole per mole of the cannabinoid.

[0052] In some embodiments, the cyclodextrin is in an amount of from about 1 to 2 mole per mole of the cannabinoid. In some embodiments, the cyclodextrin is in an amount of from about 1.1 mole to 1.9 mole, from about 1.2 mole to 1.8 mole, from about 1.3 mole to 1.7 mole, from about 1.4 mole to 1.6 mole, per mole of the cannabinoid.

[0053] In some embodiments, the cyclodextrin is in an amount of from about 1 mole to 1 .2 mole, from about 1.2 mole to 1.4 mole, from about 1.4 mole to 1.6 mole, from about 1.6 mole to 1.8 mole, or from about 1.8 mole to 2 mole, per mole of the cannabinoid.

[0054] In some embodiments, the cyclodextrin is in an amount of about 1 mole, about 1.05 mole, about 1.1 mole, about 1.15 mole, about 1.2 mole, about 1.25 mole, about 1.3 mole, about 1.35 mole, about 1.4 mole, about 1.45 mole, about 1.5 mole, about 1.55 mole, about 1.6 mole, about 1.65 mole, about 1.7 mole, about 1.75 mole, about 1.8 mole, about 1.85 mole, about 1.9 mole, about 1.95 mole, about 2 mole, per mole of the cannabinoid.

[0055] In some embodiments, the amount of the cyclodextrin is about 1 mole per mole of the cannabinoid. In some embodiments, the amount of the micelle-forming agent is about 0.81 mole per mole of the cannabinoid. In some embodiments, the cannabinoid, the cyclodextrin, and the micelle-forming agent have a molar ratio of 1 : 1 : 0.81.

[0056] In some embodiments, the cannabinoid inclusion complex is formed prior to entrapment in the nano-micelles by the micelle-forming agent. Cyclodextrins possess an internal non-polar cavity and hydroxyl groups located on their outer surface. The inclusion of hydrophobic compounds occurs primarily through hydrophobic interactions between the guest molecules and the interior walls of the cyclodextrin cavity. In some embodiments, a “cannabinoid inclusion complex” may refer to a complex formed between cyclodextrins and cannabinoids, for example, through hydrophobic interactions between cannabinoid molecules and the interior surfaces of the cyclodextrin cavity.

[0057] As used herein, a “micelle” refers to an aggregation, assembly, or shell of surfactant molecules (e.g, micelle-forming agents) dispersed in a liquid phase to form a colloidal suspension. The critical micelle concentration (CMC) is defined as the concentration of surfactant at which micelle formation is first observed in the solution. The CMC represents the point at which surfactant monomers begin to self-assemble into aggregates to minimize the system’s electrostatic and hydrophobic energy. At concentrations above the CMC, additional surfactant molecules are no longer available at the interface but instead exist as aggregates within the bulk of the solution.

[0058] In some embodiments, the cannabinoid is present in an amount ranging from about 0% to about 22.8% by weight of the nano-micelle composition. In some embodiments, the cannabinoid is present in an amount ranging from about 0.2% to about 22.8%, from about 0.4% to about 22.6%, from about 0.6% to about 22.4%, from about 0.8% to about 22.2%, from about 1% to about 22%, from about 1.2% to about 21.8%, from about 1.4% to about 21.6%, from about 1.6% to about 21.4%, from about 1.8% to about 21.2%, from about 2% to about 21%, from about 2.2% to about 20.8%, from about 2.4% to about 20.6%, from about 2.6% to about 20.4%, from about 2.8% to about 20.2%, from about 3% to about 20%, from about 3.2% to about 19.8%, from about 3.4% to about 19.6%, from about 3.6% to about 19.4%, from about 3.8% to about 19.2%, from about 4% to about 19%, from about 4.2% to about 18.8%, from about 4.4% to about 18.6%, from about 4.6% to about 18.4%, from about 4.8% to about 18.2%, from about 5% to about 18%, from about 5.2% to about 17.8%, from about 5.4% to about 17.6%, from about 5.6% to about 17.4%, from about 5.8% to about 17.2%, from about 6% to about 17%, from about 6.2% to about 16.8%, from about 6.4% to about 16.6%, from about 6.6% to about 16.4%, from about 6.8% to about 16.2%, from about 7% to about 16%, from about 7.2% to about 15.8%, from about 7.4% to about 15.6%, from about 7.6% to about 15.4%, from about 7.8% to about 15.2%, from about 8% to about 15%, from about 8.2% to about 14.8%, from about 8.4% to about 14.6%, from about 8.6% to about 14.4%, from about 8.8% to about 14.2%, from about 9% to about 14%, from about 9.2% to about 13.8%, from about 9.4% to about 13.6%, from about 9.6% to about 13.4%, from about 9.8% to about 13.2%, or from about 10% to about 13% by weight of the nano-micelle composition.

[0059] In some embodiments, the cyclodextrin is present in an amount ranging from about 0% to about 56.7% by weight of the nano-micelle composition. In some embodiments, the cyclodextrin is present in an amount ranging from about 0.2% to about 56.7%, from about 0.5% to about 56.4%, from about 0.8% to about 56.1%, from about 1.1% to about 55.8%, from about 1.4% to about 55.5%, from about 1.7% to about 55.2%, from about 2% to about 54.9%, from about 2.3% to about 54.6%, from about 2.6% to about 54.3%, from about 2.9% to about 54%, from about 3.2% to about 53.7%, from about 3.5% to about 53.4%, from about 3.8% to about 53.1%, from about 4.1% to about 52.8%, from about 4.4% to about 52.5%, from about 4.7% to about 52.2%, from about 5% to about 51.9%, from about 5.3% to about 51.6%, from about 5.6% to about 51.3%, from about 5.9% to about 51%, from about 6.2% to about 50.7%, from about 6.5% to about 50.4%, from about 6.8% to about 50.1%, from about 7.1% to about 49.8%, from about 7.4% to about 49.5%, from about 7.7% to about 49.2%, from about 8% to about 48.9%, from about 8.3% to about 48.6%, from about 8.6% to about 48.3%, from about 8.9% to about 48%, from about 9.2% to about 47.7%, from about 9.5% to about 47.4%, from about 9.8% to about 47.1%, from about 10.1% to about 46.8%, from about 10.4% to about 46.5%, from about 10.7% to about 46.2%, from about 11% to about 45.9%, from about 11.3% to about 45.6%, from about 11.6% to about 45.3%, from about 11.9% to about 45%, from about 12.2% to about 44.7%, from about 12.5% to about 44.4%, from about 12.8% to about 44.1%, from about 13.1% to about 43.8%, from about 13.4% to about 43.5%, from about 13.7% to about 43.2%, from about 14% to about 42.9%, from about 14.3% to about 42.6%, from about 14.6% to about 42.3%, from about 14.9% to about 42%, from about 15.2% to about 41.7%, from about 15.5% to about 41.4%, from about 15.8% to about 41.1%, from about 16.1% to about 40.8%, from about 16.4% to about 40.5%, from about 16.7% to about 40.2%, from about 17% to about 39.9%, from about 17.3% to about 39.6%, from about 17.6% to about 39.3%, from about 17.9% to about 39%, from about 18.2% to about 38.7%, from about 18.5% to about 38.4%, from about 18.8% to about 38.1%, from about 19.1% to about 37.8%, from about 19.4% to about 37.5%, from about 19.7% to about 37.2%, from about 20% to about 36.9%, from about 20.3% to about 36.6%, from about 20.6% to about 36.3%, from about 20.9% to about 36%, from about 21.2% to about 35.7%, from about 21.5% to about 35.4%, from about 21.8% to about 35.1%, from about 22.1% to about 34.8%, from about 22.4% to about 34.5%, from about 22.7% to about 34.2%, from about 23% to about 33.9%, from about 23.3% to about 33.6%, from about 23.6% to about 33.3%, or from about 23.9% to about 33% by weight of the nano-micelle composition.

[0060] In some embodiments, the micelle-forming agent is present in an amount ranging from about 38.4% to about 100% by weight of the nano-micelle composition. In some embodiments, the micelle-forming agent is present in an amount ranging from about 38.4% to about 100%, from about 38.8% to about 99.6%, from about 39.2% to about 99.2%, from about 39.6% to about 98.8%, from about 40% to about 98.4%, from about 40.4% to about 98%, from about 40.8% to about 97.6%, from about 41.2% to about 97.2%, from about 41.6% to about 96.8%, from about 42% to about 96.4%, from about 42.4% to about 96%, from about 42.8% to about 95.6%, from about 43.2% to about 95.2%, from about 43.6% to about 94.8%, from about 44% to about 94.4%, from about 44.4% to about 94%, from about 44.8% to about 93.6%, from about 45.2% to about 93.2%, from about 45.6% to about 92.8%, from about 46% to about 92.4%, from about 46.4% to about 92%, from about 46.8% to about 91.6%, from about 47.2% to about 91.2%, from about 47.6% to about 90.8%, from about 48% to about 90.4%, from about 48.4% to about 90%, from about 48.8% to about 89.6%, from about 49.2% to about 89.2%, from about 49.6% to about 88.8%, from about 50% to about 88.4%, from about 50.4% to about 88%, from about 50.8% to about 87.6%, from about 51.2% to about 87.2%, from about 51.6% to about 86.8%, from about 52% to about 86.4%, from about 52.4% to about 86%, from about 52.8% to about 85.6%, from about 53.2% to about 85.2%, from about 53.6% to about 84.8%, from about 54% to about 84.4%, from about 54.4% to about 84%, from about 54.8% to about 83.6%, from about 55.2% to about 83.2%, from about 55.6% to about 82.8%, from about 56% to about 82.4%, from about 56.4% to about 82%, from about 56.8% to about 81.6%, from about 57.2% to about 81.2%, from about 57.6% to about 80.8%, from about 58% to about 80.4%, from about 58.4% to about 80%, from about 58.8% to about 79.6%, from about 59.2% to about 79.2%, from about 59.6% to about 78.8%, from about 60% to about 78.4%, from about 60.4% to about 78%, from about 60.8% to about 77.6%, from about 61.2% to about 77.2%, from about 61.6% to about 76.8%, from about 62% to about 76.4%, from about 62.4% to about 76%, from about 62.8% to about 75.6%, from about 63.2% to about 75.2%, from about 63.6% to about 74.8%, from about 64% to about 74.4%, from about 64.4% to about 74%, from about 64.8% to about 73.6%, or from about 65.2% to about 73.2% by weight of the nano-micelle composition.

[0061] In some embodiments, the nano-micelle composition comprises: about 11.3% by weight of the cannabinoid; about 50.3% by weight of the cyclodextrin; and about 38.4% by weight of the micelle-forming agent.

[0062] In some embodiments, the mean particle diameter of the nano-micelles is from about 16 nm to about 32 nm. In some embodiments, the mean particle diameter of the nano-micelles is from about 18 nm to about 32 nm. In some embodiments, the mean particle diameter of the nanomicelles is from about 20 nm to about 32 nm. In some embodiments, the mean particle diameter of the nano-micelles is from about 24 nm to about 32 nm. In some embodiments, the mean particle diameter of the nano-micelles is from about 26 nm to about 32 nm. In some embodiments, the mean particle diameter of the nano-micelles is from about 28 nm to about 32 nm. In some embodiments, the mean particle diameter of the nano-micelles is from about 16 nm to about 30 nm. In some embodiments, the mean particle diameter of the nano-micelles is from about 16 nm to about 28 nm. In some embodiments, the mean particle diameter of the nano-micelles is from about 16 nm to about 26 nm. In some embodiments, the mean particle diameter of the nanomicelles is from about 16 nm to about 24 nm. In some embodiments, the mean particle diameter of the nano-micelles is from about 16 nm to about 22 nm. In some embodiments, the mean particle diameter of the nano-micelles is from about 16 nm to about 20 nm.

[0063] In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nano-micelles have a particle diameter ranging from about 16 nm to about 32 nm, from about 18 nm to about 32 nm, from about 20 nm to about 32 nm, from about 22 nm to about 32 nm, from about 24 nm to about 32 nm, from about 26 nm to about 32 nm, or from about 28 nm to about 32 nm. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nano-micelles have a particle diameter ranging from about 16 nm to about 32 nm, from about 16 nm to about 30 nm, from about 16 nm to about 28 nm, from about 16 nm to about 26 nm, from about 16 nm to about 24 nm, from about 16 nm to about 22 nm, or from about 16 nm to about 20 nm. In some embodiments, the nano-micelles have an overall size distribution ranging from about 5 nm to about 100 nm. In some embodiments, the nano-micelles have an overall size distribution ranging from about 5 nm to about 100 nm, from about 10 nm to about 100 nm, from about 15 nm to about 100 nm, from about 20 nm to about 100 nm, from about 25 nm to about 100 nm, from about 30 nm to about 100 nm, from about 35 nm to about 100 nm, from about 40 nm to about 100 nm, from about 45 nm to about 100 nm, or from about 50 nm to about 100 nm.

[0064] In some embodiments, the nano-micelles have an overall size distribution ranging from about 5 nm to about 95 nm, from about 5 nm to about 90 nm, from about 5 nm to about 85 nm, from about 5 nm to about 80 nm, from about 5 nm to about 75 nm, from about 5 nm to about 70 nm, from about 5 nm to about 65 nm, from about 5 nm to about 60 nm, from about 5 nm to about 55 nm, or from about 5 nm to about 50 nm.

[0065] In some embodiments, the nano-micelles have an overall size distribution ranging from about 5 nm to about 100 nm, from about 10 nm to about 95 nm, from about 15 nm to about 90 nm, from about 20 nm to about 85 nm, from about 25 nm to about 80 nm, from about 30 nm to about 75 nm, from about 35 nm to about 70 nm, from about 40 nm to about 65 nm, or from about 45 nm to about 60 nm.

[0066] In some embodiments, the cannabinoid has a Cmax of 6 ± 3 pg / g (e.g., about 6 pg / g) in brain when the nano-micelle composition is administered to a mouse or rat at 100 mg / kg intraperitoneal condition.

[0067] In some embodiments, the cannabinoid has a Cmax of 7.8 ± 3 pg / ml (e.g., about 7.8 pg / ml) in serum when the nano-micelle composition is administered to a mouse or rat at 100 mg / kg intraperitoneal condition.

[0068] In some embodiments, the cannabinoid has a half life of 2 ± 0.5 hours (e.g., about 2 hours) in brain after the nano-micelle composition is administered to a mouse or rat.

[0069] Cannabinoids

[0070] Cannabinoids are compounds that act on cannabinoid receptors in cells, which can alter neurotransmitter release in the brain. Cannabinoids were originally identified in Cannabis sativa L., the source of marijuana and hashish. Marijuana or its components have been reported in the scientific literature to alleviate symptoms associated with a broad range of conditions, including multiple sclerosis and various forms of muscular spasm (e.g, uterine and bowel cramps), movement disorders, pain (including migraine headache), glaucoma, asthma, inflammation, insomnia, and high blood pressure. Cannabinoids may also have utility as antioxidants, anxiolytics, anticonvulsants, antidepressants, antipsychotics, or anticancer agents, as well as appetite stimulants.

[0071] Many chemically related compounds, collectively classified as cannabinoids, have been isolated from Cannabis sativa L., Cannabis Mica, and Cannabis ruderalis, including tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN). In addition, various synthetic ligands for cannabinoid receptors have been developed. Cannabinoids are commonly categorized into groups including classical cannabinoids, non-classical cannabinoids, aminoalkylindole derivatives, and eicosanoids. Classical cannabinoids are isolated from Cannabis sativa L. or may comprise synthetic analogs of such compounds. Non-classical cannabinoids include bi- or tricyclic analogs of THC, whereas aminoalkylindoles form a structurally distinct class from both classical and non-classical cannabinoids.

[0072] A cannabinoid may be a classical, non-classical, or synthetic analog that is insoluble or sparingly soluble in water and that may provide a medicinal, nutritional, or recreational benefit to a user. As used herein, the term “insoluble” refers to compounds in which not more than 0.1 g dissolves in 100 mL of water, while the term “sparingly soluble” refers to compounds in which not more than 3.3 g dissolves in 100 mL of water.

[0073] In some embodiments, the cannabinoid component may comprise a single cannabinoid compound or a plurality of cannabinoid compounds, either in substantially pure form or in combination with other compounds. The cannabinoid component may be isolated or purified from a natural source, such as a cannabis plant, or may be a chemically synthesized cannabinoid compound. The cannabinoid component can include, but is not limited to, cannabinoid compounds that naturally occur in varying combinations and relative quantities in the plant tissues of various species, subspecies, hybrids, strains, chemovars, and other genetic variants of the genus Cannabis, including material that may be classified as “marijuana” or “hemp” in accordance with various legal or technical definitions and standards.

[0074] In some embodiments, the cannabinoid component can comprise a cannabinoid molecular distillate that includes a plurality of cannabinoid compounds. In certain embodiments, the cannabinoid molecular distillate can comprise at least about 80% cannabinoid compounds by weight or more.

[0075] Cannabidiol (CBD) IUPAC: 2-[(lR,6R)-6-isopropenyl-3-methylcyclohex-2-en-l-yl]-5- pentylbenzene-l,3-diol, having the following formula: is a cannabinoid that can be used in this disclosure. Although CBD is not known to have the psychotropic effects of THC, it is still considered to have a wide scope of potential medical and therapeutic applications. CBD may be derived from industrial hemp which has negligible amounts of THC, and may be legally grown and consumed in Canada and the United States.

[0076] An exemplary water-insoluble cannabinoid comprises tetrahydrocannabinol (THC), including delta-9-tetrahydrocannabinol (A9-THC), which is acknowledged as the primary psychoactive compound in marijuana. The cannabinoid component may also include various other cannabinoids, such as tetrahydrocannabinolic acid (THCA), delta-8-tetrahydrocannabinol (A8- THC), cannabidiolic acid (CBDA), cannabinol (CBN), cannabinolic acid (CBNA), tetrahydrocannabivarin (THCV), tetrahydrocannabivarinic acid (THCVA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), cannabigerol (CBG), cannabigerolic acid (CBGA), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabinodiol (CBND), and cannabinodiolic acid (CBND A). In some embodiments, the cannabinoid component may comprise both decarboxylated cannabinoid compounds and their corresponding carboxylic acid forms, such as both THC and THCA. A cannabis extract or cannabinoid component may be decarboxylated, for example by heating, and in various embodiments, the cannabinoid component may be substantially devoid of the acid forms of cannabinoid compounds. As used herein, the term “substantially devoid” refers to a composition containing either an undetectable amount of a substance or less than about 0.1% by weight. In some embodiments, the cannabinoid is selected from the group consisting of A9- tetrahydrocannabinol (THC), A8-tetrahydrocannabinol, A9-tetrahydrocannabinol propyl analog (THCV), cannabidiol (CBD), cannabidiol propyl analog (CBDV), cannabinol (CBN), cannabichromene, cannabichromene propyl analog, and cannabigerol, or derivatives and combinations thereof. In some embodiments, the cannabinoid is cannabidiol (CBD).

[0077] In some embodiments, the cannabinoid component may comprise isomers, stereoisomers, homologs, salts, or other forms or variants of the cannabinoid compounds disclosed herein. “Isomers” are compounds that share the same molecular formula but differ in structure. “Stereoisomers” are isomers that differ only in the spatial arrangement of atoms, i.e., they possess different stereochemical configurations. “Enantiomers” are pairs of stereoisomers that are non- superimposable mirror images of one another. A 1 : 1 mixture of enantiomers is referred to as a “racemic” mixture. The term “(±)” is used to designate a racemic mixture where applicable. “Diastereoisomers” are stereoisomers with at least two asymmetric centers that are not mirror images of each other. Absolute stereochemistry is denoted according to the Cahn-Ingold-Prelog R / S system. When a compound exists as a pure enantiomer, the configuration at each chiral center may be specified as either (R) or (S). Compounds of unknown absolute configuration that have been resolved may be designated as (+) or (-), depending on whether they rotate plane-polarized light in the dextrorotatory or levorotatory direction, respectively, at the sodium D line wavelength. Certain compounds described herein may contain one or more asymmetric centers and may exist as enantiomers, diastereomers, or other stereoisomeric forms. Such compounds may be defined by their absolute stereochemistry as (R) or (S). The present chemical entities, pharmaceutical compositions, and methods are intended to encompass all such isomeric forms, including racemic mixtures, optically pure forms, and intermediate mixtures. Optically active (R)- and (S)-isomers may be prepared using chiral synthons or reagents or resolved using conventional techniques. Where the described compounds contain olefinic double bonds or other geometrically asymmetric centers, and unless otherwise specified, both E and Z geometric isomers are intended to be included.

[0078] Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are a type of stereoisomer consisting of two molecules that are non-superimposable mirror images of each other, most commonly due to the presence of an asymmetrically substituted carbon atom that serves as a chiral center. The term “enantiomer” refers to one of a pair of such mirror-image molecules. Diastereomers are stereoisomers that are not related as mirror images, typically because they contain two or more asymmetrically substituted carbon atoms.

[0079] The designations “R” and “S” denote the absolute configuration of substituents around one or more chiral carbon atoms, while “R*” and “S*” denote relative configurations. The symbol in a structural formula indicates the presence of a chiral carbon center.

[0080] The term “racemate” or “racemic mixture” refers to a composition comprising equimolar quantities of two enantiomers. Such mixtures are optically inactive, meaning they do not rotate the plane of polarized light.

[0081] The term “geometric isomer” refers to isomers that differ in the spatial orientation of substituent atoms relative to a carbon-carbon double bond, a cycloalkyl ring, or a bridged bicyclic system. For example, substituents on each side of a carbon-carbon double bond may be arranged in either an E configuration (substituents on opposite sides) or a Z configuration (substituents on the same side). The terms “R,” “S,” “St,” “R*,” “E,” “Z,” “cis,” and “trans” indicate specific configurations relative to the core molecule.

[0082] A “derivative,” as used herein, refers to a chemical substance that is structurally related to another, typically referred to as a “parent” compound. A derivative may be formed from the parent compound in one or more synthetic steps. The phrase “closely related derivative” refers to a derivative whose molecular weight does not exceed that of the parent compound by more than 50%, and which possesses general physical and chemical properties similar to those of the parent compound. The term “pharmaceutically active derivative” refers to any compound that, upon administration to a subject, is capable of providing, either directly or indirectly, a therapeutic activity as disclosed herein.

[0083] The term “analog” refers to a small organic compound, nucleotide, protein, or polypeptide that possesses similar or identical activity or function(s) as a compound, nucleotide, protein, or polypeptide having the desired activity described herein. An analog need not necessarily include a sequence or structure that is similar or identical to that of the preferred embodiments.

[0084] The term “prodrug” refers to a compound that may be converted under physiological conditions or by solvolysis into a biologically active compound as described herein. Thus, a prodrug is a pharmaceutically acceptable precursor of a biologically active compound. A prodrug may be inactive when administered to a subject but is converted in vivo to an active compound, for example, by hydrolysis. Prodrugs may offer advantages such as improved solubility, enhanced tissue compatibility, or delayed release in a mammalian subject (see, e.g., Bundgaard, H., Design of Prodrugs, Elsevier, Amsterdam, 1985). The term “prodrug” also encompasses covalently bonded carriers that release the active compound in vivo upon administration to a subject. Prodrugs of an active compound may be prepared by modifying functional groups present in the active compound such that the modifications are cleaved, either during routine manipulation or in vivo, to yield the active parent compound. Prodrugs include, for example, compounds in which a hydroxy, amino, or mercapto group is bonded to any group that, upon administration to a mammalian subject, is cleaved to form a free hydroxy, amino, or mercapto group, respectively. Non-limiting examples of prodrugs include acetates, formates, and benzoate derivatives of alcohols; various ester derivatives of carboxylic acids; and acetamide, formamide, and benzamide derivatives of amine functional groups. Various prodrug forms are well known in the art and are described in: (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., Chapter 31, Academic Press, 1996; (b) Design of Prodrugs, edited by H. Bundgaard, Elsevier, 1985; (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larsen and H. Bundgaard, eds., Chapter 5, pages 113-191, Harwood Academic Publishers, 1991; and (d) Hydrolysis in Drug and Prodrug Metabolism, Bernard Testa and Joachim M. Mayer, Wiley-VCH, 2003.

[0085] As used herein, the term “pharmaceutically acceptable” refers to compounds, materials, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other adverse effects, commensurate with a reasonable benefit-tori sk ratio.

[0086] As used herein, the term “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable, non-toxic acids, including inorganic acids, organic acids, or their respective solvates, hydrates, or clathrates.

[0087] Cyclodextrin

[0088] Cyclodextrins (CDs) are cyclic oligosaccharides composed of (a-l,4)-linked a-D- glucopyranose units, featuring a lipophilic central cavity and a hydrophilic outer surface (Frdmming and Szejtli, 1994). Cyclodextrins are capable of forming inclusion complexes with various drugs by accommodating either the entire drug molecule or, more commonly, the lipophilic portion of the molecule within their cavity. The most abundant natural cyclodextrins are a- cyclodextrin (a-CD), P-cyclodextrin (P-CD), and y-cyclodextrin (y-CD), which contain six, seven, and eight glucopyranose units, respectively. Among these, P-CD is considered the most pharmaceutically useful complexing agent due to its optimal cavity size, commercial availability, low cost, and favorable physicochemical properties.

[0089] However, the aqueous solubility of P-CD is limited. To address this limitation, several water-soluble derivatives of P-CD have been developed, including hydroxypropyl-P-cyclodextrin (HPpCD), sulfobutylether-P-cyclodextrin (SBE-P-CD), maltosyl-P-cyclodextrin (ML-P-CD), and methylated P-cyclodextrins such as dimethyl-P-cyclodextrin (DM-P-CD), trimethyl-P- cyclodextrin (TM-P-CD), and randomly methylated P-cyclodextrin (RM-P-CD).

[0090] Cyclodextrin molecules exhibit a toroidal ring structure formed from sugar moieties, producing a lipophilic internal cavity and a hydrophilic external shell. P-Cyclodextrins consist of seven-membered rings, and 2-hydroxypropyl-P-cyclodextrin (HPpCD) is a chemically modified, more water-soluble derivative of P-cyclodextrin. The chemical structures of a-cyclodextrin, P- cyclodextrin, and y-cyclodextrin are shown below.

[0091] In some embodiments, the cyclodextrin is selected from the group consisting of a- cyclodextrin, P-cyclodextrin, y-cyclodextrin, 2-hydroxypropyl-P-cyclodextrin (HPpCD), sulfobutylether P-cyclodextrin sodium salt, randomly methylated P-cyclodextrin, branched P- cyclodextrin, and derivatives thereof. In some embodiments, the cyclodextrin is 2-hydroxypropyl- P-cyclodextrin (HPpCD). In the disclosed nano-micelle composition, cannabinoids are complexed within the cyclodextrin ring structure. As a result, the complex exhibits increased water solubility due to the externally facing hydroxyl groups, while preserving the biological activity of the cannabinoid encapsulated within the ring. P-Cyclodextrins are preferred because, without being bound to any particular theory, it is believed that the relative sizes of the cannabinoids and the cyclodextrin ring are closely matched. Furthermore, P-cyclodextrins, and in particular HPPCD, are among the safer cyclodextrins for human ingestion or application, thereby enabling higher active ingredient loading per tolerable dose. Accordingly, HPpCD is considered a superior complexation agent. HPpCD has also been approved for use in ocular formulations and is a water-soluble cyclodextrin recommended for rectal administration.

[0092] At the molecular level, the inclusion complex may exhibit a 1 : 1 molar ratio between the cannabinoid and the cyclodextrin. In some embodiments, the inclusion complex is formed by mixing a molar excess of cyclodextrin with the cannabinoid. The resulting inclusion complex may be in the form of a white, semi-opaque fine powder at room temperature that is water-soluble and suitable for use in stabilized, water-based formulations at high dosages.

[0093] Micelle-forming agents

[0094] Micelles are typically formed from amphiphilic molecules. These molecules possess a charged or polar head group linked to one or more long hydrocarbon chains. Common examples include lipids and surfactants. In an aqueous environment, the hydrophobic tails of amphiphilic molecules cluster together, forming a core within the micelle, while the hydrophilic heads orient outward to interact with the surrounding water. Micelle formation is favored above a critical micelle concentration (CMC); below this concentration, the solution primarily contains monomeric amphiphiles. However, once the CMC is exceeded, micelles spontaneously form, typically involving 30 to 100 molecules.

[0095] In some embodiments, a micelle-forming agent may be a surfactant, including but not limited to polysorbate 20 (Tween™ 20), polysorbate 40 (Tween™ 40), polysorbate 60 (Tween™ 60), polysorbate 65 (Tween™ 65), polysorbate 80 (Tween™ 80), polysorbate 85 (Tween™ 85), Triton™ N-101, Triton™ X-100, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol Hl 5), poly oxy ethylene-35 ricinoleate (Cremophor EL™), soy lecithin, pol oxamers, hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, methoxyhexadecylglycerol, Pluronic polyols, polyamines (e.g., pyran, dextran sulfate, poly IC, carbopol), peptides (e.g., muramyl peptide and dipeptide, dimethylglycine, tuftsin), oil emulsions, mineral gels (e.g., aluminum phosphate), and immune-stimulating complexes (ISCOMs).

[0096] In some embodiments, the surfactant may be selected from the group consisting of long- chain triglycerides, such as C16-C18 triglycerides, and linoleic acid. In some embodiments, the surfactant is advantageously selected for its safety (e.g., food-grade status), perceived natural origin, taste, and consumer acceptability.

[0097] In some embodiments, the micelle-forming agent comprises stearoyl polyoxyl-32 glycerides, lauroyl polyoxyl-32 glycerides, polyoxyethylene alkyl ethers, polysorbate 80, polysorbate 60, polysorbate 20, or a combination thereof. In some embodiments, the micelleforming agent comprises polysorbate 80.

[0098] Pharmaceutical Compositions and Kits

[0099] As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture comprising at least one component useful within the disclosure and one or more additional components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates the administration of one or more components of the disclosed compositions to an organism.

[0100] In some embodiments, the composition (e.g., an injectable composition) may include nanomicelles in an amount ranging from about 5% to about 90% by weight (e.g., about 5%, 8%, 11%, 14%, 17%, 20%, 23%, 26%, 29%, 32%, 35%, 38%, 41%, 44%, 47%, 50%, 53%, 56%, 59%, 62%, 65%, 68%, 71%, 74%, 77%, 80%, 83%, 86%, 89%, or 90%) or a pharmaceutically acceptable carrier in an amount ranging from about 10% to about 90% by weight (e.g., about 10%, 13%, 16%, 19%, 22%, 25%, 28%, 31%, 34%, 37%, 40%, 43%, 46%, 49%, 52%, 55%, 58%, 61%, 64%, 67%, 70%, 73%, 76%, 79%, 82%, 85%, 88%, or 90%). The relative amounts of the nano-micelles and the carrier may vary depending on the specific use case, including factors such as the size of the needle used, the types of nano-micelles and carriers employed, the area of injection, and whether cells are associated with the nano-micelles prior to injection. In some embodiments, the composition may further include at least one anti -cryogenic or anti-freeze agent. Suitable anti-cryogenic or anti-freeze agents may include polyhydric alcohols, including polyol esters composed of diols such as ethylene glycol, 1,3 -propanediol, propylene glycol, 1,4-butanediol, 1,2-butanediol, 2-methyl-l,3-propanediol, 1,5-pentanediol, neopentyl glycol, 1,6-hexanediol, 2-ethyl-2-methyl-l,3-propanediol, 1,7-heptanediol, 2-methyl-2-propyl- 1,3-propanediol, 2, 2-di ethyl- 1,3 -propanediol, 1,8-octanediol, 1,9-nonanediol, 1,10-decanediol, 1,11 -undecanediol, and 1,12-dodecanediol. Other polyols having 3 to 20 hydroxyl groups may also be used, such as trimethylol ethane, trimethylolpropane, trimethylolbutane, ditrimethylolpropane, tri-trimethylolpropane, pentaerythritol, di-pentaerythritol, tri-pentaerythritol, glycerol, polyglycerol (e.g., glycerol trimer), 1,3,5-pentanetriol, sorbitol, sorbitan, sorbitolglycerin condensate, and other polyhydric alcohols including adonitol, arabitol, xylitol, mannitol, xylose, arabinose, ribose, rhamnose, glucose, fructose, galactose, mannose, sorbose, and disaccharides and oligosaccharides such as cellobiose, maltose, isomaltose, trehalose, sucrose, raffinose, gentianose, and melezitose.

[0101] Techniques and formulations for preparing pharmaceutical compositions may generally be found in Remington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous routes. For injection, the agents may be formulated in liquid solutions, for example, in physiologically compatible buffers such as Hank’s solution or Ringer’s solution. In addition, the agents may be formulated in solid form and reconstituted or suspended immediately prior to use. Lyophilized formulations are also contemplated.

[0102] A composition may be prepared, for example, by admixing the disclosed nano-micelles and / or at least one pharmaceutically acceptable salt thereof with at least one excipient suitable for the preparation of an aqueous suspension. Non-limiting examples of such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, alginic acid, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents such as naturally occurring phosphatides e.g., lecithin); condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate); condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethylene oxycetanol); condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate); and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g, polyethylene sorbitan monooleate). The aqueous suspension may also contain at least one preservative (e.g, ethyl or n-propyl p-hydroxybenzoate), one or more coloring agents, flavoring agents, and / or sweetening agents such as sucrose, saccharin, and aspartame.

[0103] Formulations for parenteral administration may be provided in the form of aqueous or nonaqueous isotonic sterile injection solutions or suspensions. Such solutions and suspensions may be prepared from sterile powders or granules using any of the carriers or diluents described herein for oral formulations, or using other suitable dispersing or wetting agents and suspending agents. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, com oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, gum tragacanth, and / or various buffer systems. Other adjuvants and routes of administration well known in the pharmaceutical arts may also be employed. The active ingredient may additionally be administered by injection in compositions containing suitable carriers, such as saline, dextrose, or water, or using solubilization approaches including cyclodextrins (e.g., Captisol), cosolvent systems (e.g., propylene glycol), or micellar solubilization systems (e.g, Tween 80).

[0104] The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, for example, a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid, may also be used in the preparation of injectables.

[0105] Pharmaceutically acceptable carriers, adjuvants, and vehicles that may be used in the pharmaceutical compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as alpha-tocopherol polyethylene glycol 1000 succinate, and surfactants commonly used in pharmaceutical dosage forms, such as Tweens and poly ethoxylated castor oil (e.g., Cremophor surfactant, BASF), as well as other similar polymeric delivery matrices. Additional carriers may include serum proteins such as human serum albumin, buffer substances such as phosphates and glycine, preservatives such as sorbic acid and potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts. Other suitable materials include colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, and wool fat. Cyclodextrins, including alpha-, beta-, and gamma-cyclodextrins, or chemically modified derivatives such as hydroxyalkyl cyclodextrins (e.g., 2-hydroxypropyl- and 3-hydroxypropyl-cyclodextrins), may also be advantageously used to enhance the delivery of the compounds described herein.

[0106] The pharmaceutically active compounds of this disclosure may be processed in accordance with conventional pharmaceutical methods to produce medicinal agents for administration to patients, including humans and other mammals. The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and may include conventional adjuvants, such as additives, preservatives, stabilizers, wetting agents, emulsifiers, and buffers. Tablets and pills may additionally be prepared with enteric coatings. Such compositions may also include adjuvants such as wetting, sweetening, flavoring, and perfuming agents.

[0107] Pharmaceutical compositions of this disclosure may include at least one compound and / or at least one pharmaceutically acceptable salt thereof, and optionally an additional agent selected from any pharmaceutically acceptable carrier, adjuvant, or vehicle. Alternative compositions may include a compound described herein, or a prodrug thereof, in combination with a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0108] The nano-micelles, or pharmaceutical compositions thereof as described herein, may be provided in a kit. In some embodiments, the kit includes a container that contains the cannabinoid nano-micelles, or a composition thereof, and optionally informational material. The informational material may be descriptive, instructional, marketing-related, or otherwise related to the methods described herein and / or the therapeutic use of the agents. For example, the kit may include instructions for manufacturing, therapeutic regimens, and recommended periods of administration. In some embodiments, the kit may also include an additional therapeutic agent. The kit may include one or more containers, each containing a different reagent. For example, the kit may include a first container that contains the composition and a second container that contains the additional therapeutic agent.

[0109] The containers may include unit dosages of the pharmaceutical composition. In addition to the composition, the kit may also include other components such as a solvent or buffer, an adjuvant, a stabilizer, or a preservative. The kit may optionally include a device suitable for administering the composition, such as a syringe or another suitable delivery device. The device may be pre-loaded with one or both of the agents, or it may be provided empty but suitable for loading.

[0110] Methods of Preparing Cannabinoid Nano-micelles

[0111] In another aspect, this disclosure provides a method for preparing a nano-micelle composition as described herein. In some embodiments, the method comprises: preparing a cannabinoid solution by dissolving a cannabinoid in a solvent; gradually adding cyclodextrin to the cannabinoid solution with agitation to obtain a mixture; incubating the mixture at a predetermined temperature for a predetermined time period to form a cannabinoid inclusion complex in which the cannabinoid is encapsulated within the cyclodextrin; and adding a micelleforming agent to the mixture to form nano-micelles that encapsulate the cannabinoid inclusion complex.

[0112] In some embodiments, the solvent comprises ethanol. In some embodiments, the cannabinoid and the solvent are present in a molar ratio of about 1:20 e.g., 1, 1.2, 1.4, 1.6, 1.8, 2,

[0113] 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8, 10, 10.2, 10.4, 10.6, 10.8, 11, 11.2,

[0114] 11.4, 11.6, 11.8, 12, 12.2, 12.4, 12.6, 12.8, 13, 13.2, 13.4, 13.6, 13.8, 14, 14.2, 14.4, 14.6, 14.8, 15,

[0115] 15.2, 15.4, 15.6, 15.8, 16, 16.2, 16.4, 16.6, 16.8, 17, 17.2, 17.4, 17.6, 17.8, 18, 18.2, 18.4, 18.6, 18.8, 19, 19.2, 19.4, 19.6, 19.8, 20).

[0116] In some embodiments, the predetermined temperature is from about 25°C to about 45°C (e.g., about 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44,

[0117] 44.5, or 45°C). In some embodiments, the predetermined time period is from about 1 hour to about 96 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,

[0118] 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,

[0119] 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours).

[0120] As an example, cannabinoid nano-micelles may be prepared using the following process. Fifty milligrams of CBD (e.g., Cayman Chemical, #90080) was dissolved in 185 pL of ethanol (e.g., molar ratio 1 : 1), and 222 mg of HPpCD (e.g., Millipore Sigma, C0926-5G) was dissolved in 143 pL of water (e.g., molar ratio 1: 1). The HP CD solution was gradually added to the CBD ethanol solution with vortexing. After mixing, the tube was sealed and covered with aluminum foil. The mixture was incubated at room temperature (e.g., 25°C) for 48 hours. Following incubation, the resulting CBD-HPPCD inclusion complex was diluted with water, and 160 pL of polysorbate 80 (PS80) was added to reach a final volume of 8 mb (PS80 at 2% v / v).

[0121] Methods of Treatment

[0122] The disclosed cannabinoid nano-micelle compositions can be administered to a subject for the treatment or prophylaxis of a variety of diseases and / or medical conditions. In one aspect, the present disclosure provides a method for treating a disease or disorder in a subject in need thereof. In some embodiments, the method includes administering to the subject a therapeutically effective amount of the nano-micelle composition as described herein. In certain embodiments, the disease or disorder comprises neuropathic pain.

[0123] In another aspect, the present disclosure provides for the use of the nano-micelle composition described herein in the manufacture of a medicament for the treatment of neuropathic pain. In yet another aspect, the disclosure provides the nano-micelle composition described herein for use in treating neuropathic pain.

[0124] As used herein, the terms “treating,” “treat,” and “treatment” include preventing a disease, pathological condition, or medical condition from occurring (e.g., prophylaxis); inhibiting the disease, pathological condition, or medical condition or arresting its development; relieving or ameliorating the disease, pathological condition, or medical condition; and / or diminishing symptoms associated with the disease or condition. Accordingly, the terms “treat,” “treatment,” and “treating” encompass prophylactic, therapeutic, and palliative approaches, including preventing, reducing, stopping, or reversing the progression or severity of a condition or symptoms. The term “treatment” thus includes medical, therapeutic, and / or prophylactic administration. The term “treating” or “treatment” also includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner that improves or stabilizes the subject’s condition.

[0125] As used herein, the term “administering” refers to the delivery of the composition by any suitable route, including but not limited to oral, intranasal, intraocular, intravenous, intraosseous, intraperitoneal, intraspinal, intramuscular, intra-articular, intraventricular, intracranial, intralesional, intratracheal, intrathecal, subcutaneous, intradermal, transdermal, or transmucosal administration. In some embodiments, the composition is administered intratumorally, intravenously, subcutaneously, intraosseously, orally, transdermally, sublingually, in sustained release, in controlled release, in delayed release, or in the form of a suppository.

[0126] In some embodiments, the diseases or medical conditions include, but are not limited to, acquired hypothyroidism, acute gastritis, acute pain, agoraphobia, AIDS-related illness, alcohol abuse, alcoholism, alopecia areata, Alzheimer’s disease, amphetamine dependency, amyloidosis, amyotrophic lateral sclerosis (ALS), angina pectoris, ankylosis, anorexia, anorexia nervosa, anxiety disorders, any chronic medical symptom that limits major life activities, arteriosclerotic heart disease, arthritis (including rheumatoid arthritis), arthropathy, gout, asthma, attention deficit hyperactivity disorder (ADHD), autism spectrum disorders, autoimmune disease, back pain, back sprain, Bell’s palsy, bipolar disorder, brain tumor, breakthrough pain (malignant), bruxism, bulimia, cachexia, cancer, carpal tunnel syndrome, cerebral palsy, cervical disc disease, cervicobrachial syndrome, chemotherapy-induced conditions, chronic fatigue syndrome, chronic pain, chronic renal failure, cocaine dependence, colitis, conjunctivitis, constipation, Crohn’s disease, cystic fibrosis, damage to spinal cord nervous tissue, Darier’s disease, degenerative arthritis, degenerative arthropathy, delirium tremens, dermatomyositis, diabetes, diabetic neuropathy, diabetic peripheral vascular disease, diarrhea, diverticulitis, dysthymic disorder, eczema, emphysema, endometriosis, epidermolysis bullosa, epididymitis, epilepsy, Felty’s syndrome, fibromyalgia, Friedreich’s ataxia, gastritis, genital herpes, glaucoma, glioblastoma multiforme, Graves’ disease, cluster headaches, migraine headaches, tension headaches, hemophilia A, Henoch-Schbnlein purpura, hepatitis C, hereditary spinal ataxia, HIV / AIDS, conditions affecting hospice patients, Huntington’s disease, hypertension, hyperventilation, hypoglycemia, impotence, inflammatory autoimmune-mediated arthritis, inflammatory bowel disease (IBD), insomnia, intermittent explosive disorder (IED), intractable pain, intractable vomiting, lipomatosis, Lou Gehrig’s disease, Lyme disease, lymphoma, major depression, malignant melanoma, mania, melorheostosis, Meniere’s disease, motion sickness, mucopolysaccharidosis (MPS), multiple sclerosis (MS), muscle spasms, muscular dystrophy, myeloid leukemia, nail-patella syndrome, nightmares, obesity, obsessive-compulsive disorder (OCD), opiate dependence, osteoarthritis, panic disorder, Parkinson’s disease, peripheral neuropathy, peritoneal pain, persistent insomnia, porphyria, post-polio syndrome (PPS), post- traumatic arthritis, post-traumatic stress disorder (PTSD), premenstrual syndrome (PMS), prostatitis, psoriasis, pulmonary fibrosis, quadriplegia, radiation therapy side effects, Raynaud’s disease, Reiter’s syndrome, restless legs syndrome (RLS), rosacea, schizoaffective disorder, schizophrenia, scoliosis, sedative dependence, seizures, senile dementia, severe nausea, shingles (herpes zoster), sinusitis, skeletal muscle spasticity, sleep apnea, sleep disorders, spasticity, spinal stenosis, Sturge-Weber syndrome (SWS), stuttering, tardive dyskinesia (TD), temporomandibular joint disorder (TMJ), tenosynovitis, terminal illness, thyroiditis, tic douloureux, Tietze’s syndrome, tinnitus, tobacco dependence, Tourette’s syndrome, trichotillomania, viral hepatitis, wasting syndrome, whiplash, Wittmaack-Ekbom’s syndrome, writer’s cramp, nausea, vomiting, unintentional weight loss, lack of appetite, painful conditions including neurogenic pain, movement disorders, adrenal disease, migraines, fibromyalgia, related autoimmune or inflammatory conditions, spinal cord injuries, and various cancers including hematologic malignancies and solid tumors.

[0127] In some embodiments, the nano-micelle composition suppresses neuropathic pain-related activation of neurons in somatosensory pathways. In certain embodiments, the nano-micelle composition suppresses al lodynia-r elated hyperactivity of somatosensory corticospinal neurons in the subject. In some embodiments, the composition suppresses tactile allodynia or hyperalgesia in a subject with a spared nerve injury (SN1), without affecting normal tactile or nociceptive responses in healthy subjects. In other embodiments, the composition selectively suppresses hyperactive responses of somatosensory corticospinal neurons (CSNs) in a neuropathic pain state, but not in a physiologically intact condition. In further embodiments, the nano-micelle composition exerts its therapeutic effects by modulating hyperactive circuits in the dorsal horn of the spinal cord. In some embodiments, the method comprises administering the nano-micelle composition to the subject via oral, intravenous, intraperitoneal, ophthalmic, parenteral, topical, transdermal, subcutaneous, subdural, intramuscular, intradermal, intrathecal, intracerebral, intraarterial, intralesional, or pulmonary routes. In some embodiments, the cannabinoid nano-micelles or composition is administered intramuscularly or subcutaneously. In other embodiments, the cannabinoid nano-micelles or composition is administered as part of an implant.

[0128] An “effective amount” refers to an amount effective to treat a disease, disorder, or condition, or to bring about a recited effect. For example, an effective amount may be an amount sufficient to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is within the skill of the ordinary artisan. The term “effective amount” is intended to encompass an amount of a compound described herein, or an amount of a combination of compounds described herein, that is effective to treat or prevent a disease or disorder, or to treat the symptoms thereof, in a subject. Thus, an “effective amount” generally refers to an amount that produces the desired effect. A “therapeutically effective amount” of a compound, with respect to the present methods of treatment, refers to an amount of the compound in a pharmaceutical preparation which, when administered as part of a desired dosage regimen to a mammal (e.g., a human), alleviates a symptom, ameliorates a condition, or slows the progression of disease in accordance with clinically acceptable standards for the condition being treated, and provides a reasonable benefit-to-risk ratio.

[0129] The actual dosage of a composition administered to a subject may depend on various physical and physiological factors, including body weight, severity of the condition, type of disease, prior or concurrent therapeutic interventions, patient-specific factors (e.g., idiopathy), and the route of administration. The medical professional responsible for treatment will determine the appropriate concentration of the active ingredient(s) and the suitable dosage for the individual subject.

[0130] In some embodiments, the nano-micelles or compositions comprising the same may be administered in one or more doses at amounts ranging from about 0.1 mg / kg to about 100 mg / kg body weight of the subject (e.g., human). In certain embodiments, the nano-micelles or the composition may be administered in incremental ranges within this broader range, such as from about 0.1 mg / kg to about 100 mg / kg, from about 0.2 mg / kg to about 99 mg / kg, from about 0.3 mg / kg to about 98 mg / kg, and so forth, increasing in 0.1 mg / kg increments up to about 8.1 mg / kg to about 20 mg / kg, as applicable.

[0131] In additional embodiments, the method comprises administering the nano-micelle composition in one or more doses to provide a dose of cannabinoid (e. ., CBD) equivalent to from about 0.05 mg / kg to about 25 mg / kg body weight of the subject (e.g., human), such as from about 0.2 mg / kg to about 8 mg / kg. Further embodiments may include incremental ranges within this spectrum, including from about 0.05 mg / kg to about 25 mg / kg, from about 0.1 mg / kg to about 24.8 mg / kg, from about 0.2 mg / kg to about 24.6 mg / kg, and so on, in 0.1 mg / kg increments down to about 7.5 mg / kg to about 10 mg / kg.

[0132] In some embodiments, a dose may be administered as a single dose or divided into multiple sub-doses delivered at appropriate intervals, such as two, three, four, or more times per day. Each sub-dose may also be further divided into a series of discrete administrations spaced apart by short intervals.

[0133] In certain embodiments, the nano-micelles or compositions thereof may be administered at a frequency of at least once every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every week, every 2 weeks, every 3 weeks, every 4 weeks, and so forth, in weekly intervals up to every 28 weeks or more.

[0134] In some embodiments, the cannabinoid nano-micelles or compositions thereof may provide a sustained release of the active agent (e.g., oxybutynin) for a period ranging from about 4 weeks to about 8 weeks, including, for example, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks.

[0135] Combination Therapies

[0136] In some embodiments, the method may further include administering to the subject a second therapeutic agent. In some embodiments, the method may include administering to the subject an effective amount of the cannabinoid nano-micelles or the composition described herein in combination with a second therapeutic agent.

[0137] As used herein, the term “combination therapy,” unless otherwise indicated by the context, is intended to encompass the administration of two or more therapeutic agents in a coordinated fashion, including, but not limited to, concurrent dosing. Specifically, combination therapy includes both co-admini strati on e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that the administration of one therapeutic agent is conditioned in some manner upon the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period. See, e.g., Kohrt etal. (2011) Blood 117:2423.

[0138] As used herein, the terms “co-administration” or “co-administered” refer to the administration of at least two agents or therapies to a subject. In some embodiments, the coadministration of two or more agents or therapies is concurrent. In other embodiments, a first agent or therapy is administered prior to a second agent or therapy. Those skilled in the art will recognize that the formulations and / or routes of administration of the various agents or therapies may vary.

[0139] In certain embodiments, an agent (e.g., nano-micelles) may be combined with one or more other active ingredients in a unitary dosage form for simultaneous or sequential administration to a subject. The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be delivered in two or more separate administrations.

[0140] The combination therapy may provide synergistic effects, i.e., effects wherein the therapeutic benefit achieved when the active ingredients are used together exceeds the sum of their individual effects when used separately. A synergistic effect may be achieved when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered in alternation or in parallel as separate formulations; or (3) administered by some other regimen. In alternation therapy, a synergistic effect may be achieved when the compounds are administered sequentially, such as in separate tablets, pills, capsules, or by different injections delivered using separate syringes. Typically, during alternation therapy, an effective dosage of each active ingredient is administered serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together. A synergistic effect refers to an outcome greater than the predicted purely additive effects of the individual compounds of the combination.

[0141] Combination therapy is further described in U.S. Patent Nos. 11,103,514; 10,702,495; 9,382,215; and 6,833,373, which disclose additional active agents that may be combined with the compounds described herein, as well as additional conditions that may be treated with such combinations.

[0142] An active agent may be administered before or after administration of another agent by intervals ranging from minutes to weeks. In embodiments where the agents are applied separately to a cell, tissue, or organism, it is generally preferred that the interval between deliveries does not exceed a time frame that would diminish the combined therapeutic benefit. For example, the agents may be administered substantially simultaneously (i.e., within less than about one minute), or within a suitable time frame to achieve a desired combined effect.

[0143] In some embodiments, one or more agents may be administered within about 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 28 hours, 31 hours, 35 hours, 38 hours, 42 hours, 45 hours, or up to about 48 hours before and / or after administration of the disclosed active agent. In certain other embodiments, an agent may be administered within about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 8 days, 9 days, 12 days, 15 days, 16 days, 18 days, 20 days, or up to about 21 days before and / or after administration of the disclosed active agent. In some embodiments, it may be desirable to extend the time period for treatment significantly, such that several weeks (e.g., about 1, 2, 3, 4, 6, or 8 weeks or more) may elapse between respective administrations.

[0144] Administration of the compositions to a patient may follow standard protocols for therapeutic administration, taking into consideration the potential toxicity, if any. It is anticipated that treatment cycles may be repeated as needed. It is also contemplated that the disclosed active agent may be administered in conjunction with standard therapies or adjunct treatments, including, but not limited to, chemotherapy, radiotherapy, immunotherapy, gene therapy, and surgical intervention.

[0145] Additional Definitions

[0146] To aid in understanding the detailed description of the compositions and methods according to the present disclosure, a number of express definitions are provided to facilitate unambiguous interpretation of the various aspects of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. As used herein, the term “subject” or “subject in need thereof’ refers to both human and non-human animals. Examples of non-human animals include all vertebrates, such as mammals (e.g., non-human mammals, non-human primates, particularly higher primates, dogs, rodents such as mice or rats, guinea pigs, cats, and rabbits), as well as non-mammals e.g., birds, amphibians, and reptiles). In some embodiments, the subject is a human. In other embodiments, the subject is an experimental animal or an animal suitable as a disease model.

[0147] The term “disease,” as used herein, is intended to be generally synonymous with, and is used interchangeably with, the terms “disorder” and “condition” (as in “medical condition”), in that all refer to an abnormal condition of the human or animal body, or of one of its parts, that impairs normal function, is typically manifested by distinguishing signs and symptoms, and reduces the duration or quality of life.

[0148] The term “agent,” as used herein, denotes a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, antibody, protein, or portion thereof, e.g., a peptide), or an extract derived from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render them suitable as “therapeutic agents,” which are biologically, physiologically, or pharmacologically active substances that act locally or systemically in a subject.

[0149] The terms “therapeutic agent,” “therapeutically capable agent,” and “treatment agent” are used interchangeably and refer to a molecule or compound that provides a beneficial effect upon administration to a subject. Such beneficial effects include enabling diagnostic determinations; ameliorating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally counteracting a disease, symptom, disorder, or pathological condition.

[0150] Doses are often expressed in relation to body weight. Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) generally refers to [g, mg, or other unit] per kilogram (or gram, milligram, etc.) of body weight, even if the term “body weight” is not explicitly stated.

[0151] As used herein, the term “z ? vitro'’' refers to events occurring in an artificial environment, such as in a test tube, reaction vessel, or cell culture, rather than within a multicellular organism. As used herein, the term “ / / ? vivo” refers to events occurring within a multicellular organism, such as a non-human animal.

[0152] It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0153] The terms “including,” “comprising,” “containing,” and “having,” and variations thereof, are intended to encompass the listed items and equivalents thereof, as well as additional subject matter, unless otherwise specified.

[0154] The phrases “in some embodiments,” “in various embodiments,” and similar expressions may be used repeatedly throughout the disclosure. Such phrases do not necessarily refer to the same embodiment, although they may, unless the context dictates otherwise.

[0155] The terms “and / or” or “ / ” refer to any one of the listed items, any combination of the listed items, or all of the listed items.

[0156] The term “substantially” does not exclude “completely.” For example, a composition described as “substantially free” of component Y may, in fact, be completely free of Y. Where appropriate, the term “substantially” may be omitted from the definition of the invention.

[0157] As used herein, the terms “approximately” or “about,” when applied to one or more values of interest, refer to values that are similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value, unless otherwise indicated or evident from the context (except where such a range would exceed 100% of a possible value). Unless indicated otherwise herein, the term “about” is intended to include values proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

[0158] It is to be understood that wherever numerical values or ranges are provided herein, all values and ranges encompassed thereby are intended to fall within the scope of the invention. Moreover, all values that fall within such ranges, including the endpoints, are contemplated by the present disclosure. As used herein, the term “each,” when referring to a collection of items, identifies individual items within the collection, but does not necessarily refer to all items in the collection, unless explicitly stated or dictated by context.

[0159] The use of examples or exemplary language (e.g., “such as”) is intended solely to illuminate aspects of the invention and should not be construed as limiting the scope of the invention unless explicitly recited in the claims. The term “exemplary” is intended to mean “by way of example” and does not imply preference or requirement.

[0160] All methods described herein may be performed in any suitable order unless otherwise indicated or clearly contradicted by context. Where a method comprises multiple steps, such steps may be performed sequentially or simultaneously. Unless otherwise noted, any order of steps described is not intended to be limiting.

[0161] Where a method comprises a combination of steps, all combinations or sub-combinations of those steps are intended to be encompassed by the disclosure, unless specifically excluded.

[0162] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety, to the extent not inconsistent with the present disclosure. These references are provided solely for their disclosure as of the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such references by virtue of prior invention. Furthermore, the publication dates provided herein may differ from the actual dates of public availability and may require independent verification.

[0163] It is understood that the examples and embodiments described herein are provided for illustrative purposes only, and that modifications and variations thereof will be apparent to those skilled in the art. Such modifications are considered to fall within the scope and spirit of the present disclosure and the appended claims.

[0164] Examples

[0165] EXAMPLE 1

[0166] This example provides a detailed description of the materials, experimental procedures, and analytical methods to obtain the results discussed in Example 2.

[0167] CBD Solution Preparation For the CBD-HPpCD (CBD-I) solution, cannabidiol (CBD) (Cayman Chemical, #90080) was dissolved in ethanol, and (2-Hydroxypropyl)-P-cyclodextrin (HPpCD) (Millipore Sigma, C0926-5G) was dissolved in deionized water (diFFO). The HPPCD solution was then gradually added to the CBD ethanol solution with vortexing. After mixing, the tube was sealed and covered with aluminum foil. The mixture was incubated at room temperature (25°C) for 48 hours. Following incubation, the resulting CBD-HPPCD inclusion complex was further diluted in diFFO to a final volume of 8 mF

[0168] For the CBD-poly sorbate 80 (CBD-N) solution, CBD was dissolved in ethanol and then diluted with diFFO containing polysorbate 80 (PS80) (Millipore Sigma, Pl 754) at a final concentration of 2%. The final volume was 8 mF.

[0169] For the CBD-HPpCD-2% PS80 (CBD-IN) solution, the CBD-HPpCD inclusion complex was prepared as described for the CBD-I solution. During dilution into diFFO, PS80 was added simultaneously to achieve a final concentration of 2%. The final volume was 8 mF.

[0170] Sodium chloride (NaCl) was added to each solution following dilution to render the solution isotonic, achieving a final NaCl concentration of 0.9%. All solutions were filtered using 0.22 pm filters prior to use and stored at room temperature. All solutions were freshly prepared and used within two weeks of preparation.

[0171] For the CBD-sesame oil (CBD-O) solution, CBD was dissolved in ethanol and subsequently diluted in sesame oil (Millipore Sigma, S3547) to a final volume of 8 mb. The final ethanol concentration was 7.9% (v / v), similar to that in the formulation of Epidiolex.

[0172] Vehicle control solutions for each CBD formulation were prepared in the same manner as the corresponding CBD solutions, except that CBD was omitted.

[0173] Cryo-TEM Imaging and Data Analysis

[0174] Copper 1.2 / 1.3 300 mesh C-flat grids (Electron Microscopy Sciences) were glow- discharged for 30 seconds at 30 mA using a PELCO easiGlow system. Three microliters of nanomicelle solution were applied to the glow-discharged grids in a 95% humidified chamber at room temperature, allowed to dwell for one second, followed by a 3.5-second sensor blot and plungefreezing into liquid ethane using an EMGP2 plunge freezer (Leica). Vitrified samples were loaded into a Talos 120C transmission electron microscope (Thermo Fisher Scientific) using an ELSA698 specimen holder (Gatan) and imaged with a CETA 16 detector (Thermo Fisher Scientific) at an accelerating voltage of 120 kV. Manual imaging of specimens was performed using low-dose imaging with TIA software (Thermo Fisher Scientific), while automated imaging was performed using EPU software (Thermo Fisher Scientific). Cryogenic transmission electron microscopy was conducted at the Electron Microscopy Resource (EMR) within the Center for Advanced Research Technologies (CART) at the University of Rochester Medical Center. Images were processed using FIJI software, and micelle number and size were quantified using Trainable Weka Segmentation (Arganda-Carreras, I., et al., Bioinformatics, 2017, 33: 2424-2426). Three representative images from each solution (PS80 vehicle, CBD-N, and CBD-IN) were used for model training, and ten images from each solution were used for quantification.

[0175] Mass Spectrometry Analysis

[0176] Mouse pharmacokinetic studies of CBD-IN and other comparative formulations were conducted using intraperitoneal and oral administration routes. Groups of 4 to 6 mice were sacrificed at 0.5, 1, 1.5, and 2 hours following intraperitoneal administration of CBD-IN or CBD- N at 100 mg / kg, or at 0.5, 1, 2, and 4 hours following oral administration of CBD-IN or CBD-0 at 50 mg / kg, using a 5% isoflurane overdose. Following terminal anesthesia, blood and brain samples were collected. Blood was drawn via cardiac puncture using a 27G syringe and allowed to coagulate at room temperature for 30 minutes in Eppendorf (EP) tubes. After coagulation, blood samples were centrifuged at 3,000 RPM for 10 minutes at 4°C. The resulting serum was then stored at -80°C until analysis. After cardiac puncture, 0.9% saline was perfused through the circulatory system to flush the brain and liver tissues of residual blood. The entire brain and the left lobe of the liver were collected and stored at -80°C in plastic universal vials for subsequent analysis.

[0177] Sample Preparation

[0178] Liquid chromatography-mass spectrometry (LC-MS) was performed by the Harvard Center for Mass Spectrometry (HCMS). The preparation procedures for the following sample types are described below: (1) whole brain, (2) left lobe of the liver, (3) serum, and (4) CBD dissolved in solution.

[0179] The whole brain and the left lobe of the liver were prepared as follows. Samples were transferred to bead-beater vials and weighed. Either 1 mL of 70% acetonitrile or an extraction solution consisting of 70% acetonitrile and 0.1% formic acid was added. A 150 nM concentration of CBD-D3 internal standard (Cambridge Isotope Laboratories, Inc., MA, USA) was included, along with 3 to 10 steel beads (2 mm in diameter). The tissues were homogenized for 10 minutes at 50 Hz using a TissueLyser LT (Qiagen, Germantown, MD, USA). Brief centrifugation was performed to reduce foam, followed by vortexing to resuspend the biomass. Supernatants were then transferred to clean centrifuge tubes. To clean the bead-beater vials, either 1 mL of extraction solution or 500 pL of 70% acetonitrile in water was added, and the resulting wash was combined with the sample. The samples were incubated in an ultrasound bath for 10 minutes and then centrifuged at 18,000xg at 4°C for 10 minutes. The resulting supernatants were transferred to clean microcentrifuge tubes, dried under nitrogen (N2) flow, resuspended in 100-200 pL of 50% methanol in water, centrifuged, and transferred to glass microinserts for analysis.

[0180] Serum samples were prepared by transferring 100 pL of serum and 1 mL of the extraction solution into microcentrifuge tubes. The samples were vortexed for 1 minute and centrifuged at 18,000 x g at -9°C for 10 minutes. Supernatants were transferred to new microcentrifuge tubes, dried under N2 flow, resuspended in 100 pL of 50% methanol in water, centrifuged again, and transferred to glass microinserts.

[0181] CBD-IN, CBD-I, or CBD-N samples dissolved in solution were prepared using a two-step dilution series (1: 10, 1 : 100) with 50% methanol in water and 1 pM CBD-D3 in glass autosampler vials. This approach was employed to minimize variability arising from ion suppression or sample viscosity.

[0182] Calibration Curve

[0183] Standard curves were generated for each batch of brain, liver, and serum samples processed. A 12-point curve with a 1 / 3 dilution series was prepared using a CBD stock (Caymen Chemical, Ann Arbor, MI, USA) starting at 100 pM. Each standard (1 mL) was dried under N2 flow, resuspended in 100 pL of 50% methanol in water, centrifuged, and transferred to glass microinserts.

[0184] Samples with CBD-IN, CBD-I, or CBD-N dissolved in solution alone were assessed via a separate curve. Here, a stock CBD solution diluted with 50% methanol in water containing 1 pM of CBD-D3 was used to generate standards. Starting from a 100 pM concentration, the subsequent standards (1.25 mL) were prepared with a 1 / 5 dilution series in glass vials and briefly vortexed. Liquid Chromatography-Mass Spectrometry (LC-MS)

[0185] All samples were run through liquid chromatography (LC) in tandem with one of two mass spectrometers (MS). LC was accomplished with a Force Fluorophenyl column (150x2.1 mm, 3 pM, Restek, Bellefonte, PA, USA) maintained at 30 °C with two mobile phases: (phase A) water, 0.1% ammonium Fluoride and (phase B) methanol, 0.1 mM ammonium Fluoride. Each sample (5 pL injected) transited the column at 0.2 mL / min across a gradient of 40% phase B for 4 minutes, 90% phase B for 4 minutes, and re-equilibrated at 40% phase B for 3 minutes.

[0186] Subsequently, one of the following MS was used: 1) a high-resolution orbitrap, ThermoFisher QE+ coupled with an Ultimate 3000 LC (ThermoFisher Scientific, Waltham, MA, USA), or 2) a triple quad mass spectrometer, Agilent 6460 MS coupled with a 1290 LC (Agilent Technologies, Santa Clara, CA, USA). The high-resolution orbitrap achieved ionization with a heated ESI source in negative mode and the full MS 70k resolution data was acquired. From the extracted chromatograms, targets were then integrated on the accurate mass for their [M-H]- ions within 5 ppm. The triple quad MS achieved ionization with an AJS ESI source in negative mode with a nebulizer at 40 psi, sheath gas flow of 10 L / min at 350 °C, nozzle at 700 V, and capillary at 2800 V. CBD was detected between 245.1 and 313.2 m / z using a 200 ms Dwell time, fragmentor at 120, collision energy at 14 and cell accelerator at 5 V. CBD-D3 was detected between 107 and 316.2 m / z with a 200 ms Dwell time, fragmentor at 120, collision energy at 30 and cell accelerator at 5 V. MassHunter software (Agilent Technologies, Santa Clara, CA, USA) was used to analyze all data.

[0187] SNI Surgery

[0188] The Spared Nerve Injury (SNI) surgery was performed on adult mice aged 8-12 weeks under isoflurane anesthesia (3% for induction, 2% for maintenance). Briefly, the tibial and common peroneal branches of the sciatic nerve were tightly ligated using a 5.0 silk suture and transected distally while preserving the integrity of the sural nerve. Subsequently, the incision was sutured, and the mice were allowed to recover on heated pads before being returned to their home cages. Assessments of behavior and physiology following various drug treatments were conducted between 7 and 15 days post-SNI.

[0189] Behavioral Tests Within each set of behavioral measurements, animals allocated to both experimental and control groups were littermates. Body weight and sex were randomized and then assigned to various treatment groups, with no additional specific randomization utilized for the animal studies. Behavioral tests were conducted in a blinded manner to the experimental conditions. Sample sizes were not predetermined using statistical methods but were based on conventions in the literature, with effect sizes reported in this study.

[0190] Drug Administration

[0191] CBD-IN (4, 20, and 100 mg / kg), CBD-N (100 mg / kg), and matching vehicle solutions were delivered through intraperitoneal injection 30 minutes before the intraperitoneal dose effect test. CBD-IN (2, 10, and 50 mg / kg), CBD-0 (50 mg / kg), and matching vehicle solutions were delivered through oral gavage 30 minutes before the oral dose effect test.

[0192] Buprenorphine HCL (Buprenex™, Indivior Inc) (25 pg / kg), CBD-IN (100 mg / kg), and vehicle solutions were delivered through intraperitoneal injection 30 minutes before von Frey spectrum test in intact animals. CBD-IN (100 mg / kg) and vehicle solutions were delivered through intraperitoneal injection 30 minutes before all the other experiments.

[0193] For the long-term therapeutic effect tests, CBD-IN (100 mg / kg) was administered through intraperitoneal injection every other day for 10 days. Von Frey pain threshold was measured every day for 10 days. von Frey Filaments (Punctate Mechanical Stimuli)

[0194] After habituation in a small plastic cage (7.5 x 7.5 x 15 cm) for 30 minutes, mechanical sensitivity was assessed using von Frey filaments (bending forces: 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1, 1.4, and 2.0 g) targeting the sciatic nerve territory (lateral part of the hind paw). Each filament was applied ten times in ascending order of force. The minimum force filament that caused a brisk paw withdrawal or escape attempt in response to at least five out of ten stimulations determined the threshold for mechanical response and tactile allodynia.

[0195] Brush (Dynamic Mechanical Stimuli)

[0196] Each mouse was habituated in a small plastic cage (dimensions: 7.5 x 7.5 x 15 cm) for a minimum of 30 minutes before testing. The plantar hind paw was gently stroked from heel to toe using a paintbrush. For dynamic mechanical allodynia, the duration of paw flinching or licking was measured across three trials and averaged for each animal.

[0197] Acetone Test (Cold Allodynia)

[0198] Each mouse was acclimated in a small plastic cage (measuring 7.5 x 7.5 x 15 cm) for at least 30 minutes before testing. A 50 pl drop of acetone was applied to the lateral plantar surface of the hind paw, and the animal's behavior was monitored for 1 minute. Behavioral responses were scored as follows: 0 for no response; 1 for a single quick withdrawal or flick; 2 for a prolonged withdrawal or repeated flicking; and 3 for repeated flicking accompanied by licking of the paw. The acetone stimulus was administered three times to the injured hind paw, and each response was scored. The cumulative score for each mouse was calculated by summing the three individual scores.

[0199] Hot Plate Test (Contact Heat Pain)

[0200] Mice were placed on a heated metallic plate set to a constant temperature of 56°C inside an acrylic container (Bioseb) and video recorded. The time taken for the mice to exhibit a flinch was determined through blind analysis of the recorded videos.

[0201] Open Field Test

[0202] The open field test was utilized to evaluate locomotor activity in mice. The test arena was a square chamber (50 cm x 50 cm x 40 cm) with opaque walls. The arena floor was divided into a central zone (25 cm x 25 cm) and a peripheral zone. Prior to testing, mice underwent a 30-minute habituation period in the testing room. Each mouse was then placed individually in the center of the arena and allowed to freely explore for 5 minutes, while their movements were recorded using a video tracking system (EthoVision XT, Noldus). Parameters recorded included total distance traveled (cm) and average velocity (cm / s). Data analysis was conducted using the EthoVision XT software for automated tracking.

[0203] Rotarod

[0204] Mice were placed on the rotarod (IITC Life Sciences Inc., CA, USA) at rest, facing forward until stable. Over 300 s the rod increased from 3-30 rpm with a forward rotation. Magnetic switches recorded the moment each animal dropped, and the time, rotational speed, and distance traveled were recorded. Trials were repeated three times, and an average was taken. TRAP2 Labeling of Neurons

[0205] 4-Hydroxytamoxifen (4-OHT; Sigma, Cat# H6278) was dissolved in ethanol at a concentration of 20 mg / mL by shaking at 37°C for 15 minutes. The solution was then aliquoted and stored at -20°C for up to several weeks. Before use, 4-OHT was redissolved in ethanol by shaking at 37°C for 15 minutes. Subsequently, a 1 :4 mixture of castor oil and sunflower seed oil (Sigma, Cat# S259853, and S5007) was added to achieve a final concentration of 10 mg / mL 4- OHT. The ethanol was evaporated by vacuum centrifugation, and the resulting 10 mg / mL 4-OHT solutions were used on the day of preparation. All injections were administered intraperitoneally (i p ).

[0206] To induce c-Fos expression through mechanical (dynamic brush) stimulation, SNI animals treated with either CBD or vehicle were habituated in the testing chamber for 30 minutes. A paintbrush was used to stroke the hind paw, with each touch lasting 2 seconds and moving from the middle of the foot to the distal foot pad along the peripheral side within the sural nerve territory. This was done once every 2 seconds for three 10-minute sessions with a 1 -minute gap between sessions. The 4-OHT solution was injected at the end of the brush session, and the animals were sacrificed and perfused 7 days after c-Fos induction. Brain and spinal cord tissues were sliced with a compresstome to a thickness of 80 qm, collected, and mounted for imaging. Neuroinfo software (MBF Bioscience) was used for slice atlas alignment and cell number quantification.

[0207] Slice Calcium Imaging

[0208] Nine Vglut2:GCaMP6f mice (four female, five male) were used for calcium imaging. Following cardiac perfusion with ice-cold slicing solution, the lumbar region of the spinal cord was extracted by laminectomy in the same solution. The slicing solution had the following composition (in mM): NaCl 80, sucrose 75, NaFLPCL 1.25, NaHCCh 26, CaCL 0.5, MgCL 3.5, D- glucose 10, sodium ascorbate 1.3, sodium pyruvate 3, and myo-inositol 3.

[0209] Transverse spinal cord slices, 250 qm in thickness, were sectioned from the lumbar toward the cervical region. The slices were incubated in artificial cerebrospinal fluid (ACSF) at 35-36 °C for 60 minutes, followed by incubation at room temperature (23-24 °C) for at least one hour. The ACSF solution had the following composition (in mM): NaCl 120, KC1 2.5, NaFLPCL 1.25, D- glucose 20, NaHCCh 26, CaCL 2, MgCL 1, sodium ascorbate 1.3, sodium pyruvate 3, and myoinositol 3. The boundary between lumbar segments L6 and L5 was estimated based on the anatomical location of the sacral dorsal commissural nucleus (SDCom). Lumbar segments L5 through L3 were subsequently estimated based on the slicing order. Individual slices were transferred to a recording chamber (RC-27, Warner Instruments) continuously perfused with ACSF at approximately 2 mL / min and imaged using a confocal microscope (LSM980, Zeiss).

[0210] Pharmacological treatments were applied by bath perfusion, including 100 pM 4- aminopyridine (4-AP; Tocris, #0940) and 20 pM cannabidiol (CBD) dissolved in 0.1% DMSO.

[0211] GCaMP6f signals were excited using a 488 nm laser and imaged with a 10x objective (EC Plan-Neofluar 10* / 0.30) using an Airy scan-equipped LSM980 confocal microscope (Zeiss). Each image frame measured 668 x 596 pixels (258.81 x 233.41 pm, 0.397 pm / pixel). Images were acquired at 9.80 frames per second, with a pixel dwell time of 1.76 ps.

[0212] For data analysis, image sequences were imported and preprocessed using Inscopix data processing software (INSCOPIX), and neuronal calcium signals were extracted using the CNMF_E algorithm. Because neuronal spontaneous activity is typically low in slice preparations, AF / Noise was reported as the primary metric.

[0213] In vivo Calcium Imaging of Corticospinal Neurons

[0214] Calcium imaging experiments were conducted on mice that were injected with AAV2- retro-syn-GCaMP6s at a titer of 1.4 x 1012genome copies per milliliter (gc / mL) (Boston Children’s Hospital Viral Core). The injections were administered into the dorsal horn of the spinal cord on postnatal day 56 (P56). Two weeks following the injection, a cranial window was installed over the primary somatosensory cortex, specifically targeting the hindlimb region.

[0215] During the surgical procedures, the mice were anesthetized using isoflurane (3% for induction, 2% for maintenance) and treated with dexamethasone (0.2 mg / kg, subcutaneously) and carprofen (5 mg / kg, subcutaneously) to minimize brain swelling and inflammation. A circular portion of the skull (3.5 mm in diameter) above the somatosensory cortex was removed using a high-speed dental drill, and a 3 -mm coverslip was used to seal the cranial opening. Seven days later, the baseplate of a miniaturized integrated fluorescence microscope (Inscopix) was affixed on top of the coverslip. Following spared nerve injury (SNT), animals were habituated to microscope attachment in a behavioral chamber over three consecutive days, beginning on day seven post-injury. Calcium imaging was subsequently performed on freely moving mice, both intact and SNI models, under conditions with or without sensory stimulation. For stimulation, a von Frey fdament (0.04 g) was applied to the hind paw. Each imaging session included two experimental blocks — one following vehicle (VEH) injection and the other following cannabidiol (CBD) injection — after a 30-minute habituation period in the testing chamber. Each block consisted of 5 minutes of free movement followed by 10 von Frey stimulation trials, with a 30-second interval between trials conducted over a 5-minute period.

[0216] Calcium imaging was performed using a head-mounted microscope (Inscopix) with LED power set between 0.6-1.0 mW and a camera resolution of 1280 x 800 pixels. Images were acquired at 30 Hz using Inscopix data acquisition software. At the start of each session, the protective cap on the implanted baseplate was removed and the microscope was attached. The imaging field of view was approximately 1050 x 650 pm2at a spatial resolution of 0.82 pm per pixel. The imaging depth was adjusted until clearly defined dendritic trunk signals — appearing as bright spots — were visible. The focal plane was typically 150-250 pm below the lens. Concurrent video recordings of mouse behavior were captured using a Logitech C920 camera and synchronized with calcium imaging via Bonsai video recording software (Open Ephys) and an Arduino Uno microcontroller, which initiated and terminated both video and calcium imaging recordings.

[0217] Video data from calcium imaging were analyzed using Inscopix data analysis software and custom MATLAB scripts, based on previously described algorithms (Liu, Y., et al., Nature, 2018, 561 :547-550). To visualize dendritic activity during stimulation, calcium event traces from each corticospinal neuron (CSN) were aligned to the moment the von Frey fdament was applied to the plantar surface of the left hindlimb. Traces from all CSNs were then sorted by their peak activation times and displayed as temporal raster plots.

[0218] To quantify differences in CSN activity between VEH- and CBD-treated conditions in both intact and SNI animals, tri al -averaged responses of CSN dendrites to sensory stimuli were calculated. Responses occurring within 1 second of stimulation onset were compared using a paired t-test. A statistically significant increase in CSN activity in response to von Frey stimulation was observed following SNI.

[0219] To determine the proportion of neurons activated by von Frey stimulation following VEH and CBD injection in both intact and SNI animals, neurons were classified as responsive if they exhibited a peak trial-averaged activity within 1 second of stimulation onset.

[0220] To assess changes in the spontaneous activity of CSNs following VEH and CBD injection, non-moving behavior was defined as the condition in which all four paws of the mouse remained on the grid for a minimum of 2 seconds. Calcium activity recorded during these non-moving periods was quantified, and mean activity levels were compared between VEH- and CBD-injected groups.

[0221] Quantification and Statistical Analysis

[0222] Statistical comparisons between experimental groups were performed using appropriate parametric or non-parametric tests, selected based on assessments of data normality and variance homogeneity. Normality and variance equivalence were verified using Prism software (GraphPad, version 10.1.0) prior to applying any parametric tests. For comparisons between two groups, two- sided t-tests, Wilcoxon matched-pairs signed rank tests, or Mann-Whitney tests were employed, and effect sizes were calculated using Cohen’s d or Cliffs delta, as applicable. Kolmogorov- Smirnov tests were used to compare cumulative micelle size distributions. For comparisons involving multiple groups, data were analyzed using one-way or two-way analysis of variance (ANOVA), as appropriate to the experimental design. Post hoc comparisons were conducted only when the main effect was determined to be statistically significant. P-values for multiple comparisons were adjusted using either the Bonferroni or Sidak correction method. Error bars in all figures represent the mean ± standard error of the mean (s.e.m.). Statistical significance levels are indicated as follows: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and n.s. (not significant) for P > 0.05.

[0223] EXAMPLE 2

[0224] Development of an Inclusion-Complex-Enhanced Nano-micelle CBD Formulation

[0225] To enhance the solubility of CBD in aqueous solutions for systemic drug delivery, two pharmaceutical techniques commonly employed individually for hydrophobic drugs — namely, cyclodextrin-based inclusion complex formation and surfactant-based nano-micelle assembly — were investigated. A CBD-IN nano-formulation was prepared using a two-step process, as illustrated in Figure 1 A. Initially, CBD was dissolved in ethanol and subsequently encapsulated in hydroxypropyl-P-cyclodextrin (HPpCD) in an aqueous medium. Thereafter, the resulting CBD- HPpCD inclusion complex solution was diluted with deionized water and combined with PS80 at a final concentration of 2% v / v. The formation of a clear aqueous solution, in which no precipitated CBD crystals were observed, indicated an enhancement in CBD solubility achieved through this formulation.

[0226] To directly compare the dissolving capacity of the CBD-IN formulation with the two single-solvent methods previously described in the literature (CBD-HPpCD (CBD-I) and CBD- PS80 (CBD-N)), mass spectrometry analysis was performed, and the CBD concentration was quantified in solutions containing an excess amount of CBD in a standardized volume. Consistent with earlier studies, the CBD-I and CBD-N formulations were found to exhibit limited water solubility. In contrast, the CBD-IN formulation was observed to significantly increase the aqueous concentration of CBD relative to the single- solvent preparations (Figure IB) and to remain stable at room temperature for at least 28 days (Figure IE). The enhanced dissolving effect observed with the CBD-IN formulation indicates that HPpCD may have facilitated the incorporation of CBD into PS80-based nano-micelles.

[0227] To evaluate this, Cryogenic Transmission Electron Microscopy (Cryo-TEM) imaging was performed. Image analysis indicated that the diameters of PS80 micelles without cannabidiol (CBD) loading ranged from approximately 8 to 14 nm. In the CBD-N and CBD-IN solutions, larger micelles with diameters of approximately 16 to 32 nm were observed, indicating that these micelles had been loaded with CBD (Figure 1C). Upon comparing the size distributions of the nano-micelles in the three solutions (PS80 vehicle, CBD-N, and CBD-IN), it was determined that the CBD-IN formulation contained the highest proportion of drug-loaded micelles within the 16- 32 nm range (Figure ID). These results confirmed that hydroxypropyl-P-cyclodextrin (HPpCD) enhanced CBD loading into PS80 micelles and improved the aqueous solubility of CBD.

[0228] CBD-In Enhances Brain Delivery and Analgesic Efficacy

[0229] Next, it was evaluated whether the increased water solubility of cannabidiol (CBD) achieved through the CBD-IN formulation could enhance its pharmacokinetic profile and result in a more rapid and potent analgesic effect in murine models. A range of CBD doses was administered, with the highest dose being 100 mg / kg. This dosage range was selected based on (1) its equivalence to FDA-approved human doses for the treatment of epilepsy, as determined using standard human-to-mouse dose conversion methodologies, and (2) its consistency with preclinical studies investigating the anti-epileptic mechanisms of CBD in mice. However, mass spectrometry analysis of the CBD-IN solution, prepared by dissolving 50 mg of CBD in 8 mL of solution, indicated that only 43.1 ± 1.7% of the CBD remained in the aqueous phase. The remainder was presumed to have been lost due to adsorption to plastic containers. Accordingly, the actual CBD dose delivered to the mice was estimated to be approximately 43% of the intended amount.

[0230] The well-established sciatic spared nerve injury (SNI) mouse model of neuropathic pain was employed to evaluate the analgesic effects of CBD. Assessment was conducted using the von Frey assay, as described in the literature (Liu, Y., et al. (2018). Nature 561, 547-550; Decosterd, I., and Woolf, C.J. (2000). Pain 87, 149-158). A dose-dependent increase in the von Frey threshold was observed following intraperitoneal (i.p.) administration of CBD-IN, with a statistically significant elevation detected at various doses (Figure 2A, measured at 30 minutes post-injection). At a dose of 100 mg / kg, CBD-IN raised the von Frey threshold in SNI mice to approximately 1.0 g, which corresponds to the baseline sensitivity observed in intact mice, thereby indicating a complete reversal of neuropathic pain symptoms. Moreover, the analgesic efficacy of CBD-IN at the 100 mg / kg dose was found to be significantly greater than that of CBD-N, as demonstrated in Figure 2A. In parallel, the pharmacokinetic profile of CBD-IN was characterized by mass spectrometry following administration at 100 mg / kg (i.p.). The maximum concentration (Cmax) of CBD was measured at 6.05 ± 1.03 pg / g in brain tissue (Figure 2B) and 7.81 ± 3.71 pg / mL in serum (Figure 2E), both attained 30 minutes post-injection (Tmax). Notably, the concentration of CBD in the brain was significantly higher in the CBD-IN group as compared to the CBD-N group (Figure 2B). These results collectively demonstrated that the CBD-IN nanoformulations enhanced both the central nervous system delivery and the analgesic potency of CBD in the SNI model of neuropathic pain.

[0231] Because the two co-solvents used in CBD-IN, HPpCD and PS80, are commonly used in the food and pharmaceutical industries and have been approved as safe oral additives, the analgesic effect and pharmacokinetic profile of CBD-IN were evaluated following oral administration (p.o.) to assess the translational potential of the formulation. A dose-response analysis demonstrated that a 50 mg / kg oral dose of CBD-IN elicited a rapid-onset analgesic effect comparable to that of a 100 mg / kg intraperitoneal dose, with full recovery of the von Frey threshold observed within 30 minutes post-administration (Figure 2C).

[0232] Furthermore, the analgesic efficacy of orally administered CBD-IN was found to be significantly greater than that of CBD formulated in sesame oil (CBD-O), a clinically approved oral formulation (Figure 2C). Pharmacokinetic analysis performed using mass spectrometry indicated that oral administration of CBD-IN resulted in higher CBD concentrations in both brain and serum, and at an accelerated rate, relative to CBD-0 (Figures 2D and 2F). Notably, CBD-IN reached peak concentration more rapidly and exhibited a shorter hepatic half-life compared to CBD-O, with complete clearance from liver tissue occurring within four hours (Figure 2G). This rapid hepatic clearance may reduce the risk of liver toxicity during repeated dosing for long-term pain management. Accordingly, these findings support the efficient brain delivery and robust analgesic efficacy of CBD-IN via the oral route and highlight its potential for clinical translation

[0233] To broaden the therapeutic applicability of CBD-IN formulation, this strategy was applied to other water-insoluble minor cannabinoids, cannabinol (CBN) and cannabigerol (CBG). Mass spectrometry analysis confirmed that the aqueous solubility of both CBN and CBG was significantly increased following formulation (Figure 2H). The analgesic potential of these compounds was evaluated in a sciatic nerve injury (SNI) model of neuropathic pain using von Frey threshold testing. Notably, this represents the first study to assess the efficacy of CBN and CBG in the SNI model. The results show that CBN produces analgesic effects comparable to those of CBD, whereas CBG did not yield a significant effect (Figure 21). These findings underscore the utility of CBD-IN formulation not only enhancing CNS delivery and therapeutic screening of CBD, but also for extending clinical exploration to other poorly soluble cannabinoids and hydrophobic drug candidates.

[0234] CBD Suppresses Neuropathic Pain While Preserving Normal Somatosensory Responses

[0235] To further characterize the therapeutic effects of CBD-IN, the von Frey response was assessed across a range of innocuous (0.008-0.4 g) and noxious (0.6-1.4 g) mechanical stimuli in both intact and spared nerve injury (SNI) mice, 30 minutes following intraperitoneal (i.p.) administration of CBD at a dose of 100 mg / kg. Buprenorphine, a standard opioid analgesic commonly used in mice and also administered to humans for the management of severe pain, was employed as a positive control. In intact animals, administration of CBD did not alter tactile perception or pain threshold, as both psychometric curves and withdrawal thresholds remained comparable to those observed in untreated controls (Figures 3A and 3C). In contrast, administration of Buprenorphine (25 pg / kg, i.p.) resulted in a significant reduction in responses to noxious stimuli (Figure 3A). These results indicate that, at the administered dose, CBD did not interfere with normal tactile or nociceptive responses in intact animals.

[0236] In SNI mice without CBD treatment, the tactile response curve was significantly shifted compared to intact mice (Figures 3A and 3B), reflecting increased sensitivity to both innocuous (allodynia) and noxious (hyperalgesia) stimuli. Administration of CBD-IN significantly suppressed both mechanical allodynia and hyperalgesia, as shown by reduced withdrawal percentages and improved response thresholds in von Frey tests (Figures 3B and 3D). CBD also reduced dynamic allodynia, evident from shorter flinching durations in the brush test (Figure 3E). Additionally, cold allodynia assessed by the acetone test was significantly alleviated (Figure 3F), whereas heat nociception measured in the 56°C hot plate test was unaffected (Figure 3G). This differential effect aligns with prior reports that SNI models typically produce strong cold allodynia but comparatively less heat hypersensitivity. Together, these results indicate that CBD selectively suppresses neuropathic pain-related hypersensitivity while preserving normal somatosensory function.

[0237] To rule out motor impairment as a confounding factor, rotarod and open field tests were conducted in SNI animals. A range of parameters reflecting motor coordination and locomotion were assessed, including latency to fall, distance run, and maximum revolutions per minute (RPM) in the rotarod test (Figures 3H-J), as well as total distance traveled and average velocity in the open field test (Figures 3K-L). No significant differences were observed between vehicle (VEH) and CBD-treated groups, indicating that CBD (100 mg / kg) does not produce motor-related side effects in SNI animals.

[0238] To assess the long-term therapeutic effects of CBD-IN, SNI mice received five doses on alternate days, with von Frey thresholds measured daily. Repeated administration maintained consistent analgesic efficacy throughout the treatment period. No signs of cumulative effects or drug tolerance were observed, as pain thresholds remained stable across sessions (Figure 3M). These results suggest that CBD-IN provides sustained analgesia without tolerance development, supporting its potential for repeated use in managing chronic neuropathic pain.

[0239] CBD Reduces Neuropathic Pain-Related Neuronal Activation in Somatosensory Pathways

[0240] The CBD-IN formulation with fast-acting and potent analgesic effects opens up the possibility to study the in vivo actions of CBD in the CNS. To identify potential targets involved in CBD analgesia, neuronal activation patterns in response to repetitive tactile stimulation in the SNI condition were mapped throughout the CNS, using an activity-dependent genetic reporter generated by crossing Fos2A'1CreER(TRAP2) driver mice with Ail4-tdTomato reporter mice (FosTRAP2:Ail4 ) (Figure 4A). At 7 days post-SNI, mice were injected with VEH or CBD (lOOmg / kg, i.p.) and habituated in the testing chamber for 30 minutes, consistent with the CBD peak time found in pharmacokinetic results (Figure 2B). Light brush stimulation was applied to the injured paw in three 10-minute sessions, each separated by a 1 -minute rest period. 4- Hydroxytamoxifen (4-OHT) was injected immediately after tactile stimulation to induce reporter expression in activated neurons, and brain and spinal cord tissue were harvested 7 days later for histological analysis.

[0241] The analysis was primarily focused on the somatosensory pathways, given that their functional significance in neuropathic pain had been established in prior studies (see, e.g., Todd, A.J. (2010). Nat Rev Neurosci 11, 823-836). In accordance with previously reported findings, repetitive brush stimulation administered to spared nerve injury (SNI) mice was found to activate neurons located in laminae I— II of the dorsal horn at the lumbar IV segment of the spinal cord unilaterally, as indicated by the presence of Fos-positive cells in the vehicle (VEH) group (Figure 4B). Administration of CBD prior to tactile stimulation was observed to reduce activation of laminae I— II neurons, which are positioned within the spinal nociceptive pathway. By contrast, activation of neurons located in laminae III-V remained unchanged following CBD treatment (Figure 4B).

[0242] Along the ascending somatosensory pathway, the ventrobasal (VB) thalamus, including both the ventral posterolateral (VPL) and ventral posteromedial (VPM) nuclei, has been recognized as a critical relay center for nociceptive information en route to the cerebral cortex. Following administration of CBD, bilateral reduction in Fos-positive labeling in the VB region was observed (Figure 4B). Moreover, a marked bilateral reduction in Fos-positive labeling was detected in the hindlimb region of the primary somatosensory cortex (HLS1) in the CBD-treated group (Figure 4B). A comparable reduction in Fos expression was also observed in the insular cortex (Figure 4B), a region believed to be involved in the processing of the affective dimension of pain.

[0243] Quantification of Fos-positive labeling was also performed in the prelimbic / infralimbic cortices (PL / IL) and the anterior cingulate cortex (ACC), regions that have been implicated in the top-down regulation of pain responses. However, no significant effect was observed in these regions following administration of CBD as compared to the vehicle control (VEH). This lack of effect suggests that neuronal activity in these areas may be modulated by a range of behavioral and environmental variables, not all of which are directly associated with neuropathic pain or the analgesic effects of CBD. In additional brain regions that are not believed to be directly involved in neuropathic pain, such as the dentate gyrus (DG), Fos-positive labeling was similarly unaffected by CBD treatment (Figure 4B).

[0244] Collectively, these findings indicate that CBD selectively attenuated neuropathic pain- associated neuronal hyperactivity within somatosensory pathways.

[0245] CBD Attenuates Neuronal Hyperactivity in Spinal Dorsal Horn Slices ex vivo

[0246] The anatomical mapping results above show that CBD reduces neuronal activity in both the laminae I-II of the spinal cord and the somatosensory cortex of SNI animals receiving tactile stimulation. However, it is not known whether CBD is acting on one region or both, as they are reciprocally connected via a polysynaptic spinal-cortical-spinal loop. To evaluate whether CBD exerted a direct effect on the spinal cord, the initial site of nociceptive input processing within the central nervous system, transverse spinal cord slices were prepared for ex vivo drug application and neural activity imaging (Figures 5A and 5B).

[0247] Because spontaneous neural activity is generally low in isolated spinal cord slices, previous slice electrophysiological studies have used the potassium channel blocker 4-Aminopyridine (4- AP) (100 pM) to induce epileptiform activity in the superficial laminae of the spinal cord, thereby modeling the disinhibited dorsal horn circuits characteristic of neuropathic pain. To evaluate the effects of CBD under the described conditions, an established ex vivo pharmacological model was adopted to assess hyperactivity in lamina I-II neurons. Although CBD-IN had been formulated with HPpCD and PS80 to enhance systemic absorption, these excipients were expected to degrade during uptake, thereby releasing free CBD for distribution within the central nervous system (CNS). In order to isolate the effects attributable to CBD alone, established ex vivo protocols were followed, wherein CBD was solubilized using 0.1% dimethyl sulfoxide (DMSO) for direct tissue perfusion. This approach permitted the controlled administration of CBD to spinal cord slices, in a manner consistent with prior electrophysiological and calcium imaging studies conducted in comparable ex vivo preparations.

[0248] Spinal cord slices were prepared from Vglut2-GCaMP6 transgenic mice to enable labeling of excitatory neurons in the dorsal horn using a calcium indicator for cell-type-specific calcium imaging. 4-aminopyridine (4-AP, 100 pM) was perfused into the slices concurrently with either vehicle (VEH) or cannabidiol (CBD) at a concentration of 20 pM, corresponding to levels measured in vivo (Figure 2B). Incubation with 4-AP was observed to induce pronounced neuronal hyperactivity within the superficial laminae (I— II) of the dorsal horn. Administration of CBD (20 pM) was found to delay the onset and reduce the amplitude of 4-AP-induced hyperactivity (Figures 5C and 5D). These findings indicate that CBD is capable of directly modulating spinal cord activity to attenuate hyperexcitability within nociceptive circuits of the dorsal horn.

[0249] CBD Suppresses Allodynia-Related Hyperactivity of Somatosensory Corticospinal Neurons in vivo

[0250] In a previous study, it was reported that corticospinal neurons (CSNs) located in the somatosensory cortex were found to project to the dorsal horn of the spinal cord and were implicated in the regulation of touch and tactile neuropathic pain sensitivity. In mice subjected to spared nerve injury (SNI), the activity of somatosensory CSNs in response to light tactile stimulation (von Frey 0.04 g) was observed to be abnormally amplified. Chemogenetic suppression of CSN activity was shown to result in a reduction of neuropathic pain. Accordingly, somatosensory CSN responses to tactile stimulation were identified as a potential neural marker indicative of the disinhibited pathological state of the spinal-cortical-spinal loop associated with neuropathic pain.

[0251] To examine the in vivo actions of CBD in both intact and SNI mice by CSN imaging, a retrograde adeno-associated virus (AAV) was injected in the lumbar dorsal horn of the spinal cord to label somatosensory CSNs with GCaMP6. Calcium imaging of CSNs was carried out using a head-attached miniscope through a cranial window in freely behaving mice). In intact animals, around 10% of CSNs in the somatosensory area responded to light tactile stimulation with a relatively low average evoked response (Figures 6A and 6B). CBD treatment did not alter the number of responding neurons or the average amplitude of responses. These results are consistent with the von Frey spectrum behavioral readout in intact animals (Figures 3A and 3C) and suggest that CBD does not affect normal tactile sensation.

[0252] In the neuropathic pain condition, CSN responses to von Frey (0.04 g) stimuli were amplified, showing an increased number of responding neurons and an elevated average amplitude of evoked responses (Figures 6C and 6D). CBD treatment significantly suppressed allodynia- related hyperactivity of somatosensory CSNs in terms of both responding neuron numbers and average response amplitude. On the other hand, although spontaneous movements in the test chamber were reduced in both intact and SNI mice after CBD treatment, CBD did not disturb the spontaneous calcium activities of CSNs in both conditions. Taken together, these results suggest that CBD specifically suppresses the hyperactive response of somatosensory CSNs in the neuropathic pain condition in a disease-state-dependent manner.

[0253] Discussion

[0254] Nano-formulated CBD for Enhanced Brain Delivery

[0255] The CBD-IN nano-formulation was developed by integrating two complementary dissolution strategies for hydrophobic compounds: (i) the formation of an inclusion complex using HPPCD and (ii) the assembly of nano-micelles using PS80. PS80 has been widely utilized as an excipient in food and pharmaceutical formulations. However, self-assembled nano-micelles formed from PS80 have been reported to exhibit low drug loading efficiency and limited stability, thereby constraining the concentration of CBD achievable in aqueous solutions.

[0256] Similarly, HPPCD has been recognized as a well-established solubilizing agent in the pharmaceutical industry. It has been shown to form inclusion complexes with CBD and to promote a supersaturated state by inhibiting nucleation and crystallization. Despite these advantages, the resulting supersaturated liquid state has been observed to become unstable upon subsequent dilution with water — a necessary condition for systemic drug delivery. To address these limitations, the supersaturated CBD / HPpCD inclusion complex and PS80 were introduced simultaneously into an aqueous medium. PS80, functioning as a surface-active polymeric precipitation inhibitor, was found to physically entrap the supersaturated CBD / HPPCD inclusion complex within nano-micelles. This approach facilitated the formation of a stable and efficient CBD-IN nano-formulation.

[0257] The size of CBD-IN nano-micelles (16-32 nm) falls within the optimal range (10-100 nm) for systemic nanoparticle delivery, supporting efficient gastrointestinal absorption, reducing rapid renal elimination and macrophage clearance, and facilitating passage across the blood-brain barrier. This characteristic likely underlies the present findings of high brain CBD concentrations and robust analgesic effect observed 30 minutes after intraperitoneal or oral administration of the CBD-IN formulation. While nanoparticle-based packaging strategies for cannabinoids have recently been explored, potential concerns such as nanomaterial toxicity, particle size-related pharmacokinetic changes, and their impact on therapeutic efficacy remain insufficiently characterized. The present study addresses these gaps by integrating chemical (LC / MS), structural (Cryo-TEM), neural, and behavioral analyses to elucidate the relationship between CBD-IN dosage, brain pharmacokinetics, and therapeutic outcomes in neuropathic pain.

[0258] CBD-mediated Suppression of Neuropathic Pain-Related Hyperactivity Across CNS

[0259] It was demonstrated that CBD suppressed neuropathic pain-associated neuronal activation throughout the CNS. Neuropathic pain has been associated with dysfunctions across both brain and spinal cord circuits, and a number of molecular targets — such as cannabinoid, glycine, gamma- aminobutyric acid (GABA), and 5-hydroxytryptamine 1A (5-HT1A) receptors, as well as sodium and calcium channels — have been implicated in the modulation of pain signaling across these circuits. However, prior studies of effects of CBD were primarily limited to investigations involving individual CNS regions.

[0260] By employing the FosTRAP2 neuronal activity labeling system in combination with a fastacting intranasal (CBD-IN) delivery method, multiple CNS regions were identified as being modulated by CBD in the context of neuropathic pain. These regions included the spinal dorsal horn, the ventrobasal (VB) nucleus of the thalamus, the primary somatosensory cortex, and the insular cortex. Notably, CBD did not reduce neuronal activity in regions not associated with neuropathic pain, such as the dentate gyrus (DG) of the hippocampus or higher-order cortical areas such as the prelimbic / infralimbic cortex (PL / IL) and the anterior cingulate cortex (ACC), which are only partially involved in pain regulation.

[0261] Accordingly, CBD was found to exert therapeutic effects through selective action within hyperactive somatosensory pathways, while being broadly distributed across the CNS. In contrast to conventional pain interventions that typically target a single molecular mechanism or anatomical region, the use of a selectively acting yet broadly distributed CBD formulation was shown to provide significant potential for effective neuropathic pain relief.

[0262] The calcium imaging studies provide further evidence that CBD suppresses aberrant neural activity in a disease-state-dependent manner. Specifically, CBD application rapidly reduced hyperactivity in spinal dorsal horn and somatosensory corticospinal circuits in neuropathic pain models while preserving normal neural activity in these same circuits. This selective neural modulation likely explains why CBD did not disturb normal somatosensory responses or nociception in intact animals — a desirable property for minimizing off-target side effects. While current first-line pharmacotherapies for neuropathic pain, such as gabapentinoids, are partially effective, they broadly reduce neuronal excitability. This non-specific mechanism of action can lead to cognitive and motor impairments, as well as altered pain perception. In contrast, the hyperactivity-dependent mechanism observed with CBD-IN may offer a more targeted therapeutic approach, avoiding widespread suppression of normal neural function. This form of selective neural suppression has not been previously described and may expand the therapeutic potential of CBD in treating neurological disorders associated with circuit hyperexcitability. Notably, the neuronal hyperexcitability and molecular changes observed in neuropathic pain share features with certain forms of epilepsy. Exploring the shared and distinct molecular mechanisms through which CBD suppresses pathological activity in these conditions presents an exciting direction for future research.

[0263] Translational Implications of Potent, Fast-Acting CBD Nano-formulations

[0264] It was demonstrated that nano-formulated cannabidiol (CBD-IN) rapidly and completely suppressed allodynia and hyperalgesia following a single administration in a fully established murine model of chronic neuropathic pain. In contrast, previously reported rodent studies described a delayed onset of analgesic effect, which typically required repeated administration of low doses of cannabidiol over multiple day. Such effects were believed to result from gradual systemic accumulation of cannabidiol, which may be associated with an increased risk of adverse events, including hepatotoxicity. Furthermore, in studies wherein cannabidiol treatment was initiated at the onset of neuropathy, observed improvements in pain thresholds may have reflected neuroprotective effects as opposed to direct analgesic action. In the present study, a single dose of CBD-IN was shown to restore normal somatosensory responses within 30 minutes in spared nerve injury (SNI) mice, thereby supporting the compound’s rapid and potent analgesic properties.

[0265] Notably, the CBD-IN formulation is suitable for oral administration, highlighting its strong translational potential. Its vehicle components, PS80 and HP0CD, are generally recognized as safe (GRAS) by the United States Food and Drug Administration (FDA) and are listed as pharmaceutical excipients in the Inactive Ingredient Database (Rowe, R.C., Sheskey, P., and Quinn, M. (2009). Handbook of pharmaceutical excipients (Libros Digitales-Pharmaceutical Press)). Compared to the current FDA-approved CBD formulation (Epidiolex), which uses sesame oil as a solvent, oral CBD-IN achieves significantly faster and greater brain delivery when administered orally, accompanied by enhanced analgesic efficacy. Unlike oil-based formulations that depend on lipid digestion and bile salt-mediated micellization for intestinal absorption, the nano-micelle system in CBD-IN mimics the natural micellization process of dietary fats, facilitating more efficient and consistent uptake. As a result, CBD-IN absorption is less likely to be affected by dietary variables. Additionally, its relatively rapid hepatic clearance may reduce the risk of liver toxicity. Taken together, the combination of improved CNS delivery for enhanced therapeutic efficacy and rapid systemic clearance to limit adverse effects suggests that CBD-IN may offer a broader therapeutic window than conventional oil-based CBD formulations.

[0266] While Epidiolex’ s slow pharmacokinetic profile supports long-term seizure management, its low bioavailability appears insufficient for pain relief, as shown in several clinical trials. Neuropathic pain, unlike seizures that are often localized to specific brain regions, involves widespread dysfunction in the somatosensory system. Breakthrough pain triggered by sensory stimuli often exceeds the relief provided by first-line treatments, increasing the risk of patients resorting to stronger, potentially harmful drugs like opioids. The rapid onset of analgesia with CBD-IN could improve quality of life, support treatment adherence, and reduce reliance on addictive medications. This example demonstrated an efficient nano-formulation enabling effective systematic administration of CBD and showed robust suppression of hyperactive somatosensory circuits and analgesic effects against neuropathic pain in mouse models, which provides a foundation for translating CBD treatment into clinical neuropathic pain management.

[0267] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A nano-micelle composition comprising a cannabinoid inclusion complex encapsulated in nano-micelles formed by a micelle-forming agent, wherein the cannabinoid inclusion complex comprises a cannabinoid encapsulated in cyclodextrin, and the cannabinoid inclusion complex slows down precipitation and / or crystallization of the cannabinoid during entrapment in the nano-micelles by the micelle-forming agent, wherein the micelle-forming agent is in an amount above a critical micelle concentration of the micelle-forming agent and in an amount of from about 0.2 to about 1.6 mole per mole of the cannabinoid, and wherein the cyclodextrin is in an amount of from about 1 to 2 mole per mole of the cannabinoid.

2. The nano-micelle composition of claim 1, wherein a mean particle diameter of the nanomicelles is from about 16 nm to about 32 nm.

3. The nano-micelle composition of any one of the preceding claims, wherein the amount of the cyclodextrin is about 1 mole per mole of the cannabinoid.

4. The nano-micelle composition of any one of the preceding claims, wherein the amount of the micelle-forming agent is about 0.81 mole per mole of the cannabinoid.

5. The nano-micelle composition of any one of the preceding claims, wherein the cannabinoid, the cyclodextrin, and the micelle-forming agent have a molar ratio of 1 : 1 : 0.81.

6. The nano-micelle composition of any one of the preceding claims, wherein the cannabinoid is an amount of from about 0% to about 22.8% by weight of the nano-micelle composition.

7. The nano-micelle composition of any one of the preceding claims, wherein the cyclodextrin is an amount of from about 0% to about 56.7% by weight of the nano-micelle composition.

8. The nano-micelle composition of any one of the preceding claims, wherein the micelleforming agent is an amount of from about 38.4%% to about 100% by weight of the nano-micelle composition.

9. The nano-micelle composition of any one of the preceding claims, wherein the nanomicelle composition comprises: about 11.3% by weight of the cannabinoid; about 50.3% by weight of the cyclodextrin; and about 38.4% by weight of the micelle-forming agent.

10. The nano-micelle composition of any one of the preceding claims, wherein the cannabinoid is selected from the group consisting of A9-tetrahydrocannabinol (THC), A8- tetrahydrocannabinol, A9-tetrahydrocannabinol propyl analog (THCV), cannabidiol (CBD), cannabidiol propyl analog (CBDV), cannabinol (CBN), cannabichromene, cannabichromene propyl analog, and cannabigerol or derivatives, and combinations thereof.

11. The nano-micelle composition of any one of the preceding claims, wherein the cannabinoid is cannabidiol (CBD).

12. The nano-micelle composition of any one of the preceding claims, wherein the cyclodextrin is selected from the group consisting of a-cyclodextrin, y-cyclodextrin, P- cyclodextrin, 2-hydroxypropyl-P-cyclodextrin, sulfobutylether -cyclodextrin sodium salt, randomly methylated P-cyclodextrin, branched P-cyclodextrin, y-cyclodextrin, and derivatives thereof.

13. The nano-micelle composition of any one of the preceding claims, wherein the cyclodextrin is 2-hydroxypropyl-P-cyclodextrin.

14. The nano-micelle composition of any one of the preceding claims, wherein the micelleforming agent comprises stearoyl polyoxyl-32 glycerides, lauroyl polyoxyl-32 glycerides, polysorbate 80, polysorbate 60, polysorbate 20, or a combination thereof.

15. The nano-micelle composition of any one of the preceding claims, wherein the micelleforming agent comprises polysorbate 80.

16. The nano-micelle composition of any one of the preceding claims, further comprising a pharmaceutically acceptable carrier.

17. The nano-micelle composition of any one of the preceding claims, wherein the cannabinoid inclusion complex is formed before entrapment in the nano-micelles by the micelleforming agent.

18. The nano-micelle composition of any one of the preceding claims, wherein the nanomicelle composition is a fluid pharmaceutical formulation for parenteral or oral use.

19. The nano-micelle composition of any one of the preceding claims, wherein the cannabinoid has a Cmax of about 6 pg / g in brain when the nano-micelle composition is administered to a mouse at 100 mg / kg intraperitoneal condition.

20. The nano-micelle composition of any one of the preceding claims, wherein the cannabinoid has a Cmax of about 7.8 pg / ml in serum when the nano-micelle composition is administered to a mouse at 100 mg / kg intraperitoneal condition.

21. The nano-micelle composition of any one of the preceding claims, wherein the cannabinoid has a half life of about 2 hours in brain after the nano-micelle composition is administered to a mouse.

22. A kit comprising the nano-micelle composition of any one of the preceding claims.

23. A method for preparing a nano-micelle composition according to any one of the preceding claims, comprising: preparing a cannabinoid solution by dissolving the cannabinoid in a solvent; gradually adding the cyclodextrin to the cannabinoid solution with agitation to obtain a mixture, incubating the mixture at a predetermined temperature for a predetermined time period to form the cannabinoid inclusion complex in which the cannabinoid is encapsulated in the cyclodextrin; and adding the micelle-forming agent to the mixture to form nano-micelles that encapsulate the cannabinoid inclusion complex.

24. The method of claim 23, wherein the solvent comprises ethanol.

25. The method of claim 23, wherein the cannabinoid and the solvent have a molar ratio of about 1 :20.

26. The method of any one of claims 23-25, wherein the predetermined temperature is 25-45° Celsius.

27. The method of any one of claims 23-26, wherein the predetermined time period is from about 1 hour to about 96 hours.

28. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the nano-micelle composition of any one of claims 1-21.

29. The method of claim 28, wherein the disease or disorder comprises neuropathic pain.

30. The method of claim 29, wherein the nano-micelle composition suppresses neuropathic pain-related activation of neurons in somatosensory pathways.31 . The method of claim 29, wherein the nano-micelle composition suppresses allodynia- related hyperactivity of somatosensory corticospinal neurons in the subject.

32. The method of claim 29, wherein the nano-micelle composition suppresses tactile allodynia or hyperalgesia in a subject having a spared nerve injury (SNI) but does not disturb tactile and nociceptive responses in a healthy subject.

33. The method of claim 29, wherein the nano-micelle composition only suppresses hyperactive responses of somatosensory corticospinal neurons (CSNs) in a neuropathic pain state, but not in an intact healthy state.

34. The method of any one of claims 28-33, wherein the nano-micelle composition exerts effects through spinal cord dorsal horn hyperactive circuits in the subject.

35. The method of any one of claims 28-34, comprising administering to the subject the nano-micelle composition in one or more doses to provide a dose of cannabinoid equivalent to from about 0.2 mg / kg to about 8 mg / kg by body weight of the subject.

36. The method of any one of claims 28-35, comprising administering to the subject the nano-micelle composition via an oral, intravenous, intraperitoneal, ophthalmic, parenteral, topical, transdermal, subcutaneous, subdural, intravenous, intramuscular, intradermal, intrathecal, intraperitoneal, intracerebral, intraarterial, intralesional, pulmonary, nasal spray, sublingual, or mucosal route.

37. Use of the nano-micelle composition of any one of claims 1-21 in the manufacture of a medicament for the treatment of neuropathic pain.

38. A nano-micelle composition according to any one of claims 1-21 for use in treating neuropathic pain.

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