Compositions and methods of use of cannabinoids for neuroprotection

Abstract

Provided herein are methods and compositions for neuroprotection. The neuroprotective composition may be or include cannabinol or a derivative thereof. The neuroprotective composition may be used in the treatment of neurodegenerative diseases. The neuroprotective composition may be used to protect retinal neurons from degeneration in a subject in need thereof (such as for the treatment of glaucoma).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 838,216, filed April 24, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] Neurodegeneration is a phenomenon that is responsible for a wide variety of central and peripheral nervous system diseases. Neurodegeneration includes neuronal atrophy, axonal degeneration (e.g., Wallerian and / or Wallerian-like degeneration), and induction of necrotic or programmed cell death mechanisms. Different types of programmed cell death, such as apoptosis, autophagy, pyroptosis, and oncosis, have all been demonstrated in neurons. Stimuli, such as physical injury, oxidative stress, excitotoxicity, mitochondrial dysfunction, inflammation, iron accumulation, and protein aggregation, have all been shown to contribute to neurodegenerative mechanisms.

[0003] Glaucoma is a form of optic nerve degeneration characterized by progressive degeneration of retinal ganglion cells, which are central nervous system cells arranged with their cell bodies located in the retina and their axons in the optic nerve. The degeneration of these neurons is associated with progressive loss of vision and a characteristic optic nerve head morphology referred to as "cupping."

[0004] The pathogenesis of glaucoma is poorly understood, and the factors that contribute to its progression have yet to be fully characterized. Glaucoma affects over 70 million people worldwide, 10% of whom lose their vision due to the disease. Because glaucoma symptoms are not apparent in the early and middle stages, the number of people affected by glaucoma may be much higher than the number of people diagnosed with the disease. In fact, some studies have shown that less than 50% of people diagnosed with glaucoma were aware that they had the disease. Glaucoma can be broadly divided into two categories: open-angle glaucoma and closed-angle glaucoma. In the United States, more than 80% of glaucoma cases are open-angle cases.

[0005] Glaucoma can be primary (i.e., without a clearly defined cause) or secondary (due to trauma, glucocorticoids, pigment scattering syndrome, or pseudoexfoliation syndrome). Thus, although the pathogenesis of glaucoma is not fully understood, it is known that elevated intraocular pressure is associated with neurodegeneration of retinal ganglion cells. Intraocular pressure is determined by the balance between the secretion of aqueous humor by the ciliary body and its drainage via the trabecular meshwork and uveoscleral outflow pathways.

[0006] Patients with open-angle glaucoma experience reduced aqueous humor outflow due to partial obstruction of the trabecular meshwork and uveoscleral outflow pathways. Intraocular pressure can induce stress and mechanical strain on the posterior structures of the eye, specifically the lamina cribrosa and adjacent tissues. Stress and mechanical strain induced by elevated intraocular pressure can cause compression, deformation, and remodeling of the lamina cribrosa, resulting in impaired axonal transport of essential trophic factors to retinal ganglion cells. It has been shown that the death of retinal ganglion cells can induce neurodegeneration of surrounding neurons, resulting in secondary and transsynaptic neuronal damage, which can have a significant impact on disease progression.

[0007] Elevated intraocular pressure is not the only risk factor, as some individuals with high intraocular pressure may not develop the disease, and in some cases intraocular pressure-lowering treatment alone has proven ineffective in slowing or halting disease progression. Alterations in the microcirculation and immune system, excitotoxicity, and oxidative stress may also contribute to the development of optic nerve degeneration, which may or may not be associated with elevated intraocular pressure levels.

[0008] One of the few currently known effective methods for treating neurodegeneration in glaucoma is lowering intraocular pressure. Multicenter clinical trials have shown that lowering intraocular pressure has beneficial results in preventing the onset and slowing the progression of the disease. However, lowering intraocular pressure is not always effective. Furthermore, even in subjects who benefit from lowering intraocular pressure, disease progression may not be halted and existing damage may not be reversed.

[0009] Other forms of eye disease associated with neurodegeneration include age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa. The worldwide prevalence of age-related macular degeneration (AMD) is approximately 14-24% in people aged 65-74 years and approximately 35% in people over 75 years of age, and AMD results in impaired vision or loss of the central visual field (macula) due to damage to the retina and / or associated neurons. AMD is the leading cause of vision loss and potential blindness in people over the age of 50. The two main forms of AMD are atrophic (non-exudative or "dry") AMD and neovascular (exudative or "wet") AMD. Atrophic AMD is characterized by the development of geographic atrophy (GA) in the center of the macula during advanced stages of AMD, and vision may gradually deteriorate over many years due to photoreceptor loss and the development of GA. Neovascular AMD is a more severe form of AMD and is characterized by the development of angiogenesis (e.g., choroidal neovascularization) during the advanced stages of AMD, which can rapidly lead to blindness. Neovascular AMD affects more than 30 million people worldwide and is the leading cause of vision loss in people over 60 years of age; if untreated, patients can lose central vision in the affected eye within 24 months of disease onset. Approximately 90% of AMD patients have the dry form, and approximately 10% develop neovascular AMD.

[0010] Diabetic retinopathy is a complication of diabetes that is caused by hyperglycemia-induced dysfunction of the vascular wall, resulting in retinal microvascular changes, such as impaired blood-retinal barrier function and increased permeability of the capillary circulation. Both capillary and neurodegeneration then occur, resulting in severe visual impairment. Diabetic retinopathy is the leading cause of blindness in diabetic patients.

[0011] Conventional methods for alleviating the symptoms of diabetic retinopathy include laser surgery, vitrectomy, and intraocular injection of corticosteroids. However, all of these conventional methods are invasive treatments and cannot completely cure diabetic retinopathy. Therefore, patients with diabetic retinopathy need to constantly monitor their blood glucose levels to ensure that normal blood glucose levels (euglycemia) are maintained. In addition, intraocular injection of corticosteroids may also cause side effects (such as steroid-induced disorders). In view of this, it is necessary to improve conventional methods for alleviating the symptoms of diabetic retinopathy.

[0012] Retinitis pigmentosa is a slowly progressive bilateral degeneration of the retina, retinal neurons, and retinal pigment epithelium caused by a variety of genetic mutations. Symptoms include night blindness and loss of peripheral vision.

[0013] Neuroprotection is the act of rescuing or restoring the nervous system, its cells, structures, and / or functions, or rendering it resistant to neurodegenerative stimuli. Neuroprotective compositions may be useful in treating or alleviating symptoms of various diseases that induce or induce neurodegeneration, such as glaucoma. Although there has been considerable progress in understanding the underlying neurodegenerative mechanisms, there remains a need in the art for improved methods and compositions for neuroprotection.

[0014] Cannabinoids and their derivatives have several properties that make them therapeutically promising. Activation or blocking of CB1 and / or CB2 receptors with cannabinoids can modulate downstream signaling and metabolic pathways, which in turn can affect synaptic transmission (including transmission of pain and other sensory signals) in the periphery, immune responses, and inflammation. Thus, there is interest in using natural or synthetic cannabinoids for therapeutic purposes. However, although there are anecdotal reports of therapeutic effects of cannabinoids, it has been demonstrated that the neuroprotective effects of many cannabinoids and their derivatives are undetectable at physiological concentrations. Furthermore, some cannabinoids and their derivatives have been shown to contribute to excitotoxicity at physiological concentrations. Summary of the Invention

[0015] Described herein are neuroprotective compositions and formulations, as well as methods for their preparation and use. The neuroprotective composition, or a formulation comprising the neuroprotective composition, can be contacted with neurons, thereby providing neuroprotection. In certain embodiments, the contacting is performed by administering the neuroprotective composition, or a formulation comprising the composition, to a subject in need thereof. The neuroprotective compositions, formulations, and related methods are useful in treating various neurodegenerative diseases. In certain embodiments, neuroprotective compositions are provided for use in inducing neuroprotection in retinal neurons. For example, the neuroprotective composition can be administered locally or systemically to a subject to induce neuroprotection in retinal neurons, thereby treating, for example, optic neurodegenerative diseases (such as glaucoma) or inhibiting neurodegeneration associated with diabetic retinopathy, AMD, and / or retinitis pigmentosa.

[0016] In one aspect, the present invention provides a method of protecting neurons from neurodegeneration, comprising contacting the neurons with a composition comprising cannabinol or a derivative thereof in an amount sufficient to inhibit neurodegeneration. In some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed in vivo. In some embodiments, the contacting comprises administering the composition to a subject in need thereof. In some embodiments, the neuron is a retinal neuron (e.g., a retinal ganglion cell).

[0017] In some embodiments, the contacting comprises administering the composition locally to a subject in need thereof. For example, the composition is administered to a subject suffering from neurodegeneration (e.g., ocular neurodegeneration). In some cases, the contacting comprises administering the composition to a subject suffering from a neurodegenerative disease (e.g., ocular neurodegenerative disease). In some cases, the contacting comprises administering the composition to a subject suffering from glaucoma. In some cases, the contacting comprises administering the composition to a subject diagnosed with glaucoma. In some embodiments, the method comprises co-administering or sequentially administering an additional active agent for the treatment of glaucoma.

[0018] In some cases, the contacting comprises administering the composition to a subject suffering from AMD. In some cases, the contacting comprises administering the composition to a subject diagnosed with AMD. In some embodiments, the method comprises simultaneously or sequentially administering an additional active agent for the treatment of AMD.

[0019] In some cases, the contacting comprises administering the composition to a subject suffering from diabetic retinopathy. In some cases, the contacting comprises administering the composition to a subject diagnosed with diabetic retinopathy. In some embodiments, the method comprises co-administering or sequentially administering an additional active agent for the treatment of diabetic retinopathy.

[0020] In some cases, the contacting comprises administering the composition to a subject suffering from retinitis pigmentosa. In some cases, the contacting comprises administering the composition to a subject diagnosed with retinitis pigmentosa. In some embodiments, the method comprises co-administering or sequentially administering an additional active agent for the treatment of retinitis pigmentosa.

[0021] In some embodiments, the amount sufficient to inhibit neurodegeneration is an amount sufficient to reduce the amount or rate of apoptosis of a population of neurons contacted with the composition. In some embodiments, the neurons are exposed to high hydrostatic pressure and the method comprises contacting the neurons with a composition comprising cannabinol or a derivative thereof in an amount sufficient to reduce pressure-induced neurodegeneration.

[0022] In some embodiments, an amount sufficient to inhibit neurodegeneration is an amount that provides a concentration of cannabinol (or a derivative thereof, such as cannabinolic acid or a prodrug thereof) of about 0.15 μM to less than about 15 μM upon contact with a target neuron or target neuron population, or in ocular tissue of the eye. In some embodiments, an amount sufficient to inhibit neurodegeneration is an amount that provides a concentration of cannabinol of about 0.5 μM to less than 15 μM upon contact with a target neuron or target neuron population, or in ocular tissue of the eye.

[0023] In some embodiments, an amount sufficient to inhibit neurodegeneration is an amount that provides a concentration of cannabinol (or a derivative thereof, such as cannabinol acid or a prodrug thereof) of about 0.5 μM to less than 15 μM upon contact with a target neuron or population of neurons or in ocular tissue of the eye, preferably an amount that provides a concentration of cannabinol (or a derivative thereof, such as cannabinol acid or a prodrug thereof) of about 0.5 μM to less than 12 μM. In some embodiments, an amount sufficient to inhibit neurodegeneration is an amount that provides a concentration of cannabinol (or a derivative thereof, such as cannabinol acid or a prodrug thereof) of about 1.5 μM to 10 μM upon contact with a target neuron or population of neurons or in ocular tissue of the eye.

[0024] In some embodiments, the cannabinol is provided in a sustained release formulation. In some embodiments, the formulation comprises a) a delivery vehicle comprising a cellulose polymer and an anionic polysaccharide, and b) nanoparticles comprising an amphiphilic non-ionic block copolymer and cannabinol, and the gel point of the formulation is about 30°C to about 37°C.

[0025] In some embodiments, the contacting comprises systemically administering a composition comprising cannabinol or a prodrug or derivative thereof (such as cannabinolic acid or a prodrug thereof). In some embodiments, the systemic administration comprises intravenous injection. In some embodiments, the systemic administration comprises oral administration. In some embodiments, the systemic administration comprises transdermal administration.

[0026] In some embodiments, the contacting comprises topically administering a composition comprising cannabinol or a prodrug or derivative thereof (such as cannabinolic acid or a prodrug thereof). In some cases, the contacting comprises administering a composition comprising cannabinol or a prodrug or derivative thereof (such as cannabinolic acid or a prodrug thereof) directly to the eye. For example, the contacting may comprise administering a composition comprising cannabinol or a prodrug or derivative thereof (such as cannabinolic acid or a prodrug thereof) to the eye (e.g., in the form of eye drops (such as microemulsion eye drops) or as an ophthalmic gel). As another example, the contacting may comprise administering a composition comprising cannabinol or a prodrug or derivative thereof (such as cannabinolic acid or a prodrug thereof) directly into the eye (e.g., via intravitreal injection or pump).

[0027] In another aspect, described herein is a composition for treating neurodegeneration in a subject, the composition comprising cannabinol or a derivative thereof. In some embodiments, the composition is a pharmaceutical formulation suitable for achieving a neuroprotective dose of cannabinol or a derivative thereof. In some embodiments, the composition is formulated for administration to the eye. In some embodiments, the composition is formulated to provide a concentration of cannabinol or a derivative thereof in the ocular tissue of the eye and / or upon contact with retinal ganglion cells of the eye of less than about 0.15 μM to about 15 μM.

[0028] In another aspect, a composition comprising cannabinol or a derivative thereof is described herein for use in treating neurodegeneration in a subject (such as hydrostatic pressure induced neurodegeneration), preferably according to one or more of the preceding aspects, embodiments, cases, or examples using a composition described herein, or according to a method described herein.

[0029] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0030] [Figure 1] In vitro neuroprotection of differentiated 661W retinal ganglion progenitor-like cells cultured under atmospheric pressure was demonstrated by contacting the cells with CBD, CBDA, CBC, CBG, CBGA, CBN, CBND, Δ9-THC (0.5 μM, 1.5 μM, and 5 μM for each cannabinoid, respectively). Vehicle control (VC) contained 0.15% ethanol. Data are presented as % cell death relative to vehicle control (set at 0%). Treatment conditions: different cannabinoids (0.5 μM, 1.5 μM, and 5 μM concentrations) or vehicle control (VC) for 72 hours. [Diagram 2]We show that there is no significant neuroprotection in differentiated 661W cells when cultured in a pressurized chamber with elevated hydrostatic pressure of approximately 20-40 mmHg by exposing the cells to CBD, CBDA, Δ9-THC, CBGA, or CBND (treatment concentrations of 0.5 μM, 1.5 μM, and 5 μM for each cannabinoid, respectively) or a vehicle control (VC) for 72 hours. The vehicle control (VC) contained 0.15% ethanol. Data are presented as % cell death relative to normal pressure vehicle control (cell death is considered 0%). [Diagram 3] We show statistically significant neuroprotection of differentiated 661W cells when cultured in a pressurized chamber with elevated hydrostatic pressure of approximately 20-40 mmHg by exposing the cells to a vehicle control (VC) containing 0.15% ethanol or CBN (0.015 μM, 0.05 μM, 0.15 μM, 0.5 μM, 1.5 μM, 5 μM, 10 μM, and 15 μM) (as indicated) for 72 hours. Data are presented as % cell death relative to the normopressure vehicle control (cell death taken as 0%). Tests for statistically significant differences were performed by comparison with the vehicle control (VC) by one-way ANOVA (Dunnett's multiple comparison test). [Figure 4] Figure 1 shows the significant neuroprotective effect of CBN compared to Δ9-THC on differentiated 661W cells when cultured for 72 hours under hydrostatic pressure of approximately 20-40 mmHg. Respective treatment concentrations were 0.5 μM, 1.5 μM, 5 μM, 10 μM, and 15 μM for CBN and 0.5 μM, 1.5 μM, and 5 μM for Δ9-THC. Vehicle control (VC) contained 0.15% ethanol. Data are presented as % cell death relative to the normostatic vehicle control (cell death taken as 0%). Statistically significant differences were tested by comparison with vehicle control (VC) by one-way ANOVA (Dunnett's multiple comparison test). [Diagram 5]We show a significant neuroprotective effect of CBN compared to CBD on differentiated 661W cells when cultured for 72 h under hydrostatic pressure of approximately 20-40 mmHg. Here, CBN and CBD (0.5 μM, 1.5 μM, 5 μM, 10 μM, and 15 μM, respectively, and 0.5 μM, 1.5 μM, and 5 μM) and a vehicle control (VC) were used. The vehicle control (VC) contained 0.15% ethanol. Data are presented as cell death (%) relative to the normobaric vehicle control (cell death is considered to be 0%). Statistically significant differences were tested by comparison with the vehicle control (VC) by one-way ANOVA (Dunnett's multiple comparison test). [Figure 6] Statistically significant neuroprotection of differentiated 661W cells when cultured in a pressurized chamber with elevated hydrostatic pressure of approximately 10-25 mmHg. Data are presented as % cell death relative to vehicle control (defined as 0%). Treatment conditions: 0.015 μM, 0.05 μM, 0.15 μM, 0.5 μM, 1.5 μM, 5 μM, 10 μM, and 15 μM concentrations, or vehicle control for 72 hours. Testing for statistically significant differences was performed by comparison with vehicle control (VC) by one-way ANOVA (Dunnett's multiple comparison test). [Figure 7] The effect of cannabinol on protecting 661W cells from apoptosis when cultured under hyperbaric pressure is shown. Treatment of 661W cells under hyperbaric pressure with a cannabinoid-free vehicle control (VC) induced apoptosis by approximately 35%. Treatment of 661W cells with cannabinol was able to protect neurons from apoptosis at concentrations >0.015μM to <5μM, with statistically significant protection (p<0.05 and p<0.01) in the range of 0.05-1.5μM. The vehicle control (VC) contained 0.15% ethanol. Data are presented as apoptosis (%) relative to the normobaric vehicle control (taken as 0%). Tests for statistically significant differences were performed by comparison with the vehicle control (VC) by one-way ANOVA (Dunnett's multiple comparison test). [Figure 8]Neuroprotective effect of cannabinol on RGCs as measured by pattern-evoked electroretinogram (pERG) amplitude in a rat episcleral venous laser photocoagulation glaucoma model. After induction of IOP elevation by laser treatment, the functional response of RGCs was measured by attenuation of the decrease in pERG amplitude in all treatment groups. Statistically significant impairment of RGC function was observed in the vehicle treatment group on day 21 and in the CBN (high dose) group on days 14 and 21 (two-way ANOVA followed by Tukey's multiple comparison test (*p<0.05)). There was no significant difference from baseline in pERG amplitude in the CBN (low dose) group (final intraocular concentration 5 μM) and brimonidine (ALPHAGAN) groups on both days 14 and 21 of follow-up, indicating that low dose CBN provides similar neuroprotective effect on RGCs as ALPHAGAN. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Described herein are methods and compositions for protecting neurons from one or more cytotoxic stimuli. In some embodiments, the method includes contacting the neurons with a neuroprotective composition, such as by administering the composition to a subject in need thereof. The methods and compositions described herein are particularly useful, but not limited to, for protecting retinal neurons. In some cases, the methods and compositions described herein can be used for neuroprotection of retinal neurons in a subject, such as a subject suffering from glaucoma or a subject suffering from elevated intraocular pressure compared to normal intraocular pressure (e.g., in a normal subject). In certain embodiments, the neuroprotective composition is a cannabinoid, such as cannabinol. In some cases, the method includes contacting the retinal neurons with a neuroprotective agent, such as cannabinol, such as by administering the neuroprotective agent to a subject in need thereof. In some cases, the neuroprotective composition is or includes cannabinol or a solvate thereof.

[0032] In some cases, the neuroprotective composition is or comprises a cannabinol derivative (such as those described in US 2003 / 0158191), a salt, or a solvate thereof. In one embodiment, the neuroprotective composition is or comprises a cannabinol derivative compound as claimed in US 7,105,685. In one embodiment, the neuroprotective composition is or comprises a cannabinol derivative compound selected from the group consisting of the cannabinol-type (CBN-type) cannabinoids described in ElSohly & Slade, Life Sciences, 78 (2005), p. 539-48. For example, the neuroprotective composition may comprise a cannabinol or a compound of formula I:

[0033] [ka]

[0034] or may comprise cannabinol or a derivative thereof having the formula In the formula: R 1 is H and R 2 is COOH and R 3 is n-CH 11 or R 1 is H and R 2 is H and R 3 is n-CH 11 or R 1 is CH3, R 2 is H and R 3 is n-CH 11 or R 1 is H and R 2 is H and R 3 is n-C4H9 or R 1 is H and R 2 is H and R 3 is n-C3H7 or R1 is H and R 2 is H and R 3 is C2H5, or R 1 is H and R 2 is H and R 3 is CH3.

[0035] In some embodiments, the neuroprotective composition may be or include a derivative having the formula of Formula I, where R 1 is H and R 2 is COOH, R 3 is n-CH 11 It is.

[0036] In some embodiments, the neuroprotective composition may include a prodrug of any one of the cannabinols or derivatives thereof described herein. For example, the neuroprotective composition may include a prodrug of a cannabinol or derivative thereof. As another example, the neuroprotective composition may include a prodrug of a derivative having the formula of Formula I. As another example, the neuroprotective composition may include a prodrug of a derivative having the formula of Formula I, where R 1 is H and R 2 is COOH and R 3 is n-CH 11 or R 1 is H and R 2 is H and R 3 is n-CH 11 or R 1 is CH3, R 2 is H and R 3 is n-CH 11 or R 1 is H and R 2 is H and R 3 is n-C4H9 or R 1 is H and R 2 is H and R 3 is n-C3H7 or R 1 is H and R 2 is H and R3 is C2H5 or R 1 is H and R 2 is H and R 3 is CH3. In some cases, the neuroprotective composition includes a prodrug of a derivative having the formula of Formula I, where R 1 is H and R 2 is COOH, R 3 is n-CH 11 It is.

[0037] In some cases, the prodrug is an ester of cannabinol or a derivative thereof. In some cases, the prodrug is an ester of a derivative having the formula of formula I (such as one of the derivatives described herein). In some cases, the prodrug is a D-(-)-glyceric acid ester of cannabinol or a derivative thereof. In some cases, the prodrug is a D-(-)-glyceric acid ester of cannabinolic acid or a derivative thereof. In some cases, the prodrug is a D-(-)-glyceric acid ester of a derivative having the formula of formula I (such as one of the derivatives described herein). For additional prodrug guidance for the neuroprotective compounds described herein, see U.S. Patent Publication Nos. 2016 / 0228490, 2011 / 0052694, 2015 / 0197484, 2008 / 0076789, 2009 / 0143462, 2012 / 0289484, 2009 / 0036523, 2009 / 0156814, and 2008 / 0008745, as well as Adelli et al., Investigative Ophthalmology & Visual Science, April 2017, Vol. 58, No. 4, p. 2168, and Upadhye et al., AAPS PharmSciTech, Vol. 11, No. 2, June 2017, and U.S. Patent Publication Nos. 2012 / 0289484, 2009 / 0036523, 2009 / 0156814, and 2008 / 0008745. 2010, p. 509, the contents of which are incorporated herein in their entirety for all purposes, and specifically incorporated herein with respect to the cannabinoid prodrug compositions and formulations, and methods of preparing, using, and / or administering such prodrug compositions described therein.

[0038] The neuroprotective composition may include an additional active agent. In some embodiments, the neuroprotective composition may include cannabinol or a derivative thereof and an additional cannabinoid or terpenoid. In some embodiments, the neuroprotective composition may include an additional active pharmaceutical agent for the treatment of glaucoma or an additional active pharmaceutical agent for the treatment of elevated intraocular pressure.

[0039] Currently, different classes of therapeutic agents are used to lower intraocular pressure and / or treat glaucoma, including, but not limited to, prostaglandin analogues, β-adrenergic antagonists, α-adrenergic agonists, carbonic anhydrase inhibitors, and / or cholinergic agonists.

[0040] Prostaglandin analogues include, but are not limited to, latanoprost, travoprost, tafluprost, unoprostone, or bimatoprost. Prostaglandin analogues are therapeutic agents that increase aqueous humor outflow through the uveoscleral outflow pathway. These drugs are typically administered once a day at night, thereby limiting their pressure-reducing effect to nighttime only. These drugs cause many side effects, both locally and systemically, including conjunctival hyperemia, thickening of eyelashes, iris discoloration, uveitis, macular edema, and headache.

[0041] β-adrenergic antagonists include, but are not limited to, timolol, levobunolol, carteolol, metipranolol, or betaxolol.The mechanism of action of these drugs is to reduce aqueous humor production.These drugs are typically administered once a day in the morning, and cause severe systemic side effects due to their antagonistic effect on β-adrenergic receptors.This limits their usability in patients with asthma, chronic obstructive pulmonary disease, and bradycardia.

[0042] Alpha-adrenergic agonists include, but are not limited to, brimonidine or apraclonidine. These drugs first reduce aqueous humor production, then increase aqueous humor outflow. Again, this causes many side effects, both local and systemic, including irritation and dryness of the eye, allergic reactions, effects on the central nervous system, respiratory arrest, postural hypotension, cerebral or coronary insufficiency, liver damage, and kidney damage. These drugs typically must be administered three times a day, which reduces patient compliance.

[0043] Carbonic anhydrase inhibitors include, but are not limited to, dorzolamide, brinzolamide, or acetazolamide. These drugs reduce aqueous humor production. Side effects include ocular irritation, eye burning, paresthesia, nausea, diarrhea, and loss of appetite.

[0044] Cholinergic agonists include, but are not limited to, pilocarpine or carbachol. These drugs increase aqueous humor outflow. These drugs are typically administered 5 times or more per day, which significantly reduces patient compliance, resulting in reduced efficacy due to poor adherence to treatment scheme. Again, side effects occur, including ocular irritation, myopia, ciliary spasm, miosis, blurred or reduced vision, and myopia with resulting headache and reduced vision.

[0045] In some embodiments, the neuroprotective compositions described herein (e.g., those comprising cannabinol or a derivative thereof) allow for a reduction in the dose or dosing frequency of one or more therapeutic agents for the treatment of glaucoma.

[0046] definition As used herein, "subject in need thereof" and similar terms refer to a mammal, preferably a human.

[0047] As used herein, "cannabinol" or "CBN" refers to 6,6,9-trimethyl-3-pentylbenzo[c]chromen-1-ol.

[0048] "Salt" refers to the acid addition salt or base addition salt of the compound used in the method of the present invention. Examples of pharmaceutically acceptable salts are salts of mineral acids (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like), salts of organic acids (acetic acid, propionic acid, glutamic acid, citric acid, and the like), salts of quaternary ammonium (methyl iodide, ethyl iodide, and the like). It is understood that pharmaceutically acceptable salts are non-toxic. Additional information regarding suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference.

[0049] The term "solvate" as used herein refers to a compound formed by solvation (a combination of a solvent molecule with a molecule or ion of a solute) or an aggregate of a solute ion or molecule (i.e., a compound of the present invention) with one or more solvent molecules. When water is the solvent, the corresponding solvate is a "hydrate". Examples of hydrates include, but are not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, hexahydrate, and other water-containing species. Those skilled in the art will appreciate that pharma-ceutically acceptable salts and / or prodrugs of a compound may also exist in the form of a solvate. Solvates are typically formed via hydration that is part of the preparation of the compound or formed by the natural absorption of water by anhydrous compounds of the present invention. In general, all physical forms are intended to be within the scope of the present invention.

[0050] Thus, when a therapeutically active agent prepared in a method according to the invention or included in a composition according to the invention (such as, but not limited to, a cannabinol derivative) has a sufficiently acidic functional group, a sufficiently basic functional group, or both a sufficiently acidic and a sufficiently basic functional group, such group(s) can react with any of a number of inorganic or organic bases and inorganic and organic acids, depending on the conditions, to form a pharma- ceutically acceptable salt. Examples of pharma- ceutically acceptable salts include salts prepared by reaction of a pharmacologically active compound with a mineral or organic acid or an inorganic base, such as sulfate, pyrosulfate, bisulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, or the like. Examples of suitable salts include phenylacetate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate. When the pharmacologically active compound possesses one or more basic functional groups, the desired pharma- ceutically acceptable salt may be prepared by any suitable method available in the art.Such methods include, for example, treating the free base with an inorganic acid (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like), or treating the free base with an organic acid (such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, and the like), or treating the free base with a pyranosidyl acid (such as glucuronic acid or galacturonic acid), or treating the free base with an alpha-hydroxy acid (such as citric acid, tartaric acid), or treating the free base with an amino acid (such as aspartic acid, glutamic acid), or treating the free base with an aromatic acid (such as benzoic acid, cinnamic acid), or treating the free base with a sulfonic acid (such as p-toluenesulfonic acid or ethanesulfonic acid), or the like. When the pharmacologically active compound has one or more acidic functional groups, the desired pharma-ceutically acceptable salt may be prepared by any suitable method available in the art. Such methods include, for example, treating the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary), an alkali or alkaline earth metal hydroxide, or the like. Examples of suitable salts include organic salts derived from amino acids (such as glycine and arginine), ammonia, primary, secondary, and tertiary amines, and cyclic amines (such as piperidine, morpholine, and piperazine), as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

[0051] As used herein, a "composition" is intended to encompass a product containing the specified ingredients in the specified amounts, as well as any product obtained by combining the specified ingredients in the specified amounts. "Pharmaceutically acceptable" is intended to mean that the carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0052] "Pharmaceutically acceptable excipient" refers to a substance that aids in the administration of and / or absorption by a subject of an active agent. Excipients useful in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, and coloring agents. One of ordinary skill in the art will recognize that other excipients are also useful in the present invention.

[0053] In some cases, the compounds used in the methods according to the invention or included in the compositions according to the invention may contain a protecting group. Such a protecting group is used to prevent hydrolysis or other reactions that may occur later in vivo and may degrade the compound. Groups that may be protected include alcohols, amines, carbonyls, carboxylic acids, phosphates, and terminal alkynes. Protecting groups useful for protecting alcohols include, but are not limited to, acetyl, benzoyl, benzyl, β-methoxyethoxyethyl ether, dimethoxytrityl, methoxymethyl ether, methoxytrityl, p-methoxybenzyl ether, methylthiomethyl ether, pivaloyl, tetrahydropyranyl, tetrahydrofuran, trityl, silyl ether, methyl ether, and ethoxyethyl ether. Protective groups useful for protecting amines include carbobenzyloxy, p-methoxybenzylcarbonyl, t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, tosyl, trichloroethyl chloroformate, and sulfonamide. Protective groups useful for protecting carbonyls include acetals, ketals, acylars, and dithianes. Protective groups useful for protecting carboxylic acids include methyl esters, benzyl esters, t-butyl esters, esters of 2,6-disubstituted phenols, silyl esters, orthoesters, and oxazolines. Protective groups useful for protecting phosphate groups include 2-cyanoethyl and methyl. Protective groups useful for protecting terminal alkynes include propargyl alcohol and silyl groups. Other protecting groups are known in the art.

[0054] The term "prodrug" as used herein refers to a derivative that is a precursor compound that releases a biologically active compound in vivo through some chemical or physiological process after administration (e.g., a prodrug is converted to a biologically active compound when the pH reaches a physiological pH or through enzymatic action). A prodrug itself may or may not have the desired biological activity. Thus, the term "prodrug" refers to a precursor of a biologically active compound that is pharma- ceutical acceptable. In certain cases, prodrugs have improved physical and / or delivery properties over the parent compound from which they are derived. The advantages often derived from prodrugs are solubility, tissue compatibility, or delayed release in mammalian organisms (H. Bundgard, Design of Prodrugs (Elsevier, Amsterdam, 1988), pp. 7-9, 21-24). T. Higuchi et al., "Pro-Drugs as Novel Delivery Systems," ACS Symposium Series, Vol. 14 and EB Roche, ed., Bioreversible Carriers in Drug Design (American Pharmaceutical Association & Pergamon Press, 1987) provide a discussion of prodrugs. Examples of advantages of prodrugs include, but are not limited to, their physical properties, such as enhanced drug stability for long-term storage.

[0055] The term "prodrug" is also intended to include any carrier covalently bonded to an active compound that liberates the active compound in vivo when the prodrug is administered to a subject. Prodrugs of the therapeutically active compounds described herein may be prepared by modifying one or more functional groups present in the therapeutically active compounds (including cannabinoids (such as cannabinol) or cannabinol derivatives) and other therapeutically active compounds used in the methods according to the invention or included in the compositions according to the invention in such a manner that the modification is cleaved either in a routine operation or in vivo to yield the therapeutically active parent compound. Prodrugs include compounds in which a hydroxy, amino, or mercapto group is covalently bonded to any group that is cleaved to form a free hydroxy, amino, or mercapto group, respectively, when the prodrug of the active compound is administered to a subject. Examples of prodrugs include, but are not limited to, formate or benzoate derivatives of alcohols or acetamides, formamide or benzamide derivatives of therapeutically active agents having an amine functional group available for reaction, and the like. In some cases, the prodrug is a derivative of a neuroprotective compound modified with a protecting group (such as a cannabinol modified with a protecting group or a cannabinol derivative modified with a protecting group).

[0056] For example, if the therapeutically active agent, or a pharma- ceutically acceptable form of the therapeutically active agent, contains a carboxylic acid functional group, the prodrug may include an ester formed by replacing the hydrogen atom of the carboxylic acid group with a group. Such a group replacing the hydrogen atom of the carboxylic acid group may be C 1-8 Alkyl, C 2-12Alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)ethyl having 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having 3 to 9 carbon atoms, N-(alkoxycarbonyl)aminomethyl having 4 to 10 carbon atoms and 1-(N-(alkoxycarbonyl)amino)ethyl, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolactone-4-yl, di-N,N(C1-C2)alkylamino(C2-C3)alkyl (such as (3-dimethylaminoethyl)), carbamoyl-(C1-C2)alkyl, N,N-di(C1-C2)alkylcarbamoyl-(C1-C2)alkyl, and piperidino(C2-C3)alkyl, pyrrolidino(C2-C3)alkyl, or morpholino(C2-C3)alkyl.

[0057] In some cases, the therapeutically active agent, or a pharma- ceutical acceptable form of the therapeutically active agent, may be a hydroxyl group that is selected from the group consisting of H and R. 2 and the prodrug is a cannabinoid comprising 3,6,9,12-tetraoxatridecanoyl ester, N,N-dimethyl glycyl ester, 3,6,9,12-tetraoxatridecyl carbonate, N-formyl glycyl ester, N-formyl sarkosyl ester, 3,6,9,12-tetraoxatridecyl oxalate, hemisuccinate, 4-aminobutyl carbamate, prolyl ester, 3-dimethylaminopropionate, glycolate, (D)-ribonate, ammonium phosphate salt, (R)-2,3-dihydroxypropyl carbonate, 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate, glycinate, β-alaninate, (S)-2,3-dihydroxypropanoate, (S)-2,3-dihydroxypropyl carbonate, or (R)-2,3-dihydroxypropyl carbonate in the R group. 1 Included.

[0058] In some cases, the therapeutically active agent, or a pharma- ceutical acceptable form of the therapeutically active agent, may have a carboxylic acid functional group R 2 and the prodrug is a cannabinoid comprising a 3,6,9,12-tetraoxatridecanoyl ester, a N,N-dimethyl glycyl ester, a 3,6,9,12-tetraoxatridecyl carbonate, a N-formyl glycyl ester, a N-formyl sarkosyl ester, a 3,6,9,12-tetraoxatridecyl oxalate, a hemisuccinate, a 4-aminobutyl carbamate, a prolyl ester, a 3-dimethylaminopropionate, a glycolate, a (D)-ribonate, an ammonium phosphate salt, an (R)-2,3-dihydroxypropyl carbonate, a 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate, a glycinate, a β-alaninate, an (S)-2,3-dihydroxypropanoate, an (S)-2,3-dihydroxypropyl carbonate, or an (R)-2,3-dihydroxypropyl carbonate derivative in R. 1 Included.

[0059] In some cases, the prodrug may be an ester, carbonate, carbamate, or phosphate (such as one of the esters, carbonates, carbamates, or phosphates described above) in R. 1 Prodrugs of CBNA (R of Formula I) 2 is COOH, R 3 is n-CH 11 (That is.)

[0060] Similarly, when a disclosed compound, or a pharma- ceutically acceptable form of such a compound, contains an alcohol functional group, a prodrug can be formed by replacing the hydrogen atom of the alcohol group with a group. Such groups replacing the hydrogen atom of the alcohol group are (C1-C6)alkanoyloxymethyl, 1-((C1-C6))alkanoyloxy)ethyl, 1-methyl-1-((C1-C6)alkanoyloxy)ethyl(C1-C6)alkoxycarbonyloxymethyl, N(C1-C6)alkoxycarbonylaminomethyl, succinoyl, (C1-C6)alkanoyl, α-amino(C1-C4)alkanoyl, arylacyl and α-aminoacyl or α-aminoacyl-α-aminoacyl (each α-aminoacyl group is independently selected from naturally occurring L-amino acids), P(O)(OH)2, P(O)(O(C1-C6)alkyl)2 or glycosyl (the radical obtained by removing the hydroxyl group of the hemiacetal form of a carbohydrate).

[0061] When the disclosed compounds, or pharma- ceutically acceptable forms of such compounds, contain an amine functional group, prodrugs can be formed by replacing a hydrogen atom in the amine group with a group. Such groups replacing a hydrogen atom in the amine group include R-carbonyl, RO-carbonyl, NRR'-carbonyl, where R and R' are each independently (C1-C 10 )alkyl, (C3-C7)cycloalkyl, benzyl, or R-carbonyl is natural α-aminoacyl or natural α-aminoacyl-natural α-aminoacyl), C(OH)C(O)OY 1 (Y 1 is H, (C1-C6) alkyl, or benzyl), C(OY 2 )Y 3 (Y 2 is (C1-C4) alkyl; Y 3 is (C1-C6)alkyl, carboxy(C1-C6)alkyl, amino(C1-C4)alkyl, or mono-N(C1-C6)alkylaminoalkyl or di-N,N(C1-C6)alkylaminoalkyl), C(Y 4 )Y 5 (Y4 is H or methyl, and Y 5 is mono-N(C1-C6)alkylamino or di-N,N(C1-C6)alkylamino, morpholino, piperidin-1-yl, or pyrrolidin-1-yl).

[0062] The use of prodrug systems is described in T. Jarvinen et al., "Design and Pharmaceutical Applications of Prodrugs," Drug Discovery Handbook (SCGad, ed., Wiley-Interscience, Hoboken, NJ, 2005), ch. 17, pp. 733-796. Other alternatives for constructing and using prodrugs are known in the art. Where a method or pharmaceutical composition according to the invention uses or contains a prodrug of cannabinol or another therapeutically active agent, the prodrug and active metabolites of the compound may be identified using routine techniques known in the art. For example, Bertolini et al., J.Med.Chem.,40,2011-2016(1997), Shan et al.,J.Pharm.Sci.,86(7),765-767, Bagshawe,Drug Dev.Res.,34,220-230(1995), Bodor,Advances in Drug Res., 13, 224-331 (1984), Bundgaard, Design of Prodrugs (Elsevier Press 1985), Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991), Dear et al. al., J. Chromatogr. B, 748, 281-293 (2000), Spraul et al., J. Pharmaceutical & See Biomedical Analysis, 10, 601-605 (1992), and Prox et al., Xenobiol., 3, 103-112 (1992).

[0063] As used herein, the term "therapeutically effective quantity," "therapeutically effective dose," or "therapeutically effective amount" refers to a dose of one or more compositions described herein that provides the therapeutic effect for which it is administered. The exact dose will depend on the therapeutic purpose and can be ascertained by one of skill in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0064] Cannabinoids Cannabinoids are a group of chemicals known to activate cannabinoid receptors in cells throughout the human body, including the skin. Phytocannabinoids are cannabinoids obtained from the cannabis plant. Phytocannabinoids can be isolated from plants or produced synthetically. Endocannabinoids are endogenous cannabinoids that are naturally produced by cells in the human body. Standard phytocannabinoids are ABC tricyclic terpenoid compounds with a benzopyran moiety.

[0065] Cannabinoids exert their effects by interacting with cannabinoid receptors present on the cell surface. Currently, two types of cannabinoid receptors have been identified: CB1 and CB2 receptors. These two receptors share approximately 48% amino acid sequence identity, are distributed in different tissues, and have different cell signaling mechanisms. These two receptors also differ in their agonist and antagonist sensitivity.

[0066] In some cases, the cannabinoid or precursor thereof may be purified, derivatized (e.g., to form a prodrug, solvate, or salt, or to form the target cannabinoid from the precursor), and / or formulated into a pharmaceutical composition.

[0067] Cannabinoids include, but are not limited to, phytocannabinoids. In some cases, cannabinoids include, but are not limited to, cannabinol, cannabidiol, Δ 9 -Tetrahydrocannabinol (Δ 9 -THC), the synthetic cannabinoid HU-210 (6aR,10aR)-9-(hydroxymethyl)-6,6-dimethyl-3-(2-methyloctan-2-yl)-6H,6aH,7H,10H,10aH-benzo[c]isochromen-1-ol), and HU-308 ([(1R,2R,5R)-2-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-7,7-dimethyl-4-bicyclo[3. 1.1]hept-3-enyl]methanol), HU-433 (enantiomer of HU-308), cannabidivarin (CBDV), cannabichromene (CBC), cannabichromevarin (CBCV), cannabigerol (CBG), cannabigerovarin (CBGV), cannabielsoin (CBE), cannabicyclol (CBL), cannabivarin (CBV), and cannabinotriol (CBT). Further cannabinoids include tetrahydrocannabivarin (THCV) and cannabigerol monomethyl ether (CBGM). Additional cannabinoids include cannabichromenic acid (CBCA), Δ 9-tetrahydrocannabinolic acid (THCA), and cannabidiol acid (CBDA); these additional cannabinoids are characterized by the presence of a carboxylic acid group in their structure.

[0068] Further cannabinoids include nabilone, rimonabant, JWH-018 (naphthalen-1-yl-(1-pentylindol-3-yl)methanone), JWH-073 naphthalen-1-yl-(1-butylindol-3-yl)methanone, CP-55940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol), dimethylheptylpyran, HU-331 (3-hydroxy-2-[(1R)-6-isopropenyl-3-methyl -cyclohex-2-en-1-yl]-5-pentyl-1,4-benzoquinone), SR144528 (5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-1H-pyrazole-3-carboxamide), WIN55,212-2 ((11R)-2-methyl-11-[(morpholin-4-yl)methyl]-3-(naphthalene-1-carbonyl)-9-oxa-1-azatricyclo[6.3.1.0 4 , 12 ]dodeca-2,4(12),5,7-tetraene), JWH-133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran), levonatradol, and AM-2201 (1-[(5-fluoropentyl)-1H-indol-3-yl]-(naphthalen-1-yl)methanone). Other cannabinoids include Δ 8 -Tetrahydrocannabinol (Δ 8 -THC), 11-hydroxy-Δ 9 -Tetrahydrocannabinol, Δ 11 -tetrahydrocannabinol, and 11-hydroxy-tetracannabinol.

[0069] Alternatively, analogues or derivatives of such cannabinoids may be obtained by obtaining a precursor cannabinoid and further derivatizing it (eg, by synthetic means). Synthetic cannabinoids include, but are not limited to, those described in U.S. Pat. No. 9,394,267 to Attala et al., those described in U.S. Pat. No. 9,376,367 to Herkenroth et al., those described in U.S. Pat. No. 9,284,303 to Gijsen et al., those described in U.S. Pat. No. 9,173,867 to Travis, those described in U.S. Pat. No. 9,133,128 to Fulp et al., those described in U.S. Pat. No. 8,778,950 to Jones et al., those described in U.S. Pat. No. 7,700,634 to Adam-Worrall et al., those described in U.S. Pat. No. 7,504,522 to Davidson et al., those described in U.S. Pat. No. 7,294,645 to Barth et al., those described in U.S. Pat. No. 7,109,216 to Kruse et al., those described in U.S. Pat. No. 7,109,216 to Thomas et al., and those described in U.S. Pat. No. 7,109,216 to Thomas et al. al., and U.S. Pat. No. 6,284,788 to Mittendorf et al.

[0070] Neuroprotective cannabinoids according to the present invention may be at least partially selective for binding to either the CB2 or CB1 cannabinoid receptor. In some embodiments, the neuroprotective cannabinoids bind to both the CB1 and CB2 cannabinoid receptors. In some cases, the neuroprotective cannabinoids according to the present invention are selective for the CB1 cannabinoid receptor and act as partial agonists. In some other cases, the neuroprotective cannabinoids according to the present invention are selective for the CB2 cannabinoid receptor and act as partial agonists. In some cases, the neuroprotective cannabinoids bind to both the CB1 and CB2 receptors and act as partial agonists for both receptors, but have a higher affinity for the CB2 receptor and a Δ 9 -THC. In some cases, the cannabinoid, or one of the cannabinoids in the mixture of neuroprotective cannabinoids, is an inverse agonist of the CB2 receptor. As an inverse agonist, the neuroprotective cannabinoid may bind to the CB2 receptor, but induce a pharmacological response opposite to that of an agonist. In some cases, the cannabinoid in the neuroprotective composition and method according to the invention is partially selective for the CB2 cannabinoid receptor. In some cases, the neuroprotective cannabinoid or mixture of cannabinoids has a K 1 receptor, e.g., in an in vitro competitive assay, for the CB1 receptor. i At least 3-fold lower K for the CB2 receptor compared to i , with an overall higher binding affinity to CB2.

[0071] An example of a cannabinoid is cannabinol or cannabinolic acid.

[0072] Examples of prodrugs useful in the present invention include, but are not limited to, the following prodrug of cannabinol (left) and the following prodrug of cannabinolic acid (right):

[0073] [ka]

[0074] wherein X and Y may be the same or different and are selected from the group consisting of hydrogen, alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., calcium and magnesium), and cations of pharma- ceutically acceptable organic amines (e.g., quaternized or protonated amines, including alkylamines, hydroxyalkylamines, monoamines, diamines, and naturally occurring amines). Examples of such pharma- ceutically acceptable organic bases include choline, betaine, caffeine, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, hydrabamine, isopropylamine, methylglucamine, morpholine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, tetramethylammonium hydroxide, benzyltrimethylammonium hydroxide, tris(hydroxymethyl)aminomethane (TRIS), N-(2-hydroxyethyl)pyrrolidine, piperazine, glucosamine, arginine, lysine, and histidine. In another embodiment, X and Y are different substituents. In another embodiment, X and Y are the same substituent. In another embodiment, X and Y can both be part of the same functional group, such as piperazine. In another embodiment, the phosphate is selected from the group consisting of diphosphates and triphosphates, hi another embodiment, the compound is in the form of a diphosphate or triphosphate salt.

[0075] [ka]

[0076] In the formula, R 4is a linear or branched, substituted or unsubstituted alkyl or alkoxyalkyl, alkylamine, hydroxyalkyl, or hydroxyalkylamine, preferably R 4 contains 1 to 12 carbons and optionally contains up to 4 substitutions, more preferably R 4 contains 1 to 6 carbons and optionally contains up to 2 substitutions.

[0077] [ka]

[0078] In the formula, R 4 is a linear or branched, substituted or unsubstituted alkyl or alkoxyalkyl, alkylamine, hydroxyalkyl, or hydroxyalkylamine, preferably R 4 contains 1 to 12 carbons and optionally contains up to 4 substitutions, more preferably R 4 contains 1 to 6 carbons and optionally contains up to 2 substitutions.

[0079] [ka]

[0080] In the formula, R 4 is a linear or branched, substituted or unsubstituted alkyl or alkoxyalkyl, alkylamine, hydroxyalkyl, or hydroxyalkylamine, preferably R 4 contains 1 to 12 carbons and optionally contains up to 4 substitutions, more preferably R 4 contains 1 to 6 carbons and optionally contains up to 2 substitutions.

[0081] [ka]

[0082] In the formula, R 4is a linear or branched, substituted or unsubstituted alkyl or alkoxyalkyl, alkylamine, hydroxyalkyl, or hydroxyalkylamine, preferably R 4 contains 1 to 12 carbons and optionally contains up to 4 substitutions, more preferably R 4 contains 1 to 6 carbons and optionally contains up to 2 substitutions.

[0083] In some embodiments, prodrugs useful in the present invention include, but are not limited to, the following prodrug of cannabinol (left) and the following prodrug of cannabinolic acid (right):

[0084] [ka]

[0085] In some embodiments, the aforementioned prodrugs may be advantageously formulated with a cyclodextrin, such as randomly methylated beta-cyclodextrin, 2-hydroxypropyl beta-cyclodextrin, or sulfobutyl ether beta-cyclodextrin.

[0086] In some embodiments, prodrugs useful in the present invention include, but are not limited to, the following prodrug of cannabinol (left) and the following prodrug of cannabinolic acid (right):

[0087] [ka]

[0088] In some embodiments, prodrugs useful in the present invention include, but are not limited to, the following prodrugs of cannabinol:

[0089] [ka]

[0090] In some embodiments, prodrugs useful in the present invention include, but are not limited to, the following prodrugs of cannabinolic acid:

[0091] [ka]

[0092] Pharmaceutical Compositions The compositions described herein are typically formulated for administration.Thus, the present specification describes cannabinol-containing compositions that are formulated for administration with one or more pharma- ceutically acceptable carrier(s), diluent(s), or excipient(s).

[0093] Pharmaceutical compositions can be prepared by known procedures using well-known and readily available ingredients.

[0094] Pharmaceutical compositions containing cannabinol can be formulated for administration to a subject by one of a variety of standard routes, such as ophthalmic, oral, topical, parenteral, by inhalation or spray, rectal, or vaginal, in unit dose formulations containing non-toxic conventional pharma- ceutical acceptable carriers, adjuvants, excipients, and / or vehicles.

[0095] The term parenteral as used herein includes subcutaneous injections, intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection and infusion techniques in various embodiments. Pharmaceutical compositions will typically be formulated in a form suitable for administration to a subject by a selected route, for example, as eye drops, ophthalmic depots, syrups, elixirs, tablets, lozenges, lozenges, hard or soft capsules, pills, suppositories, oily or aqueous suspensions, dispersible powders or granules, emulsions, injectable solutions, or solutions.

[0096] In certain embodiments, the cannabinol composition is formulated for administration via a systemic route (e.g., intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, or oral route).

[0097] Compositions intended for oral use may be prepared in solid or liquid unit dosage forms. Liquid unit dosage forms may be prepared according to procedures known in the art for the manufacture of pharmaceutical compositions, and one or more agents selected from the group consisting of sweeteners, flavorings, colorings, and preservatives may be included in such compositions to obtain medicamentously elegant and palatable preparations. Elixirs are prepared by using a hydroalcoholic (e.g., ethanol) vehicle containing a suitable sweetener (such as sugar or saccharin) together with an aromatic flavoring agent. Suspensions may be prepared using an aqueous vehicle with the aid of suspending agents (such as acacia, tragacanth, methylcellulose, and the like).

[0098] Solid formulations (such as tablets) contain the active ingredient in admixture with non-toxic pharma- ceutically acceptable excipients suitable for the manufacture of tablets. Such excipients may be, for example, inert diluents (such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate), granulating and disintegrating agents (such as corn starch or alginic acid), binders (such as starch, gelatin, or acacia), and lubricants (such as magnesium stearate, stearic acid, or talc), as well as other conventional ingredients (such as dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, methylcellulose, and functionally similar materials). Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. For example, a time-delay material (such as glyceryl monostearate or glyceryl distearate) may be used.

[0099] Formulations for oral use may also be provided as hard gelatin capsules (wherein the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin) or soft gelatin capsules (wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil). Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compound with an acceptable vegetable oil, light liquid petrolatum, or other inert oil.

[0100] Aqueous suspensions contain the active ingredient in admixture with one or more excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia), and dispersing or wetting agents (such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearates), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., hepta-decaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate)). The aqueous suspensions may also contain one or more preservatives, for example, ethyl-p-hydroxybenzoate or n-propyl-p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, or one or more sweetening agents, such as sucrose or saccharin.

[0101] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or in a mineral oil (such as liquid paraffin). Oily suspensions may contain a thickening agent (e.g., beeswax, hard paraffin, or cetyl alcohol). Sweetening agents (such as those set forth above) and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant (such as ascorbic acid).

[0102] Dispersible powders and granules suitable for preparation of an aqueous suspension by adding water provide the active ingredient in a mixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents include those already mentioned above. Additional pharmaceutical additives (e.g., sweeteners, flavorings, and coloring agents) may also be present.

[0103] The pharmaceutical composition may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil (e.g., olive oil or peanut oil) or a mineral oil (e.g., liquid paraffin), or a mixture thereof. Suitable emulsifiers may be naturally occurring gums (e.g., gum acacia or gum tragacanth), naturally occurring phosphatides (e.g., soybean lecithin), and esters or partial esters obtained from fatty acids and hexitol anhydrides (e.g., sorbitan monooleate), and the condensation products of such partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). The emulsion may also contain optional sweeteners and flavoring agents.

[0104] The pharmaceutical composition may be in the form of an aqueous or oily sterile injectable suspension. Such suspensions may be formulated as known in the art using suitable dispersing or wetting agents and suspending agents (such as those mentioned above). The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (such as a 1,3-butanediol solution). Other acceptable vehicles and solvents that may be used include, for example, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils may also be used as a solvent or suspending medium. Various non-irritating fixed oils known to be suitable for this purpose may be used, including synthetic mono- or diglycerides. In addition, fatty acids (such as oleic acid) are also useful in the preparation of injectable solutions. Injectable solutions or suspensions may also optionally contain auxiliary agents (such as local anesthetics, preservatives, and buffers).

[0105] Pharmaceutical compositions and methods for preparing pharmaceutical compositions are otherwise known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remington's Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, Pa. (2000).

[0106] The concentration of the neuroprotective compound (eg, cannabinol) in the formulation will vary depending on the condition to be treated and / or the mode of administration.

[0107] method Described herein are methods of protecting neurons from neurodegenerative stimuli. In general, the methods include contacting neurons with an effective amount of a composition comprising cannabinol or a cannabinol derivative. The methods can be in vitro methods. Alternatively, the methods can be methods that are performed at least in part in vivo, such as by administering a neuroprotective composition to a subject. The administration can be performed by systemic injection (e.g., intravenous or subcutaneous injection) or local injection. For example, local injection can include intravitreal injection. The administration can be performed by non-invasive local administration methods. For example, local administration to retinal neurons (such as retinal ganglion cells) can include administration of an eye drop formulation, such as a hydrogel (see, e.g., WO2018 / 205022) or a microemulsion (see, e.g., U.S. Pat. No. 9,149,453).

[0108] In certain embodiments, the compound is administered for less than 6 weeks. In certain embodiments, the compound is administered for about 1-4 weeks. In other embodiments, the compound will be administered for an extended period of time (such as years) or for the remaining lifespan of the patient, such as to treat neurodegenerative diseases (such as glaucoma). The compound may be administered once a week, once every other day, once a day, twice a day, or three times a day.

[0109] The neuroprotective compound may be administered to treat the eye of a subject in need of treatment to protect retinal neurons (e.g., optic nerve fibers). For example, the subject may have suffered an injury (such as a physical injury) that affects optic nerve fibers. As another example, the subject may be in a situation where they have glaucoma or have been diagnosed with glaucoma. When the neuroprotective compound is administered to protect neurons (such as retinal neurons), the neuroprotective compound may be administered at a dose that results in a peak, median, or trough (preferably, a peak) neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative thereof) upon contact with the target neuron or target neuron population. In some embodiments, the target neuron is a retinal neuron. In some embodiments, the target neuron is a peripheral neuron. In some embodiments, the target neuron is a central neuron.

[0110] In one embodiment, the neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative or prodrug thereof) upon contact with a target neuron or target neuronal population is less than about 25 μM, less than about 20 μM, less than about 15 μM, less than about 14 μM, less than about 13 μM, less than about 12 μM, or less than about 10 μM. In one embodiment, the neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative thereof) upon contact with a target neuron or target neuronal population is greater than about 0.15 μM to less than about 25 μM, or greater than 0.15 μM to less than 25 μM, or at least about 0.15 μM to less than about 25 μM, or at least about 0.15 μM to less than 25 μM, or greater than 0.15 μM to less than about 20 μM, or greater than 0.15 μM to less than 20 μM, or at least about 0.15 μM to less than 20 μM.

[0111] In one embodiment, the neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative thereof) upon contact with the target neuron or target neuron population is greater than about 0.15 μM to less than about 15 μM, or greater than 0.15 μM to less than 15 μM, or at least about 0.15 μM to less than about 15 μM, or at least 0.15 μM to less than 15 μM, or greater than about 0.15 μM to less than 12 μM, or greater than 0.15 μM to less than 12 μM, or at least about 0.15 μM to less than 12 μM. In one embodiment, the neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative thereof) is at least about 0.5 μM to less than about 25 μM, or at least about 0.5 μM to less than 25 μM, or at least about 0.5 μM to less than 20 μM, or at least 0.5 μM to less than 20 μM. In one embodiment, the neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative thereof) is at least about 0.5 μM to less than about 15 μM, or at least about 0.5 μM to less than 15 μM, or at least about 0.5 μM to less than about 12 μM, or at least about 0.5 μM to less than 12 μM.

[0112] In one embodiment, the neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative thereof) in the ocular tissue within the eye is less than about 25 μM, less than about 20 μM, less than about 15 μM, less than about 14 μM, less than about 13 μM, less than about 12 μM, or less than about 10 μM. In one embodiment, the neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative thereof) in the ocular tissue within the eye is greater than about 0.15 μM to less than about 25 μM, or greater than 0.15 μM to less than 25 μM, or at least about 0.15 μM to less than about 25 μM, or at least about 0.15 μM to less than 25 μM, or greater than 0.15 μM to less than about 20 μM, or greater than 0.15 μM to less than 20 μM, or at least about 0.15 μM to less than 20 μM.

[0113] In one embodiment, the neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative thereof) in the ocular tissue within the eye is greater than about 0.15 μM to less than about 15 μM, or greater than 0.15 μM to less than 15 μM, or at least about 0.15 μM to less than about 15 μM, or at least 0.15 μM to less than 15 μM, or greater than 0.15 μM to less than 12 μM, or greater than 0.15 μM to less than 12 μM, or at least about 0.15 μM to less than 12 μM. In one embodiment, the neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative thereof) is at least about 0.5 μM to less than about 25 μM, or at least about 0.5 μM to less than 25 μM, or at least about 0.5 μM to less than 20 μM, or at least 0.5 μM to less than 20 μM. In one embodiment, the neuroprotective effective concentration of the neuroprotective compound (e.g., cannabinol or a derivative thereof) is at least about 0.5 μM to less than about 15 μM, or at least about 0.5 μM to less than 15 μM, or at least about 0.5 μM to less than about 12 μM, or at least about 0.5 μM to less than 12 μM.

[0114] In one embodiment, the neuroprotective effective concentration of a neuroprotective compound (e.g., cannabinol or a derivative thereof) in an ocular tissue within the eye is greater than about 0.15 μM to less than about 10 μM, or greater than 0.15 μM to less than 7.5 μM, or at least about 0.15 μM to less than about 10 μM, or at least 0.15 μM to less than 7.5 μM, or greater than about 0.15 μM to about 5 μM, or greater than 0.15 μM to 5 μM, or at least about 0.15 μM to about 5 μM, or at least 0.15 μM to 5 μM.

[0115] For example, the neuroprotective compound may be administered orally, intrathecally, intravenously, locally, or by injection, and / or directly at the site of the target neuron or target neuronal population. In one embodiment, a neuroprotective effective concentration is achieved by a systemic dose of about 1 mg / kg to about 100 mg / kg, preferably about 1 mg / kg to about 20 mg / kg, more preferably about 1 mg / kg to about 15 mg / kg, even more preferably about 1 mg / kg to about 10 mg / kg, and most preferably about 1 mg / kg to about 13 mg / kg. The dose may be administered repeatedly, for example, once a week, once every other day, once a day, or twice a day.

[0116] The neuroprotective compound may be administered intrathecally, intravenously, or injected, or directly into the eye (such as by local eye instillation or intravitreal injection or pump administration). In one embodiment, for ocular administration in ocular indications (e.g., for treating glaucoma), the systemic dose may be 1 mg / kg to 20 mg / kg. The dose may be repeated, for example, once per week, once every other day, once per day, or twice per day. In one embodiment, for systemic administration in ocular indications (e.g., for treating glaucoma), the systemic dose may be 1 mg / kg to 15 mg / kg, 1 mg / kg to 13 mg / kg, or 1 mg / kg to 10 mg / kg. The dose may be repeated, for example, once per week, once every other day, once per day, or twice per day. In one embodiment, for systemic administration in peripheral indications (e.g., for treating peripheral neuropathy and / or peripheral nerve injury or damage), the dose may be 1 mg / kg to 20 mg / kg, 1 mg / kg to 15 mg / kg, 1 mg / kg to 13 mg / kg, or 1 mg / kg to 10 mg / kg. The dose may be repeated, for example, once per week, once every other day, once per day, or twice per day. In one embodiment, for systemic administration in central indications (e.g., for treating central nerve injury or damage), the dose may be 1 mg / kg to 20 mg / kg, 1 mg / kg to 15 mg / kg, 1 mg / kg to 13 mg / kg, or 1 mg / kg to 10 mg / kg. The dose may be repeated, for example, once per week, once per day, or twice per day.

[0117] In one embodiment, for ocular administration in ocular indications (e.g., to treat glaucoma), the ocular dose applied to the eye (such as in the form of eye drops or ophthalmic gel) can be 0.5 mg to 20 mg, 0.5 mg to 15 mg, 0.5 mg to 10 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 10 mg, 0.5 mg to 5 mg, or 1 mg to 5 mg. Doses can be repeated, for example, once a week, once every other day, once a day, or twice a day. In one embodiment, for ocular administration in ocular indications (e.g., to treat glaucoma), the ocular dose applied to the eye (such as in the form of an intravitreal injection or pump) can be 0.05 mg to 2 mg, 0.05 mg to 1.5 mg, 0.05 mg to 1 mg, 0.1 mg to 2 mg, 0.1 mg to 1.5 mg, 0.1 mg to 1 mg, 0.05 mg to 0.5 mg, or 0.1 mg to 0.5 mg. Doses can be repeated, for example, once a week, once every other day, once a day, or twice a day.

[0118] In certain embodiments, the neuroprotective compound is administered within about 0-48 hours after the injury affecting the retinal neurons occurs. In certain embodiments, the neuroprotective compound is administered within about 2-24 hours after the injury affecting the retinal neurons occurs. In certain embodiments, the neuroprotective compound is administered within about 3-12 hours after the injury affecting the retinal neurons occurs. In certain embodiments, the neuroprotective compound is administered within about 3-5 hours after the injury affecting the retinal neurons occurs.

[0119] In certain embodiments, the neuroprotective compound or its formulation is administered to the subject with diabetic retinal neuropathy.In certain embodiments, the neuroprotective compound or its formulation is administered to the subject with AMD.In certain embodiments, the neuroprotective compound or its formulation is administered to the subject with retinitis pigmentosa.In certain embodiments, the neuroprotective compound or its formulation is administered to the subject with glaucoma.

[0120] Neuroprotective compounds can be administered to treat the subject who needs to be treated to protect peripheral neurons.For example, the subject may be suffering from an injury (such as physical injury) that affects one or more peripheral nerves.As another example, the subject may have a disease or condition that is characterized by peripheral nerve degeneration.

[0121] In certain embodiments, the neuroprotective compound is administered within about 0-48 hours after the injury affecting the peripheral neuron occurs. In certain embodiments, the neuroprotective compound is administered within about 2-24 hours after the injury affecting the peripheral neuron occurs. In certain embodiments, the neuroprotective compound is administered within about 3-12 hours after the injury affecting the peripheral neuron occurs. In certain embodiments, the neuroprotective compound is administered within about 3-5 hours after the injury affecting the peripheral neuron occurs.

[0122] Neuroprotective compounds can be administered to treat subjects who need treatment to protect central neurons.For example, subjects can be affected by injury that affects neurons in the central nervous system (CNS).As another example, subjects can have diseases or conditions that are characterized by central neurodegeneration.

[0123] In certain embodiments, the neuroprotective compound is administered within about 0-48 hours after an insult (e.g., a physical injury) affecting the CNS. In certain embodiments, the neuroprotective compound is administered within about 2-24 hours after an insult affecting the CNS. In certain embodiments, the neuroprotective compound is administered within about 3-12 hours after an insult affecting the CNS. In certain embodiments, the neuroprotective compound is administered within about 3-5 hours after an insult affecting the CNS.

[0124] The method can include or further include combining with neuroprotective composition a second medicament active agent and administering them simultaneously or sequentially.In some cases, the second medicament active agent is a therapeutic agent for treating glaucoma.For example, the method can include administering a drug to a subject in need thereof to reduce intraocular pressure. EXAMPLES

[0125] Example 1: Protection of neuronal cells with cannabinol at atmospheric pressure Cell culture and differentiation: Mouse 661W (RGC-5) cell line was maintained in DMEM cell culture medium (growth medium) supplemented with 10% FBS and 1% antibiotic-antimycotic (penicillin / streptomycin) at 37° C. in a humidified atmosphere of 5% CO2. To induce neuronal differentiation of 661W cells, the culture medium was replaced by growth medium containing 321 nM staurosporine (STSR) and the cells were incubated for 24 h at 37° C. in a humidified atmosphere of 5% CO2.

[0126] Compounds and Dosage Formulations: Selected cannabinoids: CBN, CBD, CBGA, CBDA, CBND, and Δ 9 -THC was obtained from Cayman and Toronto Research Chemicals. Stock solutions of 1 mM and 10 mM were prepared using ethanol as the solvent. For CBN, treatment concentrations of 0.015 μM, 0.05 μM, 0.15 μM, 0.5 μM, 1.5 μM, 5 μM, 10 μM, and 15 μM were prepared. For other cannabinoids, treatment concentrations of 0.5 μM, 1.5 μM, and 5 μM were prepared directly in control medium (DMEM + 5% FBS + 1% antibiotic-antimycotic) by using the appropriate stock solutions.

[0127] Cytotoxicity and Neuroprotection Assessment: The cytotoxicity of cannabinoids on differentiated 661W cells at ambient pressure was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were seeded (4,000 cells / well) in 96-well plates containing DMEM complete medium and allowed to reach approximately 70% confluence for 24 hours. After 24 hours, the cell culture medium was replaced by growth medium containing 321 nM staurosporine (STSR) and incubated for 1 day to induce differentiation towards a neuronal phenotype.

[0128] MTT assay: For the MTT assay, differentiated 661W cells were treated with various concentrations of cannabinoids to determine cytotoxicity. Briefly, a 5 mg / mL stock solution of methylthiazolyldiphenyltetrazolium bromide (Sigma-Aldrich) was prepared in PBS. After 72 h of cannabinoid treatment, 661W cells were incubated in 200 μL of DMEM with 20 μL of MTT stock solution for 2 h at 37°C. After subsequent washing with PBS, 200 μL of isopropanol was added to each well and the color change resulting from dissolving the formazan salt was rapidly quantified at a wavelength of 570 nm using a spectrophotometer (BMG Labtech). Data were normalized to vehicle control (VC) containing 0.15% ethanol and presented as % cell death. Cell death (%) in VC at atmospheric pressure was considered as 0%. Results are shown in Figure 1.

[0129] Example 2: Neuroprotection of 661W cells with cannabinol under high hydrostatic pressure Except where indicated, all procedures were carried out as described in Example 1. Differentiated 661W cells treated with the respective concentrations of cannabinoids were placed in a pressure chamber, which was maintained at an increased hydrostatic pressure of 20-40 mmHg for 72 hours. At the end of the incubation, 661W cells were processed for MTT assay to determine cytotoxicity. The results are shown in Figures 2-5.

[0130] In another study, the cannabinol derivative CBNA was applied to differentiated 661W cells in a pressure chamber at concentrations of 0.015 μM, 0.05 μM, 0.15 μM, 0.5 μM, 1.5 μM, 5 μM, 10 μM, and 15 μM, and the pressure chamber was maintained at an elevated hydrostatic pressure of about 10-25 mmHg for 72 hours.

[0131] [ka]

[0132] This was carried out by contacting the cells with a cannabinol derivative CBNA having the formula: In the formula: R 1 is H and R 2 is COOH, R 3 is n-CH 11 Vehicle control (VC) contained 0.15% ethanol. At the end of the incubation, 661W cells were processed for MTT assay to determine cytotoxicity. The results are shown in FIG. 6.

[0133] Example 3: Neuroprotection of 661W cells with cannabinol as detected using an apoptosis assay Apoptosis was assessed using the Cell-APO Percentage™ Apoptosis Kit, which detects and measures apoptosis by colorimetric methods. In this assay, the onset of apoptosis in anchorage-dependent mammalian cells during in vitro culture is monitored using the Cell-APO Percentage™ Apoptosis System. This assay measures the execution phase of apoptosis, which is associated with the translocation of phosphatidylserine from the inner to the outer surface of the mammalian cell membrane (experimentally demonstrated by the binding of Annexin V to phosphatidylserine). The movement of phosphatidylserine across the membrane results in the uptake of APO Percentage dye by apoptotic cells. The dye is continuously taken up and selectively transferred to apoptotic cells until blebbing occurs. Necrotic cells are unable to retain the dye and therefore do not stain.

[0134] Differentiated 661W cells placed under high pressure (approximately 20-25 mmHg) in a pressure chamber were treated with cannabinol at concentrations of 0.015 μM, 0.05 μM, 0.15 μM, 0.5 μM, 1.5 μM, and 5 μM and assessed for apoptosis according to the manufacturer's instructions. Briefly, 661W cells (4 × 10 cells) were plated in 24-well tissue culture plates in 500 μl culture medium. 4 ) were seeded and incubated at 37°C / 5% CO2 until confluence was reached (approximately 24 hours). Test cannabinol samples and vehicle controls were added to the cells at selected concentrations and incubated for 6 hours.

[0135] Thirty minutes before the end of the incubation period, the initial treatment medium was replaced with treatment medium containing 5% dye for all wells except the blank wells, and then incubated for an additional 30 minutes at 37°C / 5% CO2. All medium was then removed from each well, and cells were gently washed twice with PBS (1000 μl / well) to remove non-cell-bound dye. Trypsin (50 μl) was added to each well and incubated for 10 minutes at 37°C / 5% CO2. After detaching the cells from the plastic and surface treating the cell culture, 200 μl of dye-releasing reagent was added to each well on the shaker plate and shaken for 10 minutes. During the 30 minutes, the dye that had accumulated within the labeled cells was released into solution, and the concentration of this released intracellular dye was measured using a microplate colorimeter. The contents of each well (250 μl) were transferred to a 96-well flat-bottom plate, and its absorbance was read at 550 nm (blue-green filter) using a microplate reader. Blank values ​​were subtracted from values ​​for all other conditions. Mean absorbance values ​​± standard error of the mean were plotted as a percentage of vehicle control absorbance values.

[0136] As shown in Figure 7, when exposed to neurons under hyperbaric conditions, cannabinol provided effective protection against apoptosis at concentrations >0.015μM to <5μM, with statistically significant protection (p<0.05 and p<0.01) at concentrations between 0.05 and 1.5μM. The hyperbaric conditions in the pressure chamber mimic the clinical situation of elevated intraocular pressure in glaucoma patients, and the observed inhibition of apoptosis by cannabinol under these conditions may lead to the amelioration of retinal cell degeneration and optic nerve damage.

[0137] As shown in these examples, cannabinol exhibits surprisingly effective neuroprotective effects when in contact with neurons in the concentration range of about 0.15 μM to less than about 15 μM. It has also been demonstrated that effective protection against apoptosis occurs under high pressure conditions in the concentration range of more than 0.015 μM to less than 5 μM. In contrast, other cannabinoids (such as CBD and THC) exhibit high toxicity when in contact with neurons at about 0.5 μM concentrations (CBD, CBG, CBGA), about 1.5 μM concentrations (CBD, CBC, CBG, CBGA, CBND), or about 5 μM concentrations (CBD, CBDA, CBC, CBG, CBGA, CBND, and THC). These effects are surprising in light of the disclosure of neurotoxicity when administered with cannabinol in an earlier publication by Colasanti et al., Exp. Eye Res. (1984) 39, 251-59. The neuroprotective effect of cannabinol was also observed under hyperbaric conditions in a pressure chamber, which mimics the clinical situation of elevated intraocular pressure in glaucoma. 9 The cannabinol derivative CBNA (Formula I, where R 1 is H and R 2 is COOH, R 3 is n-CH 11 Similar neuroprotective effects were observed in 114 patients with 10-HT2A1-induced glaucoma.

[0138] Example 4: Neuroprotection by cannabinol detected using a rat episcleral venous laser photocoagulation glaucoma model Pattern-stimulated electroretinogram (pERG) amplitude, a parameter measuring retinal ganglion cell (RGC) activity, was assessed in the eyes of anesthetized animals. Eyes were kept moist during pERG recordings with instillation of 2.5% GonioVisc ophthalmic lubricating solution (Hub Pharmaceuticals), which also ensured electrical contact between the cornea and the ERG electrode. The ERG electrodes were circular silver / silver chloride fine wires. Recordings were made from one eye while the other was mechanically closed. The closed eye was used as a reference for the ERG recordings to minimize physiological noise, and a ground electrode was placed caudal. Only signals from the right eye (OD) were recorded. pERG stimuli were generated on a gamma-linearized monitor screen, resulting in a vertical sinusoidal pattern with 0.10 waves per degree of visual angle at the viewing distance. The mean stimulus luminance was 45 lux, and the black-and-white contrast was 99%. The pattern was reversed every 300 ms, with 1,200 reversals per recording. Two recordings were made for each eye. For pERG analysis, pERG waveforms were overlaid, checked for consistency, and averaged for the final pERG response. Baseline pERG responses were recorded 4 days prior to laser treatment (day 0), and pERG responses were recorded on days 7, 14, and 21 after laser treatment. At baseline, the initial pERG baseline amplitude was recorded for all animal eyes. Animals were then randomized into control and treatment groups based on their pERG response.

[0139] The following treatment groups were used in this study: Group 1: Vehicle (0.5% DMSO-PBS) (IVT, days 0, 7, and 15) n=11 Group 2: Low dose CBN (5 μM in 0.05% DMSO-PBS) (IVT, days 0, 7, and 15) n=13 Group 3: High dose CBN (50 μM in 0.5% DMSO-PBS) (IVT, days 0, 7, and 15) n=11 Group 4: Brimonidine (topical administration, 5 μl per eye twice daily, days -3 to 21) n=14

[0140] FIG. 8 and Table 1 show the effect of cannabinol on pERG activity. After laser treatment, the functional response of RGCs was measured by a reduced decrease in pERG amplitude in all treatment groups. However, statistically significant differences were observed only in the vehicle group on day 21 ([1.92 μV] vs. baseline [3.84 μV]) and in the high dose CBN group on both days 14 and 21 ([1.87 μV] on day 14 and [2.19 μV] vs. baseline [3.83 μV] on day 21 (Table 1)). There were no significant differences in pERG amplitude from baseline after disease induction in the brimonidine (ALPHAGAN) and low dose CBN groups on both follow-up days 14 and 21. The data indicate that low dose CBN at a final intraocular concentration of 5 μM provides neuroprotection to RGCs similar to ALPHAGAN.

[0141] [Table 1]

[0142] - pERG amplitude (μV) was reduced in all treatment groups at follow-up days 7, 14, and 21. - Statistically significant differences were observed in the vehicle group on day 21 compared to baseline, and in the high-dose CBN group on both days 14 and 21 compared to baseline (two-way ANOVA followed by Tukey's multiple comparison test ( * p<0.05, ** p<0.01). In the -ALPHAGAN group and the low-dose CBN group (final intraocular concentration 5 μM), there were no significant differences from baseline in pERG values ​​(%) on the 7th, 14th, and 21st days of follow-up.

[0143] References: Kalesnykas G,Uusitalo H.Comparison of simultaneous readings of intraocular pressure in rabbits using Perkins handheld,Tono-Pen XL,and TonoVet tonometers.Graefes Arch Clin Exp Ophthalmol.2007 May;245(5):761-2. * * *

[0144] The present invention illustratively described herein can be suitably practiced in the absence of any of the element(s) or limitation(s) not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are to be interpreted openly and without limitation. Furthermore, the terms and expressions used herein are used as terms of description and not as terms of limitation, and there is no intention by the use of such terms and expressions to exclude any equivalents or portions thereof shown and described in the future, and it will be recognized that various modifications are possible within the scope of the invention as claimed.

[0145] Thus, although the present invention has been specifically disclosed by preferred embodiments and optional features, it is to be understood that those skilled in the art may make modifications and variations to the invention disclosed herein, and such modifications and variations are deemed to be within the scope of the invention disclosed herein. The inventions have been described broadly and generically herein. Each narrower species and subclassification falling within the scope of such generic disclosure also forms part of such invention. This includes any condition or negative limitation that removes any subject matter from the genus applied to each generic description of the invention, regardless of whether such excluded material is expressly attributed thereto.

[0146] Moreover, those skilled in the art will recognize that when features or aspects of the invention are described in terms of Markush groups, such description also describes the invention in terms of any individual members or sub-members of such Markush groups. It is also understood that the above description is intended to be illustrative, and not limiting. Many embodiments will become apparent to those skilled in the art upon reviewing the above description. Thus, the scope of the invention should not be determined with reference to the above description, but rather with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all literature and references, including patent publications, are incorporated herein by reference.

Claims

1. A pharmaceutical composition for use in protecting retinal neurons from neurodegeneration or treating neurodegeneration of retinal neurons, wherein the retinal neurons are contacted with the pharmaceutical composition, the pharmaceutical composition comprising a neuroprotective compound in an amount sufficient to inhibit neurodegeneration, the neuroprotective compound having a structure represented by formula I: 【Chemistry 1】 wherein R 1 is H, and R 2 is COOH, and R 3 But n-C 5 H 11 or R 1 is H, and R 2 is H and R 3 But n-C 5 H 11 or R 1 But, CH 3 and R 2 is H and R 3 But n-C 5 H 11 or R 1 is H, and R 2 is H and R 3 But n-C 4 H 9 or R 1 is H, and R 2 is H and R 3 But n-C 3 H 7 or R 1 is H, and R 2 is H and R 3 But, C 2 H 5 or R 1 is H, and R 2 is H and R 3 But, CH 3 and wherein the amount sufficient to inhibit neurodegeneration is an amount that results in a concentration of the neuroprotective compound of between 0.15 μM and less than 15 μM upon contact with the target retinal neuron.

2. The pharmaceutical composition of claim 1 , administered to a subject in need thereof.

3. The pharmaceutical composition of claim 2 , wherein the subject is suffering from a neurodegenerative disease.

4. 4. The pharmaceutical composition of claim 3, wherein the neurodegenerative disease is a neurodegenerative disease affecting the eye.

5. The pharmaceutical composition described in claim 4, wherein the neurodegenerative disease is selected from the group consisting of glaucoma, age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy.

6. The pharmaceutical composition according to claim 3 , wherein the neurodegenerative disease is glaucoma.

7. 7. The pharmaceutical composition of claim 6 for simultaneous or sequential administration of an additional active agent for the treatment of glaucoma.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the amount sufficient to inhibit neurodegeneration is an amount sufficient to reduce the amount or rate of apoptosis in a population of retinal neurons contacted with the pharmaceutical composition.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the retinal neurons are exposed to high hydrostatic pressure, and wherein the amount sufficient to inhibit neurodegeneration is an amount sufficient to reduce pressure-induced neurodegeneration.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition comprising the neuroprotective compound is provided as a microemulsion.

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition comprising the neuroprotective compound is provided as a sustained release formulation.

12. The sustained release formulation comprises: a. a delivery vehicle comprising a cellulose polymer and an anionic polysaccharide; b. nanoparticles comprising an amphiphilic non-ionic block copolymer and said neuroprotective compound; Including, The pharmaceutical composition of claim 11, wherein the gel point of the formulation is from about 30°C to about 37°C.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the amount sufficient to inhibit neurodegeneration is an amount that provides a concentration of the neuroprotective compound upon contact with the retinal neurons of greater than about 0.5 μM to less than about 15 μM.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the amount sufficient to inhibit neurodegeneration is an amount that provides a concentration of the neuroprotective compound upon contact with the retinal neurons of greater than about 0.5 μM to less than about 12 μM.

15. 15. The pharmaceutical composition of claim 14, wherein the amount sufficient to inhibit neurodegeneration is an amount that provides a concentration of the neuroprotective compound upon contact with the retinal neurons of about 1.5 μM to 10 μM.

16. A pharmaceutical composition according to any one of claims 1 to 15 for systemic administration.

17. The pharmaceutical composition of claim 16 , wherein the systemic administration comprises intravenous injection or oral administration.

18. A pharmaceutical composition according to any one of claims 1 to 15 for topical administration.

19. A pharmaceutical composition according to any one of claims 1 to 15 for direct administration to the eye.

20. The pharmaceutical composition of claim 19, for topical administration as an eye drop formulation or for intravitreal administration to the eye.

21. 21. The pharmaceutical composition of claim 20, wherein the ophthalmic formulation is to be administered once a week, once a day, or twice a day.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein the neuroprotective compound is cannabinol or cannabinolic acid.

23. 23. The pharmaceutical composition of claim 22, wherein the neuroprotective compound is cannabinol.

24. A pharmaceutical composition according to any one of claims 1 to 23 for the treatment of neurodegeneration in a subject in need thereof.

25. 1. A pharmaceutical composition comprising a neuroprotective compound in an ophthalmic formulation, the neuroprotective compound being represented by Formula I: 【Chemistry 2】 wherein R 1 is H, and R 2 is COOH, and R 3 But n-C 5 H 11 or R 1 is H, and R 2 is H and R 3 But n-C 5 H 11 or R 1 But, CH 3 and R 2 is H and R 3 But n-C 5 H 11 or R 1 is H, and R 2 is H and R 3 But n-C 4 H 9 or R 1 is H, and R 2 is H and R 3 But n-C 3 H 7 or R 1 is H, and R 2 is H and R 3 But, C 2 H 5 or R 1 is H, and R 2 is H and R 3 But, CH 3 and wherein the amount of the neuroprotective compound is sufficient to provide a concentration of the neuroprotective compound of from about 0.15 μM to less than about 15 μM upon contact with the target retinal neuron.

26. 26. The pharmaceutical composition of claim 25, wherein the neuroprotective compound is present in a concentration of 0.1% to 0.5% w / w.

27. 27. The pharmaceutical composition of claim 25 or claim 26, wherein the neuroprotective compound is cannabinol or cannabinolic acid.

28. 28. The pharmaceutical composition of claim 25, claim 26, or claim 27, wherein the neuroprotective compound is cannabinol.

29. The pharmaceutical composition according to any one of claims 25 to 28, wherein the eye drop formulation is a microemulsion or hydrogel formulation.

30. 30. The pharmaceutical composition of claim 29, wherein the ophthalmic formulation is a hydrogel formulation comprising: a) a delivery vehicle comprising a cellulose polymer and an anionic polysaccharide; and b) nanoparticles comprising an amphiphilic non-ionic block copolymer and the neuroprotective compound, and wherein the gel point of the formulation is from about 30°C to about 37°C.

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