FIELD OF THE DISCLOSURE

[0001] Described herein are novel topical compositions for treating peripheral neuropathic pain. The topical compositions include a cannabinoid and a fatty acid amide hydrolase inhibitor or a monoacylglycerol lipase inhibitor. The topical compositions may be used in the management of peripheral neuropathic pain.

BACKGROUND OF THE DISCLOSURE:

[0002] Peripheral neuropathy is a condition of peripheral nerve damage. It can be caused by a variety of sources, including diabetes, infections, chemotherapy, or trauma.

[0003] Peripheral neuropathy can present as a variety of conditions. For example, vulvodynia is a painful peripheral neuropathic condition involving chronic vulvar pain, physical disability, sexual dysfunction, and affective distress. Vulvodynia can be subdivided into two types: i) generalized vulvodynia (GVD), when pain is felt in the entire vulvar region, and ii) localized vulvodynia (LVD), when pain is localized to vestibule (vestibulodynia), clitoris (clitorodynia), or a portion of the vulva (hemi vulvodynia). Although the etiology of vulvodynia remains multi-factorial, the commonly accepted hypothesis is the neuropathic pain hypothesis, which involves an inflammatory process or abnormal neural activity of the peripheral or central nervous system.

[0004] Symptomatic approaches are primarily used to manage peripheral neuropathy. Oral medications like tricyclic antidepressants (amitriptyline), serotonin-norepinephrine reuptake inhibitors (duloxetine), and calcium channel alpha-2-delta ligands (gabapentin and pregabalin) are conventionally used as a first line of therapy. However, these medications provide pain relief to only 30 to 40% of patients. The second line of therapy includes tramadol via oral route, lidocaine, and capsaicin as topical formulations. Yet the pain management efficacy with all these approaches may be relatively poor. [0005] Cannabinoids are the most effective anti -nociceptive agents. Synthetic and natural cannabinoids are highly lipophilic, cross the blood-brain barrier, and are associated with abuse potential.

[0006] Endocannabinoids are potent anti -nociceptive agents that act by binding to CB1 and CB2 receptors. Apart from their major role in pain reduction, these endocannabinoids activate the CB1 receptors on peripheral sensory neurons and afferent nociceptors in skin and produce an analgesic effect in somatic, visceral, inflammatory, and neuropathic pain.

[0007] Previous studies show that the endocannabinoid anandamide (N- arachidonoylethanolamine) possesses significant anti -nociceptive activity in peripheral neuropathy. Studies have also shown that one of the possible mechanisms for peripheral pain sensation is involvement of transient receptor potential ion channel vanilloid 1 receptors (TRP- VI), formerly called VR-1 receptors. Anandamide has been shown to exhibit agonistic potential for this receptor, in addition to capsaicin, resiniferatoxin, lipoxygenase products, and camphor.

[0008] However, the effectiveness of endocannabinoids such as anandamide can be limited by their metabolic uptake. Many endocannabinoids, including anandamide, are metabolized by the enzyme fatty acid amide hydrolase (FAAH). To counteract this metabolic effect, the levels of endocannabinoids can be increased by inhibiting FAAH. However, the development of potent and safe FAAH inhibitors has been delayed due to their off-target mediated side effects leading to coma and brain cell death.

[0009] Similarly, many endocannabinoids are metabolized by monoacylglycerol lipase (MAGL). MAGL are enzymes found abundantly in the brain and cerebrospinal fluid (CSF). To counteract this metabolic effect, the levels of endocannabinoids can be increased by inhibiting MAGL. However, chronic MAGL inhibition is known to cause CB-1 receptor desensitization, physical dependence, and decrease endocannabinoid dependent synaptic plasticity.

[0010] Further, the effectiveness of endocannabinoids such as anandamide can be limited by their delivery. Topical compositions including anandamide are known to be unstable, and anandamide is difficult to retain in skin because it is a small molecule. [0011] Accordingly, it would be desirable to provide an effective and safe topical product available for the management of peripheral neuropathic pain.

BRIEF DESCRIPTION OF THE DISCLOSURE

[0012] In one embodiment, the present disclosure is directed to a topical composition including a cannabinoid, a fatty acid amide hydrolase inhibitor or a monoacylglycerol lipase inhibitor, a solubility enhancer, optionally a water-soluble polymer, and optionally a permeation enhancer.

[0013] In another embodiment, the present disclosure is directed to a method for treating peripheral neuropathic pain, the method including administering a topical composition including a cannabinoid, a fatty acid amide hydrolase inhibitor or a monoacylglycerol lipase inhibitor, a solubility enhancer, optionally a water-soluble polymer, and optionally a permeation enhancer.

[0014] In yet another embodiment, the present disclosure is directed to a method for preparing a topical gel composition, the method including forming a first dispersion including a water-soluble polymer in an aqueous medium, adding to the first dispersion a composition including a second dispersion including a cannabinoid, a fatty acid amide hydrolase inhibitor or a monoacylglycerol lipase inhibitor, and a solubility enhancer, optionally adding to the first dispersion a composition including a third dispersion including a permeation enhancer, and forming the gel.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Fig. 1 is a graphical depiction of an example embodiment of the percentage inhibition of FAAH in the presence of JZL-195 (20 mM) and silymarin constituents (n=3±sd).

[0016] Figs. 2A-2E are a graphical depiction of an example embodiment of anandamide metabolism in human epidermal keratinocyte cells (n=3) for a variety of constituents. Fig. 2A demonstrates taxifolin (TX) as the constituent, Fig. 2B demonstrates silychristin (SC) as the constituent, Fig. 2C demonstrates silydinain (SD) as the constituent, Fig. 2D demonstrates silybin (SB) as the constituent, and Fig. 2E demonstrates isosilybin (IS) as the constituent. [0017] Fig. 3 is a graphical depiction of an example embodiment of a phase solubility curve of anandamide and silybin.

[0018] Figs. 4A and 4B are a graphical depiction of an example embodiment of the permeation of anandamide and silybin across a porcine epidermis (n=3). Fig. 4A demonstrates permeation of anandamide, and Fig. 4B demonstrates permeation of silybin.

[0019] Figs. 5A and 5B are a graphical depiction of an example embodiment of the induction of peripheral neuropathic pain assessment in rats (n=6). Fig. 5A demonstrates induction of thermal hyperalgesia, and Fig. 5B demonstrates induction of mechanical allodynia.

[0020] Figs. 6A and 6B are a graphical depiction of an example embodiment of the assessment of anandamide gel in peripheral neuropathic pain induced rats (n=6). Fig. 6A demonstrates assessment using thermal hyperalgesia, and Fig. 6B demonstrates assessment using mechanical allodynia.

[0021] Fig. 7 is a graphical depiction of an example embodiment of induction of vulvodynia in rats.

[0022] Fig. 8 is a graphical depiction of an example embodiment of the anti-vulvodynic activity of anandamide topical gel in rats

DETAILED DESCRIPTION OF THE DISCLOSURE

[0023] Described herein, novel topical formulations have been developed for the treatment of peripheral neuropathic pain. The topical formulations include a topical composition including a cannabinoid, a fatty acid amide hydrolase inhibitor or a monoacylglycerol lipase inhibitor, a solubility enhancer, optionally a water-soluble polymer, and optionally a permeation enhancer. Such delivery formulations and methods can increase tissue levels of delivered cannabinoids to increase their pharmaceutical effects.

[0024] In some embodiments, the cannabinoid is selected from an endocannabinoid, a phytocannabinoid, a synthetic cannabinoid, and combinations thereof. In some embodiments, the cannabinoid is an endocannabinoid. [0025] In some embodiments, the cannabinoid is selected from endocannabinoids metabolized by FAAH, anandamide, N-Oleoyl ethanolamine (OEA), N-Palmitoyl ethanolamine (PEA), N-Arachidonoyl taurine, Oleamide, N-Arachidonoyl glycine, N-Arachidonoyl dopamine, and combinations thereof. In some embodiments, the cannabinoid is anandamide.

[0026] In some embodiments, the cannabinoid is selected from endocannabinoids metabolized by monoacylglycerol lipase (MAGL), 2-arachidonoyl glycerol, 2-oleoyl glycerol, 2- palmitoyl glycerol, and combinations thereof.

[0027] In some embodiments, the novel topical formulations further include at least one cannabinoid selected from delta 8-tetrahydrocannabinol, delta 9-tetrahydrocannabinol, cannabidiol, cannabinol, cannabigerol, cannabichromene, dronabinol, nabilone, and combinations thereof. In these embodiments, the cannabinoids may produce a synergistic effect to treat peripheral neuropathic pain.

[0028] In some embodiments, the fatty acid amide hydrolase inhibitor is selected from silymarin, silybin, silybin A, silybin B, isosilybin, isosilybin A, isosilybin B, silychrsitin, silydianin, taxifolin, myricetin, isorhamnetin, kaempferol, pristimerin, biochanin A, genistein, daidzen, nutmeg extracts, 1-Monomyristin, 7-hydroxyflavone, 3,7-dihydroxyflavone, amyrin, celastrol, chelerythrine, euphol, salvinorin A, Corydalis yanhusuo plant extract, formononetin, myristicin, pelargonidir, Lepidium meyenii (Maca) extracts, tanshinone IIA, macamides, N-benzyl stearamides, and combinations thereof. In some embodiments, the fatty acid amide hydrolase inhibitor is selected from silymarin, taxifolin, silychristin, silydianin, silybin, isosilybin, and combinations thereof. In some embodiments, the fatty acid amide hydrolase inhibitor is silymarin.

[0029] In some embodiments, the monoacylglycerol lipase inhibitor is selected from natural plant-based compounds with MAGL inhibition activity, synthetic compounds with MAGL inhibition activity, and combinations thereof. In some embodiments, the monoacylglycerol lipase inhibitor is selected from pristimerin, euphol, amyrin, tanshinone IIA, celastrol, pristimerol, dihydrocelastrol, dihydrocelastryl diacetate, orlistat, and combinations thereof.

[0030] In some embodiments, the novel topical formulations include a fatty acid amide hydrolase inhibitor and a monoacylglycerol lipase inhibitor. [0031] In some embodiments, the water-soluble polymer is a hydrogel polymer selected from celluloses, gums, polymers, semisynthetic polymeric derivatives, adhesives, proteins, other gelling or semisolid/viscosity enhancing materials, and combinations thereof. In some embodiments, the water-soluble polymer is selected from homopolymers and copolymers. In some embodiments, the water-soluble polymer is selected from a polyacrylic acid polymer, cellulose derivatives, starch derivatives, carbohydrate polymers, poloxamers, gums, carboxyvinyl polymers, and combinations thereof. In some embodiments, the water-soluble polymer is selected from polyethylene glycols, surfactant gels, fatty acid gels, alcohol gels, cellulose polymers, carbomer based gels, carbopols, xanthan, pectin, karaya, guargum, starch-based gels, hyaluronic acid, and combinations thereof. In some embodiments, the water-soluble polymer is a polyacrylic acid polymer. In some embodiments, the water-soluble polymer is Carbopol ^® 940.

[0032] In some embodiments, the solubility enhancer is selected from solvents, complexing agents, cyclodextrins, polymers, surfactants, and combinations thereof. In some embodiments, the solubility enhancer is a complexing agent selected from alpha cyclodextrins, beta cyclodextrins, gamma cyclodextrins, hydroxypropyl, carboxy methyl, methyl, sulfobutyl ether derivatives of alpha, beta, or gamma cyclodextrins, and combinations thereof. In some embodiments, the solubility enhancer is a polymer selected from pluronic polymers, polyacrylates, polyethylene glycol, and combinations thereof. In some embodiments, the solubility enhancer is a surfactant selected from Tweens, Spans, Brij, sodium lauryl sulfate, fatty acid esters, and combinations thereof. In some embodiments, the solubility enhancer is a solvent selected from dimethyl sulfoxide, glycols, n-methyl pyrrolidinone, and combinations thereof.

[0033] In some embodiments, the solubility enhancer is selected from hydroxypropyl-b- cyclodextrin (HR-b-CD), cyclodextrins, and combinations thereof. In some embodiments, the solubility enhancer is hydroxypropyl^-cyclodextrin.

[0034] In some embodiments, the solubility enhancer is present in an amount sufficient to reduce turbidity of the topical composition. At high concentrations of cyclodextrin, the topical compositions exhibit turbidity, but this turbidity can be reduced by use of a sulfobutyl ether cyclodextrin. When turbidity requires reduction in this way, the sulfobutyl ether cyclodextrin is a critical element. [0035] In some embodiments, the cannabinoid or fatty acid amide hydrolase inhibitor or monoacylglycerol lipase inhibitor may form a complex with the permeation enhancer. In some embodiments, the cannabinoid and fatty acid amide hydrolase inhibitor or monoacylglycerol lipase inhibitor form a complex with the permeation enhancer.

[0036] In some embodiments, the topical composition optionally includes a permeation enhancer. In some embodiments, the permeation enhancer is selected from solvents, terpenes, lipids, surfactants, complexing agents, carriers, nanomaterials, vesicles, azones, pyrrolidone, polymers, fatty alcohols, fatty acids, esters of fatty acids, and combinations thereof. In some embodiments, the permeation enhancer is selected from propylene glycol, glycerol, ethanol, dimethyl sulfoxide, and combinations thereof. In some embodiments, the permeation enhancer is selected from propylene glycol, Tween 80, transcutol, and combinations thereof. In some embodiments, the permeation enhancer is propylene glycol.

[0037] In some embodiments, the topical composition is in a form selected from the group consisting of gels, ointments, creams, lotions, foams, liquids applied to the skin, and combinations thereof. In some embodiments, the topical composition is a gel. In some embodiments, the topical composition is in the form of an organogel or a hydrogel. In some embodiments, the topical composition is in the form of a hydrogel.

[0038] The topical compositions described herein may be used to treat peripheral neuropathic pain according to the methods of the present disclosure. In some embodiments, the method for treating peripheral neuropathic pain comprises administering a topical composition to a patient. In some embodiments, the patient is selected from a human and an animal.

EXAMPLES

[0039] Example 1: FAAH inhibition assay.

[0040] Method

[0041] Silymarin is an extract of Milk Thistle (Silybum marianum) that includes a mixture of flavonolignans and flavanols. There are no reports of silymarin acting as an FAAH inhibitor. Silymarin extract is a safe herbal product, as there are no severe adverse events reported. [0042] Silymarin constituents were screened to identify their potential FAAH inhibition activity using a fluorescence-based fatty acid amide hydrolase inhibitor screening assay kit. The assay was carried out as described in the manual. Briefly, different concentrations of silymarin constituents taxifolin (TX), silychristin (SC), silydianin (SD), silybin (SB), isosilybin (IS), as well as known FAAH inhibitor JZL-195, were incubated with FAAH enzyme and probe substrate 7- amino-4-methoxy coumarin (AMC) arachidonoyl amide at 37 °C for 30 min. At the end of incubation, the hydrolyzed product AMC was measured for fluorescence intensity at excitation and emission wave lengths of 350 and 455 nm, respectively.

[0043] Results

[0044] The fluorescence intensity of AMC was measured in presence of different concentrations of silymarin constituents. Percentage inhibition of FAAH enzyme was calculated based on the following formula. 100

[0045] The inhibition of FAAH enzyme by silymarin constituents was concentration dependent with IC50 values listed in the Table 1. FAAH inhibition activity of silymarin constituents is shown in Fig. 1. Maximum inhibition of FAAH enzyme 72.6% was found to occur in the presence of 200 mM silybin. The FAAH inhibition activity is in the order of SB > SD > SC > IS > TX.

[0046] Table 1. The ICso of FAAH inhibitors (n=3±sd). [0047] Example 2: In-vitro skin metabolism studies.

[0048] Method

[0049] The immortal human epidermal keratinocytes (HaCaT) were used to study in- vitro skin metabolism of anandamide. The cells were maintained at 37 °C in 5% CO2 and 90% relative humidity with culture media that consisted of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For in-vitro keratinocyte clearance studies, 20,000 HaCaT cells per well were used. The assay was performed by incubating anandamide at a concentration of 10 mM both individually and with potential FAAH inhibitors, silymarin constituents at 20, 10, 5 and 2.5 pM concentrations in growth medium at 37 °C with 5% CO2. The cells were treated with acidified ice-cold acetonitrile to terminate the reaction at 0, 1, 2, 4 and 6 hour time intervals. The anandamide was extracted from the cells and analyzed by LC- MS/MS. A substrate depletion method was used to evaluate the clearance of anandamide in HaCaT cells.

[0050] Results

[0051] Human epidermal keratinocyte clearance protocol was developed to study the metabolism of anandamide in human skin. The percentage of anandamide metabolized, half-life, and intrinsic clearance is presented in Table 2. Anandamide is highly cleared in human epidermal keratinocytes with half-life of 1.1 hours. In the presence of silymarin constituents, clearance of anandamide was decreased in human epidermal keratinocytes indicating potential FAAH inhibition effect of silymarin constituents. Anandamide metabolism decreased with increasing concentrations of silymarin constituents as shown in Figs. 2A-2E. Maximum inhibition of anandamide metabolism was observed in the presence of silybin at 20pM concentration. The anandamide metabolism inhibition activity is in the order of SB > IS > SC > TX > SD. Any of these constituents could be used to inhibit FAAH.

[0052] Table 2. Percentage metabolized, half-life, and intrinsic clearance rates of anandamide in human epidermal keratinocyte cells.

[0053] Example 3: Formulation development.

[0054] Potent FAAH inhibitor molecule silybin was incorporated along with anandamide in a hydrogel formulation. The topical hydrogel formulation was developed using water-soluble polymer Carbopol ^® 940 along with water-soluble permeation enhancers. As anandamide is a highly lipophilic drug having log P value of 6.3, the water solubility of anandamide was increased by complexation with Hydroxy propyl -b-Cy cl odextri n (HR-b-CD).

[0055] Solubility enhancement by complexation

[0056] The solubility of anandamide and silybin was enhanced by complexation with HR-b-CD using Higuchi and Connors method. The phase solubility profile was determined by adding excess of the anandamide and silymarin to different concentrations 10, 20, 40, 60, 80 and 100 mM of HR-b-CD in water, respectively. These samples were shaken at room temperature for 24 hours to assess the saturation solubility. After equilibrium, samples were centrifuged at 10,000 rpm and filtered using Millipore (0.22pm) syringe filter. The filtrates were analyzed using LC- MS/MS and HPLC for anandamide and silymarin, respectively after appropriate dilution. Phase solubility study curves were used to calculate complexation efficiency (CE) and stability constants (Kl: l).

[0057] Results

[0058] The phase solubility curve, shown in Fig. 3, demonstrates a linear increase in solubility of anandamide and silybin as a function of increase in concentration of HR-b-CD, indicating an AL type curve. The aqueous solubility of anandamide and silybin was increased >25,000 and 112-fold in the presence of 100 mM of HR-b-CD compared to the intrinsic solubility of anandamide and silybin in water, respectively (Table 3). The affinity (stability) constants (Km) Eq. (1) and complexation efficiencies (CE) Eq. (2) of HR-b-CD-anandamide and HR-b-CD- silybin was calculated based on the parameters of the phase solubility plot and results are shown in Table 3.

[0059] Equation 1. Stability constant (Km). Variable m is the slope of the curve obtained by plotting the drug solubility versus cyclodextrin concentration, determined by linear regression and So is intrinsic solubility of drug. m

^{Kl 1} 5 ₀ (l - m)

[0060] Equation 2. Complexation efficiency (CE). Variable m is the slope of the curve obtained by plotting the drug solubility versus cyclodextrin concentration, determined by linear regression. m

CE =

(1 - m)

[0061] Table 3. Complexation parameters of anandamide and silybin.

[0062] Preparation of hydrogel formulation

[0063] The hydrophilic polymer Carbopol ^® 940 was used in hydrogel preparations. Hydrogels were prepared using 0.5% of Carbopol ^® 940 polymeric dispersions in water with HP- b-CD-drug complex. 0.05% of HR-b-CD-anandamide complex and HR-b-CD-silybin complex were used in hydrogel formulations. After uniform dispersion of polymer in aqueous medium other chemical permeation enhancers were dispersed separately in water and added to the primary polymeric dispersion under continuous stirring. Then triethanolamine was added dropwise to form the gel. The final composition was set aside for complete swelling. Different permeation enhancers propylene glycol (PG), Tween 80 and transcutol were studied to increase drug permeation across epidermis. Multiple hydrogel formulations (F-l to F-6) were prepared using different composition of permeation enhancers as listed in Table 4.

[0064] Table 4. Composition of hydrogel formulations.

[0065] Example 4: In vitro skin permeation and skin deposition studies.

[0066] Method

[0067] Ex-vivo porcine epidermis permeation studies were performed using Franz diffusion cells. The hydrogel formulations (F-l to F-6) were studied for the permeation of HR-b- CD enabled anandamide and silybin across porcine epidermis along with penetration into porcine epidermis. On the day of experiment, the stored porcine epidermis were thawed at room temperature. Porcine epidermis was sandwiched between two chambers of a Franz diffusion cell with an active diffusion area of 0.64 cm ² , and the stratum corneum surface was exposed to the donor chambers. The resistance across porcine epidermis was measured using a wave form generator to ensure the integrity of epidermal segment used for the permeation study. Porcine epidermis with resistance of >10 KW.ah ² was used for permeation studies. To the donor compartment, 10 mg of anandamide topical gel was applied, and a receiver chamber was filled with 20 mM HR-b-CD in phosphate-buffered saline (PBS), pH 7.4, which was stirred at 600 rpm with a 3 mm magnetic stir bar and the temperature was maintained at 37 °C with a circulating water bath. After different time points 1, 2, 4, 6, 8, and 24 h, 200 pL of sample was removed from the receiver chamber for analysis and replaced with 200 pL of fresh 20 mM HR-b-CD in PBS. After 24 hours, the donor chamber was emptied out and porcine epidermis was removed. The porcine epidermis was washed with water and methanol thoroughly to ensure complete removal of the anandamide and silybin from the surface. After washing, porcine epidermis was dried at room temperature for 4 hour and to 1 mg of porcine epidermis 200 pL of PBS was added. The porcine epidermis was homogenized using tissue homogenizer and anandamide and silybin was extracted from homogenate. The extracted samples from different time points and skin were transferred into vials and were subjected to LC-MS/MS or HPLC analysis.

[0068] Results

[0069] The drugs should penetrate into the skin to produce its intended therapeutic activity on topical delivery. The cumulative amount of anandamide and silybin permeation across porcine epidermis in 24 hours was calculated and amount of anandamide and silybin penetrated epidermis after 24 hours was analyzed and the results are shown in Table 5. Anandamide and silybin permeation across porcine epidermis at different time intervals from formulations F-l to F-6 are shown in Figs. 4A and 4B. Permeation of anandamide and silybin was increased with time up to 24 hours from different formulations. The permeation and penetration study results demonstrate that F-3 formulation with 20% of PG increased permeation of anandamide and silybin across porcine epidermis compared to other permeation enhancers. In the presence of 20 % of PG, skin penetration of anandamide and silybin was increased by 2.5 and 2.76-fold, respectively compared to HR-b-CD-drug complex without enhancer.

[0070] Table 5. Permeation of hydrogel formulations.

[0071] Example 5: LC-MS/MS analysis.

[0072] The analysis of anandamide was performed using a Waters Xevo® TQD (Waters, Milford, MA, USA) mass spectrometer, equipped with standard electrospray ionization (ESI). The source parameters viz., source temperature, desolvation temperature, cone gas flow, and desolvation gas flow, were 150 °C, 500 °C, 25 L/h, and 600 L/h, respectively. Nitrogen was used as the nebulizer and cone gas, while argon was employed as collision gas. The mass spectrometer was coupled with Acquity™ Ultra Performance LC equipped with binary solvent and sample manager (Waters, Milford, MA, USA). Separation was achieved using a Waters Acquity UPLC BEH C18 (2.1 mmx50 mm, 1.7 pm) column along with a Waters Acquity C18 guard column (2.1 mmx 10 mm, 1.5 pm). An isocratic elution protocol was adapted using mobile phase A consisting of 0.1 % formic acid (v/v) in water and mobile phase B consisting of 100% methanol. Mobile phase conditions were set at 70% A and 30% B with a flow rate of 0.4 mL/min. ESI conditions were optimized for anandamide and phenacetin (internal standard) in positive mode. The optimized compound parameters used for anandamide are parent m/z 349.13, fragment m/z 61.9, cone voltage 46 V, collision voltage 22 V and dwell times of 0.3 s for each ion. The analytical data were processed by Masslynx.

[0073] Example 6: HPLC analysis.

[0074] Silybin was analyzed using high performance liquid chromatography (HPLC). The HPLC method developed using a Shimadzu UFLC system, equipped with prominence SPD- M20A (Diode array detector). The chromatographic separation of silybin and the internal standard (IS) dapsone was achieved on a Symmetry Shield RP18 column (150 x 4.6 mm, 5 pm); which was maintained at ambient room temperature. The binary mobile phase system reservoir A (Methanol: lOmM Ammonium acetate pH 5 [65:35 v/v]) and reservoir B (10 mM ammonium acetate pH 5) were run as per gradient program (0-1.9 min: 25% A and 75% B; 2.0-14.9 min: 80 % A and 20 % B and 15-37.9 min: 80 % A and 20% B and 38-40 min: 25 % A and 75 % B) at a flow rate of 1 mL/min was used throughout the analytical run of 40 min. The ex-vivo samples were subjected to HPLC analysis without any further extraction procedure.

[0075] Animal Studies.

[0076] Example 7: Induction of peripheral neuropathic pain using chemotherapeutic agent in rat model.

[0077] Paclitaxel dose preparation

[0078] The 1 mg/mL of paclitaxel solution was prepared by weighing 10 mg of paclitaxel and dissolved in 1 ml of solvent ethanol (10%), to this solution 1 ml of cremophor EL (10%) was added, and the volume was made up to 10 mL with normal saline (80%).

[0079] Peripheral neuropathic pain induction procedure

[0080] The peripheral neuropathic pain was induced using chemotherapeutic agent. In the present study paclitaxel was administered for induction of neuropathic pain in Sprague Dawley (SD) rats. The 30 SD rats on arrival were housed in cages (2 animals in each cage) at animal care facility in a temperature and humidity-controlled room with a 12:12 hour light: dark cycles, provided with free access to food and water. The animals were acclimatized for a week prior to experimentation and baseline nociceptive responses were recorded. The animals were randomly divided into two different groups: Group I consisting of six animals (naive) and Group II consisting of 24 animals (neuropathic pain induction). The solvent (10% ethanol + 10% cremphor EL and 80% normal saline) was administered intraperitoneally on alternate days to Group I animals. The paclitaxel 2 mg/kg was administered intraperitoneally on four alternate days (0, 2, 4 and 6th day) to Group II animals till induction of pain. The nociceptive responses were recorded in both Group I and II animals on every alternate day before administering of drug/vehicle to respective groups, to ensure the neuropathic pain induction. [0081] Example 8: Evaluation of anandamide hydrogel analgesic effect in neuropathic pain model.

[0082] Method

[0083] After induction of neuropathy, animals were used to evaluate anandamide hydrogel for analgesic activity. Group I animals were used as naive; Group II of peripheral neuropathy induced animals were randomly divided into four subgroups with six animals each. Group Ila animals were used as negative control and the placebo gel effect was assessed, Group lib animals were used to assess positive control gel (gabapentin gel 10 %), Group lie animals were used for anandamide hydrogel (0.05 %) and Group lid animals were used for anandamide with silybin hydrogel (0.05 %). Baseline hyperalgesic and allodynic responses of all animals were recorded before applying the gels using Hargreaves test and Dynamic plantar aesthesiometer, respectively. To Group II animals, respective gels were applied to the right hind paws with the help of patches and left paws served as sham control in each group. After 2 hours, gels were washed off and three nociceptive responses, each at 5 min interval, were recorded using Hargreaves test and Dynamic plantar aesthesiometer at 0, 2, 3, 4 and 6 hours by a blinded observer.

[0084] Assessment of nociceptive threshold

[0085] All behavioral assays were conducted by an observer who was blinded to drug conditions. The nociceptive threshold of animals was assessed using behavioral studies. Each rat’s plantar surface of the hind paws was tested for heat-hyperalgesia and mechano-allodynia.

[0086] Hind paw thermal-hyperalgesia

[0087] Thermal-hyperalgesia of the hind paw was assessed using Hargreaves test. In this study, a rat was placed into a plexiglass chamber mounted on a heated (35 °C) plexiglass platform. Animal acclimatization was allowed for 30 min at this condition prior to experimentation. The intensity of the radiant beam was adjusted before applying to paws by setting average baseline latencies to 10-12 seconds and a cut-off period of 20 seconds. The radiant heat to plantar surface of hind paw was applied and nocifensive paw withdrawal response was recorded. At an interval of 5 min, three paw withdrawal responses to radiant beam exposure were recorded for both hind paws.

[0088] Hind paw mechano-allodynia

[0089] The hind paw mechano-allodynia response was assessed in rats using dynamic plantar aesthesiometer. The rats were placed in chambers on an elevated grid floor and mechanical stimulus was applied on the plantar surface of hind paws using a blunt microfilament. The force to produce mechanical stimulus using blunt microfilament was set at a ramp of 3 g/s for a period of 10 s and a cut-off period of 30 s. At interval of 5 min, three paw withdrawal latency and force (g) required to produce nocifensive response to mechanical stimulus were recorded for both hind paws.

[0090] Statistical analysis

[0091] The data were calculated for mean and standard error of the mean (SEM) of each study. The data from hind paw thermal hyperalgesia and mechano-allodynia was analyzed using analysis of variance (ANOVA) followed by Dunnett’s post hoc analyses. The level of significance of the data was considered if the P<0.05 with confidential interval of 95%.

[0092] Results

[0093] Induction of peripheral neuropathic pain

[0094] The animals in Group II were used to induce peripheral neuropathic pain. Peripheral neuropathic pain induction was assessed by thermal hyperalgesia evaluated using Hargreaves test and mechanical allodynia evaluated using Dynamic plantar aesthesiometer. After administration of paclitaxel 2 mg/kg on alternate days to Group II animals, nociceptive responses such as paw withdrawal time after thermal stimulation and response time after the mechanical stimulation was recorded from both paws of each animal. As shown in Figs. 5A and 5B, the response time after thermal and mechanical stimuli was decreased significantly after 8 ^th day, indicating induction of peripheral neuropathic pain.

[0095] Evaluation of anandamide hydrogel analgesic effect in neuropathic pain model [0096] After the induction of peripheral neuropathic pain in rats, the animals were applied with placebo gel as negative control, gabapentin gel as positive control, or anandamide gel or anandamide-silybin gel to respective groups. After 2 hours of gel application, the nociceptive responses were recorded at 2 hours, 3 hours, 4 hours, and 6 hours. Percentage increases in paw withdrawal threshold response time were calculated in comparison with untreated paw from thermal hyperalgesia and mechanical allodynia and are shown in Figs. 6A and 6B. The results indicate that at 2 hours, 3 hours, and 4 hours after gel application, there was a significant increase in the pain threshold for allodynic and hyperalgesic responses. The anandamide-silybin hydrogel showed maximum antinociceptive activity until 4 hours compared to anandamide gel. These results indicate the FAAH inhibitor compound along with anandamide improves the analgesic efficacy of endocannabinoid anandamide in peripheral neuropathic pain conditions.

[0097] Example 9: Evaluation of anti-vulvodynic activity of anandamide gel.

[0098] Method

[0099] Adult female Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing 250 - 300 g were used in this study. All the animals were housed in cages (2 animals in each cage) at animal care facility in a temperature and humidity-controlled room with a 12:12 hour light: dark cycles, provided with free access to food and water.

[0100] The animals were acclimatized for a week prior to experimentation and baseline nociceptive responses were recorded. The animals were administered with paclitaxel 2 mg/kg intraperitoneally on alternate days for four days for induction of pain. The nociceptive responses were recorded in all the animals on every alternate day to ensure the induction of peripheral neuropathy. Simultaneously, the animals were evaluated for induction of vulvodynia.

[0101] On each day of behavioral testing, animals were allowed to habituate for 1 hour to the testing environment. A day prior to experimentation, each rat’s anogenital region was shaved for good visibility. The animal’s vulva was applied with a calibrated series of von Frey filaments (Plantar aesthesiometer) to the target tissue using the up-down psychophysical method of Dixon (Chaplan et ak, 1994; Farmer et ah, 2015). A gentle pressure was applied on the vulva to each filament until it bowed and held for 2 s and the flinch response (lifting of pelvic region) was recorded. Each testing session consisted of three threshold determinations separated by 5 min; these three thresholds responses were averaged. A series of eight von Frey filaments (0.06 to 3.9 g; filaments #4 to #11) was applied to the vulva beginning with the #7 filament. Filaments were wiped with aseptic alcohol between recordings to avoid the risk of cross-contamination (Farmer et al., 2015).

[0102] Upon confirmation of induction of vulvodynia, the animals were evaluated for anti-vulvodynic activity of anandamide gel. The rats were grouped into four groups of 6 animals each as follows: Group I - Negative control (placebo gel), Group II - Positive control (Gabapentine-75mg/kg i.p), Group III - Anandamide gel, and Group IV - Anadamide gel + Gabapentine 75mg/kg, i.p. Basal flinch response threshold was recorded in all the animals on day 10 after induction of vulvodynia. After 2 hours of gel application, the flinch responses were recorded at 1 hour, 2 hours and 3 hours.

[0103] Statistical analysis

[0104] The data were calculated for mean and SEM of each study. Student’s t-test was applied for vulvodynia. The significance levels of the data were considered at P<0.05.

[0105] Results

[0106] Induction of Vulvodynia

[0107] Evaluation for vulvodynia was performed from Day 1 until Day 11 on alternate days as described above. All the animals that were injected with paclitaxel 2mg/kg, i.p., developed the signs of peripheral neuropathy. The nociceptive response evaluated by the presence of hyperalgesia and allodynia, confirmed the induction of neuropathic pain in all the animals. Simultaneous to the evaluation of nociceptive response, the animals were also observed for the presence of vulvodynic pain. All the animals starting from Day 3 significantly showed the signs of vulvodynia. The vulvodynic pain continued to exist until Day 11 (Fig. 7). This could be possibly attributed to the peripheral neuropathic condition.

[0108] Evaluation of anandamide topical gel for anti-vulvodynic activity [0109] Induction of vulvodynia was clearly established from Day 3 onwards as observed and described above. Vulvodynia was induced in all the groups. Upon induction, the animals’ baseline flinch response threshold was recorded. Gabapentin group significantly potentiated the flinch response threshold immediately within one hour after injection. The activity subsided thereon gradually over three hours. However, in the anandamide gel treated group, there was a sustained potentiation of the flinch response threshold. The most surprising observation of the study was seen in Group IV, where gabapentin was given systemically, and anandamide was applied topically. In this group there was a significant potentiation of the flinch response threshold compared to the gabapentin and anandamide alone groups, indicating a possible synergistic anti- vulvodynic activity (Fig. 8). This interaction may be attributed to the simultaneous activation of TRP-V1 receptors by both these agonists.

[0110] The results disclosed herein are surprising and provide topical compositions with substantial benefits. In particular, the ability to deliver anandamide topically is novel and surprising. The topical application of anandamide has never been reported in the art. Further, the ability to use anandamide to treat vulvodynial pain is novel and surprising. The application of anandamide to treat vulvodynial pain has never been reported in the art.

[0111] These results demonstrate that the novel compositions in accordance with the present disclosure allow for increased delivery and effectiveness of cannabinoids.

[0112] Example 10: Additional Studies

[0113] The following planned studies will demonstrate the MAGL inhibition potential of silymarin, demonstrate the stability of topical gel formulations, further enhance delivery of cannabinoids in topical gel formulations, and increase the load of FAAH inhibitors in topical gel formulations.

[0114] MAGL Inhibition

[0115] Silymarin’ s potential to inhibit MAGL and enhance 2-AG levels in the dermal region to alleviate neuropathic pain will be assessed.

[0116] Formulation Stability [0117] Topical formulations will be stored in a stability chamber under two conditions, at 40 °C and 75% RH and at 25 °C and 60% RH. After period of 3, 6 and 12 months, the stability of gel will be assessed by evaluating anandamide and silybin content in product.

[0118] Increased Anandamide Drug Load in Topical Formulation

[0119] To enhance the delivery of anandamide into skin, the amount of anandamide loaded in the formulation will be increased to produce maximum analgesic activity of topical compositions.

[0120] Inhibition of anandamide cutaneous metabolism

[0121] The silybin load in formulation will be increased to enhance the silybin drug delivery and thereby produce maximum inhibition of cutaneous anandamide metabolism.

[0122] This written description uses examples to illustrate the present disclosure, including the best mode, and to enable any person skilled in the art to practice the disclosure, including making and using any compositions or systems and performing any incorporated methods. The patentable scope of the disclosure is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have elements that do not differ from the literal language of the claims, or if they include equivalent elements with insubstantial differences from the literal language of the claims.

[0123] As various changes could be made in the above embodiments without departing from the scope of the disclosure, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

[0124] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any limitation explicitly indicated. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process or method.

[0125] The transitional phrase “consisting of’ excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0126] The transitional phrase “consisting essentially of’ is used to define a composition or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0127] Where an invention or a portion thereof is defined with an open-ended term such as “comprising,” it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms “consisting essentially of’ or “consisting of.”

[0128] Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0129] Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular. [0130] As used herein, the term “about” means plus or minus 10% of the value.