PPAR-α agonist loaded microemulsion for oral delivery: compositions and methods of use thereof

Abstract

Provided herein are microemulsions that solubilize hydrophobic drugs, increasing their solubility and bioavailability. In exemplary aspects, the microemulsion comprises the peroxisome proliferator- activated receptor alpha (PPAR-α) agonist A 190 (IUPAC name 3-((4-((4-fluorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid, and methods for its use to treat pain such as chemotherapy-induced peripheral neuropathy (CIPN).

Description

[0001] PPAR-a AGONIST LOADED MICROEMULSION FOR ORAL DELIVERY: COMPOSITIONS AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims benefit of United States patent application 63 / 706,994, filed October 14, 2024.

[0003] STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] This invention was made with government support under grants R21EY028279, R01EY030472 and R01CA219637 awarded by the National Institutes of Health. The United States government has certain rights in the invention.

[0005] BACKGROUND OF THE INVENTION

[0006] Technical Field

[0007] The invention generally relates to microemulsions that solubilize hydrophobic drugs, increasing their solubility and bioavailability. In an aspect, the microemulsion comprises the peroxisome proliferator- activated receptor alpha (PPAR-a) agonist A 190 (IUPAC name 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid) and methods for its use to treat pain such as chemotherapy-induced peripheral neuropathy (CIPN).

[0008] Description of Related Art

[0009] Chemotherapy-induced peripheral neuropathy (CIPN) is a serious potential side effect of anticancer drugs, including taxanes- and platinum-based compounds, vinca alkaloids, epothilones, and bortezomib. CIPN may initially present as an acute pain syndrome with sensory symptoms and can develop into chronic neuropathy with repeated treatment cycles. The pathophysiology of CIPN is highly complex, involving multiple mechanisms unique to each anticancer drug class. This complexity makes the treatment and prevention of CIPN extremely challenging. Presently, there are no approved or effective preventive agents for the CIPN and several previous clinical trials involving antiepileptic and antidepressant drugs have shown mixed results. Following the first report by Devchand et al. in 1996, which indicated an increased inflammatory response in peroxisome proliferator- activated receptor-a (PPAR-a) deficient mice, a large number of studies have investigated and highlighted the significant role of PPARs in reducing peripheral neuropathic pain. Fenofibrate is a Food and Drug Administration (FDA) approved hyperlipidemia drug, rapidly hydrolyzed by esterase to the active metabolite, fenofibric acid, which is then transported to the tissues expressing PPAR-a. However, fenofibric acid faces several challenges, including low affinity for PPAR-a, poor selectivity among PPAR subtypes, dose-limiting toxicides, and limited bioavailability. Recently, we reported a potent and highly isotype selective non-fibrate PPAR-a agonistic chemotype based on the 4-benzyloxy-benzylamino scaffold. Given the premise described previously, we became interested in potential of this series for CIPN indications. The most advanced compound in this series is A190 (Figure 10), which exhibits an EC50 <40 nM in cell-based assays and a >2, 700-fold selectivity for PPAR-a over PPAR-5 and PPAR-y. A190, however, exhibits poor aqueous solubility (0.028 mg / mL), resulting in reduced oral bioavailability, expected to dampen therapeutic efficacy in CIPN relevant contexts. The lack of solubility was anticipated to lead to lack of dose proportionality, difficulty in maintaining steady-state plasma concentrations, and undesirable side effects. These limitations underscored the need to develop drug delivery systems that could address these issues.

[0010] SUMMARY OF THE INVENTION

[0011] Provided herein are new microemulsion compositions that successfully solubilize hydrophobic drugs, thereby increasing their solubility and bioavailability. In one aspect, an exemplary hydrophobic drug is the peroxisome proliferator- activated receptor alpha (PPAR-a) agonist A190 (IUPAC: 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino) benzoic acid). In a further exemplary aspect, the microemulsion comprising A 190 is used to treat pain, such as CIPN.

[0012] Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

[0013] It is an object of this invention to provide a microemulsion comprising 5.0 to 30.0% of an oil phase comprising one or more oils, 20.0 to 75.0% an aqueous phase, 20 to 50% of a mixture of a surfactant and a cosurfactant, and a hydrophobic drug solubilized in the oil phase. In some aspects, the oil phase comprises one or more of soybean oil, cottonseed oil, com oil, acaprylic / capric triglyceride, a medium-chain mono and / or di-glyceride, a long-chain mono glyceride, a fatty acid (e.g., oleic acid, caprylic acid), a fatty acid ester, linseed oil, sunflower oil, fish oil, avocado oil or coconut oil. In further aspects, the oil comprises one or more of oleic acid, caprylic acid and propylene glycol monocaprylate type II. In additional aspects, the aqueous phase comprises one or more of water, one or more biocompatible salts and, optionally, one or more biocompatible buffering agents. In yet further aspects, the mixture of a surfactant and a cosurfactant comprises one or more nonionic, cationic, or anionic surfactants. And in further aspects, the mixture of a surfactant and a cosurfactant comprises one or more of: polyoxyethylene sorbitan monooleate, polyethylene glycol 400, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, (polyethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)), polyoxyl 35 castor oil, polyethoxylated castor oil, PEG-35 castor oil, polyethoxylated alcohol, polyoxyl lauryl ether, polyoxyethylated castor oil, lecithin, polyethylene glycol, a glycerol ester of a fatty acid, a sugar ester, glyceryl monocaprylate, caprylocaproyl macrogol-8 glyceride, a lauroyl polyoxyl-6 glyceride, a lauroyl macrogol-6 glyceride, caprylic / capric triglyceride, glyceryl trioctanoate / tricaprate, or a mixture thereof. In additional aspects the hydrophobic drug is 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid, the oil phase comprises oleic acid, the surfactant is polyoxyethylene sorbitan monooleate, the cosurfactant is polyethylene glycol having an average molecular weight of 400 and the aqueous phase is phosphate buffered saline. In other aspects, the microemulsion comprises 7.5% oleic acid, 52.5% phosphate buffered saline, 40% of a 1:1, v:v ratio mixture of polyoxyethylene sorbitan monooleate and polyethylene glycol having an average molecular weight of 400, and 7.5 mg of 3-((4-((4-fluorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid. In an additional aspect, the microemulsion further comprises a stabilizer. In other aspects, the stabilizer is selected from the group consisting of: an antioxidant, a preservative, a buffering agent, an organic buffer, a polymer, a sugar and a sugar alcohol. In alternative aspects, the microemulsion has a droplet size of 100 nm and a drug loading efficiency of 95% or more.

[0014] Also provided herein is a method of preventing and / or treating pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the microemulsion described in the paragraph above. In some aspects, the pain is peripheral pain, neuropathic pain and / or inflammatory pain. In further aspects, the neuropathic pain is chemotherapy-induced peripheral neuropathy (CIPN) pain. In additional aspects, the pain is inflammatory pain. In some aspects, the pain is chronic pain. Also provided is a method of activating PPARa in a cell, comprising contacting the cell with the microemulsion of the invention under conditions which allow the 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino) benzoic acid to enter to the cell and bind to PPARa. In some aspects, the cell is in vitro or in vivo.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1. Solubility of A190 in various oils, surfactants, and co- surfactants. Each value represents the mean ± SD (n = 4 for each group).

[0017] Figure 2. 3D response surface plots including contour plots generated by Box-Behnken design (BBD) representing the effect of independent variables on dependent variables. (A, D, G, J) Effect of oil and Smix ratio on mean droplet size, PDI, zeta potential, and drug content, respectively; (B, E, H, K) Effect of oil and sonication time on mean droplet size, PDI, zeta potential, and drug content, respectively, and (C, F, I, L) Effect of Smix ratio and sonication time on mean droplet size, PDI, zeta potential, and drug content, respectively.

[0018] Figure 3 A and B. (A) Cryo-TEM image of A 190 microemulsion. (B) In vitro cumulative percentage release profiles of A 190 dispersion (in 10% PEG 400 + 5% DMSO + 85% of 10% HP-P CD mixture) or A190 microemulsion in pH 1.2 or pH 6.8 media. Each value represents the mean ± SD (n=3 for each group).

[0019] Figure 4A and B. In vitro cytotoxic effects of A190 in 0.2% dimethyl sulfoxide (DMSO), vehicle microemulsion, and A190 microemulsion on (A) HepG2 cells and (B) Caco-2 cells after incubation for 24 h. Values are mean ± SD (n=5). *p < 0.05, **p < 0.01, ***p < 0.001 compared to respective DMEM or 0.2% DMSO controls.

[0020] Figure 5. In vitro cumulative percentage release profiles of A 190 dispersion (in 10% PEG 400 + 5% DMSO + 85% of 10% HP-P CD mixture) or A190 microemulsion in pH 1.2 or pH 6.8 media. Each value represents the mean ± SD (n=3 for each group).

[0021] Figure 6. The mean plasma concentration-time profiles of A 190 after oral administration of A 190 dispersion (20 mg / kg) and A 190 microemulsion (20 mg / kg) to rats. Each value represents the mean ± SEM (n=4-5 for each group). A 190 dispersion was prepared in the mixture of 10% PEG 400 + 5% DMSO + 85% of 10% HP-P CD mixture in deionized water.

[0022] Figure 7A-C. Effects of A190 microemulsion in the CIPN Model. A) The effects of systemic A 190 microemulsion in the chemotherapy-induced peripheral neuropathy (CIPN) pain model. Animals began behavioral testing 14 days following the final injection of paclitaxel. Antiallodynic effects after oral administration of A 190 microemulsion (20 mg / kg) were measured. The mechanical paw withdrawal thresholds were determined at 1, 3, 6, 24, 48 and 72 h after the drug administration. B) A190 microemulsion did not significantly impact paw withdraw thresholds with mice pretreated with MK886 compared to their vehicle group (P <0.0001) in the CIPN model. C) At the highest dose tested, A190 microemulsion (20 mg / kg) did not significantly alter locomotor activity compared to vehicle-treated animals (P = 0.5947). Data are expressed as mean ± S.E.M. (n = 8 / group; 50% male and 50% female). #p<0.05 vs. veh / veh group, *p<0.05 vs. PAC / veh group. Veh= vehicle; PAC = paclitaxel; ME = Microemulsion.

[0023] Figure 8A and B. Effects of A 190 microemulsion in the Chronic Inflammatory CFA Model. A) The effects of systemic A 190 microemulsion in the CFA model. Antiallodynic effects after oral administration of A 190 (20 mg / kg). The mechanical paw withdrawal thresholds were determined 3 days after intraplantar injection of CFA (100%) at 1, 3, 6, 24, 48 and 72 h after the drug administration, (n = 8 / group; 50% male and 50% female). A190 fully reversed the mechanical hypersensitivity in a time-related manner. B) Systemic A 190 microemulsion (20 mg / kg) reduces paw edema at 48 h after administration (P<0.0001). APaw diameter= Ipsilateral paw diameter- Contralateral paw diameter. Data are expressed as mean ± S.E.M. #p<0.001 vs. veh / veh group and *#p<0.05 vs. CFA / veh group. Veh= vehicle; ME = Microemulsion.

[0024] Figure 9. Representative hematoxylin and eosin (H&E)-stained sections of collected tissues (liver, heart, spleen, kidney, duodenum, jejunum, and ileum) after oral administration of vehicle microemulsion and A 190 microemulsion (once daily for 3 days).

[0025] Figure 10. Structure of PPAR-a agonist A 190 (IUPAC name 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid.

[0026] DETAILED DESCRIPTION

[0027] Microemulsions are thermodynamically stable disperse systems that offer numerous advantages such as the ability to load both hydrophilic and hydrophobic drugs, smaller droplet size, high drug loading, self-dispersing / emulsifying, ease of preparation, and scale-up. Provided herein are microemulsion compositions comprising hydrophobic drugs solubilized therein.

[0028] Given the promise of the PPAR-a agonist A 190 and the difficulties of solubilizing this drug, a microemulsion comprising this exemplary hydrophobic drug was developed as a vehicle for delivery (e.g. oral delivery). The microemulsion surprisingly provides high drug loading, formulation stability and bioavailability. Thus, in one aspect, provided herein is a new microemulsion composition comprising the non-opioid drug (3-((4-((4-fluorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid (A190) as a novel therapeutic approach to treat pain, such as neuropathic and inflammatory pain. A 190 exhibits an EC50 <40 nM in cell-based assays and a >2, 700-fold selectivity for PPAR-a over PPAR-5 and PPAR-y. However, A190 unfortunately exhibits poor aqueous solubility (0.009 mg / mL), resulting in reduced oral bioavailability that dampens therapeutic efficacy. This problem is overcome using the novel microemulsion formulations disclosed herein. The microemulsions were developed by studying the effect of changing the type and percentage of excipients (e.g. oil, surfactants, and co-surfactants) on the droplet size, polydispersity index, zeta potential, and drug loading. The new formulations use a low percentage of biocompatible, non-toxic (GRAS) components, and biodegradable excipients to achieve high solubility, high drug loading, long-term storage stability, and high absorption.

[0029] In particular, the exemplary A 190 microemulsions exhibited physical stability with a droplet size of approximately 100 nm and a drug loading efficiency greater than 95%. The effective and apparent permeability of A 190 from the microemulsion was significantly higher compared to free A 190 dispersion. Additionally, no significant impact on cell viability was observed, indicating low toxicity and good biocompatibility. The oral bioavailability of A 190 microemulsion was approximately 5 -fold higher than that of a control A 190 dispersion, demonstrating the microemulsion’s potential to greatly enhance the oral bioavailability of this exemplary hydrophobic drug. Furthermore, the findings revealed that the orally administered A 190 microemulsion effectively reduced CIPN-induced mechanical hypersensitivity, likely mediated through PPARa activation. The A 190 microemulsion was found to be equally effective in reducing the chronic inflammatory Complete Freund's Adjuvant (CFA)-induced pain. These results underscore A190's potential as a non-opioid therapeutic candidate, utilizing a novel microemulsion formulation, for the management of pain, e.g. chemotherapy-induced neuropathic pain and chronic inflammatory pain.

[0030] DEFINITIONS

[0031] Hydrophobic drugs are difficult to dissolve in water and require special formulations or drug delivery systems. Examples of hydrophobic drugs used for treating pain include but are not limited to A 190, propofol fentanyl, oxycodone, hydrocodone, buprenorphine, methadone, celecoxib, diclofenac, ibuprofen, naproxen, amitriptyline and lidocaine.

[0032] Peroxisome proliferator- activated receptor alpha (PPAR-a), also known as NR1C1 (nuclear receptor subfamily 1, group C, member 1), is a nuclear receptor protein functioning as a transcription factor that in humans is encoded by the PPARA gene. Together with peroxisome proliferator- activated receptor delta and peroxisome proliferator- activated receptor gamma, PPAR-a is part of the subfamily of peroxisome proliferator- activated receptors. An agonist is a chemical substance that binds to and activates a certain or certain receptors on cells, causing a biological response.

[0033] A microemulsion is a thermodynamically stable, optically transparent dispersion of an oil phase and an aqueous phase, stabilized by e.g. surfactants and co- surfactants. Microemulsions exhibit droplet sizes in the range of 10-100 nanometers. Unlike conventional emulsions, microemulsions are single-phase systems that are clear, have a smaller droplet size, and possess unique properties that make them valuable in applications like drug delivery and as solubilization enhancers. Microemulsions are stable over time, meaning they do not spontaneously separate into distinct oil and water phases. They appear clear rather than cloudy because their dispersed droplets are much smaller than the wavelength of visible light. They comprise very small droplets of one liquid (e.g. oil or water) dispersed within the other, stabilized by surfactants and co-surfactants. Surfactants and co-surfactants are essential components that reduce the interfacial tension between oil and water, allowing for the formation of the stable dispersions. Microemulsions enhance the oral bioavailability of hydrophobic drugs by stimulating pancreatic and biliary secretions, increased gastrointestinal transit time, stimulation of lymphatic transport, increased intestinal membrane permeability, and reduced activity of metabolism and efflux pumps.

[0034] MICROEMULSION COMPOSITIONS

[0035] The microemulsion formulations disclosed herein comprise at least the following components:

[0036] An oil phase makes up from about 5.0 to about 30.0%, such as about 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, 27.7.5 or 30.0%, including all decimal fractions in between these values. In some aspect, the percentage of oil ranges from about 7.5 to about 22.5%, or about 10.0 to 20.0%, such as about 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0 or 20.0%, including all decimal fractions in between these values. In some aspects, the percentage of oil is 7.5%. The hydrophobic drug is suspended in the oil phase.

[0037] The types of oils that are used in the microemulsions include but are not limited to: oleic acid. In some embodiments, the one or more oils are selected from the group consisting of vegetable oils, animal oils, and synthetic or semisynthetic oils, or a mixture thereof. In some embodiments, the one or more oils are selected from the group consisting of soybean oil, cottonseed oil, com oil, caprylic / capric triglycerides (e.g,, Miglyol® 810, a triglyceride ester of saturated caprylic and capric fatty acids and glycerol, Captex® 355, glyceryl tricaprylate / tricaprate), medium-chain mono and di-glycerides (e.g., Capmul® MEM, mono-diglyceride of medium chain fatty acids (mainly caprylic and capric), long-chain mono glycerides (e.g., Peceol™, glyceryl monooleate (GMO)) fatty acids (e.g., oleic acid, caprylic acid), and fatty acid esters (e.g., ethyl oleate, ethyl butyrate, isopropyl mysistate, isopropyl palmitate), or a mixture thereof. In some embodiments, the one or more oils are selected from the group consisting of linseed oil, sunflower oil, fish oil, avocado oil, and coconut oil, or a mixture thereof. In some embodiments, the one or more oils is Capryol® 90 (propylene glycol monocaprylate type II).

[0038] A surfactant:cosurfactant mix makes up from about 20 to 50% of the microemulsion, such as about 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, or 50.0%. In some aspects, the surfactant:cosurfactant mix is present in a range of from about 35.0, 35.5, 40.0, 40.5 or 50.0%, including all decimal fractions in between these values. In some aspects, the surfactant:cosurfactant mix makes up 40% of the microemulsion.

[0039] The surfactant and cosurfactant in the surfactant:cosurfactant mix (Smix) are generally present in a ratio of from about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1 ,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1, volume to volume (v / v), including all decimal fractions in between these values. In some aspects, the ratio is 1:1 v / v.

[0040] Examples of surfactants and cosurfactants that can be paired in the practice of the invention include but are not limited to: polyoxyethylene sorbitan monooleate (commercial name Tween® 80), polyethylene glycol 400, where the "400" refers to its average molecular weight (PEG 400). In some embodiments, the one or more surfactants are selected from the group consisting of nonionic, cationic, and anionic surfactants. In some embodiments, the one or more surfactants is a nonionic surfactant selected from the group consisting of Polysorbates (such as polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate), Pluronic® (polyethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)), Cremophor® (polyoxyl 35 castor oil or polyethoxylated castor oil or PEG-35 castor oil), polyethoxylated alcohol, polyoxyl lauryl ether, Brij®, polyoxyethylated castor oil, lecithin, polyethylene glycol, glycerol esters of fatty acids, sugar esters, glyceryl monocaprylate, caprylocaproyl macrogol-8 glycerides, lauroyl polyoxyl-6 glycerides or lauroyl macrogol-6 glycerides, and caprylic / capric triglyceride or glyceryl trioctanoate / tricaprate, or a mixture thereof.

[0041] The microemulsion also comprises an aqueous phase. In some aspects, the aqueous phase makes up from about 20.0 to about 75.0% of the total volume, such as about 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, or 75.0 %. In some aspect, the aqueous phase makes up from about 40.0 to about 60.0%, such as about 40.0, 42.5, 45.0, 47.5, 50.0, 52.5, 55.0, 57.5, 60.0 %. In some aspects, the aqueous phase makes up 52.5% of the microemulsion.

[0042] The aqueous phase generally comprises, e.g. water, one or more biocompatible salts, biocompatible buffering agents, etc., that are pharmaceutically acceptable.

[0043] "Pharmaceutically acceptable salts" refers to relatively non-toxic, inorganic and organic acid addition salts and base addition salts. Exemplary acid salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-P-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. Base salts can also be utilized, including pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. Suitable inorganic base addition salts include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts include amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like. In some aspects, the salt is NaCl.

[0044] Various buffers and means for adjusting pH can be used to prepare a pharmaceutical composition disclosed herein, provided that the resulting preparation is pharmaceutically acceptable. Biocompatible buffering agents include, without limitation, acetate buffers, citrate buffers, phosphate buffers, neutral buffered saline, phosphate buffered saline and borate buffers. It is understood that acids or bases can be used to adjust the pH of a composition as needed. In some aspects, the buffer is phosphate buffer and the aqueous phase is phosphate-buffered saline (PBS).

[0045] The hydrophobic drug (e.g. A190) loaded microemulsion can be susceptible to changes in pH and ionic strength within the gastrointestinal tract, which may compromise stability and reduce oral bioavailability. This is addressed by incorporating a stabilizer that enhances the structural integrity of the microemulsion structure during gastrointestinal transit. Thus, in further aspects, the microemulsions also comprise at least one stabilizer. Examples of stabilizers that may be used include but are not limited to: antioxidants such as butylated hydroxytoluene (BHT), ascorbic acid, and citric acid; preservatives such as benzoic acid, methylparaben, and propylparaben; various buffering agents used to control the pH of the formulation such as carbonates and bicarbonates, phosphates (e.g. dibasic potassium phosphate and monopotassium phosphate), citrates (e.g. sodium citrate); acetates; organic buffers like Tris (Tris(hydroxymethyl)aminomethane) and CHES (N-cyclohexyl-2-aminoethanesulfonic acid), and ammonium chloride paired with ammonia; and the like; one or more additional surfactants such as polysorbates and lecithins; various polymers such as polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and celluloses like hydroxypropyl methylcellulose (HPMC); and sugars and sugar alcohols such as sucrose, trehalose, and mannitol, often used for lyophilized products.

[0046] Typical stabilizers and the typical amounts that are included include but are not limited to: Antioxidants: 0.001% to 0.5%; Preservatives: 0.01% to 1.0%; Buffering agents: 0.05% to 1.0%; Organic buffers: 0.05% to 1.0%; Polymers: 0.1% to 5.0%; Sugar and Sugar alcohols: 1% to 40%.

[0047] The microemulsions also comprise at least one hydrophobic drug. The amount of hydrophobic drug that is present in a microemulsion generally ranges from about 0.1 to about 2.0%. In some aspects, the hydrophobic drug is A190 and the microemulsions are generally loaded with from about 1.0 mg / mL to about 20.0 mg / ml or microemulsion. In some aspects, the loading of A 190 is 7.5 mg / mL.

[0048] The microemulsions may also be formulated for delayed, long-acting and / or sustained release. For example, the addition of water to microemulsion formulations results in phase transition to e.g. liquid crystal (LC) or coarse emulsion (CE), or combinations of both, as described by Wi et al. (Journal of Controlled Release, Volume 174, 28 January 2014, Pages 188-194). Hamad (Pharmaceutics, 2022 Sep 19; 14(9): 1975) describes the use of microgels for this purpose. In addition, various lyotropic crystals are also used to manufacture sustained release formulations. io MICROEMULSION CHARACTERISTICS

[0049] The microemulsions generally have a mean droplet size of from about 90 to 130 nm, such as about 90.0, 95.0, 100.0, 105.0, 110.0, 115.0, 120.0, 125.0 or 130.0 nm. In some aspects, the means droplet size ranges from about 110 to about 125, such as about 115.0, 116.0, 117.0, 118.0, 119.0, 120.0, 121.0, 122.0, 123.0, 124.0 or 125.0 including all decimal fractions in between these values. In some aspects, the mean diameter is 119.8 + 1.64 nm.

[0050] In some aspects, the microemulsions have a Polydispersity Index (PDI) ranging from about 0.10 to about 0.15, such as about 0.11, 0.12, 0.13, 0.14, or 0.15. In some aspects, the PDI is 0.131 ± 0.007, including all decimal fractions in between these values

[0051] In additional aspects, the microemulsions have a zeta potential ranging from about -1.0 to about -10.0, such as about -1.0, -2.0, -3.0, -4.0, -5.0, -6.0, -7.0, -8.0, -9.0 or -10.0. In some aspects, the zeta potential ranges from about -5.5 to about 6.5, such as about -5.55, -5.56, 05.57, -5.58, -5.59, -6.00, -6.10, -6.20, -6.30, -6.40 or -6.50. In some aspects, the zeta potential is about -5.92 + 0.87 mV.

[0052] The hydrophobic drug is suspended in the oil phase. In some aspects, the drug content is at least about 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% or even more, up to e.g. 95, 96, 97, 98 or 99%. For example, the A 190 content in the microemulsion, as measured using HPLC, showed a drug content of >95%.

[0053] The microemulsions exhibit a permeability across intestinal walls of at least about 10’15Papp, xl0“6, cm / s, such as about 10, 11, 12, 13, 14, or 15 Papp, xl0“6, cm / s. In some aspects, the permeability is from about 12.00, 12.10, 12.20, 12.30, 12.40, 12.50 or 13.00.

[0054] The area under the curve” (AUC) of the microemulsions generally ranges from about 10,000 to about 60,000, such as about 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50, 000 55, 000 or 60,000 ng / ml*h. In some aspects, the range is from about 30,000 to about 60,000, such as about 30,000, 35,000, 40,000, 45,000, 50,000, 55,000 or 60,000. As an example, the A190 microemulsion exhibited an AUC of 47434.9 + 4824.9 ng / ml*h, which was significantly (4.7-fold) higher compared with that of an A190 dispersion (9980.9 + 2621.0 ng / ml*h).

[0055] The Relative Bioavailability (%) of a hydrophobic drug in a microemulsion as described herein compared to a simple dispersion of the same hydrophobic drug ranges from e.g. about 2.5-25.0 fold, e.g. about 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5 or 25.0-fold. As an example, the A 190 microemulsion exhibited an approximately 5 -fold greater bioavailability.

[0056] The present microemulsions exhibit a stability of at least about 3-12 months, when n stored at room temperature under sterile conditions. That is, they are stable and do not lose potency or break down for at least about 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or more. Generally, microemulsions are stored as liquid formulations.

[0057] METHODS

[0058] Provided herein are methods of preventing and / or treating and / or reducing pain in a subject in need thereof. The methods comprise administering to the subject a therapeutically effective amount of at least one microemulsion as disclosed herein. A “therapeutically effective amount” refers to an amount that entirely prevents or decreases pain in the subject, e.g. no pain occurs or all pain disappears, or at least ameliorates pain, e.g. the level of pain that occurs is less than would occur in the absence of administration of the microemulsion or if the pain is already present, the level of pain is decreased compared to the level that was present before or in the absence of administration of the microemulsion.

[0059] As shown in the Examples below, in some aspects, the amount of microemulsion that is administered generally ranges from about 0.05 to about 0.3 ml per dose for mice and 0.5 to about 2.5 mL per dose for rats. In the exemplary case where A190 is the drug that is administered, the amount of A 190 that is administered per dose generally ranges from about 1 to about 5 mg / kg of body weight for other species such as humans, companion pets, etc.

[0060] Those of skill in the art will recognize that the dosing schedule or regimen will vary based on several factors. For example, it will vary depending on the type of hydrophobic drug that is administered, the characteristics of the patient (e.g. gender, age, weight, overall health, genetic background, etc.) and on the disease or condition being treated. The details of recommended dosing are typically worked out in clinical trials. However, in general, the microemulsion is administered from about 1-6 times daily (e.g. 1, 2, 3, 4, 5 or 6 times), e.g. every hour, every 2 hours, very 4 hours, every 6 hours, every 8 hours, etc. Alternatively, administration may be once daily in the AM or PM, or twice daily e.g. in the AM and PM (12 hours apart). The gaps between administration may be longer, e.g. once per week, once per month, etc. Any dosing protocol that brings relief of pain to the patent may be used. In some aspects, administration is coordinated with, for example, other medical procedures such chemotherapy, surgery, etc. In such instances, administration may be before, during or after the procedure, or two or more of these, e.g. before and after, or before during and after, etc.

[0061] In some aspects, the microemulsions disclosed herein have a pain preventing / treating / reducing activity of e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% or more, e.g. at least about 100%. In yet other aspects the pain is prevented / treated / reduced in a range of from, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0062] The present microemulsions are particularly advantageous for oral administration, i.e. the microemulsion is taken by mouth and swallowed by the patient. The present oral microemulsion formulations offer significant advantages for treating diseases and conditions (maladies) where the drug either exhibits increased systemic toxicity when administered parenterally or suffers from poor bioavailability when taken orally in another form. Thus, the pharmaceutically suitable microemulsions disclosed herein are generally administered orally.

[0063] In some aspects, the microemulsions are administered as a liquid. However, “oral administration” also includes aspects in which the microemulsions are encapsulated within a delivery vehicle, e.g. a gel cap (e.g. a soft gel capsule that is dissolved in the intestine thereby releasing the active agents) or other form that contains the liquid microemulsion. In such aspects, the microemulsion is contained within the delivery vehicle, swallowed by the subject and the delivery vehicle is dissolved, e.g. in the stomach, releasing the microemulsion and the active agent contained therein

[0064] A 190 is an exemplary hydrophobic drug but the present microemulsion formulation strategy also applies to other drugs that have solubility and permeability issues, providing a generalizable approach to overcome formulation challenges in oral drug delivery.

[0065] In addition, the compositions may be administered in conjunction with other treatment modalities. For example, they are administered with other analgesic agents various chemotherapy agents, etc. In particular, for the treatment of cancer patients undergoing chemotherapy, they are administered in conjunction with the chemotherapy agents. The microemulsions are administered before, during or after the chemotherapy agents are administered.

[0066] TREATMENTS The microemulsions disclosed herein are used in methods to prevent and / or treat a wide variety of types of pain. Any type of pain that is caused by or associated with overactivity of PPAR-a can be treated by the microemulsions disclosed herein.

[0067] Types of pain that are treated include but are not limited to:

[0068] Acute, short-term pain that comes on suddenly and usually has a clear cause, such as tissue injury. It typically lasts fewer than 6 months and goes away once the underlying issue is treated. Common causes of acute pain include: broken bones, surgery, dental work, labor and childbirth, cuts, burns, etc.

[0069] Chronic pain which lasts e.g. for years and ranges from mild to severe on any given day. Past injuries or damage can cause chronic pain, but sometimes, there’s no clear cause. Symptoms that can accompany chronic pain include: muscle tension, low energy and limited mobility. Some common examples of chronic pain include: frequent headaches, nerve damage pain, low back pain, arthritis pain and fibromyalgia pain.

[0070] Nociceptive pain is the most common type of pain. It occurs when potential or actual tissue damage stimulates nociceptors, which are specialized pain receptors located throughout the body, especially in the skin and internal organs. When activated (for example, by a cut, bruise, or inflammation), nociceptors send electrical signals through the nervous system to the brain, which interprets them as pain. Nociceptive pain is the type you usually feel with injuries or inflammation. It can be acute or chronic and is also further classified as visceral or somatic.

[0071] Visceral pain comes from injury or damage to the internal organs. It’s usually felt in the trunk of the body, such as the chest, abdomen, or pelvis. It can be hard to localize to one exact spot. Symptoms include: pressure, aching, squeezing, cramping, nausea or vomiting, and changes in body temperature, heart rate, or blood pressure. Examples of causes of visceral pain include: gallstones, appendicitis, irritable bowel syndrome, etc.

[0072] Somatic pain comes from stimulation of pain receptors in the body’s tissues, such as the skin, muscles, joints, connective tissues, and bones (rather than the internal organs). Somatic pain usually feels like a constant aching or gnawing sensation and can be further classified as either superficial somatic pain (from the skin or surface tissues such as a canker sore or a scrape) and deep somatic pain (from deeper structures like muscles, tendons, or bones such as a tom tendon or a bone fracture). Examples of somatic pain include: bone fractures, strained muscles, connective tissue diseases, such as osteoporosis, cancer that affects the skin or bones, skin cuts, scrapes, and burns and joint pain, including arthritis pain.

[0073] Neuropathic pain results from damage to, or dysfunction of, the nervous system. When nerves are injured or not working properly, they can misfire and send pain signals even in the absence of a specific injury or pain in response to things that aren’t usually painful, such as cold air or clothing against your skin. Symptoms of neuropathic pain include but are not limited to: burning, freezing, numbness, tingling, shooting, stabbing, electric shocks, etc. Diabetes is a common cause of neuropathic pain. Other sources of nerve injury or dysfunction that can lead to neuropathic pain include: chronic alcohol consumption, accidents, infections, facial nerve problems, such as Bell’s palsy, spinal nerve inflammation or compression, shingles, carpal tunnel syndrome, HIV, central nervous system disorders, such as multiple sclerosis or Parkinson’s disease, radiation, chemotherapy drugs, etc.

[0074] Inflammatory pain is a type of pain caused by an inflammatory response in the body. It occurs when tissues are damaged or infected, leading to the release of chemicals called inflammatory mediators. These mediators activate pain receptors, causing a burning, throbbing, or aching sensation. Causes of inflammatory pain include but are not limited to: trauma (e.g., injuries, surgery); infections (e.g., bacterial, viral); autoimmune disorders (e.g., rheumatoid arthritis, polymyalgia rheumatica, giant cell arteritis); chronic conditions (e.g., obesity, heart disease); allergic reactions; and the like. Aspects of the present specification disclose, in part, a method of treating an individual with a chronic inflammation. In one embodiment, the method comprises the step of administering to an individual in need thereof a pharmaceutical composition disclosed herein, wherein administration reduces a symptom associated with the chronic inflammation, thereby treating the individual.

[0075] Peripheral pain In some aspects, the pain is peripheral pain. Peripheral pain refers to pain that originates in the peripheral nervous system, which includes the nerves outside the brain and spinal cord. It can affect various parts of the body, such as the hands, feet, legs, arms, and face. Peripheral pain can be caused by a wide range of factors, including but not limited to: diabetes, trauma (e.g., injuries, surgeries), infections, autoimmune disorders (e.g., Guillain-Barre syndrome, rheumatoid arthritis, polymyalgia rheumatica, giant cell arteritis, etc.), certain medications (especially chemotherapy drugs), vitamin deficiencies, and genetic disorders.

[0076] In some aspects, the peripheral pain that is treated is CIPN. Presently, there are no approved or effective preventive agents for the CIPN and several previous clinical trials involving antiepileptic and antidepressant drugs have shown mixed results. One recent double-blind, randomized controlled trial showed an improvement in CIPN symptoms after treatment with duloxetine for 5 weeks. However, the American Society of Cancer (ASC) and American Society of Clinical Oncology (ASCO) guidelines request that clinicians gradually taper the dose of duloxetine before discontinuing treatment, as abrupt discontinuation might cause withdrawal symptoms similar to other antidepressant drugs. Thus, the present novel oral microemulsion formulation of a non-opioid drug, such as A190, incorporating excipients classified as Generally Recognized As Safe (GRAS), advantageously demonstrates a favorable safety profile and does not induce withdrawal symptoms commonly associated with antidepressant medications.

[0077] Any type of pain may be treated using the microemulsions described herein, e.g. those loaded with A 190. By “treating” we mean that at least one symptom of the pain is alleviated or lessened. While a laudable goal is to eliminate pain and all symptoms of pain, those of skill in the art will also recognize that much benefit can accrue if one of more symptoms are lessened, even if they are not eliminated.

[0078] Further provided are methods of activating PPARa in a cell. The methods comprise contacting the cell with a microemulsion comprising 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid under conditions which allow the 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid to enter to the cell and bind to PPARa. The cell may be in vitro or in vivo. The conditions which allow the 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid to enter to the cell and bind to PPARa generally include physiological conditions, such as when the external and / or internal milieu of the cell is at a temperature range of 20-40 degrees Celsius, preferably about 37 °C, atmospheric pressure of 1, pH of 6-8, glucose concentration of e.g. 1-20 mM, atmospheric oxygen concentration, etc.

[0079] It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0080] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Representative illustrative methods and materials are herein described; methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0081] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual dates of public availability and may need to be independently confirmed.

[0082] It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as support for the recitation in the claims of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitations, such as "wherein [a particular feature or element] is absent", or "except for [a particular feature or element]", or "wherein [a particular feature or element] is not present (included, etc.)...".

[0083] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0084] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention. EXAMPLE

[0085] A novel PPARa agonist loaded microemulsion that attenuates neuropathic and inflammatory pain

[0086] Microemulsions are thermodynamically stable disperse systems that offer numerous advantages such as the ability to load both hydrophilic and hydrophobic drugs, smaller droplet size, high drug loading, self-dispersing / emulsifying, ease of preparation, and scale-up. These oil-based formulations were developed using a selected concentrations of oils or their derivatives and were effectively emulsified using a mixture of surfactant and co- surfactant, yield a stable microemulsion system. This system enhances the oral bioavailability of hydrophobic drugs through various mechanisms, including stimulation of pancreatic and biliary secretions, increased gastrointestinal transit time, stimulation of lymphatic transport, increased intestinal membrane permeability, and reduced activity of metabolism and efflux pumps. Additionally, the droplet size of microemulsions (<200 nm) plays a key role in enhancing intestinal drug absorption and distribution due to their larger surface area and improved permeability.

[0087] Considering these premises and potential of microemulsions, we hypothesized that a new oil-in-water (o / w) microemulsion of A 190 could improve solubility and intestinal membrane permeability of the A 190, increase oral bioavailability, and enhance therapeutic efficacy against CIPN. To test this hypothesis, we first selected the microemulsion components based on drug solubility and excipient miscibility and then developed the A 190 microemulsion formulation. Subsequently, extensive studies were conducted to analyze the physicochemical characteristics of the formulation, assess in vitro and ex vivo permeability, evaluate pharmacokinetic profiles in healthy rats, and determine the in vivo efficacy of A 190 microemulsion in CIPN in mice. We also further evaluated the efficacy on another important Complete Freund's Adjuvant (CFA)-induced chronic inflammatory pain mouse model.

[0088] 2. Materials and methods

[0089] 2.1. Materials

[0090] Paclitaxel was purchased from Athenex, NDC 70860-200-50, Richmond, USA. Paclitaxel was dissolved in a 1:1:18 mixture of 200 proof ethanol, Kolliphor®, distilled water, Tween® 80, polyethylene glycol 400 (PEG 400), propylene glycol, Complete Freund’s Adjuvant (CFA), dimethyl sulfoxide (DMSO), and (2-Hydroxypropyl)-P-cyclodextrin (HP-P-CD) was purchased from Sigma- Aldrich, MO, USA. Fenofibrate d6 (internal standard, IS) was purchased from Toronto Research Chemicals, New York, ON, Canada. MK886 was purchased from Tocris® (1311, Bristol, United Kingdom and dissolved in a 1:1:18 mixture of 200 proof ethanol, Kolliphor®, and distilled water (Sigma-Aldrich). Caprylocaproyl macrogol-8-glycerides (Labrasol®), diethylene glycol monoethyl ether (Transcutol® HP), and propylene glycol monocaprylate (Capryol® 90) was provided as a gift by Gattefosse, Saint-Priest, France. Caco-2 cells (human colon cancer) and HepG2 cells (human hepatocellular carcinoma) obtained from ATCC® (American Type Culture Collection, Manassas, VA, USA).

[0091] 2.2. Animals

[0092] Sprague Dawley rats (males, 250-300 g) were purchased from Charles River Laboratories (Wilmington, MA) for in vivo pharmacokinetic study. Pain model experiments were conducted using adult (10-15 weeks) male and female C57BL / 6J mice from Jackson Laboratory (Bar Harbor ME, USA). Initially, they were maintained in a temperature- and humidity-controlled vivarium space (21 ± 3 °C, 55 ± 10%) on a 12 h light / dark cycle (lights on at 7:00 AM) with free access to food (Teklad LM-485 mouse sterilized diet, Harlan Laboratories Inc., Indianapolis IN, USA) and water until needed. Then mice were retrieved from the vivarium and housed (4-5 mice per cage) for the duration of the study in a temperature- and humidity-controlled out-of-vivarium space on the same light / dark cycle. They were given ad libitum food and water. All experiments were performed during the light cycle. This study was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University and carried out in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.

[0093] 2.3. Preparation and characterization of Al 90 loaded microemulsions

[0094] 2.3.1. Solubility based selection of microemulsion components

[0095] An essential criterion for selecting microemulsion components is the solubility of the poorly water-soluble drugs. An excess amount of A190 was added to 1 mL of various aqueous phase (deionized water, PBS, PBS with 0.2% Tween® 80, and PBS with 0.2% Sodium lauryl sulfate (SLS)), oils (oleic acid, Capryol® 90, Maisine® oil), and surfactants or co-surfactants (Labrafil® M 1944 CS, PEG 400, Tween® 80, Transcutol® HP, Labrasol®, Propylene Glycol, Cremophor) in stoppered glass vials. Each sample was vortexed and kept in an isothermal shaker maintained at 25 ± 1.0°C to reach equilibrium for 48 h. The resulting mixture was centrifuged at 4000 x g (Eppendorf 5424 R Refrigerated Centrifuge) for 15 minutes, and the supernatant was collected and diluted with acetonitrile followed by filtration with 0.22 pm (PTFE, hydrophobic, ThermoScientific) membrane filters. The concentration of A190 in the filtrates was quantified by HPLC-UV (Shimadzu Prominence LC system). Chromatographic separation was achieved using an Agilent Pursuit XRs 5 C18 column (250 x 4.6 mm) with UV-detector system. The mobile phase consisted of acetonitrile and water, both containing 0.1% trifluoroacetic acid (TFA) in a ratio of 70:30 v / v, with the flow rate of 1 mL / min at 25 °C. The injection volume was 10 pL. The detection was performed at 227 nm wavelength. After selection of oils and surfactants / co- surfactants with higher A 190 solubility, miscibility studies was performed as previously described.30In brief, selected excipients were mixed with each other in equal volumes (1 mL), vortexed for 10 min, and left to equilibrate for around 30 minutes. The resulting mixture was visually inspected for transparency / turbidity and phase separation.

[0096] 2.3.2. Development using Box-Behnken experimental design

[0097] Several process and formulation parameters integral to microemulsion formulation might influence the final physicochemical characteristics, drug loading, and stability of the microemulsion. Therefore, a Box-Behnken design (BBD) with 3-factor and 3-level (33) was conducted to screen the most relevant process parameters and determine the minimum number of experiments needed to develop the A 190 microemulsion. The total of 15 experimental runs were generated and evaluated using JMP Pro 14 software (SAS Institute, Cary, NC, USA), as shown in Table 1. Based on preliminary studies, the three key parameters influencing the physicochemical properties of A 190 microemulsion, namely percentages of oil (Xi), surfactant / co-surfactant ratio (X2), and sonication time (X3) were selected as independent variables. All the independent variables were adjusted at three different levels: -1 (lower level), 0 (medium level), and +1 (higher level). Additionally, mean microemulsion droplet size (Yi), polydispersity index (PDI; Y2), zeta potential (Y3), and drug loading (Y4) were taken as the responses for the design experiment. In addition, physical stability of the prepared microemulsions such as phase separation and transparency / turbidity were evaluated. To evaluate the reproducibility of the design, 3-center points were included, whereas the impact of unexplained variability was minimized by conducting all experiments in a random order. Statistical model for each response was chosen based on the highest order polynomial where additional terms are significant, and the model is not aliased. The A 190 microemulsion was prepared according to the predictor profile for maximum desirability within the design space. The model was based on achieving A 190 microemulsion with minimum droplet size and PDI with maximum drug loading percentage, and zeta potential values between -10 mV to +10 mV.

[0098] A190 microemulsion with different concentration of oils (7.5-22.5%), surfactant / co-surfactant mixture of 40% with 3:1, 1:1, and 1:3 v / v ratio, and deionized water (37.5%— 52.5%) were prepared using high energy ultrasonication method. In brief, A190 was dissolved in oleic acid (oil) under sonication (Branson CPX2800H, USA) for 30 minutes and a fixed weight ratio of Tween® 80 / PEG 400 (surfactant / co-surfactant mixture) were added. After vigorous mixing, deionized water was slowly added with continuous stirring at 1500 x g for 15 min. The emulsions formed were sonicated by ultrasonic Liquid Processor VCX 500 (Sonics & Materials, Inc., CT, USA) at an amplitude of 40%. Finally, each A190 microemulsion was subjected to physicochemical characterization.

[0099] Table 1. List of dependent and independent variables in Box-Behnken design for A190 microemulsion.

[0100] 2.3.3. Characterization of the A190 microemulsions

[0101] The mean droplet size, PDI, and zeta potential of the A 190 microemulsion were measured using a dynamic laser light scattering analyzer (Malvern Zetasizer Nano ZS90; Malvern Instruments, Malvern, UK). All A190 microemulsion formulations were diluted with 10 mM NaCl solution at a ratio of 1:100 v / v and sonicated for 1 min to minimize multiple scattering effects. Each measurement carried out at 25 °C was estimated by averaging three runs and presented as mean ± standard deviation (SD). The drug content in the A 190 microemulsion was determined by diluting each formulation with acetonitrile, filtered using a 0.2 pm membrane filter, and quantified using the HPLC system with a UV detector as mentioned earlier in section 2.3.1. The droplet morphology and droplet size of the A190 microemulsion were confirmed using a high-resolution transmission electron microscopy (TEM). In brief, A 190 microemulsion was diluted 100 times in deionized water, and a drop of diluted microemulsion was placed onto a carbon-coated copper grid. The excess liquid on the samples was wiped off with a filter paper and one drop of 2% aqueous solution of phosphotungstic acid was added to the grid to enhance contract and allow negative staining. The dried grid containing the A190 microemulsion was observed using a TEM (JEM-200; JEOL, Tokyo, Japan).

[0102] 2.3.4 Stability ofA190 microemulsions

[0103] The formulation selected from the BBD was subjected to the thermodynamic stability tests, including centrifugation test, heating-cooling cycle test, and freeze-thaw test. To understand the effect of centrifugal force on phase separation of two immiscible phases (water and oil), A 190 microemulsion was centrifuged down at 1000 x g for 30 minutes (Eppendorf 5424 R Refrigerated Centrifuge). For the heating-cooling cycle test, A 190 microemulsion was examined at room temperature for 48 h, after being subjected to 6 cycles of heating (45 °C) and cooling (4°C). Whereas, for freeze-thaw cycles, A190 microemulsion was examined at room temperature for 48 h, after being subjected to 3 cycles of freezing (-21 °C) and thawing (25°C). In each test, A190 microemulsion was studied for phase separation, creaming or cracking. To further access the stability of the microemulsion, dispersibility study was performed using USP 2 dissolution apparatus. In brief, one mL of A190 microemulsion was dispersed in 100 mL of PBS pH 6.8 at 37°C and observed for phase separation and drug precipitation as previously described.

[0104] To assess the storage stability, A190 microemulsion was placed in glass scintillation vials and were kept at room temperature (25 ± 5°C) for 3 months. At a specified time period, the microemulsion formulation was evaluated in terms of droplet size, PDI, zeta potential, and percentage drug content remaining. In addition, A 190 microemulsion was visually inspected for signs of instability, including phase separation, turbidity, transparency, precipitation, and color change. Each experiment was performed in triplicate, and the result were presented as mean ± SD.

[0105] 2.4. In vitro and ex vivo permeability of Al 90 microemulsion

[0106] To predict the passive intestinal permeability of a drug, the parallel artificial membrane permeability assay (PAMPA) technique is used, which utilizes a phospholipid-coated lipophilic membrane that mimics the properties of the intestinal wall.33The in vitro intestinal membrane permeability of free A190 (dispersed in phosphate-buffered saline (PBS) pH 6.8), A190 dispersion (dispersed in a mixture of 10% PEG 400 + 5% DMSO + 85% of 10% HP-P CD), and A190 microemulsion were evaluated using a PAMPA (BD BioSciences, San Jose, CA, USA), as described previously.30,34Briefly, donor samples were prepared by the dilution of free A190, A190 dispersion, and A190 microemulsion with PBS pH 6.8 at a concentration of 200 pg / mL (based on A190). After loading 0.3 mL of PBS pH 6.8 to each well of the acceptor plate, both acceptor and donor plates were sandwiched, while ensuring that the membrane of donor plate was in proper contact with the media in the acceptor plates. Next, 0.2 mL of the diluted samples were loaded in each well of the donor plate. Following incubation of entire plate for 5 h at room temperature, the plate assembly was separated, and samples were collected from both the acceptor and donor plates. The concentration of A190 that permeated through the phospholipid membrane was measured by HPLC as described earlier. The effective permeability (Pe) of each drug was then calculated using the following formula:

[0107] Pe=-ln( 1 - CA[t] / Cequilibrium) / (AX [ 1 / VD+ 1 / V A] Xt) where Peis the permeability (cm / s), A is the effective filter area (0.228 cm2), VD is the volume of the donor well (0.2 mL), VA is the volume of the receptor well (0.3 mL), t is the total time of incubation in seconds, CA(1) denotes the concentration of drug in the receptor well at time t, and Cequiiibrium represents (Co[t] x VD + C \[t] x VA) / (VD + VA), where Co(t) denotes the concentration of drug in the donor well at time t.

[0108] Ex vivo intestinal permeation study of the A 190 microemulsion, A 190 dispersion, and free A 190 was carried out using freshly excised rat intestinal membrane via non-everted rat sac model. In brief, SD rats housed in standard conditions with free access to food and water were sacrificed, duodenal part of the small intestine was excised, and cleaned for facial debris and mucus using Ringer’s lactate solution (ICU Medical, Inc.). The clean intestinal segment was cut into pieces of 6 cm length and one end was tied firmly to form a sac. A 190 microemulsion, A190 dispersion, and free A190 diluted to 1 mg / mL in PBS pH 6.8 was loaded in the sac (0.5 mL) and second end of the intestinal segment tied using thread. The sac was placed in a jacketed glass with 25 mL of PBS pH 6.8, maintained at 37°C with continuous stirring (100 rpm). At predetermined time intervals, 1 mL of release media was collected and replaced with the fresh media. The concentrations of A 190 permeated through the non-everted intestinal sac was quantified using the HPLC method as described in section 2.3.1. The apparent permeability coefficient (Papp) of A190 or A190 microemulsion was calculated using the formula:

[0109] PaPP= (dQ / dt) / (A / C0) where, Pappis the apparent permeability (cm / s), dQ / dt is the rate of linear appearance of mass on the receptor side (pmoL / s), Co is the initial concentration of A190 inside the sac (pg / mL), and A is the surface area of the intestinal sac exposed to the A 190 microemulsion or dispersion. Surface area was calculated by treating intestinal segment as a cylinder and using the formula: A = 2K X radium x length.

[0110] 2.5. Cell viability studies

[0111] To evaluate the cytotoxicity of free A190 (in 0.2% DMSO in DMEM), vehicle microemulsion, and A190 microemulsion against Caco-2 cells and HepG2 cells, MTT assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was performed. Caco-2 and HepG2 cells were cultured in a 25 cm2tissue culture flask with DMEM supplemented with 10% FBS and Penicillin (100 U / mL), Streptomycin (100 pg / mL) at 37°C in a humidified incubator with 5% CO2. Once the cells reached confluence, they were trypsinized, and 100 pL of cell suspension at a density of 10 x 104cells / well were seeded in a 96-well tissue culture plates, followed by incubation at 37°C. After 48 hours, the growth medium was replaced with freshly prepared dilutions of free A 190, vehicle microemulsion, and A 190 microemulsion diluted in DMEM (0.1, 1, 2.5, 5, 7.5, 10, 25, 50, 100 pM based on A190). Untreated cells (DMEM) and cells treated with 0.2% DMSO in DMEM served as controls. After 24 h and 48 h of incubation, the media from each well were replaced with 100 pL of MTT solution (5 mg / mL in PBS, filtered). The plates were gently shaken and then incubated at 37°C in a humidified 5% CO2 incubator for 3 hours. Following the incubation, the supernatant was removed, and 100 pL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The DMSO was gently pipetted up and down DMSO to ensure complete solubilization of crystals. Absorbance was then measured using a plate reader at 540 nm. The cell viability percent of each group was calculated using the following equation: (Optical density of samples / Optical density of control) x 100.

[0112] 2.6. In vitro dissolution study

[0113] Dissolution study was performed using a USP I Apparatus (Basket) (Sotax AT Xtend, Switzerland) with 100 mL of medium containing 0.1 N HC1 solution (pH 1.2) or PBS pH 6.8 at 100 rpm and a temperature of 37°C ± 0.2°C. In each test, A190 dispersion or A190 microemulsion (3.75 mg in 1.5 mL) were filled in a hydroxypropyl methylcellulose (HPMC) capsules (size 00) from Qualicaps®. Capsules were placed inside the basket and subjected to dissolution test, and 1 mL of samples were withdrawn at 10, 20, 30, 45, 60, 90, 120, 180, 240, and 300 min. In the meantime, the same volume of fresh medium was added to keep constant volume. The dissolution study of each sample was performed in triplicate. After filtration, the amount of A 190 in the collected dissolution samples was quantified by the HPLC method as described in section 2.3.1.

[0114] 2.7. In vivo oral absorption in rats To evaluate the improvement in the oral bioavailability of A 190 after formulation as a microemulsion, A 190 microemulsion was orally administered to rats. Each rat received either A190 dispersion or A190 microemulsion at a dose of 20 mg / kg. Subsequently, 150 pL blood samples were collected via tail vein at pre-determined intervals and transferred in sodium EDTA-coated tubes. The blood was immediately centrifuged (2500 x g, 15 min, 4 °C), and plasma was separated and stored at -80 °C until analysis.

[0115] To measure the plasma concentration of A 190, a total of 100 pL of each standard solution or defrosted plasma sample was spiked with 100 pL of fenofibrate d6 (5 pg / mL, IS). Next, 400 pL of ice-cold acetonitrile was added to the mixture to precipitate protein, followed by vortex mixing and centrifugation at 9000 x g for 5 min at 4 °C. After centrifugation, the supernatant was transferred to glass vial and evaporated to dryness using a sample concentrator dry-block (Techne, Cambridge, UK) at 50 °C. Finally, residues were reconstituted with 100 pL of acetonitrile: water (1:1, v / v). The plasma concentration of A 190 was determined using a Liquid Chromatography Mass Spectrometer (LC-MS-2020) (Shimadzu Corporation) with a Phenomenex Kinetex C18 column (100 x 2.1 mm, 1.7 pm, PFP 100 A). The chromatographic separation was performed using isocratic mobile phase (acetonitrile with 0.1% formic acid: water with 0.1% formic acid, 60:40, v / v) at a flow rate of 0.3 mL / min. A 10 pL sample was injected, and A 190 was measured in positive ionization mode using an electron spray ionization (ESI) interface. The following parameters were studied for A190 analysis: interface temperature, 350 °C; DL temperature, 250°C; nebulizing gas flow, 1.5 L / min; drying gas flow, 12 L / min.

[0116] 2.8. Chemotherapy Induced Peripheral Neuropathy (CIPN) pain model

[0117] CIPN model was established as previously described.36In brief, paclitaxel was administered at a dose of 8 mg / kg intraperitonially every other day; four administrations completed one injection cycle. Control mice received paclitaxel dissolved in a mixture of 1:1:18 v / v / v (ethanol / kolliphor / distilled water) at a dose of 10 ml / kg, i.p. and followed the same injection cycle. Animals began behavioral testing 14 days following the final injection. A190 microemulsion was administered at a dose of 10 and 20 mg / kg by oral gavage and tested at different time points (1, 3, 6, 24, 48 and 72 h) after drug administration. To investigate whether paclitaxel-induced hypersensitivity reduction with oral dosing of A 190 microemulsion is mediated through PPAR-a activation, CIPN mice were pretreated with the selective PPAR-a antagonist, MK886 (6 mg / kg, i.p. 30 min pretreatment time) followed by A190 microemulsion (20 mg / kg, p.o.) and mice were tested 48 h later.

[0118] 2.9. Induction of chronic inflammatory pain by CFA We explored the effects of A 190 microemulsion in a widely used model of persistent inflammatory pain, which bases on the injection of CFA into the paw. CFA is composed of inactivated and dried Mycobacterium tuberculosis and adjuvant and was purchased from Sigma-Aldrich (St. Louis MO, USA). The CFA pain model is based on hypersensitivity, paw swelling, and nuclear factor-KB-mediated transcription of tumor necrosis factor a involved in the formation of the principal mediators of inflammation37. Mice were injected intraplantarly with 20 pL of CFA (50%, diluted in mineral oil; Sigma-Aldrich). Mechanical sensitivity (see the measurement of the von Frey test) was measured before and 3 days after CFA injection. A 190 microemulsion (20 mg / kg) or vehicle microemulsion was administered by oral gavage on day 4 after CFA injection, and mice were tested for mechanical sensitivity at different time points (1, 3, 6, 24, 48 and 72 h) after drug injection.

[0119] 2.9.1 Evaluation of mechanical sensitivity

[0120] Mechanical sensitivity thresholds were determined according to the method of Chaplan et al, and as adapted in Toma et al., 2017.36,38A series of calibrated von Frey filaments (Stoelting, Wood Dale IL, USA) with logarithmically incremental stiffness ranging from 0.07 to 3.6 expressed as diameter sensitivity (ds) log 10 of 10 x force (in milligrams) was applied to the paw with a modified up-down method.38The mechanical threshold was expressed as log 10 of 10 x force (in milligrams), indicating the force of the von Frey hair to which the animal reacted (paw withdrawn, licking, or shaking). All behavioral testing on animals was performed in a blinded manner.

[0121] 2.9.2 Paw edema measurement

[0122] Immediately following Von Frey testing, paw edema was measured. The thickness of CFA-treated paws was measured 48 h after drug administration, using a digital caliper (Traceable Calipers, Friendswood, TX). Data were recorded to the nearest ± 0.01 mm and expressed as change in paw thickness (APD = difference in the ipsilateral paw diameter - contralateral paw thickness).

[0123] 2.9.3 Locomotor activity

[0124] Mice were placed into individual Omnitech photocell activity cages (28 x 16.5 cm) (Columbus OH, USA) 24 h after administration of either vehicle or A 190 microemulsion (20 mg / kg, p.o.). Interruptions of the photocell beams (two banks of eight cells each) were then recorded for the next 60 min. Data are expressed as number of photocell interruptions.

[0125] 2.11. Histopathology evaluation

[0126] SD rats were administered daily doses of either a vehicle microemulsion or a A 190 microemulsion for 3 consecutive days. On day 4, rats were euthanized using CO2 euthanasia followed by cervical dislocation. For histological evaluation, various tissues, including the liver, heart, spleen, kidney, duodenum, jejunum, and ileum. These tissues were immediately fixed in 10% neutral buffered formalin for 24 hours before being transferred to 70% ethanol. The tissues were then embedded in paraffin wax and sectioned into 5 pm-thick slices using a microtome. Hematoxylin and eosin (H&E) staining was performed on the tissue sections. A blinded pathologist evaluated the stained slides for any signs of tissue damage, inflammation, or other histological alterations. Imaging was conducted using Phenochart, a tissue imaging software integrated with the Phenoimager (AKOYA Biosciences).

[0127] 2.11. Statistical analysis

[0128] Behavioral data are expressed as mean ± SEM (Standard Error of the Mean). Time-course data and other behavioral results were analyzed using one-way or two-way repeated measures of variance (ANOVA) followed by post-hoc Tukey test with the alpha level set at 0.05. The behavioral statistical analysis was performed with GraphPad Prism software, version 9.5 (GraphPad Software, Inc., La Jolla, CA, USA). The probability was considered significant if P<0.05. No significant sex differences were observed in all experiments, so the male and female data were pooled. For all other analysis, one-way or two-way ANOVA followed by Tukey’ s multiple-comparison test was used to compare more than two mean values. All data were expressed as mean ± SD for in vitro analysis and mean ± SEM for in vivo analysis. In all analyses, p < 0.05 was considered statistically significant.

[0129] 3. Results

[0130] 3.1. Preparation and characterization of Al 90 microemulsions

[0131] Solubility studies of A190 were conducted to identify appropriate oils, surfactant, and co-surfactant for the formulation of a stable A 190 microemulsion, as shown in Figure 1. The solubility of A190 in Capryol® 90 (12.7 ± 0.88 mg / mL) was higher compared to oleic acid (4.3 ± 0.03 mg / mL), and Maisine® oil (5.5 ± 0.67 mg / mL). However, oleic acid was selected as the oil phase, based on the solubility along with good miscibility with the chosen surfactants and co- surfactants. A190 was more soluble in PEG 400 (13.7 ± 2.56 mg / mL), Transcutol® HP (10.5 ± 0.48 mg / mL), and Tween® 80 (8.07 ± 0.53 mg / mL) compared to Labrasol® (6.39 ± 0.41 mg / mL), propylene glycol (5.47 ± 0.20 mg / mL) and Cremophor (6.62 ± 0.40 mg / mL). The surfactant and co-surfactant that demonstrated higher solubility with appropriate miscibility were subsequently selected as components for design of experiment using BBD. Besides, the solubilities of A190 in aqueous medium such as deionized water, PBS, PBS with 0.2% Tween® 80, and PBS with 0.2% SLS were 0.028 ± 0.01 mg / mL, 0.033 ± 0.005 mg / mL, 0.159 ± 0.001 mg / mL, and 0.091 ± 0.001 mg / mL, respectively.

[0132] Following the preliminary study to select oil, surf actant / co- surfactant, a 3-factor, 3-level response surface-based BBD design was run to screen and select the critical process and formulation-related factors that affects the mean droplet size, PDI, zeta potential, and drug content. The details of the experimental run with composition, process parameters, and the corresponding responses obtained are shown in Table 1. The outcomes for each dependent variables were used to determine the model’s best fit, using the corresponding F- and - values. In addition, lack of fit and regression coefficient was calculated, thereby providing insights into the reliability and precision of the selected model. The mean droplet size, PDI, zeta potential, and drug content of the A190 microemulsion ranged from 148 to 686 nm, 0.138 to 0.593, -7.66 to -3.71 mV, and 95.5 to 108.1%, respectively across the different trials, depending on the composition and levels of each factor specified by the design (Table 2). The ANOVA results for the BBD showed that the model for mean droplet size (Yi), PDI (Y2), and drug content (Y4) were significant with the / -'-values of 7.97, 3.36, and 4.68 with p-values of <0.05 each, and R2of 0.950, 0.984, and 0.957, respectively (Table 3). In addition, Yi, Y2, and Y4 were significantly affected by the percent of oil (Xi) as indicated with the p-values <0.01, <0.05, and <0.01, respectively. In addition, mean droplet size was affected by sonication time (Y3) but not by the Smix ratio (Y2). No significant effect of Smix ratio and sonication was observed on PDI. Whereas the model for zeta potential (Y3) was non- significant with F-value of 1.45, p-value of 0.356, and R2of 0.957 (Table 3). 3D response surface plots were used to study the influence of the main and interactive effects of independent variables on mean droplet size, PDI, zeta potential, and drug content as shown in Figures 2A-C, 2D-F, 2G-I, and 2J-K, respectively. When two independent variables were varied in a certain range, one variable was kept constant. The shape of the 3D response plots in Figure 2A, 2D, 2J revealed that the increase in oil concentration in the microemulsion formulation positively affected on the mean droplet size, PDI, and drug content. Besides, the nonsignificant lack of fit values for each of these models also proved that the selected model was significant (Table 3).

[0133] Table 2. Design of experiment using Box-Behnken design (BBD) to study the combined effect of independent variables on mean droplet size, PDI, zeta potential, and drug content.

[0134] Table 3. Statistical ANOVA results of BBD for mean droplet size, PDI, zeta potential, drug content of A 190 ME.

[0135] Notes: The significance of the effect of independent variables on responses: *P<0.05; **P<0.01

[0136] The A190 microemulsion was selected from the prediction profiler of the BBD. Using this profiler, we set the values of independent variables so that the ME formulation would have minimum mean droplet size and PDI, zeta potential near neutral (-10 mV to +10 mV), and drug content close to 100%. The formulation consisted of 7.5% of oil, 40% of Smix (1:1 v / v), and 52.5% of aqueous phase. The placebo ME had a mean droplet size, PDI, and zeta potential of 119.8 + 1.64 nm, 0.131 + 0.007, and -5.92 + 0.87 mV, respectively. The mean droplet size, PDI, and zeta potential of the A 190 loaded ME remained similar even after the incorporation of A190, which indicates that A190 was well distributed in the oil phase (Table 4). The A 190 content in the microemulsion as measured using HPLC showed the drug content of >95%. The surface morphology of A 190 ME as determined by TEM indicated the formation of homogenous droplets with diameters less than xxx nm, consistent with the results obtained with a laser diffraction particle analyzer (Figure 3).

[0137] Microemulsions are thermodynamically stable systems, therefore A 190 microemulsion was studied for freeze-thaw cycle test, centrifugation test, and heating-cooling cycle test. No phase separation, precipitation, creaming, and loss of drug content was observed for A 190 microemulsion, which confirmed the formation of thermodynamically stable microemulsion. Moreover, A 190 microemulsion passed the dispersibility test with no phase separation and precipitation observed in PBS media. Long-term stability of microemulsion is a critical parameter to be considered during formulation development. Therefore, we performed the long-term storage stability of the A190 microemulsion at room temperature (25 + 5°C). Upon 3 months of storage, the drug content of A 190 microemulsion was maintained at >95% and no significant change in the mean droplet size, PDI, zeta potential, and drug content was recorded (Table 4). In addition, the formulation was physically stable, with no signs of phase separation and precipitation.

[0138] Table 4. Mean droplet size, PDI, and zeta potential of vehicle microemulsion and A 190 microemulsion.

[0139] 3.2. In vitro and ex vivo permeability ofA190 microemulsion

[0140] To understand the passive diffusion of free A 190, A 190 dispersion, and A 190 microemulsion through artificial intestinal membrane, we performed in vitro permeability using artificial phospholipid membrane. The Peof A190 dispersion was higher by 1.5-fold compared to free A 190. Moreover, incorporation of A 190 into microemulsion formulation significantly increased the effective permeability of A190 by 8.6- and 13.1-fold compared to A190 dispersion and free A 190, respectively (Table 3). Moreover, the apparent permeability for the A190 microemulsion formulation was 12.2 x 10’6cm / s, which was significantly greater by 2-folds compared to that of A190 dispersion (6.0 x 10’6cm / s) (Table 5). However, free A190 did not permeate through the intestinal membrane (below the limit of quantification, 150 ng / mL).

[0141] Table 5. Effective permeability and apparent permeability of A 190, A 190 dispersion, and A 190 microemulsion.

[0142] Notes: Values are mean + SD (n=9-12). ***P<0.001, compared to A190 in dispersion;

[0143] ###P<0.001, compared to A190 in PBS pH 6.8; *P<0.05, compared to A190 in dispersion.

[0144] 3.3 In vitro cytotoxicity

[0145] To assess the potential cytotoxic effects of free A190, vehicle microemulsion, and A190 microemulsion, an MTT assay using Caco-2 and HepG2 cell lines was performed (Figure 4). No significant cytotoxicity was observed for free A190 in 0.2% DMSO, even at the highest concentration of <50 pM in both cell lines at 24 h (Figure 4 A-B) and 48 h (Figure 4). The biocompatibility of the prepared microemulsion was evaluated by assessing the cytotoxicity of vehicle alone (no drug). The vehicle microemulsion had no significant influence on the cell viability (>80%) of Caco-2 and HepG2 cells within the concentration range of 0-5 pM, indicating less toxicity and biocompatibility of the materials used, as shown in earlier studies39. In each cell line, A 190 microemulsion maintained cell viability of more than 80% at a concentration below 5 pM (Figure 4). However, concentration-dependent toxicity was observed at a higher concentration.

[0146] 3.4 In vitro dissolution study

[0147] To verify the self-emulsification property of A 190 microemulsion, we performed in vitro dissolution study of A190 dispersion and microemulsion in 0.1 N HC1 solution (pH 1.2) or phosphate buffer (pH 6.8) (Figure 5). In a dissolution medium at pH 1.2, approximately 68% of A 190 was released from the microemulsion within 120 minutes, while the dispersion formulation showed less than 3% dissolution. From the observation (Figure 5), it is clear that the drug release was faster at pH 6.8 compared to pH 1.2, highlighting the impact of pH on the release profile of the drug. At pH 6.8, only 5.5% of A190 was dissolved from the dispersion, compared to >90% released from the microemulsion at 120 minutes. These finding indicates that the microemulsion formulation released majority of the drug within 120 minutes, significantly enhancing the dissolution rate of the highly lipophilic A190. This is because the A 190 microemulsion facilitated successful self-emulsification in the dispersion medium, preventing phase separation or drug precipitation.

[0148] 3.5. Pharmacokinetics study

[0149] An in vivo absorption study was conducted to assess whether the improved solubility and enhanced permeability of A 190 could enhance its oral bioavailability. Figure 6 shows the plasma concentration profiles of A 190 after oral administration of A 190 dispersion and A 190 microemulsion to rats. The pharmacokinetic parameters are summarized in Table 6. The AUC of orally administered A190 microemulsion was 9980.9 + 2621.0 ng / ml*h, which was significantly higher by 4.7-fold compared with that of A190 dispersion (47434.9 + 4824.9 ng / ml*h). The Cmaxwas also increased by 5.06-fold in A 190 microemulsion compared to that of orally administered A190 dispersion (2024.8 + 527.9 ng / ml vs 400.8 + 105.7. ng / ml). Both the formulations demonstrated a delayed Tmaxof >24 h. In addition, the oral bioavailability of A 190 from the microemulsion was 4.9-fold higher compared to orally administered A 190 dispersion. No difference in the ti / 2 was observed between A190 microemulsion and A190 dispersion. These findings indicated that the oral bioavailability of the hydrophobic drug could be substantially enhanced by using the oil-in-water microemulsion formulation strategy.

[0150] Table 6. Plasma pharmacokinetic parameters of A 190 following oral administration of A 190 dispersion and A190 microemulsion to rats (mean + SD, n = 4-5).

[0151] AUCinf, area under the curve from time zero to infinity; AUCo >t, area under the curve from time zero to the last sampling time point; Cmax, maximum observed concentration; ti / 2, half-life.

[0152] 3.6 The Effects ofA190 microemulsion in the CIPN model

[0153] A 190 microemulsion was tested at a dose of 10 and 20 mg / kg in the CIPN model. As reported in Figure 7, A190 microemulsion reduced CIPN-induced mechanical hypersensitivity as shown in a 2-way repeated ANOVA analysis with F(treatment) (4, 35) = 71.46; P <0.0001), F(time (4.844, 169.5) = 29.11; P <0.0001) and (Ftreatment X time (28, 245) = 4.343; P <0.0001), where F(treatment) denotes the dose of A190 microemulsion administered. Post-hoc analysis (Tukey) revealed that the dose of 20 mg / kg partially reversed CIPN induced mechanical hypersensitivity 6-72 h after administration, while the lowest dose 10 mg / kg of A190 microemulsion reversed partially CIPN-induced mechanical hypersensitivity from 24-48 h after administration. The effect of A 190 microemulsion at 10 mg / kg dissipated 48 h after administration (Figure 7A). In vehicle-treated mice 20 mg / kg (no PAC), A190 did not alter von Frey responses. In addition, an ordinary one-way ANOVA analysis showed that A 190 microemulsion did not significantly impact paw withdraw thresholds (F (5,42) = 28.75; P <0.0001) (Figure 7B), with mice pretreated with a known PPAR-a inhibitor (MK886), compared to their vehicle group. Finally, at the highest doses tested, A 190 (20 mg / kg) did not significantly alter locomotor activity compared to vehicle-treated animals (F (3, 28) = 0.6416; P=0.5947) (Figure 7C).

[0154] 3.7 The activity of Al 90 in the CFA-induced inflammatory pain model

[0155] A 190 microemulsion was tested at 20 mg / kg in the CFA model. As reported in Figure 8, A 190 microemulsion reduced CFA-induced mechanical hypersensitivity as shown in a 2- way repeated ANOVA analysis with F(treatment) (3, 28) = 62.63; P <0.0001), F(time (5, 145) = 9.736; P <0.0001 ) and F(treatment) X time (21, 196) = 4.286; P <0.0001), where F(treatment) denotes the dose of A 190 microemulsion administered. Post-hoc analysis (Tukey) revealed that the dose of 20 mg / kg partially reversed CFA mechanical hypersensitivity 6 h and fully at 48 h after injection. The effect of A 190 microemulsion dissipated 72 h after administration (Figure 8A). In vehicle-treated mice 20 mg / kg, A190 did not alter von Frey responses. In addition, A 190 microemulsion significantly reduced paw edema (F (3, 28) = 81.06; P <0.0001, Figure 8B), with mice treated with 20 mg / kg dose differing from the vehicle group.

[0156] 3.8 Histological evaluation

[0157] The safety and tolerability of oral formulations are critical for ensuring minimal systemic toxicity, maintaining intestinal integrity, and achieving effective bioavailability of the drugs. The histopathological evaluations of various tissues such as liver, heart, spleen, kidney, duodenum, jejunum, and ileum showed no evidence of toxic effects after 3 days administration of vehicle microemulsion and A 190 microemulsion (Figure 9). The hepatic lobules appeared uninjured, with no signs of inflammation or pathological alterations. The myocardium showed no evidence of fibrosis, and the cardiac myocytes displayed the typical cytoarchitecture without any signs of hypertrophy. The spleen tissues exhibited normal architecture, with clearly defined red and white pulp. Renal tissues displayed intact glomerular and tubular structures, with no evidence of sclerosis or acute necrosis. In addition, duodenum, jejunum, and ileum showed no clinically significant histological or morphological alterations at the administered dose.

[0158] 4. Discussion

[0159] In the present study, we developed an A190 loaded microemulsion with drug dissolved in oil phase, which acts as a drug reservoir and prolongs drug release. All excipients used in the microemulsion formulation were classified as ‘generally recognized as safe’ (GRAS). The aqueous solubility of A190 was <30 pg / mL at 25°C (Figure 1), and thus, the formulation of a stable A 190 microemulsion system required the appropriate selection of oil, surfactant, and co-surfactant. The critical criteria for selection of oil are the solubility of the drug along with miscibility with the surfactant and co-surfactant. Whereas surfactant and co-surfactants are screened and selected based on their solubilizing capacity and the ability to stabilize the microemulsion. The solubility of A 190 in oleic acid was lower than that of Capryol® 90 (Figure 1). However, oleic acid was selected as the oil phase due to its amphiphilic nature, which contributes to the reduction of the interfacial tension, facilitating the formation of stable microemulsion. Tween® 80 was select as surfactant because it is minimally impacted by the changes in pH and ionic strength. PEG 400, when used as a co- surfactant, can serve as a bridge between the hydrophobic and hydrophilic phases in the microemulsion system. Previous studies indicated that incorporating PEG components into the microemulsion system enhances the diffusivity of the formulation. Moreover, combining surfactant with PEG cosolvents help minimize the risk of drug precipitation during dilution both in vitro or in vivo assessments.

[0160] To investigate the effects of independent variables along with sonication time on the droplet size, PDI, zeta potential, and drug content of the A 190 microemulsion, we employed a 3-factor, 3-level (33) BBD with 15 experimental runs (Table 1). As shown in Figure 2A and 2D, an increase in oil concentration resulted in increase in droplet size and PDI respectively. In addition, according to results of statistical analysis, oil concentration significantly affected the mean droplet size ( ><0.01 ; F = 48.7) and PDI (£<0.05; F = 14.9) (Table 3). The increase in mean droplet size with increased oil concentration indicates that the interfacial tension is not sufficiently reduced due to limited adsorption of the surfactant molecules on the oil surface. Additionally, insufficient coverage of oil surface by surfactant molecules leads to oil droplet convergence, thereby increasing coalescence, phase separation, and Ostwald ripening. This is also a contributing factor to the increased PDI in a microemulsion, indicating a broader size distribution. When altering the ratio of surfactant to co-surfactant, an initial decrease in surfactant concentration leads to an increase in the mean droplet size of the microemulsion due to less surfactant available to adequately reduce the interfacial tension and stabilize the droplets. However, as the surfactant concentration continues to decrease and the co-surfactant concentration increases, the mean droplet size and PDI eventually shows a non-significant decrease, likely because the co-surfactant begins to aid in reducing the interfacial tension and stabilizing the droplets, albeit not as effectively as the primary surfactant. Thus, the co-surfactant partially compensates for the reduced surfactant and improves the packing of the oil-water interface, leading to a slight and non-significant reduction in droplet size and PDI.

[0161] In Figure 2B, increase in the sonication time decreased the mean droplet size and PDI which suggest the effect of shear disruptive forces generated by the ultrasound. Initially, the larger droplets are disrupted, allowing them to coalesce until their surfaces are adequately covered by surfactant molecules for stabilization, with no remaining molecules to form micelles. All the tested microemulsions exerted a negative zeta potential ranging from -3.71 mV to -7.66 mV and none of the independent variables significantly affected the zeta potential of A 190 microemulsion (Figure 2G-I). The results are in agreement with previous studies, which also showed negative zeta potential values for similar microemulsions. The negative charge was because the surfactant system in the microemulsion formulation remained unchanged and the density of the charged species on the droplet surface stayed constant. As shown in Figure 2J, increasing the oil concentration in the microemulsion system enhanced the drug loading capacity by improving drug solubilization, facilitating greater drug partitioning into the oil phase, and expanding the oil droplet surface area, which accommodates more drug molecules. In this study, no substantial impact of Smix ratio and sonication time on drug content was observed (Figure 2K and 2L).

[0162] The prediction profiler indicated that the formulation and process parameters for A 190 microemulsion were: 7.5% of oleic acid, Tween® 80:PEG 400 ratio of 1:1 v / v, and a sonication time of 1.5 min. A190 microemulsions formulated using a high energy ultrasonication method.. The A 190 microemulsion showed a monomodal distribution with a low polydispersity index (<0.25) and a mean droplet size of <200 nm with a negative surface charge. Still, the larger average droplet size of A 190 microemulsion may be due to the presence of oleic acid and Tween® 80 as the oil and surfactant, respectively, both of which have bulkier side chains. The low surface charge values imply that A 190 microemulsion is primarily stabilized by steric hinderance rather than electrostatic interaction, due to the nonionic PEO blocks of Tween® 80 at the surface of oil droplets. TEM analysis to investigate the microscopic morphology of A 190 microemulsion revealed spherical structures (Figure 3). These spherical structures might improve cellular uptake, thereby improving the drug delivery efficiency.

[0163] Microemulsions being a thermodynamically stable system capable of effectively delivering both hydrophilic and lipophilic drugs via oral routes, the thermodynamic stability of A 190 microemulsion was evaluated. The A 190 microemulsion formulation was physically stable and remained translucent without any signs of creaming, cracking, precipitation, or phase separation. The formation of this monophasic and translucent formulation is attributed to the nonionic surfactant / co-surfactant, which lowered the interfacial tension between oil and aqueous phases. The centrifugation test, heating-cooling cycle, and freeze-thaw cycles had no significant impact on the droplet size and PDI of the microemulsions. Upon storage for three months at room temperature, there were no significant changes in the mean droplet size, PDI, and zeta potential of the microemulsion (data not shown). This indicates that the A 190 microemulsion exhibited good physical stability during long-term storage at 25 °C. This aligns with previous findings that microemulsions with smaller mean droplet sizes, and those that do not undergo flocculation, coalescence, Ostwald ripening, or gravitational separation, tend to be physically stable.

[0164] Assessing intestinal membrane permeability is crucial when developing strategies to enhance oral bioavailability of drugs, which necessitates a deeper understanding of the fundamental mechanism involved in drug translocation across the intestinal membranes.67In this study, we utilized PAMPA, non-everted rat sac model, and pharmacokinetic analysis in rats as in vitro, ex vivo, and in vivo methods, respectively, to investigate whether the permeability of a hydrophobic drug like A 190 could be improved using oil / water (o / w) microemulsion as a drug delivery system. The free A190 (dispersed in PBS pH 6.8) exhibited low permeability compared to that of A190 dispersion. This characteristic is due to the slightly increased solubilization, and improved lipid membrane fluidity caused by PEG 400 and HP-P CD. Moreover, the artificial intestinal permeability and ex vivo study showed significant increase in the permeability of A 190 microemulsion compared to that of free A 190 and A 190 dispersion. The improved intestinal permeability can be explained in several ways: (1) the A 190 microemulsion system enhances solubilization compared to 10% PEG 400 + 5% DMSO + 85% of 10% HP-P CD mixture and PBS pH 6.8; (2) the nano-size of microemulsion improves absorption; (3) the increases surface area of the droplets enhanced contact with the biological membrane, and (4) the absorption-enhancing effects of nonionic surfactants like Tween® 80 and PEG 400 in the microemulsion system improves permeability by disrupting tight junctions at the molecular level and altering the fluidity in the intestinal membrane.

[0165] In this study, we investigated the cytotoxic effects of free A 190, vehicle microemulsion, and A 190 microemulsion on Caco-2 and HepG2 cell lines across concentration ranges from 0.1 pM to 100 pM. Free A190 exhibited low toxicity (with cell viability remaining above 80% at concentrations of 50 pM or lower) when compared to the vehicle and A190 microemulsion (Figure 4). This lower cytotoxicity could be attributed to the safety of the drug molecule or the formation of drug aggregates in the cell culture medium due to A190's high hydrophobicity, which may prevent its adsorption onto the cell lines. Moreover, both Tween® 80 and PEG 400 are known to exhibit low cytotoxicity, maintaining high cell viability at lower concentrations.

[0166] The inadequate aqueous solubility of the drug presents a significant challenge in designing effective oral drug delivery system. For oral bioavailability, the drug should dissolve in gastrointestinal (GI) fluids prior to absorption into the systemic circulation. The results from the in vitro dissolution study indicated the potential of microemulsion to improve the solubility of A 190 (Figure 5), which might be one of the reasons for enhanced oral bioavailability of A 190 microemulsion. The A 190 microemulsion released the majority of the drug within 120 minutes in both pH 1.2 and pH 6.8 media. The rapid release is attributed to the smaller droplet size of the microemulsion and its spontaneous emulsification upon contact with aqueous media, facilitated by gentle agitation, a process that mimics GI motility. Similar findings have been documented in previous studies, which indicated that the formulations with smaller particle sizes exhibit faster drug release rates.

[0167] The o / w microemulsion showed promises as an oral delivery vehicle for hydrophobic drugs like A 190, as it significantly enhanced the oral bioavailability. The higher values of Cmax and AUCiast when administering A190 microemulsion compared to the aqueous dispersion is indicative of the substantial enhancement in the intestinal absorption of A 190 microemulsion, resulting in a 4.9-fold increase in AUCiast without altering its elimination kinetics. This is attributed to the improved solubility of A190 in the microemulsion, and opening of tight junctions by surfactant / cosurfactant, facilitating paracellular transport, compared to A 190 dispersion, which precipitated in the artificial membrane or intestinal membrane.73In the apical side of small intestine, oil / surfactants stimulate the secretion of pancreatic lipase, bile salts, and bile lipids, which adhere to the surface of microemulsion forming a more stable emulsion with smaller droplet size, that is further hydrolyzed into free fatty acids and monoacylglycerols. These lipid digestion products allow higher solubilization of A 190 and may further adhere to gut membrane that passively diffuse across the intestinal epithelial layer, thereby extending GI residence time. The elimination half-lives of A 190 dispersion and A 190 microemulsion were 14.5 h and 15.6 h, respectively, with corresponding Tmax of 27.6 h and 24 h. The multiple peaking in pharmacokinetic profiles can speculated to biliary secretion followed with higher enterohepatic recycling efficiency of the drug, reducing the clearance and naturally delaying the time to reach maximum concentration. The secondary absorption peak suggests higher rate of absorption at the later stage that might be due to dissociation of micelles, bile acids and active reabsorption. In order to define the contribution of biliary secretion on clearance and enterohepatic recycling, further in vivo experiments with animal models such as bile duct-ligated both in fed and fasted state need to be performed. Besides, surfactant-stabilized and well-characterized microemulsions owing to their nano size may also directly diffuse across the intestinal membrane or be absorbed into enterocytes through clathrin- or caveola-mediated endocytosis and micropinocytosis. As a result, the oral bioavailability of A 190 was significantly improved when incorporated into the o / w microemulsion compared to A 190 dispersion, with the relative bioavailability of A 190 microemulsion increasing by 4.9-fold compared to A 190 dispersion.

[0168] Chemotherapy drugs like paclitaxel are associated with peripheral neurotoxicity, particularly affecting peripheral nerves, which poses a significant limitation to their use at high or cumulative doses. The toxicity is exacerbated when paclitaxel is co-administered with other neurotoxic antineoplastic drugs, such as cisplatin, potentially leading to dose-limiting peripheral neuropathy and compromising the overall efficacy of the treatment regimen.75It has been reported that PPARa plays an important role in the regulation of neuropathic pain.76Our group showed an increase in the expression of PPAR-a in dorsal root ganglion (DRG) at day 7 in a CIPN model. However, the early increase in PPAR-a mRNA expression does not seem adequate to overcome the potential paclitaxel toxicity and alleviate neuropathic pain. Therefore, to effectively manage the inflammation, sustaining elevated PPAR-a levels over time may be crucial, suggesting that PPAR-a agonists could represent a promising therapeutic strategy.

[0169] In our previous studies, we observed that fenofibrate partially reversed paclitaxel-induced mechanical hypersensitivity and fully reversed cold hypersensitivity, but only when administered intraperitoneally. This might be due to the limited intestinal absorption and oral bioavailability of fenofibrate. When fenofibric acid, the active metabolite of fenofibrate, was administered, it similarly alleviated hypersensitivity when given either intraperitoneally or orally, although its low aqueous solubility required the use of a vehicle like sesame oil. Additionally, choline-fenofibrate, a water-soluble salt form, proved more effective in reversing both types of hypersensitivity via oral administration without signs of tolerance in animals. Based on these results, the ability of fibrates to reverse paclitaxel-induced mechanical and cold hypersensitivity appears to be PPAR-a-dependent, as the use of a PPAR-a antagonist completely blocked the effects of these fibrates.

[0170] In this study, we tested the effect of a new PPAR-a agonist, A 190 with enhanced potency and selectivity compared to fenofibric acid. A 190 microemulsion at 20 mg / kg given orally fully reversed paclitaxel-induced mechanical hypersensitivity, while fenofibric acid given orally at 90 mg / kg partially blocked it. The results showed a full reduction in mechanical hypersensitivity induced by CIPN and CFA with a single oral administration of A 190 microemulsion. Furthermore, the onset of mechanical hypersensitivity observed between 6 to 72 h after paclitaxel injection aligns with the pharmacokinetic data, which shows that paclitaxel reaches its peak plasma concentration at approximately 24 hours. In order to determine whether the effect of A 190 microemulsion in the CIPN model is mediated through activation of PPAR-a, we used a specific PPAR-a antagonist coupled with A 190 treatment. The PPAR-a antagonist completely blocked the effect of the A 190 microemulsion, suggesting that the effect of the drug is PPAR-a-dependent. The results from this study indicate that A 190 microemulsion increases the oral bioavailability of this highly lipophilic drug and reduces CIPN- and CFA-induced mechanical hypersensitivity. The histopathological analysis of collected tissues following repeated oral administration of both vehicle microemulsion and A 190 microemulsion in rats revealed no signs of toxicity, indicating that the oral delivery of A 190 loaded microemulsion is safe.

[0171] 5. Conclusion

[0172] In summary, we demonstrated that microemulsion formulation increased both the solubility and permeability of hydrophobic A 190. This led to significant increase in the oral bioavailability in vivo. In addition, orally administered A190 microemulsion reduced CIPN- and CFA-induced mechanical hypersensitivity, which is mediated by PPARa. Thus, the A 190 microemulsion is safe and effective for the management of chemotherapy induced neuropathic pain or chronic inflammatory pain. While the invention has been described in terms of its several exemplary embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

CLAIMSWe claim:

1. A microemulsion comprising5.0 to 30.0% of an oil phase comprising one or more oils, 20.0 to 75.0% an aqueous phase,20 to 50% of a mixture of a surfactant and a cosurfactant, and a hydrophobic drug solubilized in the oil phase.

2. The microemulsion of claim 1, wherein the oil phase comprises one or more of soybean oil, cottonseed oil, com oil, acaprylic / capric triglyceride, a medium-chain mono and / or di-glyceride, a long-chain mono glyceride, a fatty acid (e.g., oleic acid, caprylic acid), a fatty acid ester, linseed oil, sunflower oil, fish oil, avocado oil or coconut oil.

3. The microemulsion of claim 1 or 2, wherein the oil comprises one or more of oleic acid, caprylic acid and propylene glycol monocaprylate type II.

4. The microemulsion of any of claims 1-3, wherein the aqueous phase comprises one or more of water, one or more biocompatible salts and, optionally, one or more biocompatible buffering agents.

5. The microemulsion of any of claims 1-4, wherein the mixture of a surfactant and a cosurfactant comprises one or more nonionic, cationic, or anionic surfactants.

6. The microemulsion of any of claims 1-5, wherein the mixture of a surfactant and a cosurfactant comprises one or more of: polyoxyethylene sorbitan monooleate, polyethylene glycol 400, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, (polyethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)), polyoxyl 35 castor oil, polyethoxylated castor oil, PEG-35 castor oil, polyethoxylated alcohol, polyoxyl lauryl ether, polyoxyethylated castor oil, lecithin, polyethylene glycol, a glycerol ester of a fatty acid, a sugar ester, glyceryl monocaprylate, caprylocaproyl macrogol-8 glyceride, alauroyl polyoxyl-6 glyceride, a lauroyl macrogol-6 glyceride, caprylic / capric triglyceride, glyceryl trioctanoate / tricaprate, or a mixture thereof.

7. The microemulsion of any of claims 1-6, wherein the hydrophobic drug is 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid, the oil phase comprises oleic acid, the surfactant is polyoxyethylene sorbitan monooleate, the cosurfactant is polyethylene glycol having an average molecular weight of 400 and the aqueous phase is phosphate buffered saline.

8. The microemulsion of any of claims 1-7, wherein the microemulsion comprises7.5% oleic acid,52.5% phosphate buffered saline,40% of a 1:1, v:v ratio mixture of polyoxyethylene sorbitan monooleate and polyethylene glycol having an average molecular weight of 400, and7.5 mg of 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid.

9. The microemulsion of any of claims 1-8, further comprising a stabilizer.

10. The microemulsion of claim 9, wherein the stabilizer is selected from the group consisting of: an antioxidant, a preservative, a buffering agent, an organic buffer, a polymer, a sugar and a sugar alcohol.

11. The microemulsion of any of claims 1-10, wherein the microemulsion has a droplet size of 100 nm and a drug loading efficiency of 95% or more.

12. A method of preventing and / or treating pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the microemulsion of any of claims 1-11.

13. The method of claim 12, wherein the pain is peripheral pain, neuropathic pain and / or inflammatory pain.

14. The method of claim 12, wherein the neuropathic pain is chemotherapy-induced peripheral neuropathy (CIPN) pain.

15. The method of claim 12, wherein the pain is inflammatory pain.

16. The method of claim 12, wherein the pain is chronic pain.

17. A method of activating PPARa in a cell, comprising contacting the cell with the microemulsion of any of claims 1-11 under conditions which allow the 3-((4-((4-fhiorobenzyl)oxy)-3-methylbenzyl)amino) benzoic acid to enter to the cell and bind to PPARa.

18. The method of claim 17, wherein the cell is in vitro or in vivo.

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