PREPARATION THEREOF

TECHNICAL FIELD

[01] The present disclosure relates to pharmaceutical compositions for use as analgesic, anti-inflammatory, and/or antipyretic agents and methods of preparation thereof, and more particularly to a pharmaceutical composition that includes chitosan and gold nanoparticles as carriers to one or more non-steroidal anti-inflammatory drug (“NS AID”), and a method of preparation thereof.

BACKGROUND

[02] NS AIDs are frequently used as a therapy for relieving pain and inflammation.

Despite of NSAIDs effectiveness, they are associated with a significant risk of serious gastrointestinal adverse events especially upon its use for prolonged periods. This imposed the need of developing a potent analgesic and anti-inflammatory composition that requires a low dose of NSAIDs while having a proper pharmacological action, and at the same time possessing fewer side effects.

[03] There are different conventional approaches that increase NS AID potency and enhance its onset of action, namely, the stereochemistry approach, the formulation approach, and changing the route of administration approach.

[04] Chitosan is composed of randomly distributed D-glucosamines and N- acetylated D-glucosamines linked via beta-(l-4) glycosidic bonds. It has different pharmacological properties, including but not limited to, anti-inflammatory activity. It is reported in the art that the anti-inflammatory action of chitosan could be optimum upon using low molecular weight chitosan saccharide. The oral administration of low molecular weight chitosan saccharide would be effective in alleviating the allergic inflammation in vivo. Other studies conducted in the art examined the anti inflammatory effects low molecular weight chitosan saccharide with two different ranges of molecular weights, i.e. from about 10 kDa to about 20 kDa, and from about 1 kDa to about 3 kDa, topically on skin. Based on the results of these studies, both molecular weights were considered anti-inflammatory agents that can be applied topically. Analgesic effect of chitosan was also reported using mice model. The main analgesic effect of chitosan could be due to the ability of chitosan to absorb proton ions released in the inflammatory site. Chitosan properties were found to facilitate the development of a reduced-dose fast-release solid oral dosage form of NS AID such as ibuprofen and naproxen.

[05] Gold and gold derivatives were used in the art for the treatment of rheumatoid arthritis. Nanotechnology introduced colloidal gold nanoparticles, which were reported to have anti-inflammatory action. Colloidal gold nanoparticles have a potential to be used as a drug carrier for local delivery. It is also reported in the art that a co administration of colloidal gold with proteins which enabled the percutaneous absorption of protein drugs. This result indicated that colloidal gold nanoparticles may act as a skin penetration enhancer. Although colloidal gold nanoparticles may act as a skin penetration enhancer, the colloidal gold itself do not deeply penetrate the human skin. Studies conducted in the art investigated the colloidal gold nanoparticles ability to penetrate the skin and explored its metabolic effects using different sizes ranging from about 10 nm to about 60 nm on viable excised human skin after a twenty four- hour exposure using multiphoton tomograph-fluorescence lifetime imaging microscopy. The results of such studies indicated that there were no significant deep penetrations of gold nanoparticles below the stratum comeum. Furthermore, there were no changes in metabolic output in the viable epidermis. The results may indicate the safety of using colloidal gold nanoparticles as a skin penetration enhancer since the viable human skin resists the deep permeation of small gold particles.

[06] It is also reported in the art that the incorporation of chitosan during the synthesis of gold nanoparticles offers better penetration and uptake of therapeutic agents across the mucosal membrane, and chitosan itself acts as a reducing agent during gold nanoparticle synthesis. However, the state-of-the-art reported chitosan reduced gold nanoparticles as carriers for the delivery of anti-cancer drugs or hormones such as insulin and erythropoietin. Nevertheless, there is still a need for a composition that uses chitosan reduced gold nanoparticles as carriers for NSAIDs.

SUMMARY

[07] Aspects of the present disclosure provide a pharmaceutical composition including positively charged dispersion containing low molecular weight chitosan saccharide and colloidal gold nano-particles, at least one non-steroidal anti inflammatory drug loaded on the positively charged dispersion, and a solvent.

[08] In some aspects, the low molecular weight chitosan saccharide may have an average molecular weight of about 1 kDa to about 40 kDa.

[09] In other aspects, the low molecular weight chitosan saccharide may have an average molecular weight of about 1 kDa to about 20 kDa.

[010] In yet other aspects, the low molecular weight chitosan saccharide may have an average molecular weight of about 10 kDa to about 17 kDa.

[011] In yet other aspects, the low molecular weight chitosan saccharide may have an average molecular weight of about 12 kDa to about 14 kDa.

[012] In aspects of the present disclosure, the colloidal gold nano-particles in the positively charged dispersion may have a diameter of about 1 to about 40 nm.

[013] In aspects of the present disclosure, the colloidal gold nano-particles in the positively charged dispersion may have a zeta potential of at least about +20mV.

[014] In some aspects, the at least one non-steroidal anti-inflammatory drug may be selected from a group consisting of ibuprofen, aspirin, magnesium salicylates, choline salicylate, celecoxib, diclofenac potassium, diclofenac sodium, diflunisal, etodolac, fenoprofen calcium, flurbiprofen, indomethacin, ketoprofen, meclofenamate sodium, mefenamic acid, meloxicam, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxicam, rofecoxib, salsalate, sodium salicylate, sulindac, tolmetin sodium, and valdecoxib.

[015] In other aspects, the at least one non-steroidal anti-inflammatory drug may include ibuprofen.

[016] The pharmaceutical composition in aspects of the present disclosure may be formulated as a topical formulation.

[017] In aspects of the present disclosure, the pharmaceutical composition may be used as analgesic agent.

[018] In other aspects of the present disclosure, the pharmaceutical composition may be used as anti-inflammatory agent. [019] In yet other aspects of the present disclosure, the pharmaceutical composition may be used as antipyretic agent.

[020] Aspects of the present disclosure further provide a method of preparing a pharmaceutical composition comprising a non-steroidal anti-inflammatory drug loaded on a positively charged dispersion comprising low molecular weight chitosan saccharide and colloidal gold nano-particles, wherein the method comprises the steps of: diluting chloroauric acid with double distilled water then with an aqueous solution of pH about 6.5 and containing low molecular weight chitosan saccharide solution to obtain a first solution; heating the first solution to boiling then leaving it to equilibrate at room temperature to form a positively charged dispersion; and loading a negatively charged salt of the non-steroidal anti-inflammatory drug on the positively charged dispersion by electrostatic attractions.

[021] In aspects of the present disclosure, chitosan at the pH of about 6.5 is soluble due to the protonation of 50% of primary amine functional groups and the other amine functional group will facilitate the reduction of gold salts to gold metallic nanoparticles. This produces positively charged chitosan gold nanoparticles.

[022] In yet some aspects, in the positively charged dispersion 50% of amino groups of chitosan have a positive charge.

[023] In some aspects, chloroauric acid is reacted with low molecular weight chitosan saccharide according to a molar ratio of about 1 :6 to about 1 : 10 based on glucosamine monomer originally present in low molecular weight chitosan saccharide.

BRIEF DESCRIPTION OF THE DRAWINGS

[024] The invention will now be described with reference to the accompanying drawings, which illustrate preferred embodiments of the present disclosure, without however limiting the scope thereof, and in which:

[025] FIG. 1 illustrates a flow chart of a method of preparing a positively charged dispersion used in preparing a pharmaceutical composition for use as analgesic, anti- inflammatory, or antipyretic agent, prepared in accordance with embodiments of the present disclosure.

[026] FIG. 2 illustrates a plot comparing ultraviolet visible spectra of different positively charged dispersion containing low molecular weight chitosan saccharide and colloidal gold nanoparticles configured in accordance with embodiments of the present disclosure, wherein“A” represents a positively charged dispersion including low molecular weight chitosan saccharide and colloidal gold nanoparticles with molar ratio of 1 : 1,“B” represents a positively charged dispersion including low molecular weight chitosan saccharide and colloidal gold nanoparticles with molar ratio of 1 :2,“C” represents a positively charged dispersion including low molecular weight chitosan saccharide and colloidal gold nanoparticles complex with molar ratio of 1 :4, and“D” represents a positively charged dispersion including low molecular weight chitosan saccharide and colloidal gold nanoparticles complex with molar ratio of 1 :6.

[027] FIG. 3 A illustrates a column chart comparing median particle sizes of different positively charged dispersions including low molecular weight chitosan saccharide and colloidal gold nanoparticles prepared in accordance with embodiments of the present disclosure, each positively charged dispersion has a unique molar ratio of chloroauric acid to glucosamine monomer weight in the chitosan saccharide.

[028] FIG. 3B illustrates a column chart comparing zeta potentials of different positively charged dispersions including low molecular weight chitosan saccharide and colloidal gold nanoparticles prepared in accordance with embodiments of the present disclosure, each positively charged dispersion has a unique molar ratio of chloroauric acid to glucosamine monomer weight in the chitosan saccharide.

[029] FIG. 4A illustrates a flow chart of a method of preparing a pharmaceutical composition for use as analgesic, anti-inflammatory, or antipyretic agent, prepared in accordance with embodiments of the present disclosure.

[030] FIG. 4B illustrates a schematic diagram showing the chemical interaction between the negatively charged ibuprofen lysinate loaded on a positively charged dispersion.

[031] FIG. 5 illustrates a plot comparing absorbance and wavelength of negatively charged ibuprofen lysinate loaded on a positively charged dispersion including low molecular weight chitosan saccharide and colloidal gold nanoparticles prepared in accordance with embodiments of the present disclosure.

[032] FIG. 6 illustrates a plot showing the change in particle size and zeta potential of a positively charged dispersion including low molecular weight chitosan saccharide and colloidal gold nanoparticles prepared in accordance with embodiments of the present disclosure, upon loading a negatively charged ibuprofen lysinate to such positively charged dispersion.

[033] FIG. 7 illustrates a plot comparing diffusion rates of free negatively charged ibuprofen lysinate (IBU) and negatively charged ibuprofen lysinate loaded on a positively charged dispersion including low molecular weight chitosan saccharide (LM-COS) and colloidal gold nanoparticles (CGN) prepared in accordance with embodiments of the present disclosure, after application on a mouse skin.

[034] FIG. 8 illustrates a column chart comparing response time to hot plate of mice exposed to water, negatively charged free ibuprofen lysinate, positively charged dispersion prepared in accordance with embodiments of the present disclosure, and negatively charged ibuprofen lysinate loaded on the positively charged dispersion, wherein“IBU” refers to fee negatively charged ibuprofen lysinate,“LM-COS-CGN” refers to the positively charged dispersion, and“LM-COS-CGN-IBU” refers to the negatively charged ibuprofen lysinate loaded on the positively charged dispersion.

[035] FIG. 9 illustrates a column chart comparing percentage of increase in leg volume of mice one hour after injection of mice after injection by carrageenan, and exposure to exposed to water, negatively charged free ibuprofen lysinate, a positively charged dispersion prepared in accordance with embodiments of the present disclosure, and negatively charged ibuprofen lysinate loaded on the positively charged dispersion, wherein“IBU” refers to free negatively charged ibuprofen lysinate,“LM-COS-CGN” refers to the positively charged dispersion, and “LM-COS-CGN-IBU” refers to negatively charged ibuprofen lysinate loaded on the positively charged dispersion.

DETAILED DESCRIPTION

[036] In describing and claiming the present invention, the following terminology will be used. [037] The singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise.

[038] The term“skin” as used in the present disclosure is defined to include human or animal skin, as well as mucosal surfaces that are usually at least partially exposed to air.

[039] As used herein, the term“topical” means in the broadest sense applied outside a body, preferably on skin.

[040] As used in the present disclosure, the term“topical formulation” refers to a formulation that may be applied to skin or a mucosa. Topical formulations may, for example, be used to confer therapeutic benefit to a patient. Topical formulations can be used for both topical and transdermal administration of substances.

[041] The term“topical administration” is used in the present disclosure in its conventional sense to mean delivery of a substance, such as an NSAID, to the skin or a localized region of the body.

[042] The term“transdermal” in this disclosure means in the broadest sense through the skin.

[043] As used herein, the term “transdermal administration” is used to mean administration through the skin. Transdermal administration is often applied where systemic delivery of an active is desired, although it may also be useful for delivering an active to tissues underlying the skin with minimal systemic absorption (i.e. localized delivery).

[044] Embodiments of the present disclosure provide a pharmaceutical composition for use as analgesic, anti-inflammatory, or antipyretic agent, wherein such pharmaceutical composition may include a positively charged dispersion including low molecular weight chitosan saccharide and colloidal gold nanoparticles, at least one NSAID, and a solvent.

[045] In embodiments of the present disclosure, the colloidal gold nanoparticles of the positively charged dispersion may have a diameter of about 1 to about 40 nm.

[046] In embodiments of the present disclosure, the colloidal gold nanoparticles of the positively charged dispersion may have a zeta potential of at least about +20 mV. [047] In embodiments of the present disclosure, the low molecular weight chitosan saccharide of the positively charged dispersion may have an average molecular weight of about 1 kDa to about 40 kDa.

[048] In some embodiments, the low molecular weight chitosan saccharide of the positively charged dispersion may have an average molecular weight of about 1 kDa to about 20 kDa.

[049] In other embodiments, the low molecular weight chitosan saccharide of the positively charged dispersion may have an average molecular weight of about 10 kDa to about 17 kDa.

[050] In yet other embodiments of the present disclosure, the low molecular weight chitosan saccharide of the positively charged dispersion may have an average molecular weight of about 12 kDa to about 14 kDa.

[051] In embodiments of the present disclosure, the at least one NSAID may be selected from a group consisting of Ibuprofen, aspirin, magnesium salicylates, Choline salicylate, celecoxib, diclofenac potassium, diclofenac sodium, diflunisal, etodolac, fenoprofen calcium, flurbiprofen, Ibuprofen, indomethacin, ketoprofen, meclofenamate sodium, mefenamic acid, meloxicam, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxicam, rofecoxib, salsalate, sodium salicylate, sulindac, tolmetin sodium, or valdecoxib.

[052] The colloidal gold nanoparticles of the positively charged dispersion may be able to carry a negatively charged NSAID salt and may act as drug carriers.

[053] In embodiments of the present disclosure, the positively charged dispersion may be prepared by reacting chloroauric acid with chitosan saccharide, wherein the molar ratio of chloroauric acid to glucosamine monomer present in the chitosan saccharide may be from about 1:6 to about 1 : 10.

[054] The positively charged dispersion of the embodiments of the present disclosure may have low particle size of less than 40 nm and positive zeta potential of more than + 20 mV.

[055] In embodiments of the present disclosure, the solvent may include water, or water mixed with different cosolvents or surfactants to enhance permeation, such as, but not limited to ethanol, glycerin, propylene glycol, polyethylene glycol, polysorbates, and sorbitan esters.

[056] The loading of the NSAID on the positively charged dispersion may be based on electrostatic attraction of negatively charged NSAID molecule to the surface of positively charged surface of chitosan gold nanoparticles.

[057] In embodiments of the present invention, the pharmaceutical composition may be a topical formulation.

[058] The topical formulation of the present disclosure may be formulated by those skilled in the art as liquids, solutions, emulsions, creams, lotions, suspensions, triturates, gels, jellies, foams, pastes, ointments, shampoos, adhesives, other conventional patches without a heating component, or the like.

[059] The pharmaceutical composition in embodiments of the present disclosure for use as analgesic, anti-inflammatory, or antipyretic agent.

[060] All components of the pharmaceutical composition have to be pharmaceutically acceptable. The term "pharmaceutically acceptable" means at least non-toxic. The therapeutically active component should preferably be present in the above-mentioned pharmaceutical composition, the concentration of about 0.1 to 99.5% by weight, preferably of about 0.5 to 95% by weight of the total mixture.

[061] The disclosure is now further illustrated on the basis of Examples and a detailed description from which further features and advantages may be taken. It is to be noted that the following explanations are presented for the purpose of illustrating and description only; they are not intended to be exhaustive or to limit the disclosure to the precise form disclosed.

Example 1

Preparation of the positively charged dispersion Preparation of low molecular weight chitosan saccharide [062] The LMWC polymer was prepared by the acid hydrolysis method. Reference is now being made to FIG. 1 A, which illustrates a flowchart of a method of preparing a low molecular weight chitosan saccharide to be used in the composition of the present disclosure. The method may include the following steps: dissolving about 10 g of high molecular weight chitosan (of molecular weight of about 250 kDa) in about 830 mL 0.1 rnol L ^-1 HC1 then adding about 170 mL of concentrated HC1 (37%), leading to a concentration of HC1 equal to 2 mol ^L_1 and a concentration of chitosan equal to 10 g-L ^-1 (process block lA-1). Then, stirring the resultant solution at a speed of about 1000 rpm and maintained under reflux for about 2 hours (process block 1 A-2). Cooling the reaction mixture, and adding about 96% ethanol to precipitate the low molecular weight chitosan hydrochloride salt (process block 1 A-3). After that, Centrifuging and freeze-drying the low molecular weight chitosan hydrochloride obtained the low molecular weight chitosan saccharide (process block 1 A-4).

Preparation of low molecular weight chitosan saccharide and colloidal gold nano-particles

[063] Reference is now being made to FIG. IB, which illustrates a flowchart of a method of preparing a positively charged dispersion of a pharmaceutical composition in accordance with embodiments of the present disclosure, wherein the method may include diluting about 2 ml of 1% chloroauric acid (“HAuCL”) with about 193 ml of double distilled water then about 5ml of aqueous solution of pH about 6.5 containing low molecular weight chitosan saccharide solution to form a first solution (process block IB-1). Then, heating the first solution to boiling for a period of about 15 minutes and leaving it to equilibrate for about 24 hours at room temperature to form a dispersion (process block IB-2). Then, storing the dispersion in a closed container in refrigerator at a temperature of 4 °C till its use (process block IB-3).

[064] Different molar ratios of HAuCL to low molecular weight chitosan saccharide (13 kDa, DDA100%) were used. These molar ratios, based on glucosamine monomer weight in the low molecular weight chitosan saccharide, were 1 : 1, 1 :2, 1 :4, 1 :6, 1 :8, 1 : 10, 1 : 12 and 1 : 14. These ratios formed different colored solutions of colloidal gold nano-particles. The ratios 1 :6-1 : 14 showed clear transparent red to pink colors, indicating a nanoparticle formation and suggesting an optimal addition ratio of low molecular weight chitosan saccharide.

Example 2 Characterization of the positively charged dispersion using ultra-violet visible (“ UV-Vis”) spectrophotometer

[065] Reference in this example is made to FIG. 2. Four liquid samples of about 2 mL each of the positively charged dispersion were placed in a quartz cuvette and scanned by UV-Vis spectrophotometer (UV-1800 UV-Vis spectrophotometer, Shimadzu, Japan). The samples had the molar ratios of HAuCU to low molecular weight chitosan saccharide (based on glucosamine monomer weight present in low molecular weight chitosan saccharide) 1 : 1, 1 :2, 1 :4, and 1 :6.

[066] The spectrophotometry results are shown in FIG. 2. As indicated in FIG. 2, a first sharp peak was obtained for the ratio 1 :6, which indicates optimal interactions between the formed colloidal gold nanoparticles and low molecular weight chitosan saccharide.

Example 3

Characterization of the positively charged dispersion in terms of zeta potential and particle size

[067] In this example, reference will be made to FIGS. 3A-3B.

[068] The particle size and zeta potential and were measured for all molar ratios prepared in Example 1 using Malvern Zetasizer Nano-ZS series instrument (Malvern Instruments, U.K.).

[069] The median particle size of the positively charged dispersions prepared in accordance with Example 1 are illustrated in FIG. 3 A, and the zeta potential of the positively charged dispersions prepared in accordance with Example 1 are illustrated in FIG. 3B.

[070] As depicted form FIGS. 3A-3B, increasing the amount of the low molecular weight chitosan saccharide in the positively charged dispersion resulted in a decrease in particle size and an increase in zeta potential in the beginning, then an increase in particle size and a decrease in zeta potential occurred.

[071] The presence of high positive charge on the positively charged dispersion may have many advantages, as the low molecular weight chitosan saccharide’s positive charge may provide a high mucoadhesion to mucosal surfaces, cornea, and tight junction. Also, the positive charge present on a nanoparticle system may improve skin attachment. Nanoparticles with positive zeta potential may have the ability to be stored in uppermost skin layers, providing the tank effect of the skin. Therefore, the positively charged dispersion of the present disclosure may serve as a tool for carrying drugs deeply to skin layer, then providing drugs in a sustained release manner without gold self-penetration due to the high positive charge on gold nanoparticles surface.

Example 4

Preparation of the pharmaceutical composition

[072] In this example, reference will be made to FIGS. 4-5.

[073] FIG. 4 A illustrates a flowchart of a method for preparing the pharmaceutical composition in accordance with embodiments of the present disclosure, wherein the method may include diluting about 2 ml of 1% chloroauric acid (“HAuCU”) with about 193 ml of double distilled water then about 5ml of aqueous solution of pH about 6.5 containing low molecular weight chitosan saccharide solution to form a first solution (process block 4-1). Then, heating the first solution to boiling for a period of about 15 minutes and leaving it to equilibrate for about 24 hours at room temperature to form a positively charged dispersion (process block 4-2). After that, loading negatively charged ibuprofen lysinate on the positively charged dispersion through electrostatic interactions between negatively charged ibuprofen lysinate and the positively charged dispersion (process block 4-3).

[074] FIG. 4B illustrates a schematic diagram showing the ionic chemical interaction between the illustrates a schematic diagram showing the chemical interaction between the negatively charged ibuprofen lysinate loaded on a positively charged dispersion.

[075] Different concentrations of negatively charged ibuprofen lysinate were added to constant amount of the positively charged dispersion (of molar ratio 1 :6 prepared in the previous examples).

[076] Eight liquid samples of the positively charged dispersion, of 2 mL volume each, were placed in quartz cuvette and scanned by UV-Vis spectrophotometer (UV-1800 UV-Vis spectrophotometer, Shimadzu, Japan) at a wavelength of about 200 nm to about 800 nm. Then, negatively charged ibuprofen lysinate of different concentrations was added to the eight samples. The spectrophotometric results are shown in FIG. 5. As depicted from FIG. 5, there is a shift in wavelength upon addition of negatively charged ibuprofen lysinate to the samples accompanied with a change in the absorbance. This change may indicate an interaction between the negatively charged ibuprofen lysinate and the positively charged dispersion surface, wherein the optimal or maximum interaction could be obtained at a concentration of negatively charged ibuprofen lysinate of about 4 mg/mL since after that concentration, a decrease in absorbance happened.

Example 5

Particle size and zeta potential of pharmaceutical composition

[077] Reference in this example will be made to FIG. 6.

[078] Negatively charged ibuprofen lysinate was loaded on the positively charged dispersion as described in Example 4 to produce the pharmaceutical composition. Eight liquid samples of the pharmaceutical composition were placed in quartz cuvette. Particle size and zeta potential were measured using Malvern Zetasizer Nano-ZS series instrument (Malvern Instruments, U.K.).

[079] FIG. 6 demonstrates the accumulation of negatively charged ibuprofen lysinate on the surface of the positively charged dispersion till the addition of 4 mg/mL negatively charged ibuprofen lysinate, then a rapid increase in particle size happened, which suggests ionic interaction with the positively charged dispersion and the negatively charged ibuprofen leading to aggregation. The addition of the negatively charged ibuprofen lysinate was found to decrease the zeta potential of the positively charged dispersion, which provides another indication of surface interaction. However, at 4 mg/mL, the zeta potential became below + 20 mV, which encouraged nanoparticle aggregation. The preferred concentration of ibuprofen would be equal or less than 4 mg/ml.

Example 6

Skin diffusion study

[080] In this example, reference will be made to FIG. 7. [081] vitro Franz diffusion study was selected to conduct the skin diffusion study. Samples of the pharmaceutical composition composed of 10 mL of the positively charged dispersion and 1.3 mg/mL negatively charged ibuprofen lysinate were prepared and placed in a donor compartment. A reference negatively charged ibuprofen lysinate aqueous solution (of concentration 1.3 mg/mL) in 10 mL distilled water was used for comparison. Healthy albino mice skin was selected from dorsal region was used as a membrane of diffusion. The temperature of the Franz cells was kept at about 32 °C during all over the study period. About 27mL of sodium phosphate buffer of pH 7.4 was added to the acceptor compartment all over the study period. The pharmaceutical composition was exposed to a constant area of diffusion i.e. 4.37 cm ² . Samples of 1 mL were withdrawn at specific time interval and replaced with other 1 mL of phosphate buffer to keep the acceptor compartment volume constant.

[082] The samples were analyzed using high performance liquid chromatography (“HPLC”) (Prominence HPLC, Shimadzu, Japan). The mobile phase contains water to acetonitrile with a volume ratio of about 40 to about 60 and pH was adjusted to about 2.4 using phosphoric acid. The column used was octadecylsilane, 5 um and 4.6x150 mm. Injection Loop: 50 mL. Flow rate was about 1 mL/min. The wavelength was about 220 nm.

[083] FIG. 7 shows the diffusion behavior of negatively charged ibuprofen lysinate attached to the positively charged dispersion compared to the reference negatively charged ibuprofen lysinate aqueous solution through mouse skin. As indicated in FIG. 6, negatively charged ibuprofen lysinate was diffused rapidly through its attachment to the positively charged dispersion compared to the reference negatively charged ibuprofen lysinate aqueous solution, which showed very slow diffusion behavior.

Example 7

Transdermal analgesic and anti-inflammatory activities

[084] In this example, reference will be made to FIGS. 8 and 9.

Animals

[085] In this example, adult male mice, weighing about 20 g to about 25 g each, were used. The mice were housed in standard environmental conditions in an experimental animal room and fed laboratory diet ad libitum with free access to water and were kept for seven days with about twelve-hour light/dark cycle. The study followed the United States National Institutes of Health Guidelines for Care and Use of Laboratory Animals in Biomedical Research.

Analgesic activity using Hot plate test

[086] Eddy’s hot plate test, a test the pain response in animals, was performed at a fixed temperature of about 55 ±0.5°C. Four groups of six mice each were selected randomly for the analgesic activity study. The reaction time was recorded once the mice exhibited pain feeling by licking of paw or jumping response which appeared first. The animals were placed on jars containing different solutions, namely distilled water as control, negatively charged ibuprofen lysinate (of about 1.36 mg/mL concentration) in distilled water and negatively charged ibuprofen lysinate loaded on the positively charged dispersion with ibuprofen concentration of about 1.36 mg/mL so that only animal’s front and hind legs were exposed to the solutions in order to allow the transdermal diffusion of solutions’ ingredients. Each group was subjected to about 30 seconds exposure time to the solutions. Then, the animals allowed waiting for about one hour prior to being subjected to Eddy’s hot plate test, and the reaction time was recorded. The cut-off time (i.e. the time at which the exposure of the animal to the hot plate is ended) of 15 second was used.

[087] FIG. 8 shows response time to hot plate of mice exposed to water (control), free negatively charged buprofen lysinate, positively charged dispersion, and negatively charged ibuprofen lysinate loaded on the positively charged dispersion. The negatively charged ibuprofen lysinate loaded on the positively charged dispersion showed the maximum response time, which means that the negatively charged ibuprofen lysinate loaded on the positively charged dispersion has the maximum analgesia compared to the remaining preparations.

Anti-inflammatory test using carrageenan-induced mouse paw edema

[088] Animal legs (5 legs per group) were exposed to distilled water as control, negatively charged ibuprofen lysinate (of about 1.36 mg/mL concentration) in distilled water and negatively charged ibuprofen lysinate loaded on the positively charged dispersion with ibuprofen concentration of about 1.36 mg/mL. Immediately after that, about 0.05 mL of 1% w/v carageenan was injected in the right leg of all animals. The percent change in volume of right legs prior and after a period of about 1 hour of injection was measured. The inhibition of increase in paw edema after about 1 hour was monitored, and the results are illustrated in as shown in FIG. 7.

[089] As depicted in FIG. 9, negatively charged ibuprofen lysinate loaded on the positively charged dispersion showed the maximum inhibition of increase in mice paw edema, which means that the which means that the negatively charged ibuprofen lysinate loaded on the positively charged dispersion has the maximum anti inflammatory activity compared to the remaining preparations.

[090] While embodiments of the present disclosure have been described in detail and with reference to specific embodiments thereof, it will apparent to one skilled in the art that various additions, omissions, and modifications can be made without departing from the spirit and scope thereof.

[091] As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a defacto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[092] Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of approximately 1 to approximately 4.5 should be interpreted to include not only the explicitly recited limits of 1 to approximately 4.5, but also to include individual numerals such as 2, 3, 4, and sub-ranges such as 1 to 3, 2 to 4, etc. The same principle applies to ranges reciting only one numerical value, such as“less than approximately 4.5,” which should be interpreted to include all of the above-recited values and ranges. Further, such an interpretation should apply regardless of the breadth of the range or the characteristic being described. [093] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are now described.

[094] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[095] As used herein, the term“about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.