Edaravone dosage form

Abstract

Disclosed is an Edaravone dosage form and a use thereof in preparing a drug used for treating diseases related to oxidative stress, the dosage form being selected from a lipid-based delivery system, a solid dispersion, micelles and a co-solvent based formulation.

Claims

1. A solid phase dispersion formulation, comprising Edaravone as an active ingredient, and a polymer carrier selected from Soluplus, wherein the solid phase dispersion comprises Edaravone and Soluplus in a ratio of Edaravone:Soluplus of 1:1 to 1:16 by mass.

2. The solid phase dispersion formulation of claim 1, further comprising a surfactant.

3. The solid phase dispersion formulation of claim 2, wherein the surfactant includes an anionic, cationic, or amphoteric surfactant, selected from the group consisting of sodium dodecanesulfonate, sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), polyoxyethylene sorbitan long-chain fatty acid esters, Vitamin E-TPGS, bile salts, sodium deoxycholate, sodium glycocholate, polyoxyethylene polyoxypropylene glycols and combinations thereof.

4. The solid phase dispersion formulation of claim 2, wherein the surfactant is TPGS 1000.

5. The solid phase dispersion formulation of claim 1, comprising Edaravone, Soluplus and optionally TPGS 1000.

6. A method of preparing the solid phase dispersion formulation of claim 1, comprising a step of: dispersing Edaravone or a pharmaceutically acceptable salt thereof as an active ingredient in a polymer carrier and, optionally, a surfactant.

7. The method of claim 6, further comprising a step selected from: melting ice bath agitation, thin film cooling, liquid nitrogen, spray congealing, hot-melt extrusion, Meltrex™, melt agglomeration or solvent evaporation, including oven drying, vacuum drying, rotary evaporation, heating on hot plate, spray drying, freeze drying, supercritical anti-solvent, co-precipitation, electrostatic spinning, spray freeze drying, ultra-rapid freezing or fluid-bed coating, and solvent melting.

8. The solid phase dispersion formulation of claim 1, comprising Edaravone and Soluplus in a ratio of Edaravone:Soluplus of 1:5 by mass.

9. An oral pharmaceutical composition comprising Edaravone or a pharmaceutically acceptable salt thereof, and Soluplus in a ratio of Edaravone:Soluplus of 1:1 to 1:16 by mass.

Description

DESCRIPTION OF THE FIGURES

(1) To clearly indicate the technical solution of the present invention, a brief introduction thereto is provided below in reference to the Figures. Apparently, these Figures are merely some embodiments recorded in this application. The present invention includes but is not limited to these Figures.

(2) FIG. 1 shows the solubility of Edaravone (mg/gm) in various vehicles.

(3) FIG. 2 shows the screening of a drug carrier system (Soluplus) based on the solubility study of Edaravone.

(4) FIG. 3 shows the solubility of Edaravone in different types of formulations (Examples 1-11).

(5) FIG. 4 shows the in vitro safety study of different types of Edaravone formulations (Examples 1-11).

(6) FIG. 5 shows the particle size distribution of micellar formulation (Example 6).

(7) FIG. 6 shows the dissolution study of solid dispersion (Example 8) in different simulated body fluids.

(8) FIG. 7 shows the bioavailability study of SMEDDS (Examples 1 and 2).

(9) FIG. 8A shows the stability study of Edaravone in biorelevant media at various pHs.

(10) FIG. 8B shows the stability study of the solid dispersion (Example 8) in biorelevant media at various pHs.

EMBODIMENTS

(11) To further understand the present invention, preferable solutions of the present invention are described in details below in reference to examples.

(12) However, these examples are merely used to illustrate the characteristics and merits of the novel Edaravone formulations of the present invention, but not to limit the protection scope of the present invention.

(13) Components used in the invention with their chemical names.sup.[5]:

(14) TABLE-US-00001 Components Use Chemical names and other names Capryol ™ Oil Propylene glycol monocaprylate PGMC (type I) NF Cremophor ® Surfactant Kolliphor ® RH 40, RH 40 Macrogolglycerol hydroxystearate, PEG-40 castor oil, Polyoxyl 40 hydrogenated castor oil Labrasol Surfactant Caprylocaproyl polyoxylglycerides, Caprylocaproyl macrogol-8 glycerides EP, Caprylocaproyl polyoxyl-8 glycerides NF TPGS 1000 Surfactant D-α-Tocopherol polyethylene glycol 1000 succinate, Vitamin E polyethylene glycol succinate, Vitamin E-TPGS, Water soluble form of Vitamin E Transcutol P Co-surfactant Transcutol HP, highly purified diethylene glycol monoethyl ether EP/NP Aerosil 200 Solid Fumed silicon dioxide (fumed adsorbent silica) Soluplus Polymer Polyvinyl caprolactam-polyvinyl solubilizer acetate-polyethylene glycol graft and stabilizer copolymer (PCL-PVAc-PEG) PEG 300 Water-soluble Polyethylene glycol 300 (300 is organic molecular weight) solvent, solubilizer If desired, all aqueous buffer systems may be used to prepare liquid formulation of Edaravone Acidifying Citric acid, acetic acid, fumaric acid, hydrochloric agents acid, nitric acid Anti-oxidants Ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propylgallate, sodium ascorbate, sodium bisulfate, sodium formaldehyde sulfoxylate, sulfoxylate, sodium metabisulfite Buffering Potassium metaphosphate, potassium phosphate, agents monobasic, sodium acetate, sodium citrate, anhydrous and dihydrate

Example 1

(15) Lipid Based Self-Emulsifying Drug Delivery System (SMEDDS) Formulation

(16) TABLE-US-00002 Ingredients Quantity Edavarone 10 mg/ml Capryol ™ PGMC   30% Cremophor RH 40 23.33% Labrasol:TPGS 1000 (4:1) 23.33% Transcutol P 23.33%

Example 2

(17) Lipid Based SMEDDS Formulation

(18) TABLE-US-00003 Ingredients Quantity Edaravone 10 mg/ml Capryol ™ PGMC   30% Cremophor RH 40 23.33% Labrasol:TPGS 1000 (4:1) 23.33% Transcutol P 23.33% Aerosil 200 (Adsorbent) 5% w/v

Example 3

(19) Lipid Based SMEDDS Formulation

(20) TABLE-US-00004 Ingredients Quantity Edaravone 10 mg/ml Capryol ™ PGMC   30% Cremophor RH 40 23.33% Labrasol 23.33% Transcutol P 23.33%

Example 4

(21) Lipid Based SMEDDS Formulation

(22) TABLE-US-00005 Ingredients Quantity Edaravone 10 mg/ml Capryol ™ PGMC   30% Labrasol 46.66% Transcutol P 23.33% Aerosil 200 (Adsorbent) 5% w/v

Example 5

(23) Lipid Based Nanoemulsion Formulation

(24) TABLE-US-00006 Ingredients Quantity Edaravone 10 mg/ml Capryol ™ PGMC   30% Cremophor RH 40 23.33% Labrasol:TPGS 1000 (4:1) 23.33% Transcutol P 23.33% Water Q.S.

Example 6

(25) Micellar Formulation

(26) TABLE-US-00007 Ingredients Quantity (mg) Edaravone 100 Soluplus:TPGS 1000 500:200 PBS (pH 7.4) 10 ml

Example 7

(27) Solid Dispersion Formulation

(28) TABLE-US-00008 Ingredients Quantity (mg) Edavarone 100 Soluplus:TPGS 1000 500:75

Example 8

(29) Solid Dispersion Formulation

(30) TABLE-US-00009 Ingredients Quantity (mg) Edaravone 100 Soluplus 500

Example 9

(31) Co-Solvent-Based Formulation

(32) TABLE-US-00010 Ingredients Quantity Edaravone 20 mg/ml Cremophor RH 40 250 mg PEG 300 250 mg TPGS 1000 125 mg Water 375 mg

Example 10

(33) Co-Solvent-Based Formulation

(34) TABLE-US-00011 Ingredients Quantity Edaravone 20 mg/ml Labrasol 500 mg TPGS 1000 125 mg Water 375 mg

Example 11

(35) Co-Solvent-Based Formulation

(36) TABLE-US-00012 Ingredients Quantity Edaravone 20 mg/ml PEG 300 500 mg TPGS 1000 125 mg Water 375 mg

Preparation Example 1

(37) Preparation of Liquid Self-Micro Emulsified Drug Delivery Systems (SMEDDS, Examples 1 and 3)

(38) Referring to Examples 1 and 3, the required amounts of Oil (Capmul PGMC), surfactants (Cremophor RH 40, Labrasol and TPGS 1000) and Co-surfactant (Transcutol P) were accurately weighed into glass vials. Then, the components were mixed by gentle stirring and vortex mixing, and heated at 37° C. in an incubator. The required amount of Edaravone was added and vortex mixing was performed, until Edaravone has perfectly dissolved.

Preparation Example 2

(39) Preparation of Solid Self-Micro Emulsified Drug Delivery Systems (Examples 2 and 4)

(40) Liquid SMEDDS formulation was prepared as mentioned above. It was diluted in the minimum quantity of miliQ water and stirred at room temperature for 2 h after adding a required quantity of Aerosil 200. The resultant mixture was then allowed to stand for 15 min to attain the equilibrium and filtered through 0.45 μm syringe filter (PVDF). Before Freeze Drying, solutions were frozen at −80° C. for at least 6 h and then subjected to lyophilization in Novalyphe-NL 500 (Savant Instruments Corp., Holbrook, N.Y.) for at least 24 h at −45° C. and 7102 mbar pressure. The solid SMEDDS were then stored in a desiccator.

Preparation Example 3

(41) Preparation of Nano-Emulsion Based System (Example 5)

(42) Referring to Example 5, the required amounts of Oil (Capmul PGMC), surfactants (Cremophor RH 40, Labrasol and TPGS 1000) and Co-surfactant (Transcutol P) were accurately weighed into glass vials. Then, the components were mixed by gentle stirring and vortex mixing, and heated at 37° C. in an incubator. The required amount of Edaravone was added and vortex mixing was performed, until Edaravone has perfectly dissolved. The required quantity of miliQ water was added dropwise until a clear formulation was obtained.

Preparation Example 4

(43) Preparation of Micellar Formulation (Example 6)

(44) Referring to Example 6, a required quantity of Edaravone, Soluplus and TPGS 1000 was dissolved in Ethanol. The organic solvent was removed by Buchi Rotavap II instrument. The film formed was dried in vacuum desiccator overnight, and then hydrated with 10 mL 1× PBS buffer (pH 7.4), incubated at 37° C. for 30 min, and then sonicated for a few minutes. The resultant mixture was filtered through 0.45 μm syringe filter (PVDF).

Preparation Example 5

(45) Preparation of Solid Dispersion Based Formulations (Examples 7 and 8)

(46) Referring to Examples 7 and 8, a required quantity of Edaravone, Soluplus with and without TPGS 1000 was dissolved in ethanol. The organic solvent was removed by Buchi Rotavap II instrument. The film formed was dried in a vacuum desiccator overnight. Dried samples were scrapped off from the flask and collected in a mortar. Powders were crushed and made homogenous by using mortar pestle.

Preparation Example 6

(47) Preparation of Co-Solvent Based Formulations (Examples 9, 10 and 11)

(48) Referring to Examples 9-11, all components were accurately weighed into glass vials. Then, the components were mixed by gentle stirring and vortex mixing, and heated at 37° C. in an incubator. The required amount of Edaravone was added and vortex mixing was performed, until Edaravone has perfectly dissolved.

(49) Examples 1-11 provide a number of formulations, including lipid, micelles, solid dispersion and co-solvent based formulations. Advantages of these formulations are shown by effect examples as follows:

Effect Example 1

(50) Studies on the Solubility of Edaravone in Different vehicles

(51) The selection of pharmaceutical vehicles is the most important step for the development of Liquid Oral formulation. In separate glass vials, 1 mL of each vehicles as shown in FIG. 1 was taken. An excess amount of Edaravone was added to the above mentioned solutions followed by continuous rotation using a mechanical shaker (Axyos Technologies, Brisbane, Australia) throughout the test for 24 hours at room temperature. After reaching equilibrium, each vial was centrifuged at 3000 rpm for 5 min, and excess insoluble Edaravone was discarded by filtration through 0.45 μm PVDF syringe filter. Subsequently, the filtrates were diluted using methanol. The solubility analysis was performed in triplicate by using the previously developed and validated HPLC method. The analysis of the samples was performed on an HPLC (Shimadzu, Kyoto, Japan) system equipped with a UV-VIS detector [SPD-20 A], DGU-20A3 online degasser, CBM-20A system controller, SIL-20AHT autosampler and a LC solution Chromopac data processor. Zorbax Eclipse XDB-C18 (4.6*150*3.5 mm.sup.3) analytical column was used. Samples were analyzed with the mobile phase consisting of methanol, miliQ water and acetic acid at the ratio of 100:100:1 (v/v/v). The injection volume was 20 μL with a flow rate of 1 mL/min, and detection was performed at 240 nm.

(52) To prepare Edaravone formulations, aqueous and non-aqueous solubilizers can be used alone or in combination in the present invention. The self-micro emulsified drug delivery system (SMEDDS, i.e., lipid based drug delivery system) is prepared by selecting ingredients (oils, surfactants and co-surfactants).

(53) As can be seen from FIG. 1, Labrasol, Transcutol P, PEG 300, Caproyl PGMC and Cremophor RH 40 are preferable vehicles of Edaravone.

Effect Example 2

(54) Screening Drug Carrier Soluplus

(55) Screening Carriers

(56) The selection of polymeric carrier is the most important step for the preparation of solid dispersions (SDs). To separate glass vials, 1%, 2%, 3%, 4%, 5% and 6% w/v solutions of different carriers was added, respectively. An excess amount of Edaravone was added to the above mentioned solutions followed by continuous rotation using a mechanical shaker (Axyos Technologies, Brisbane, Australia) throughout the test for 24 hours at room temperature. After reaching equilibrium, each vial was centrifuged at 3000 rpm for 5 min, and excess insoluble Edaravone was discarded by filtration through 0.45 μm PVDF syringe filter. Subsequently, the filtrates were diluted using methanol. The solubility analysis was performed in triplicate by using the previously developed and validated HPLC method.

(57) Optimizing Solid Dispersion Based systems

(58) The inventors prepared a number of batches of solid dispersions by using different ratios of drug to polymer (1:1, 1:3, 1:5, 1:7, 1:8, 1:10, 1:13, and 1:16) in order to optimize the ratio to achieve the maximum solubility. Solvent evaporation technique and Buchi Rotavap II instrument were used to prepare solid dispersions. Required amounts of drug and soluplus were dissolved in ethanol, then the mixture was dried at 55-60° C. under vacuum (500-600 mbar). The inventors compared the solubility of the optimized ratio with physical mixture of the same ratio (1:5) of drug to polymer. Physical mixtures of Edaravone and soluplus were prepared by mixing using mortar and pestle. The product was collected and stored in a desiccator before analysis. To separate glass vials, 1 mL of water was added. An excess amount of Edaravone was added to the above mentioned solutions followed by continuous rotation using a mechanical shaker (Axyos Technologies, Brisbane, Australia) throughout the test for 24 hours at room temperature. After reaching equilibrium, each vial was centrifuged at 3000 rpm for 5 min, and excess insoluble Edaravone was discarded by filtration through 0.45 μm PVDF syringe filter. Subsequently, the filtrates were diluted using methanol. The solubility analysis was performed in triplicate by using the previously developed and validated HPLC method.

(59) As can be seen from FIG. 2, it was demonstrated from studies that solubility improvement in concentration-dependent manner was found for soluplus. Later, the inventors optimized the ratio of drug to polymer. The optimized solid dispersion with a drug to soluplus ratio of 1:5 based system has capability to improve solubility significantly (18 folds) in water. The physical mixture (PM) of optimized drug to polymer ratio of 1:5 has an ability to enhance solubility more than 2 folds.

Effect Example 3

(60) Solubility Studies of Edaravone in Different Solvents

(61) Solubility Studies of Examples 1-5 and 9-11 Formulations

(62) To separate glass vials, 1 mL of each formulation was added. An excess amount of Edaravone was added to the above mentioned solutions followed by continuous rotation using a mechanical shaker (Axyos Technologies, Brisbane, Australia) throughout the test for 24 hours at room temperature. After reaching equilibrium, each vial was centrifuged at 3000 rpm for 5 min, and excess insoluble Edaravone was discarded by filtration through 0.45 μm PVDF syringe filter. Subsequently, the filtrates were diluted using methanol. The solubility analysis was performed in triplicate by using the previously developed and validated HPLC method.

(63) Solubility Study of Example 6 Formulation

(64) An excess quantity of Edaravone, and the required quantity of Soluplus and TPGS 1000 were dissolved in ethanol. The organic solvent was removed by Buchi Rotavap II instrument. The film formed was dried in vacuum desiccator overnight, hydrated with 10 mL 1× PBS buffer (pH 7.4), incubated at 37° C. for 30 min, and then sonicated for a few minutes. Each sample was centrifuged at 3000 rpm for 5 min. The resultant mixture was filtered through 0.45 μm syringe filter (PVDF). The solubility analysis was performed in triplicate by using the previously developed and validated HPLC method.

(65) Solubility Studies of Examples 7 and 8 Formulations

(66) To separate glass vials, 1 mL of water was added. An excess amount of solid dispersions was added to the above mentioned solutions followed by continuous rotation using a mechanical shaker (Axyos Technologies, Brisbane, Australia) throughout the test for 24 hours at room temperature. After reaching equilibrium, each vial was centrifuged at 3,000 rpm for 5 min, and excess insoluble Edaravone was discarded by filtration through 0.45 μm PVDF syringe filter. Subsequently, the filtrates were diluted using methanol. The solubility analysis was performed in triplicate by using the previously developed and validated HPLC method.

(67) It was demonstrated that the solubility of Edaravone in all the formulations (Examples 1-11), especially SMEDDS of Examples 1-4, has a significant improvement than in water (FIG. 3).

Effect Example 4

(68) Cell Toxicity Studies of SH-5Y5Y Cells

(69) Cell Culture Assay

(70) SH-SY5Y cell lines were used in order to perform cell culture. The DMEM media (Dulbecco's Modified Eagle Medium): Nutrient Mixture F12 in the ratio of 1:1 was used to culture cells respectively, supplemented with 10% FBS and 1% penicillin-streptomycin solution in 25 ml cell culture flask. Cells were cultivated in an incubator at 37° C. in presence of 5% CO.sub.2.

(71) MTT Assay on Cell Viability with SH-SY5Y

(72) In the 96 well plate, SH-5YSY cells were seeded at the density of 5×10.sup.3 cells/well. The culture media was changed after 24 hours with or without formulation-containing medium. The formulations were prepared by using previously sterilized water. A cell viability assay using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] methods was performed. After 20 h, 20 μL of MTT (Sigma-Aldrich, USA, 5 mg/ml in PBS), was added to each well and incubated for 1 h. 150 μL of DMSO was added to dissolve the insoluble purple formazan product to produce a colored solution. The optical density (OD) was read at 600 nm wavelength on the multi-well scanning spectrophotometer (BIO-RAD Model 2550 EIA Reader).

(73) As can be seen from FIG. 4, the in vitro safety of all formulations was confirmed as the inventors did not observe any significant toxicity from any of the formulations. The inventors found slight improvement of cell viability in cases of micelle formulation (Example 6) and solid dispersion (Example 7) because of the presence of TPGS 1000 which was reported as neuroprotection.

Effect Example 5

(74) The Size Distribution of Edaravone Micellar Formulation

(75) The size and size distribution of micellar formulation was measured by the dynamic light scattering (DLS) (Malvern Zeta Sizer Nano ZS). The samples were prepared by diluting the micellar solution with miliQ water and sonicated for 5 min before measurement. Particle size and Poly dispersity Index (PDI) were measured in triplicate by using protocol mentioned above.

(76) It is shown in FIG. 5 that because of the particle size in nano range, it has a potential to improve the cellular uptake of drug, resulting in an improvement of efficacy. This result also supports the results mentioned in FIG. 4 (Example 6).

(77) Characteristic parameters of micellar formulation of the present invention (Example 6) were: particle size 15.68, polydispersity index 0.361, and drug loading 10.11 mg/ml.

Effect Example 6

(78) Dissolution study of Edaravone in Solid Dispersion (SD)

(79) The dissolution of SDs form was carried out by using USP type II paddle apparatus (AT 7 Smart, Sotax GmbH, Germany). The operating parameters were: 50 rpm rotation speed, 37±0.5° C. temperature, and SGF (simulated gastric fluid) 1.2 pH (USP), FaSSIF pH 5.0 (fasted state simulating Intestinal fluid), FeSSIF (fed state simulating Intestinal fluid) pH 6.5, and SIF (Simulating Intestinal fluid) 7.5 pH (USP). The formulation equivalent to 100 mg of Edaravone was filled into a size ‘2’ hard gelatin capsule. Capsules were then placed inside the sinker and put into dissolution vessel. The samples were collected at different time intervals and replaced with equal amount of fresh dissolution media each time. The samples were filtered through 0.45 μm PVDF syringe filter and analyzed by the previously developed HPLC method.

(80) As can be seen from FIG. 6, the solid dispersion of Example 8 can be used to prepare solid dosage form like a tablet or a capsule, and then the inventors have performed dissolution study to predict its behavior in various simulated biological fluids.

(81) The inventors have found that the solid dispersion based formulation can provide sustained release after an initial burst release.

Effect Example 7

(82) Bioavailability of Edaravone in SMEDDS Formulation

(83) Male Sprague-Dawley rats (250±10 g) were acquired at least 1 week before starting the experiments as they were adapted to the laboratory environment, food, and water available for them. Rats were anaesthetized before operations. A longitudinal cut was made on the neck part and the area nearer to the jugular vein. Later, filled the catheter with 20 units/ml of heparin saline solution and inserted the catheter into the jugular vein up to first silicon stopper. The catheter was fixed over there by stitching the stopper and muscle. The other end of the catheter was passed beneath the skin of neck portion and nearer to ears. Lastly, filled the catheter with 500 unit/ml of heparin saline solution and plugged into the free end of catheter. After the surgery, the rats were kept in different cages for recovery. Then, on the next day, pharmacokinetic study of every rat would be carried out. Animals were fasted for 12 h before drug administration with free access to water.

(84) Edaravone suspension was prepared by adding Edaravone into 0.5% carboxymethylcellulose sodium (CMC-Na) solution and then ultrasonicated for several minutes to obtain homogenous suspensions. Two groups of rats were orally administrated with Edaravone suspension, and SMEDDS at an equivalent dose of 30 mg/kg of Edaravone, respectively. One group of rats was administered through iv route. After administration of drug and formulation by oral gavage, 0.2 ml of blood samples were collected at time intervals of 0, 15, 30, 45, 60, 90, 120, 180, 240, 300, 360, 420, and 480 minutes. Each time, when blood samples were collected, catheter would be flushed with the same amount of heparin saline solution. After collection of blood samples, they were centrifuged at 5000 rpm for 5 min at 4° C. to separate plasma from blood. Plasma was separated and stored at −20° C. until analysis. Plasma (200 μl) was added with 40 μl of 30% HClO.sub.4 to acidify plasma and precipitate the protein. After that, centrifuged at 4° C. and 16000 rpm for 6 minutes. The contents were diluted in methanol/water (50:50) and filtrated through 0.22 μm membrane filter before injected into LC/MS/MS.

(85) The analysis of the samples was performed on Quadrapole LC/MS/MS (Shimadzu, Kyoto, Japan) system equipped with API 3000 mass spectrometer, Shimadzu SIL 20A autosampler, Shimadzu LC20AD Pump and an Analyst 1.6.2 data processor. Concentrations of the Edaravone in plasma were quantified using a newly developed and validated LC/MS/MS method. The extracts were reconstituted in methanol/water (50:50), injected into a Shimadzu Nexera HPLC system and resolved on a Kinetex C18 2.6 mm×50 mm×3 mm column (Phenomenex) with a mobile phase flow rate of 0.2 mL/min and an injection volume of 15 μL. Mobile phase A (MPA) was 5% methanol and 0.1% formic acid in water and mobile phase B (MPB) was 95% methanol and 0.1% formic acid in water. The mobile phase timetable was set as a gradient from 15% MPB initially to 70% MPB in 7.5 min, held to 100% MPB for further 0.5 min, then 15% MPB for 2 min in preparation for the next sample. The total run time for each sample analysis was 10 min. The column effluent was introduced into mass spectrometry using electro spray ionisation (ESI) in negative mode. The operating parameters of the ionisation source, including analyte-dependent parameters and source-dependent parameters were optimized to obtain the optimum performance of the mass spectrometer for the analysis. MRM analysis was conducted by monitoring the precursor ion to produce ion transitions (m/z) as follows: Edaravone 175.0/133.10 and Phenazone 189.1/147.1. Zero air was used as the source gas while nitrogen was used as both the curtain and collision gas. Peak areas were obtained from the compounds, and IS and known concentrations of calibrators were used to construct a calibration curve from compounds/IS area ratios. The limit of quantification was 5 ng/mL. The intraday and interday variability for each compound was within 15%.

(86) To study the bioavailability of self-microemulsifying drug delivery system (SMEDDS) of the present invention (Examples 1 and 2), Edaravone was orally administered in suspension form (prepared by adding Edaravone into 0.5 carboxymethylcellulose sodium (CMC-Na) solution), and Edaravone via iv route was used as a control. The bioavailability of SMEDDS was significantly improved compared to Edaravone suspension (FIG. 7). SMEDDS can also improve the half-life of the drug which shows a potential to maintain therapeutic level at a longer time.

Effect Example 8

(87) Stability Studies of Edaravone in Solid Dispersion

(88) Simulated gastrointestinal fluids (without enzymes and bile component) were prepared according to the USP methods. SGF (simulated gastric fluid) 1.2 pH (USP) and SIF (Simulating Intestinal fluid) 6.8 pH (USP) and 7.4 pH. To determine the chemical stability of SD formulations, the solutions in miliQ water was prepared and used. SD formulation was dissolved in aforesaid buffer solutions. Samples were collected at the predetermined time interval and filtered through 0.45 μm PVDF syringe filter. All samples were analysed in triplicate by HPLC.

(89) It was demonstrated in FIG. 8A that Edaravone showed significant degradation at neutral to basic pH, but it maintained substantially constant at acidic pH.

(90) It was demonstrated in FIG. 8B that the solid dispersion of the present invention (Example 8) can protect Edaravone from degradation in various biorelevant media.

(91) The above examples are merely to help understand the core spirits of the present invention. It should be noted that, for those skilled in the art, several improvements or modifications can be made to the novel formulations and preparation methods of the present invention, without departing from the principle of the present invention, which, however, fall into the scopes of the appended claims.

REFERENCES

(92) 1. Bernabeu, E., et al., Novel Soluplus®-TPGS mixed micelles for encapsulation of paclitaxel with enhanced in vitro cytotoxicity on breast and ovarian cancer cell lines. Colloids Surf B Biointerfaces, 2016. 140: p. 403-11.

(93) 2. Dian, L., et al., Enhancing oral bioavailability of quercetin using novel soluplus polymeric micelles. Nanoscale Res Lett, 2014. 9(1): p. 2406.

(94) 3. Jin, X., et al., Soluplus® micelles as a potential drug delivery system for reversal of resistant tumor. Biomed Pharmacother, 2015. 69: p. 388-95.

(95) 4. Xia, D., et al., Supersaturated polymeric micelles for oral cyclosporine A delivery: The role of Soluplus-sodium dodecyl sulfate complex. Colloids Surf B Biointerfaces, 2016. 141: p. 301-310.

(96) 5. Strickley, R. G., Solubilizing excipients in oral and injectable formulations. Pharm Res, 2004. 21(2): p. 201-30.