Sublingual pharmaceutical compositions of edaravone and (+)-2-borneol

Abstract

A sublingual tablet pharmaceutical composition containing edaravone and (+)-2-borneol, and a preparation method thereof. The sublingual tablet pharmaceutical composition includes edaravone, (+)-2-camphanol, an excipient, a filler, a binder, a disintegrant, and a lubricant. The excipient is selected from one or more of mannitol, lactose, dextran, cysteine, glycine, copovidone, and beta-cyclodextrin.

Claims

1. A sublingual tablet pharmaceutical composition comprising edaravone, (+)-2-borneol and a pharmaceutically acceptable adjuvant, wherein the pharmaceutically acceptable adjuvant comprises an excipient, and wherein the excipient comprises one or more selected from the group consisting of mannitol, lactose, dextran, cysteine, glycine, copovidone and beta-cyclodextrin.

2. The pharmaceutical composition according to claim 1, comprising mannitol and copovidone at a mass ratio of 1:5 to 5:1 as excipients.

3. The pharmaceutical composition according to claim 2, comprising mannitol and copovidone at a mass ratio of 1:1 to 5:1 as excipients.

4. The pharmaceutical composition according to claim 1, wherein the mass ratio of edaravone in terms of free base to (+)-2-borneol is greater than 4 and less than 10 or greater than 0.1 and less than 1.

5. The pharmaceutical composition according to claim 1, wherein the mass ratio of edaravone in terms of free base to (+)-2-borneol is 5:1.

6. The pharmaceutical composition according to claim 1, wherein the weight ratio of (+)-2-borneol to the excipient is from 0.1:1 to 1:1.

7. The pharmaceutical composition according to claim 1, wherein the weight ratio of (+)-2-borneol to the excipient is from 0.3:1 to 1:1.

8. A method for preparing the pharmaceutical composition according to claim 1, comprising dissolving (+)-2-borneol in an organic solution, and dissolving the excipient in an aqueous solution; combining the two solutions and stirring, allowing the mixed solution to stand; performing freeze-drying and then sieving; adding edaravone, filler, binder, disintegrant and lubricant, and mixing until homogeneous; and tableting.

9. The pharmaceutical composition according to claim 1, wherein within about 0.1 to 10 hours after administration of a unit of the sublingual tablet to a patient, edaravone concentration in blood reaches 10 to 8000 ng/mL, and (+)-2-borneol concentration in blood reaches 1 to 200 ng/mL.

10. The pharmaceutical composition according to claim 1, wherein within about 0.1 to 6 hours after administration of a unit of the sublingual tablet to a patient, edaravone concentration in blood reaches 50 to 5000 ng/mL, and (+)-2-borneol concentration in blood reaches 2 to 150 ng/mL.

11. The pharmaceutical composition according to claim 1, comprising an adjuvant selected from mannitol and copovidone.

Description

DETAILED DESCRIPTION

(1) The present disclosure discloses a sublingual administration preparation of edaravone and (+)-2-borneol. Those skilled in the art can learn from the content of the present disclosure, and based on the principle of pharmacy, appropriately improve the process parameters or the prescription ratio. It is to be noted that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present disclosure. The application of the present disclosure has been described in preferred embodiments, and it is obvious for a person skilled in the art to change or appropriately modify and combine the to method and application described herein without departing from the scope of the present disclosure, to implement and apply the technique of the present disclosure.

(2) The edaravone mentioned in the examples is 3-methyl-1-phenyl -2-pyrazolin-5-one.

(3) The present disclosure is further illustrated by the following examples, but the examples are not intended to limit the present disclosure.

Example 1

(4) TABLE-US-00001 Material Amount (g) Edaravone 30 (+)-2-Borneol 1.5 Lactose 39.7 Hypromellose 4 Croscarmellose sodium 4 Magnesium stearate 0.8

(5) Preparation method: (+)-2-borneol, edaravone, lactose, hypromellose, croscamellose sodium, magnesium stearate were uniformly mixed and then tableted.

Example 2

(6) TABLE-US-00002 Material Amount (g) Edaravone 30 (+)-2-Borneol 2 Lactose 39.2 Hypromellose 4 Croscarmellose sodium 4 Magnesium stearate 0.8

(7) Preparation method: (+)-2-borneol, edaravone, lactose, hypromellose, croscarmellose sodium, magnesium stearate were uniformly mixed and then tableted.

Example 3

(8) TABLE-US-00003 Material Amount (g) Edaravone 1.5 (+)-2-Borneol 30 Lactose 39.7 Hypromellose 4 Croscarmellose sodium 4 Magnesium stearate 0.8

(9) Preparation method: (±)-2-borneol, edaravone, lactose, hypromellose, croscarmellose sodium, magnesium stearate were uniformly mixed and then tableted.

Example 4

(10) TABLE-US-00004 Material Amount (g) Edaravone 6 (+)-2-Borneol 30 Beta-cyclodextrin 35.2 Lactose 10 Hypromellose 4 Croscarmellose sodium 4 Magnesium stearate 0.8

(11) Preparation method: (+)-2-Borneol was dissolved in an ethanol solution, and beta-cyclodextrin was dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, lactose, hypromellose, croscarmellose sodium and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 5

(12) TABLE-US-00005 Material Amount (g) Edaravone 40 (+)-2-Borneol 8 Beta-cyclodextrin 23.2 Lactose 10 Hypromellose 4 Croscarmellose sodium 4 Magnesium stearate 0.8

(13) Preparation method: (+)-2-Berneol was dissolved in an ethanol solution, and beta-cyclodextrin was dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, lactose, hypromellose, croscarmellose sodium and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 6

(14) TABLE-US-00006 Material Amount (g) Edaravone 30 (+)-2-Borneol 6 Mannitol 35.2 Copovidone 4 Croscarmellose sodium 4 Magnesium stearate 0.8

(15) Preparation method: (+)-2-Borneol was dissolved in an ethanol solution, and mannitol in an amount of 5 times the weight of (+)-2-borneol and a small amount of copolyvidone were dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, the remaining mannitol, copolyvidone, croscarmellose sodium and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 7

(16) TABLE-US-00007 Material Amount (g) Edaravone 50 (+)-2-Borneol 5 Mannitol 16.2 Copovidone 4 Croscarmellose sodium 4 Magnesium stearate 0.8

(17) Preparation method: (+)-2-Borneol was dissolved in an ethanol solution, and mannitol in an amount of 3 times the weight of (+)-2-borneol and a small amount of copolyvidone were dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, the remaining mannitol, copolyvidone, croscarmellose sodium and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 8

(18) TABLE-US-00008 Material Amount (g) Edaravone 30 (+)-2-Borneol 6 Mannitol 14 Lactose 22.2 Copovidone 3 Croscarmellose sodium 4 Magnesium stearate 0.8

(19) Preparation method: (+)-2-Borneol was dissolved in an ethanol solution, and mannitol and a small amount of copolyvidone were dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, lactose, copolyvidone, croscarmellose sodium and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 9

(20) TABLE-US-00009 Material Amount (g) Edaravone 30 (+)-2-Borneol 6 Mannitol 6 Lactose 29.2 Copovidone 4 Croscarmellose sodium 4 Magnesium stearate 0.8

(21) Preparation method: (+)-2-Borneol was dissolved in an ethanol solution, and mannitol and a small amount of copolyvidone were dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, lactose, copolyvidone, croscarmellose sodium and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 10

(22) TABLE-US-00010 Material Amount (g) Edaravone 30 (+)-2-Borneol 6 Lactose 31.2 Copovidone 8 Croscarmellose sodium 4 Magnesium stearate 0.8

(23) Preparation method: (+)-2-Borneol was dissolved in an ethanol solution, and lactose in an amount of 5 times the weight of (+)-2-borneol and a small amount of copolyvidone were dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, the remaining lactose, copolyvidone, croscarmellose sodium and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 11

(24) TABLE-US-00011 Material Amount (g) Edaravone 12 (+)-2-Borneol 24 Lactose 31.2 Copovidone 8 Croscarmellose sodium 4 Magnesium stearate 0.8

(25) Preparation method: (+)-2-Borneol was dissolved in an ethanol solution, and lactose in an amount of 1.25 times the weight of (+)-2-borneol and a small amount of copolyvidone were dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, the remaining lactose, copolyvidone, croscarmellose sodium and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 12

(26) TABLE-US-00012 Material Amount (g) Edaravone 30 (+)-2-Borneol 6 Mannitol 14 Macrocrystalline cellulose 22.2 Copovidone 3 Croscarmellose sodium 4 Magnesium stearate 0.8

(27) Preparation method: (+)-2-Borneol was dissolved in an ethanol solution, and mannitol and a small amount of copolyvidone were dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, microcrystalline cellulose, copolyvidone, croscarmellose sodium and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 13

(28) TABLE-US-00013 Material Amount (g) Edaravone 30 (+)-2-Borneol 6 Mannitol 6 Microcrystalline cellulose 16.2 Copovidone 3 Croscarmellose sodium 7 Silicon dioxide 1.1 Magnesium stearate 0.7

(29) Preparation method: (+)-2-Borneol was dissolved in an ethanol solution, and mannitol and copolyvidone were dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, microcrystalline cellulose, croscarmellose sodium, silicon dioxide and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 14

(30) TABLE-US-00014 Material Amount (g) Edaravone 30 (+)-2-Borneol 6 Mannitol 14 Microcrystalline cellulose 11.2 Copovidone 3 Croscarmellose sodium 4 Silicon dioxide 1.1 Magnesium stearate 0.7

(31) Preparation method: (+)-2-Borneol was dissolved in an ethanol solution, and mannitol and a small amount of copolyvidone were dissolved in an aqueous solution. The obtained two solutions were combined and stirred, and then left to stand. The resultant was subjected to freeze-drying and sieving. Edaravone, microcrystalline cellulose, copolyvidone, croscarmellose sodium, silicon dioxide and magnesium stearate were added and mixed until homogeneous, and the mixture was tableted.

Example 15

(32) Stability test and results: an appropriate amount of samples of examples 1 to 14 were packed as a final product. Samples were storage at 40° C. and 60° C. for 10 days, 30 days and 90 days. The properties, content and related substances in the samples were tested. The results are as follows:

(33) TABLE-US-00015 Related Storage Content (%) substances Examples conditions Properties Edaravone (+)-2-Borneol (%) Example 1 0 day White to off-white 99.8 99.5 0.35 tablet 40° C., 10 days White to off-white 99.5 69.4 0.37 tablet 60° C., 10 days While to off-white 99.4 48.2 0.45 tablet 40° C., 30 days White to off-white 99.5 57.8 0.38 tablet 60° C., 30 days White to off-white 99.4 38.1 0.44 tablet 40° C., 90 days White to off-white 99.0 15.4 0.45 tablet 60° C., 90 days White to off-white 98.9 10.1 0.51 tablet Example 2 0 day White to off-white 99.7 101.2 0.45 tablet 40° C., 10 days White to off-white 99.8 71.3 0.38 tablet 60° C., 10 days White to off-white 99.5 60.2 0.44 tablet 40° C., 30 days While to off-white 99.7 54.1 0.46 tablet 60° C., 30 days White to off-white 99.6 43.5 0.47 tablet 40° C., 90 days White to off-white 99.2 16.3 0.53 tablet 60° C., 90 days White to off-white 99.1 12.7 0.62 tablet Example 3 0 day White to off-white 99.7 100.2 0.49 tablet 40° C., 10 days White to off-white 99.5 71.1 0.56 tablet 60° C., 10 days White to off-white 99.4 62.8 0.48 tablet 40° C., 30 days White to off-white 99.2 59.7 0.52 tablet 60° C., 30 days White to off-white 99.5 34.2 0.50 tablet 40° C., 90 days White to off-white 99.1 21.3 0.62 tablet 60° C., 90 days White to off-white 99.3 18.1 0.78 tablet Example 4 0 day White to off-white 99.7 99.8 0.48 tablet 40° C., 10 days White to off-white 99.8 99.4 0.49 tablet 60° C., 10 days White to off-white 99.7 99.8 0.51 tablet 40° C., 30 days White to off-white 99.4 99.0 0.44 tablet 60° C., 30 days White to off-white 99.5 98.7 0.53 tablet 40° C., 90 days White to off-white 99.2 98.1 0.54 tablet 60° C., 90 days White to off-white 99.3 98.8 0.61 tablet Example 5 0 day White to off-white 99.7 100.8 0.47 tablet 40° C., 10 days White to off-white 99.4 101.3 0.48 tablet 60° C., 10 days White to off-white 99.6 101.1 0.48 tablet 40° C., 30 days White to off-white 99.8 100.5 0.39 tablet 60° C., 30 days White to off-white 99.5 100.3 0.44 tablet 40° C., 90 days White to off-white 99.2 99.8 0.43 tablet 60° C., 90 days White to off-white 99.1 99.1 0.54 tablet Example 6 0 day White to off-white 99.8 99.8 0.55 tablet 40° C., 10 days White to off-white 99.2 99.8 0.60 tablet 60° C., 10 days White to off-white 99.8 99.7 0.54 tablet 40° C., 30 days White to off-white 99.7 99.5 0.58 tablet 60° C., 30 days White to off-white 99.8 99.8 0.56 tablet 40° C., 90 days White to off-white 99.7 91.6 0.48 tablet 60° C., 90 days White to off-white 99.8 83.7 0.56 tablet Example 7 0 day White to off-white 99.7 99.5 0.47 tablet 40° C., 10 days White to off-white 99.2 99.6 0.48 tablet 60° C., 10 days White to off-white 99.8 99.8 0.56 tablet 40° C., 30 days White to off-white 99.1 99.8 0.47 tablet 60° C., 30 days White to off-white 99.5 99.7 0.48 tablet 40° C., 90 days White to off-white 99.6 90.8 0.46 tablet 60° C., 90 days White to off-white 99.4 87.3 0.51 tablet Example 8 0 day White to off-white 99.8 101.2 0.41 tablet 40° C., 10 days White to off-white 99.9 101.1 0.43 tablet 60° C., 10 days White to off-white 99.9 100.9 0.41 tablet 40° C., 30 days White to off-white 99.7 100.0 0.39 tablet 60° C., 30 days White to off-white 99.8 101.0 0.40 tablet 40° C., 90 days White to off-white 99.7 99.8 0.50 tablet 60° C., 90 days White to off-white 99.8 99.2 0.48 tablet Example 9 0 day White to off-white 99.7 99.7 0.46 tablet 40° C., 10 days White to off-white 99.7 99.8 0.45 tablet 60° C., 10 days White to off-white 99.5 99.6 0.47 tablet 40° C., 30 days White to off-white 99.6 99.7 0.45 tablet 60° C., 30 days While to off-white 99.7 99.8 0.44 tablet 40° C., 90 days White to off-white 99.5 98.7 0.47 tablet 60° C., 90 days White to off-white 99.4 98.7 0.51 tablet Example 10 0 day White to off-white 99.8 100.1 0.37 tablet 40° C., 10 days White to off-white 99.7 100.2 0.36 tablet 60° C., 10 days White to off-white 99.5 99.8 0.41 tablet 40° C., 30 days White to off-white 99.6 99.1 0.38 tablet 60° C., 30 days White to off-white 99.4 99.6 0.40 tablet 40° C., 90 days White to off-white 99.6 94.3 0.44 tablet 60° C., 90 days White to off-white 99.7 89.4 0.47 tablet Example 11 0 day White to off-white 98.5 99.8 0.45 tablet 40° C., 10 days White to off-white 98.2 99.5 0.39 tablet 60° C., 10 days White to off-white 98.7 99.6 0.51 tablet 40° C., 30 days White to off-white 98.6 99.7 0.38 tablet 60° C., 30 days White to off-white 98.4 99.8 0.50 tablet 40° C., 90 days White to off-white 98.3 95.7 0.51 tablet 60° C., 90 days White to off-white 98.1 90.7 0.49 tablet Example 12 0 day White to off-white 99.3 99.3 0.46 tablet 40° C., 10 days White to off-white 99.4 99.5 0.45 tablet 60° C., 10 days White to off-white 99.5 99.7 0.43 tablet 40° C., 30 days White to off-white 99.2 99.6 0.41 tablet 60° C., 30 days White to off-white 99.1 99.3 0.48 tablet 40° C., 90 days White to off-white 99.4 99.4 0.41 tablet 60° C., 90 days White to off-white 99.2 99.7 0.49 tablet Example 13 0 day White to off-white 99.5 99.5 0.35 tablet 40° C., 10 days White to off-white 99.6 99.4 0.39 tablet 60° C., 10 days White to off-white 99.7 99.7 0.41 tablet 40° C., 30 days White to off-white 99.5 99.6 0.40 tablet 60° C., 30 days White to off-white 99.4 99.4 0.42 tablet 40° C., 90 days White to off-white 99.3 99.8 0.44 tablet 60° C., 90 days White to off-white 99.5 99.4 0.44 tablet Example 14 0 day White to off-white 99.8 99.2 0.41 tablet 40° C., 10 days White to off-white 99.4 99.4 0.46 tablet 60° C., 10 days White to off-white 99.8 99.5 0.47 tablet 40° C., 30 days White to off-white 99.2 99.6 0.45 tablet 60° C., 30 days White to off-white 99.6 99.3 0.46 tablet 40° C., 90 days White to off white 99.4 99.4 0.43 tablet 60° C., 90 days White to off-white 99.5 99.7 0.47 tablet

(34) The above data of the qualitative test results of the examples show that each of examples 4 to 14 stored at a high temperature for 30 days exhibited a good stability. Among them, examples 8, 12, 13 and 14 stored at a high temperature for 90 days exhibited a good stability.

Example 16

Dissolution Rate Test

(35) According to the dissolution and release test method in “Chinese Pharmacopoeia” 2015 version (the fourth part 0931, the second method), 900 ml of water was used as dissolution medium (wherein for examples 3 and 4, 250 ml of water was used as dissolution medium), under a rotating speed of 50 rpm, test was operated according to regulations. Samples were collected at different time points, filtered through 0.8 μm filter, and the subsequent filtrates were used as the test solution. Appropriate amount of edaravone reference substance was taken and dissolved in 20 mmol/L ammonium acetate/acetonitrile (80:20) solution, and then diluted to approximately 0.02 mg/ml for use. The UV absorbance of the test solution was measured at 254 nm, and the dissolution rate of the sample was calculated. The results are shown in FIGS. 1, 2 and 3

Example 17

(36) Distribution of edaravone in plasma and brain tissue of SD rats after sublingual administration

1. Materials and Methods

1.1 Experimental Animals

(37) Sprague-Dawley (SD) rats, SPF grade, male, weight 180-200 g.

(38) Source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

(39) Certificate number: 11400700138404

(40) License number: SCXK (Beijing) 2012-0001

(41) Foods and water supply: Rats were fasted for 12 h before the test, and foods were supplied 4 h after the administration. Water was not prohibited throughout the experiment. Abnormalities of the animals were observed and recorded during drug administration and sample collecting.

1.2 Test Drugs

(42) Compound edaravone injection: specification 12.5 mg/5 mL (edaravone and (+)-2-borneol were 10 mg/5 mL and 2.5 mg/5 mL, respectively).

(43) Sublingual tablets prepared according to the formulation ratio of Example 8, each containing 5 mg of edaravone and 1 mg of borneol.

1.3 Methods

(44) Group 1: Intravenous administration of compound edaravone injection (N=4)

(45) Dosage: 16 mg/kg edaravone, 4 mg/kg, (+)-2-borneol; administration volume: 8 mL/kg. Plasma and brain tissue samples were collected at 2 min, 15 min, 30 min, 1 h, 2.5 h and 5 h. At each time point, whole blood and brain tissue were collected simultaneously.

(46) Group 2: one sublingual tablet was given under the tongue (N=6).

(47) SD rats were intraperitoneal injected with chloral hydrate to make SD rats under mild anesthesia, The mouth of rat was moistened with 50 μL of water, and one tablet was inserted under the tongue of the rat, 1 tablet/rat. The mouth was fixed for 30 min to prevent the tablet from falling off or sliding into the gastrointestinal tract. Taking the time of inserting the tablet under the tongue as the time point of 0 min, the whole blood and brain tissue were collected, at 5 min, 15 min, 30 min, 1 h, 2.5 h and 5 h.

2. Experimental Results

(48) Average of each pharmacokinetic parameter of edaravone in the plasma from SD rats after intravenous administration with compound edaravone injection and SD rats administrated with sublingual tablet are shown below.

(49) TABLE-US-00016 Intravenous Sublingual administration administration Tmax (h) 0.033 2.5 Cmax (ng/mL) 58550 4793 AUC.sub.0-5 h (h*ng/mL) 18179 15638 MRTlast (h) 0.59 2.06 F (%) / 62.6

(50) Average of each pharmacokinetic parameter of edaravone in the brain homogenate from SD rats after intravenous administration with compound edaravone injection and SD rats administrated with sublingual tablet are shown below.

(51) TABLE-US-00017 Intravenous Sublingual administration administration Tmax (h) 0.033 0.25 Cmax (ng/mL) 1405 55.3 AUC.sub.0-5 h (h*ng/mL) 270 106 MRTlast (h) 0.20 1.18 F (%) / 28.5

(52) Average of each pharmacokinetic parameter of borneol in the plasma from SD rats after intravenous administration with compound edaravone injection and SD rats administrated with sublingual tablet are shown below.

(53) TABLE-US-00018 Intravenous Sublingual administration administration Tmax (h) 0.033 1.00 Cmax (ng/mL) 1611 105 AUC.sub.0-5 h (h*ng/mL) 622 353 MRTlast (h) 0.64 1.86 F (%) / 51.6

(54) Average of each pharmacokinetic parameter of borneol in the brain homogenate from SD rats after intravenous administration with compound edaravone injection and SD rats administrated with sublingual tablet are shown below.

(55) TABLE-US-00019 Intravenous Sublingual administration administration Tmax (h) 0.033 0.5 Cmax (ng/mL) 5726 260 AUC.sub.0-5 h (h*ng/mL) 1686 529 MRTlast (h) 0.34 1.31 F (%) / 28.5

(56) The results of the distribution of the ingredients in the sublingual tablet in plasma and brain tissue of SD rats showed that the bioavailability of edaravone and (+)-2-borneol in the sublingual preparation was as follows: edaravone about 62.6%, (+)-2-borneol about 51.6%; the bioavailability in the brain was as follows: edaravone about 28.5%, (+)-2-borneol about 28.5%, indicating that the bioavailability of edaravone and borneol by sublingual administration was high, satisfying sublingual administration requirements.

(57) The sublingual tablets containing edaravone and (+)-2-borneol had advantages of good pharmacokinetic properties, high bioavailability, high brain permeability, and convenient administration.

Example 18

(58) Protective effect of sublingual administration on focal cerebral ischemia-reperfusion

1 Materials and Methods

1.1 Experimental Animals

(59) Sprague-Dawley (SD) rat, male, clean grade, weight 260-280 g

1.2 Test Drugs

(60) Sublingual tablets were prepared according to the ratio of Example 8 in three specifications: 0.67 mg of edaravone and 0.13 mg of borneol (3 mg kg dose group); 2.01 mg of edaravone and 0.39 mg of borneol (9 mg/kg dose group); 6 mg of edaravone and 1.2 mg of borneol (27 mg/kg dose group).

(61) Compound edaravone injection, specification; 5 mL:12.5 mg, produced by Nanjing Xiansheng Dongyuan Pharmaceutical Co., Ltd.

1.3 Experimental Methods

1.3.1 Preparation of Focal Cerebral Ischemia Reperfusion Model

(62) A cerebral ischemia reperfusion model was established by middle cerebral artery occlusion (MCAO) through internal carotid artery suture. After being anesthetized with 7% hydrated trichloroacetaldehyde (6 ml/kg) the animals were fixed on the operating table in prone position, the skin was disinfected, the neck was open, in the middle, and right common carotid artery, external carotid artery and internal carotid artery were separated, vagus nerve was gently removed, the external carotid artery was ligated, and by following the internal carotid artery, the pterygopalatine artery was ligated. The end of the common carotid artery proximity to the heart was clamped, and an incision was made from the distal end of the ligature line of the external carotid artery, into which a nylon catheter with an outer diameter of 0.285 mm was inserted. The nylon catheter entered into the internal carotid artery through bifurcation of the common carotid artery, and was slowly inserted until there was slight resistance (about 20 mm from the bifurcation). All blood supply to the middle cerebral artery was blocked. After the right cerebral suffered from ischemia for 2 h, the nylon catheter was gently puffed out, blood supply was restored for reperfusion, and the skin was sutured, and disinfected.

1.3.2 Animal Grouping and Administration

(63) The experimental animals were divided into 5 groups: three groups of sublingual tablet (dose 3 mg/kg, 9 mg/kg, 27 mg/kg), positive drug compound edaravone group (3 mg/kg) and model group. After the preparation of the cerebral ischemia model, the animals were randomly assigned to each group. The animals in the sublingual tablet group were administered sublingual tablets with corresponding specification simultaneously with reperfusion, 1 tablet/rat, and the mouths were fixed to prevent the tablets from falling off or sliding into the gastrointestinal tract until the tablets were completely absorbed. The animals in the positive drug group were intravenously administered once immediately after reperfusion, and the animals in the model group were administered with an equal volume of physiological saline. The symptoms of neurological deficit were evaluated 24 hours after cerebral ischemia, and then the animals were sacrificed, the brains were taken out and subjected to staining and imaging to determine the area of cerebral infarction.

13.3 Neurological Deficit Scoring and Cerebral Infarct Size Measurement

(64) Neurological deficit was evaluated using the modified Bederson 5-score scale. The symptoms of neurological deficit in rats after cerebral ischemia were evaluated by single-blind method. Specifically, the test designer marked the animals in groups. The one who scored the symptoms of neurological defects did not know the grouping of the animals. After the scoring was over, the score results of various markers were submitted to the test designer, and the designer disclosed the score for each animal of each test group.

(65) TABLE-US-00020 Annexed table: Assessment of Neurological Deficit (Bederson Scores) 0: When the tail is lifted and the body is hung in the air, the animal's forelimbs extend to the floor and there is no other behavioral deficit. 1: When the tail is lifted and the body is hung in the air, the animal's forelimbs on the contralateral (left) side of the surgery show wrist flexion, shoulder rotation, elbow abduction, and close to the chest wall. 2: The animal is placed on a smooth plate and the resistance reduces when pushed the shoulder in the surgical side to move to the contralateral side. 3: When the animal walks freely, it will circle or wind to the contralateral side of the surgery. 4: The limbs are soft and have no spontaneous activity.

(66) Determination of the degree of cerebral infarction

(67) After the animal was sacrificed, the brain were removed following decapitation, the olfactory bulb, cerebellum and low brain stem were removed. The blood on the surface of the brain was washed with physiological saline, and the residual water on the surface was removed. The brain was placed at −20° C. for 20 min. Immediately after removal, the plane intersected with the line of sight was cut vertically downwards to get a coronal section, and one slice every 2 mm backward was cut. The brain slices were incubated in 2% TTC dye solution (37° C. for 90 min) for staining. The normal brain tissue was stained dark red, and the ischemic tissue was in pale color. After washed with physiological saline, the brain slices were quickly arranged in a row from front to back, and the residual water on the surface were removed, and then photographed.

(68) The images were processed by image analysis software, and the corresponding volume of the left brain and the infarct volume were calculated according to formula, and the percentage of infarct was determined. Calculation of infarct volume:
V−t(A1+A2+A3+ . . . +An) t is the slice thickness and A is the infarct area.
% I=100%×(VC−VL)/VC % I is the percentage of infarct, volume, VC is the brain volume of the control side (left hemisphere), and VL is the volume of the non-infarct area of the infarct (right hemisphere).

2 Experimental Results

2.1 Effects on the Neurological Deficit

(69) The degree of neurological deficit in each group of animals was shown in Table 1. Compared with the model group, the three doses (3, 9, 27 mg/kg) of the sublingual tablet and the positive drug compound edaravone (3 mg/kg) both significantly ameliorated the neurological deficit.

(70) Table 1. Effects of sublingual administration of edaravone and (+)-2-borneol on the neurological deficit.

(71) TABLE-US-00021 TABLE 1 Effects of sublingual administration of edaravone and (+)-2-borneol on the neurological deficit Number of Scores of neurological Groups animals deficit Model group 12 2.58 ± 0.51  Compound edaravone group 13 1.77 ± 0.93*  Sublingual tablet (0.8 mg) 14 2.00 ± 0.68*  Sublingual tablet (2.4 mg) 12 1.75 ± 0.75** Sublingual tablet (7.2 mg) 13 1.54 ± 1.05** X ± SD. Compared to the model group, *p < 0.05, **p < 0.01.

2.2 Effects on the Area of Cerebral Infarction

(72) The effects on the area of cerebral infarction were shown in Table 2. Compared with the model group, the three doses (3, 9, 27 mg/kg) of sublingual tablet and the positive drug compound edaravone both significantly reduced the size of the cerebral infarction area in ischemia-reperfusion animal model.

(73) TABLE-US-00022 TABLE 2 Effects of sublingual administration of edaravone and (+)-2-borneol on the area of cerebral infarction Number of Area of cerebral Groups animals infarction (%) Model group 12 35.1 ± 11.5  Compound edaravone group 13 22.9 ± 13.0* Sublingual tablets (0.8 mg) 14 24.0 ± 10.0* Sublingual tablets (2.4 mg) 12 22.0 ± 11.4* Sublingual tablets (7.2 mg) 13  20.7 ± 13.1** X ± SD. Compared to the model group, *p < 0.05, **p < 0.01.

Example 19

(74) Study on the distribution of edaravone in plasma and brain tissue of SD rats after sublingual administration:

3. Materials and Methods

1.4 Experimental Animals

(75) Sprague-Dawley (SD) rats, SPF grade, male, weight 180-200 g.

(76) Source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

(77) Certificate number: 11400700138404.

(78) License number: SCXK (Beijing) 2012-0001.

(79) Foods and water supply: Rats were fasted for 12 h before the test, and foods were supplied 4 h after the administration. Water was not prohibited throughout the experiment. Abnormalities of the animals were observed and recorded during drug administration and sample collecting.

1.5 Test Drugs

(80) Compound edaravone injection: specification 12.5 mg/5 mL (edaravone and (+)-2-borneol were 10 mg/5 mL and 2.5 mg/5 mL, respectively).

(81) Sublingual tablets prepared according to the formulation ratio of Example 13, each containing 5 mg of edaravone and 1 mg of borneol.

1.6 Methods

(82) Group 1: Intravenous administration of compound edaravone injection (N=4)

(83) Dosage: 16 mg/kg edaravone, 4 mg/kg (+)-2-borneol; administration volume: 8 mL/kg. Plasma and brain tissue samples were collected at 2 min, 15 min, 30 min, 1 h, 2.5 h and 5 h. At each time point, whole blood and brain tissue were collected simultaneously.

(84) Group 2: one sublingual tablet was given under the tongue (N=6).

(85) SD rats were intraperitoneal injected with chloral hydrate to make SD rats under mild anesthesia. The mouth of rat was moistened with 50 μL of water, and one tablet was inserted under the tongue of the rat, 1 tablet/rat. The mouth was fixed for 30 min, to prevent the tablet from falling off or sliding into the gastrointestinal tract. Taking the time of inserting the tablet under the tongue as the time point of 0 min, the whole blood and brain tissue were collected at 5 min, 15 min, 30 min, 1 h, 2.5 h and 5 h.

4. Experimental Results

(86) Average of each pharmacokinetic parameter of edaravone in the plasma from SD rats after intravenous administration with compound edaravone injection and SD rats administrated with sublingual tablet are shown below.

(87) TABLE-US-00023 Intravenous Sublingual administration administration Tmax (h) 0.032 0.5 Cmax (ng/mL) 56370 5875 AUC.sub.0-5 h (h*ng/mL) 16038 17427 MRTlast (h) 0.59 2.63 F (%) / 79.9

(88) Average of each pharmacokinetic parameter of edaravone in the brain homogenate from SD rats after intravenous administration with compound edaravone injection and SD rats administrated with sublingual tablet are shown below.

(89) TABLE-US-00024 Intravenous Sublingual administration administration Tmax (h) 0.032 0.25 Cmax (ng/mL) 13.98 59.2 AUC.sub.0-5 h (h*ng/mL) 255 110 MRTlast (h) 0.20 1.16 F (%) / 30.1

(90) Average of each pharmacokinetic parameter of borneol in the plasma from SD rats after intravenous administration with compound edaravone injection and SD rats administrated with sublingual tablet are shown below.

(91) TABLE-US-00025 Intravenous Sublingual administration administration Tmax (h) 0.032 1.00 Cmax (ng/mL) 1571 114 AUC.sub.0-5 h (h*ng/mL) 593 392 MRTlast (h) 0.63 1.93 F (%) / 60.1

(92) Average of each pharmacokinetic parameter of borneol in the brain homogenate from SD rats after intravenous administration with compound edaravone injection and SD rats administrated with sublingual tablet are shown below.

(93) TABLE-US-00026 Intravenous Sublingual administration administration Tmax (h) 0.032 0.5 Cmax (ng/mL) 5430 273 AUC.sub.0-5 h (h*ng/mL) 1729 541 MRTlast (h) 0.34 1.30 F (%) / 29.6

(94) The results of the distribution of the ingredients in the sublingual tablet in plasma and brain tissue of SD rats showed that the bioavailability of edaravone and (+)-2-borneol in the sublingual preparation was as follows: edaravone about 79.9%, (+)-2-borneol about 60.1%; the bioavailability in the brain was as follows: edaravone about 30.1%, (+)-2,-borneol about 29.6%, indicating that the bioavailability of edaravone and borneol by sublingual administration was high, satisfying sublingual administration requirements.

(95) The sublingual tablets containing edaravone and (+)-2-borneol had advantages of good pharmacokinetic properties, high bioavailability, high brain permeability, and convenient administration.

Example 20

(96) Protective effect of sublingual administration on focal cerebral ischemia-reperfusion

1 Materials and Methods

1.1 Experimental Animals

(97) Sprague-Dawley (SD) rat, male, clean grade, weight 260-280 g

1.2 Test Drugs

(98) Sublingual tablets were prepared according to the ratio of Example 13 in three specifications: 0.67 mg of edaravone and 0.13 mg of borneol (3 mg/kg dose group); 2.01 mg of edaravone and 0.39 mg of borneol (9 mg/kg dose group); 6 mg of edaravone and 1.2 mg of borneol (27 mg/kg dose group).

(99) Compound edaravone injection, specification: 5 mL:12.5 mg, produced by Nanjing Xiansheng Dongyuan Pharmaceutical Co., Ltd.

1.3 Experimental Methods

1.3.1 Preparation of Focal Cerebral Ischemia Reperfusion Model

(100) A cerebral ischemia reperfusion model was established by middle cerebral artery occlusion (MCAO) through internal carotid artery suture. After being anesthetized with 7% hydrated, trichloroacetaldehyde (6 ml/kg), the animals were fixed on the operating table in prone position, the skin was disinfected, the neck was open in the middle, and right common carotid artery, external carotid artery and internal carotid artery were separated vagus nerve was gently removed, the external carotid artery was ligated, and by following the internal carotid artery, the pterygopalatine artery was ligated. The end of the common carotid artery proximity to the heart was clamped, and an incision was made from the distal end of the ligature line of the external carotid artery, into which a nylon catheter with an outer diameter of 0.285 mm was inserted. The nylon catheter entered into the internal carotid artery through bifurcation of the common carotid artery, and was slowly inserted until there was slight resistance (about 20 mm from the bifurcation). All blood supply to the middle cerebral artery was blocked. After the right cerebral suffered from ischemia for 2 h, the nylon catheter was gently pulled out, blood supply was restored for reperfusion, and the skin was sutured, and disinfected.

1.3.2 Animal Grouping and Administration

(101) The experimental animals were divided into 5 groups: three groups of sublingual tablet (dose 3 mg/kg, 9 mg/kg, 27 mg/kg), positive drug compound edaravone group (3 mg/kg) and model group. After the preparation of the cerebral ischemia model, the animals were randomly assigned to each group. The animals in the sublingual tablet group were administered sublingual tablets with corresponding specification simultaneously with reperfusion, 1 tablet/rat, and the mouths were fixed to prevent the tablets from falling off or sliding into the gastrointestinal tract until the tablets were completely absorbed. The animals in the positive drug group were intravenously administered once immediately after reperfusion, and the animals in the model group were administered with an equal volume of physiological saline. The symptoms of neurological deficit were evaluated 24 hours after cerebral ischemia, and then the animals were sacrificed, the brains were taken out and subjected to staining and imaging to determine the area of cerebral infarction.

1.3.3 Neurological Deficit Scoring and Cerebral Infarct Size Measurement

(102) Neurological deficit was evaluated using the modified Bederson 5-score scale. The symptoms of neurological deficit in rats after cerebral ischemia were evaluated by single-blind method. Specifically, the test designer marked the animals in groups. The one who scored the symptoms of neurological defects did not know the grouping of the animals. After the scoring was over, the score results of various markers were submitted to the designer, and the test designer disclosed the score for each animal of each test group.

(103) TABLE-US-00027 Annexed table: Assessment of Neurological Deficit (Bederson Scores) 0: When the tail is lifted and the body is hung in the air, the animal's forelimbs extend to the floor and there is no other behavioral deficit. 1: When the tail is lifted and the body is hung in the air, the animal's forelimbs on the contralateral (left) side of the surgery show wrist flexion, shoulder rotation, elbow abduction, and close to the chest wall. 2: The animal is placed on a smooth plate and the resistance reduces when pushed the shoulder in the surgical side to move to the contralateral side. 3: When the animal walks freely, it will circle or wind to the contralateral side of the surgery. 4: The limbs are soft and have no spontaneous activity.

(104) Determination of the Degree of Cerebral Infarction

(105) After the animal was sacrificed, the brain were removed following decapitation, the olfactory bulb, cerebellum and low brain stem were removed. The blood on the surface of the brain was washed with physiological saline, and the residual water on the surface was removed. The brain was placed at −20° C. for 20 min. Immediately after removal, the plane intersected with the line of sight was cut vertically downwards to get a coronal section, and one slice every 2 mm backward was cut. The brain slices were incubated in 2% TTC dye solution (37° C. for 90 min) for staining. The normal brain tissue was stained dark red, and the ischemic brain tissue was in to pale color. After washed with physiological saline, the brain slices were quickly arranged in a row from front to back, and the residual water on the surface were removed, and then photographed.

(106) The images were processed by image analysis software, and the corresponding volume of the left brain and the infarct volume were calculated according to formula, and the percentage of infarct volume was determined. Calculation of infarct volume;
V=t(A1+A2+A3 . . . +An) t is the slice thickness and A is the infarct area.
% I=100%×(VC−VL)/VC % I is the percentage of infarct volume, VC is the brain volume of the control side (left hemisphere), and VL is the volume of the non-infarct area of the infarct side (right hemisphere).

2 Experimental Results

2.1 Effects on the Neurological Deficit

(107) The degree of neurological deficit in each group of animals was shown in Table 3. Compared with the model group, the three doses (3, 9, 27 mg/kg) of the sublingual tablet and the positive drug compound edaravone (3 mg/kg) both significantly ameliorated the neurological deficit.

(108) TABLE-US-00028 TABLE 3 Effects of sublingual administration of edaravone and (+)-2-borneol on the neurological deficit Number of Scores of neurological Groups animals deficit Model group 11 2.64 ± 0.42  Compound edaravone group 12 1.74 ± 0.71*  Sublingual tablets (0.8 mg) 14 2.35 ± 0.73*  Sublingual tablets (2.4 mg) 14 1.83 ± 0.77** Sublingual tablets (7.2 mg) 13 1.43 ± 0.95** X ± SD. Compared to the model group, *p < 0.05, **p < 0.01.

2.2 Effects on the Area of Cerebral Infarction

(109) The effects on the area of cerebral infarction were shown in Table 4. Compared with the model group, the three doses (3, 9, 27 mg/kg) of sublingual tablet and the positive drug compound edaravone both significantly reduced the size of the cerebral infarction area in ischemia-reperfusion animal model.

(110) Table 4. Effects of sublingual administration of edaravone and (+)-2-borneol on the area of cerebral infraction.

(111) TABLE-US-00029 TABLE 4 Effects of sublingual administration of edaravone and (+)-2-borneol on the area of cerebral infarction Number of Area of cerebral Groups animals infarction (%) Model group 11 37.2 ± 12.3 Compound edaravone group 12  21.6 ± 12.5* Sublingual tablets (0.8 mg) 14 23.4 ± 9.3* Sublingual tablets (2.4 mg) 14 21.3 ± 8.5* Sublingual tablets (7.2 mg) 13  19.8 ± 10.1** X ± SD. Compared to the model group, *p < 0.05, **p < 0.01.

(112) The experimental data showed that the sublingual tablet of the present disclosure can achieve a drug effect comparable to that of an injection.