Method for analyzing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, treatment of amyotrophic lateral sclerosis and inhibition of progression of amyotrophic lateral sclerosis, and method for manufacturing a drug containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance

Abstract

To provide an analysis method for accurately and easily measuring a phenyl hydrazine included in a 3-methyl-1-phenyl-2-pyrazoline-5-on bulk drug.SOLUTION: The method for analyzing a phenyl hydrazine content in a 3-methyl-1-phenyl-2-pyrazoline-5-on bulk drug comprises: measuring a phenyl hydrazine content of a standard solution including a phenyl hydrazine or its salt, and first acidic water and first water-soluble organic solvents and showing a phenyl hydrazine concentration of 0.01 μg / mL-10 μg / mL to acquire a first measurement value; measuring a phenyl hydrazine content of a 3-methyl-1-phenyl-2-pyrazoline-5-on sample solution including a 3-methyl-1-phenyl-2-pyrazoline-5-on bulk drug and second acidic water and second water-soluble organic solvents to acquire a second measurement value; and detecting the phenyl hydrazine contents of the 3-methyl-1-phenyl-2-pyrazoline-5-on bulk drugs from the first and second measurement values. The first acidic water is at least one selected from a hydrochloric acid, an acetic acid aqueous solution, etc., and the first water-soluble organic solvent is at least one selected from the group consisting of acetonitrile and methanol. The second acidic water is at least one selected from a hydrochloric acid, an acetic acid aqueous solution, etc., and the second water-soluble organic solvent is at least one selected from the group consisting of acetonitrile and methanol.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, a method for treating and inhibiting the progression of amyotrophic lateral sclerosis (hereinafter sometimes referred to as ALS), and a method for producing a drug containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance. [Background technology]

[0002] ALS, a motor neuron disease, is an intractable disease that begins with initial symptoms such as hand weakness, finger movement disorder, and upper limb fasciculations. It progresses through muscle atrophy, muscle weakness, bulbar paralysis, and muscle fasciculations, eventually leading to respiratory failure. ALS is classified into upper limb, bulbar, lower limb, and mixed types depending on the site of onset. In all types, muscle groups throughout the body are affected as symptoms progress. Drugs containing 3-methyl-1-phenyl-2-pyrazolin-5-one, an active ingredient, have been approved and are in clinical use in Japan and the United States as effective treatments for ALS and slowing disease progression (generic name: edaravone, trade names: Radicut® and Radicava®, manufactured and sold by Mitsubishi Tanabe Pharma Corporation, etc.).

[0003] Phenylehydrazine is one of the impurities that may be contained in pharmaceuticals. A method for analyzing phenylhydrazine in 3-methyl-1-phenyl-2-pyrazolin-5-one has been reported in which a solution of ammonium dihydrogen phosphate (pH adjusted to 2.0 with phosphoric acid) in methanol is prepared as a dissolving solution, and this solution is analyzed by HPLC using a solution of ammonium dihydrogen phosphate (pH adjusted to 3.5 with phosphoric acid) in methanol as a mobile phase (Non-Patent Document 1). The disclosure of this method is incorporated herein by reference. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Central South Pharmacy, August 2014, Vol.12 No.8, 814-816. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide an analytical method for accurately and simply measuring the amount of phenylhydrazine contained in a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance. Another objective of the present invention is to provide a method for treating amyotrophic lateral sclerosis and suppressing the progression of amyotrophic lateral sclerosis using a drug containing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance in which the phenylhydrazine content is strictly controlled. Another object of the present invention is to provide a method for producing a drug containing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance in which the phenylhydrazine content is strictly controlled. [Means for solving the problem]

[0006] A method for analyzing the phenylhydrazine content in a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance according to one embodiment of the present invention includes: measuring the phenylhydrazine content of a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, a first acidic water, and a first water-soluble organic solvent and exhibiting a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL to obtain a first measurement value; measuring the phenylhydrazine content of a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, a second acidic water, and a second water-soluble organic solvent to obtain a second measurement value; and detecting the phenylhydrazine content in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first and second measurement values, wherein the first acidic water is selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, a trifluoroacetic acid solution, a formic acid solution, an aqueous phosphoric acid solution, and an aqueous perchloric acid solution. the first water-soluble organic solvent is at least one water-soluble organic solvent selected from the group consisting of acetonitrile and methanol, and when the first water-soluble organic solvent is methanol, the first acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, and an aqueous perchloric acid solution, and the second acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, an aqueous phosphoric acid solution, and an aqueous perchloric acid solution, and the second water-soluble organic solvent is at least one water-soluble organic solvent selected from the group consisting of acetonitrile and methanol, and when the second water-soluble organic solvent is methanol, the second acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, and an aqueous perchloric acid solution.

[0007] A method for treating amyotrophic lateral sclerosis according to another aspect of the present invention includes: measuring the phenylhydrazine content of a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, first acidic water, and a first water-soluble organic solvent, the phenylhydrazine standard solution having a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL, to obtain a first measurement value; measuring the phenylhydrazine content of a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, second acidic water, and a second water-soluble organic solvent, to obtain a second measurement value; detecting the phenylhydrazine content in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first and second measurement values to determine that the phenylhydrazine amount is 20 ppm or less; and administering the drug containing the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance to a patient in need thereof, wherein the first acidic water is selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, and formic acid. the first water-soluble organic solvent is at least one water-soluble organic solvent selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, a trifluoroacetic acid solution, a formic acid solution, and a perchloric acid solution; and the second acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, a trifluoroacetic acid solution, a formic acid solution, a phosphoric acid solution, and a perchloric acid solution; the second water-soluble organic solvent is at least one water-soluble organic solvent selected from the group consisting of acetonitrile and methanol; and when the second water-soluble organic solvent is methanol, the second acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, a trifluoroacetic acid solution, a formic acid solution, a phosphoric acid solution, and a perchloric acid solution.

[0008] A method for suppressing the progression of amyotrophic lateral sclerosis according to yet another aspect of the present invention includes a method for suppressing the progression of amyotrophic lateral sclerosis, the method comprising administering phenylhydrazine or a salt thereof, a first acidic water, and a first water-soluble organic solvent to a subject in a concentration of 0.01 μg / mL to 10 measuring the phenylhydrazine content of a phenylhydrazine standard solution showing a phenylhydrazine concentration of μg / mL to obtain a first measurement value; measuring the phenylhydrazine content of a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, second acidic water, and a second water-soluble organic solvent to obtain a second measurement value; detecting the phenylhydrazine content in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first measurement value and the second measurement value to determine that the phenylhydrazine amount is 20 ppm or less; and administering the drug containing the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance to a patient in need thereof, wherein the first acidic water is at least one selected from the group consisting of hydrochloric acid, an aqueous solution of acetic acid, an aqueous solution of trifluoroacetic acid, an aqueous solution of formic acid, an aqueous solution of phosphoric acid, and an aqueous solution of perchloric acid. the first water-soluble organic solvent is at least one water-soluble organic solvent selected from the group consisting of acetonitrile and methanol; when the first water-soluble organic solvent is methanol, the first acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, and an aqueous perchloric acid solution; and the second acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, a phosphoric acid solution, and an aqueous perchloric acid solution; the second water-soluble organic solvent is at least one water-soluble organic solvent selected from the group consisting of acetonitrile and methanol; when the second water-soluble organic solvent is methanol, the second acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, and an aqueous perchloric acid solution.

[0009] In yet another aspect of the present invention, a method for producing a drug containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance having a phenylhydrazine content of 20 ppm or less includes the steps of: measuring the phenylhydrazine content of a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, first acidic water, and a first water-soluble organic solvent, the phenylhydrazine standard solution exhibiting a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL to obtain a first measurement value; measuring the phenylhydrazine content of a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing an organic solvent to obtain a second measured value; detecting the phenylhydrazine content in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first and second measured values to determine that the phenylhydrazine content is 20 ppm or less; and mixing the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance with at least one pharmacologically and pharmaceutically acceptable excipient, The first acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, a trifluoroacetic acid solution, a formic acid solution, a phosphoric acid solution, and a perchloric acid solution, the first water-soluble organic solvent is at least one water-soluble organic solvent selected from the group consisting of acetonitrile and methanol. When the first water-soluble organic solvent is methanol, the first acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, a trifluoroacetic acid solution, a formic acid solution, and a perchloric acid solution, and the second acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, a trifluoroacetic acid solution, a formic acid solution, a phosphoric acid solution, and a perchloric acid solution, the second water-soluble organic solvent is at least one water-soluble organic solvent selected from the group consisting of acetonitrile and methanol. When the second water-soluble organic solvent is methanol, the second acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, a trifluoroacetic acid solution, a formic acid solution, and a perchloric acid solution. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide an analytical method for accurately and simply measuring the amount of phenylhydrazine contained in a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance. Furthermore, according to the present invention, a method for treating amyotrophic lateral sclerosis and suppressing the progression of amyotrophic lateral sclerosis can be provided using a drug containing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance in which the phenylhydrazine content is strictly controlled. Furthermore, the present invention can provide a method for producing a drug containing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance in which the phenylhydrazine content is strictly controlled. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows a chromatogram obtained by HPLC measurement of a phenylhydrazine solution prepared in a 0.1 mol / L hydrochloric acid / acetonitrile mixture (6:4). [Figure 2] FIG. 2 shows a chromatogram obtained by HPLC measurement of a phenylhydrazine solution prepared in a 0.1 mol / L hydrochloric acid / acetonitrile mixture (4:6). [Figure 3] FIG. 3 shows a chromatogram obtained by HPLC measurement of a phenylhydrazine solution prepared in a 0.1 mol / L hydrochloric acid / acetonitrile mixture (2:8). [Figure 4] FIG. 4 shows a chromatogram obtained by HPLC measurement of a phenylhydrazine solution prepared in a 1 mol / L hydrochloric acid / acetonitrile mixture (6:4). [Figure 5] FIG. 5 is a chromatogram obtained by HPLC measurement of a phenylhydrazine solution prepared in a 0.1 mol / L hydrochloric acid / acetonitrile mixture (7:3). [Figure 6] FIG. 6 is a graph showing the change over time in the ratio (%) of the peak area to the initial value of phenylhydrazine contained in an added sample solution prepared with a 0.1 mol / L hydrochloric acid / acetonitrile mixed solution (7:3). [Figure 7]FIG. 7 is a graph showing the change over time in the ratio (%) of the peak area to the initial value of phenylhydrazine contained in an added sample solution prepared with a 0.1 mol / L hydrochloric acid / methanol mixed solution (5:5). DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, with reference to the accompanying drawings, embodiments of a method for analyzing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, a method for treating amyotrophic lateral sclerosis (hereinafter also referred to as ALS), a method for inhibiting the progression of amyotrophic lateral sclerosis, and a method for producing a drug containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance will be described in detail.

[0013] The method for analyzing the phenylhydrazine content in a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance of the present invention includes measuring the phenylhydrazine content of a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, a first acidic water, and a first water-soluble organic solvent, and exhibiting a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL, to obtain a first measurement value; measuring the phenylhydrazine content of a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, a second acidic water, and a second water-soluble organic solvent, to obtain a second measurement value; and detecting the phenylhydrazine content in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first and second measurement values. The method for analyzing the phenylhydrazine content in a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance of the present invention may further include other steps as necessary.

[0014] 3-Methyl-1-phenyl-2-pyrazolin-5-one can be represented by the following structural formula: 3-Methyl-1-phenyl-2-pyrazolin-5-one exists as a tautomer shown in the following structural formula.

[0015] [ka]

[0016] The phenylhydrazine content of the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance may be determined by analyzing a sample solution of the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance by one or more quantitative analytical techniques and comparing the test results with those of a substantially pure phenylhydrazine standard solution.

[0017] The sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance and the phenylhydrazine standard solution are preferably prepared in a solvent in which the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance and phenylhydrazine or a salt thereof are completely dissolved, respectively. Use of such a solution enables accurate measurement of the phenylhydrazine contained in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance. Complete dissolution of the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance and phenylhydrazine or its salt can be confirmed visually. In addition, to evaluate trace amounts of dissolved residue, this can be confirmed by filtration and HPLC analysis.

[0018] It is desirable that the sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance and the phenylhydrazine standard solution are prepared so that 3-methyl-1-phenyl-2-pyrazolin-5-one or its physiologically acceptable salt, and / or phenylhydrazine do not substantially decompose between the time of preparation and analysis after a certain period of time has elapsed. Phenylhydrazine is one of the decomposition products of 3-methyl-1-phenyl-2-pyrazolin-5-one or its physiologically acceptable salt, and the degree of decomposition of 3-methyl-1-phenyl-2-pyrazolin-5-one or its physiologically acceptable salt can be confirmed by measuring the change in the amount of phenylhydrazine in the solution. The phenylhydrazine content in the sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance and / or the phenylhydrazine standard solution can be measured by the analytical method using HPLC described below.

[0019] Specifically, the fact that phenylhydrazine does not substantially decompose means that the measured value of phenylhydrazine measured within 30 minutes after preparing a phenylhydrazine standard solution does not change by 20% or more, more preferably by 10% or more, and even more preferably by 5% or more, between the measured value and the measured value measured after a certain time has elapsed. Furthermore, for 3-methyl-1-phenyl-2-pyrazolin-5-one or a physiologically acceptable salt thereof to not substantially decompose, specifically, the measured value of phenylhydrazine measured within 30 minutes of preparing a sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance and the measured value of phenylhydrazine measured after a certain time period preferably do not change by 4 ppm or more relative to the amount of 3-methyl-1-phenyl-2-pyrazolin-5-one, more preferably by 2 ppm or more, and even more preferably by 1 ppm or more. The certain period of time is not particularly limited, but is preferably 2 hours, 3 hours, 4 hours, 5 hours, 9 hours, 10 hours, 12 hours, or 24 hours, more preferably 2 hours, 3 hours, 4 hours, 5 hours, 9 hours, 10 hours, or 12 hours, and particularly preferably 2 hours, 3 hours, 4 hours, or 5 hours.

[0020] The sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance contains 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, acidic water, and a water-soluble organic solvent.

[0021] The acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, an aqueous phosphoric acid solution, and an aqueous perchloric acid solution. These acidic waters may be used alone, or two or more types of acidic waters may be mixed in an appropriate ratio. Preferably, at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, and an aqueous formic acid solution is used, and more preferably, hydrochloric acid.

[0022] The acid concentration of the acidic water is preferably 0.01 mol / L to 5 mol / L, more preferably 0.02 mol / L to 1 mol / L, even more preferably 0.05 mol / L to 0.2 mol / L, and particularly preferably 0.1 mol / L.

[0023] The water-soluble organic solvent is at least one selected from the group consisting of acetonitrile and methanol. These water-soluble organic solvents may be used alone or in combination in an appropriate ratio. Acetonitrile is preferred.

[0024] When the water-soluble organic solvent is methanol, the acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous solution of acetic acid, an aqueous solution of trifluoroacetic acid, an aqueous solution of formic acid, and an aqueous solution of perchloric acid.

[0025] The ratio of acidic water to water-soluble organic solvent can be changed as appropriate. The volume ratio of acidic water to water-soluble organic solvent can be 3:1 to 4:5, preferably 3:1 to 5:5, and more preferably 3:1 to 6:4. For example, the volume ratio of acidic water to water-soluble organic solvent can be 7:3, 6:4, or 5:5. When the acidic water and water-soluble organic solvent are hydrochloric acid and acetonitrile, a particularly preferred volume ratio is 7:3. When the acidic water and water-soluble organic solvent are hydrochloric acid and methanol, a particularly preferred volume ratio is 5:5.

[0026] The method for preparing the sample solution is not particularly limited as long as it contains 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, acidic water, and a water-soluble organic solvent. The sample solution may be prepared by mixing prepared acidic water with 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance and a water-soluble organic solvent, or by mixing water with 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance and / or a water-soluble organic solvent and then adding the corresponding acid.

[0027] The phenylhydrazine standard solution contains phenylhydrazine or a salt thereof, acidic water, and a water-soluble organic solvent. The phenylhydrazine standard solution may further contain 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance.

[0028] Examples of salts of phenylhydrazine include salts with mineral acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, and phosphoric acid. Of phenylhydrazine or its salts, salts of phenylhydrazine are preferred. Of the salts of phenylhydrazine, phenylhydrazine hydrochloride is preferred. In the analytical method of the present invention, phenylhydrazine hydrochloride is less likely to decompose during storage than phenylhydrazine, and therefore phenylhydrazine or its salts are most preferred for use in the standard solution of the present invention.

[0029] It is desirable that phenylhydrazine or its salt be substantially pure. Specifically, a purity of at least 90% or more is preferred, more preferably at least 95% or more, at least 98% or more, and even more preferably at least 99% or more. Phenylhydrazine hydrochloride with a purity of 98% or more or 99% or more is commercially available (e.g., phenylhydrazine hydrochloride, 99%; Alfa Aesar; code: A14645), so commercially available products may be used.

[0030] The acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, an aqueous phosphoric acid solution, and an aqueous perchloric acid solution. These acidic waters may be used alone, or two or more types of acidic waters may be mixed in an appropriate ratio. Preferably, at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, and an aqueous formic acid solution is used, and more preferably, hydrochloric acid.

[0031] The acid concentration of the acidic water is preferably 0.01 mol / L to 5 mol / L, more preferably 0.02 mol / L to 1 mol / L, even more preferably 0.05 mol / L to 0.2 mol / L, and particularly preferably 0.1 mol / L.

[0032] The water-soluble organic solvent is at least one selected from the group consisting of acetonitrile and methanol. These water-soluble organic solvents may be used alone or in combination in an appropriate ratio. Acetonitrile is preferred.

[0033] When the water-soluble organic solvent is methanol, the acidic water is at least one selected from the group consisting of hydrochloric acid, an aqueous solution of acetic acid, an aqueous solution of trifluoroacetic acid, an aqueous solution of formic acid, and an aqueous solution of perchloric acid. The phenylhydrazine concentration in the phenylhydrazine standard solution can be adjusted appropriately depending on the phenylhydrazine concentration in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance to be analyzed. For example, the concentration of phenylhydrazine in the phenylhydrazine standard solution (the concentration of phenylhydrazine in the case of free phenylhydrazine, or the concentration equivalent to phenylhydrazine in the case of a phenylhydrazine salt) can be set to 0.01 μg / mL to 10 μg / mL, preferably 0.05 μg / mL to 2 μg / mL, and particularly preferably 0.1 μg / mL to 1 μg / mL. Alternatively, the phenylhydrazine concentration of the phenylhydrazine standard solution can be adjusted to a concentration equivalent to 1 ppm to 200 ppm based on the dilution ratio of the sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance. A ppm-equivalent concentration refers to a concentration corresponding to the dilution ratio of the sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance. For example, when 0.10 g of 3-methyl-1-phenyl-2-pyrazolin-5-one is mixed with acidic water and a water-soluble organic solvent to make 10 mL, the concentration equivalent to 1 ppm is 0.01 μg / mL. The concentration is preferably equivalent to 5 ppm to 100 ppm, and more preferably equivalent to 10 ppm to 50 ppm.

[0034] In one embodiment of the present invention, a first measurement value is obtained by measuring the phenylhydrazine content of a phenylhydrazine standard solution exhibiting a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL. The number of first measurements is not particularly limited as long as it allows comparison with a second measurement value obtained by measuring the phenylhydrazine content of a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution, and may be one or more.

[0035] The ratio of acidic water to water-soluble organic solvent can be changed as appropriate. The volume ratio of acidic water to water-soluble organic solvent can be 3:1 to 4:5, preferably 3:1 to 5:5, and more preferably 3:1 to 6:4. For example, the volume ratio of acidic water to water-soluble organic solvent can be 7:3, 6:4, or 5:5. 7:3 is particularly preferred. When the acidic water and water-soluble organic solvent are hydrochloric acid and acetonitrile, a particularly preferred volume ratio is 7:3. When the acidic water and water-soluble organic solvent are hydrochloric acid and methanol, a particularly preferred volume ratio is 5:5.

[0036] The method for preparing the phenylhydrazine standard solution is not particularly limited as long as it contains phenylhydrazine or a salt thereof, acidic water, and a water-soluble organic solvent. The phenylhydrazine standard solution may be prepared by mixing prepared acidic water with phenylhydrazine or a salt thereof, and a water-soluble organic solvent, or by mixing water with phenylhydrazine or a salt thereof, and / or a water-soluble organic solvent, and then adding the corresponding acid.

[0037] The acidic water in the sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance is preferably the same as the acidic water in the phenylhydrazine standard solution. The water-soluble organic solvent in the sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance is preferably the same as the water-soluble organic solvent in the phenylhydrazine standard solution. The volume ratio of the acidic water to the water-soluble organic solvent in the sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance is preferably the same as the volume ratio of the acidic water to the water-soluble organic solvent in the phenylhydrazine standard solution. More preferably, the volume ratios of the acidic water, the water-soluble organic solvent, and the acidic water to the water-soluble organic solvent in the sample solution of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance are the same as the volume ratios of the acidic water, the water-soluble organic solvent, and the acidic water to the water-soluble organic solvent in the phenylhydrazine standard solution, respectively.

[0038] Quantitative analytical techniques include high performance liquid chromatography (HPLC). High performance liquid chromatography includes high performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC). When using high performance liquid chromatography, the number of theoretical plates of the phenylhydrazine peak is preferably as large as possible. For example, the number of theoretical plates N determined by the half-width method can be 1000 or more, more preferably 3000 or more.

[0039] formula 1 Number of theoretical plates: N = 5.54 × (tr / W 0.5 ) 2 tr: retention time W 0.5 : Peak half-height width

[0040] The symmetry coefficient S is an index that represents the degree of symmetry of a peak on a chromatogram; the closer the symmetry coefficient S is to 1, the more symmetric the peak (normal distribution). The closer the symmetry coefficient S is to 1, the higher the performance of the analytical method in terms of trueness, precision, limit of quantitation, etc., and therefore it is preferable to set the symmetry coefficient S to, for example, 2.0 or less, more preferably 1.5 or less.

[0041] formula 2 Symmetry factor: S = W 0.05h / 2f W 0.05h : Peak width at 1 / 20 of the peak height from the peak baseline 2f: W 0.05h The distance on the rising side of the peak obtained by dividing the peak width in half by a perpendicular line drawn from the peak apex to the horizontal axis

[0042] Examples of physiologically acceptable salts of 3-methyl-1-phenyl-2-pyrazolin-5-one or physiologically acceptable salts thereof include salts with mineral acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc.; salts with organic acids such as methanesulfonic acid, p-toluenesulfonic acid, acetic acid, oxalic acid, citric acid, malic acid, fumaric acid, etc.; salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as magnesium; and salts with amines such as ammonia, ethanolamine, 2-amino-2-methyl-1-propanol, etc. Physiologically acceptable salts also include hydrates and solvates.

[0043] The term "3-methyl-1-phenyl-2-pyrazolin-5-one drug substance" refers to the active ingredient 3-methyl-1-phenyl-2-pyrazolin-5-one or a physiologically acceptable salt thereof. One of the impurities that may be contained in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance is phenylhydrazine. In the manufacturing method described in European Patent Publication No. 208874 (or Japanese Patent Publication No. 5-31523), phenylhydrazine is used as a precursor for synthesizing 3-methyl-1-phenyl-2-pyrazolin-5-one.

[0044] Phenylhydrazine may increase during the storage period of solution formulations containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance. Phenylhydrazine is a mutagenic compound, and industrial hygiene exposure limits have been established in various countries. The United States has the strictest exposure limit for phenylhydrazine, at 2,860 μg / kg / year. Phenylhydrazine is easily decomposed by light and oxygen.

[0045] The amount of phenylhydrazine impurity contained in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance is preferably as small as possible, for example, preferably 20 ppm or less, more preferably 10 ppm or less. When 3-methyl-1-phenyl-2-pyrazolin-5-one is used to treat or slow the progression of ALS, it is preferable to continue drug treatment with this drug for the rest of the patient's life after starting administration of this drug, and therefore it is preferable to strictly control the amount of phenylhydrazine.

[0046] One embodiment of the present invention is a method for producing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance having a phenylhydrazine content of 20 ppm or less, comprising: measuring the phenylhydrazine content of a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, first acidic water, and a first water-soluble organic solvent, the phenylhydrazine standard solution exhibiting a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL to obtain a first measurement value; measuring the phenylhydrazine content of a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, the second acidic water, and the second water-soluble organic solvent to obtain a second measurement value; and detecting the phenylhydrazine content in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first measurement value and the second measurement value, and determining that the phenylhydrazine content is 20 ppm or less; wherein the first acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, an aqueous phosphoric acid solution, and an aqueous perchloric acid solution, the first water-soluble organic solvent is at least one water-soluble organic solvent selected from the group consisting of acetonitrile and methanol, and when the first water-soluble organic solvent is methanol, the first acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, and an aqueous perchloric acid solution, and The second acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, an aqueous phosphoric acid solution, and an aqueous perchloric acid solution, and the second water-soluble organic solvent is at least one water-soluble organic solvent selected from the group consisting of acetonitrile and methanol. When the second water-soluble organic solvent is methanol, the second acidic water is at least one acidic water selected from the group consisting of hydrochloric acid, an aqueous acetic acid solution, an aqueous trifluoroacetic acid solution, a formic acid solution, and an aqueous perchloric acid solution.

[0047] One embodiment of the present invention is a drug for treating or inhibiting the progression of amyotrophic lateral sclerosis, which contains 3-methyl-1-phenyl-2-pyrazolin-5-one, a drug substance produced by the production method of the present invention, in which the phenylhydrazine content in the drug substance is 20 ppm or less.

[0048] A further embodiment of the present invention is a drug comprising a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance for treating amyotrophic lateral sclerosis or suppressing the progression of the disease, wherein the amount of phenylhydrazine in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance is determined to be 20 ppm or less by analysis. Furthermore, it may contain 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance and pharmacologically and pharmaceutically acceptable additives, and may also contain one or more drug substances other than 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance. A drug containing a pharmacologically and pharmaceutically acceptable additive according to one embodiment of the present invention may be produced by a manufacturing method including mixing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance having a phenylhydrazine content of 20 ppm or less with a pharmacologically and pharmaceutically acceptable additive.

[0049] 3-methyl-1-phenyl-2-pyrazolin-5-one or a physiologically acceptable salt thereof, which is an active ingredient according to one embodiment of the present invention, may be administered to a patient as is, but it is preferable to add the active ingredient to pharmacologically and pharmaceutically acceptable additives and administer it as the above-mentioned drug.

[0050] Examples of pharmacologically and pharmaceutically acceptable additives that may be used include excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers or solubilization aids, isotonicity agents, pH adjusters, stabilizers, propellants, adhesives, etc. Examples of drugs suitable for oral administration include tablets, capsules, powders, fine granules, granules, liquids, and syrups, while examples of drugs suitable for parenteral administration include injections, drip infusions, patches, and suppositories.

[0051] Pharmaceuticals suitable for oral administration may contain additives such as excipients such as glucose, lactose, D-mannitol, starch, or crystalline cellulose; disintegrants or disintegration aids such as carboxymethylcellulose, starch, or carboxymethylcellulose calcium; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, or gelatin; lubricants such as magnesium stearate or talc; coating agents such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, or titanium oxide; and bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat.

[0052] Drugs suitable for injection or infusion may contain formulation additives such as solubilizers or solubilizing agents that can form aqueous or ready-to-use injections, such as distilled water for injection, physiological saline, and propylene glycol; isotonicity agents, such as glucose, sodium chloride, D-mannitol, and glycerin; and pH adjusters, such as inorganic acids, organic acids, inorganic bases, and organic bases.

[0053] The generic name "Edaravone" and the trade names "Radicut (registered trademark)" and "Radicava (registered trademark)" which contain 3-methyl-1-phenyl-2-pyrazolin-5-one as an active ingredient and are already in clinical use as a neuroprotectant and a treatment for ALS and an inhibitor of disease progression, and which are manufactured and sold by Mitsubishi Tanabe Pharma Corporation, may be used as 3-methyl-1-phenyl-2-pyrazolin-5-one or a physiologically acceptable salt thereof in the medicament and method of the present invention.

[0054] A method for treating or inhibiting the progression of amyotrophic lateral sclerosis according to one embodiment of the present invention comprises administering to a patient in need thereof a drug containing an effective amount of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, wherein the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance in the drug is analyzed for phenylhydrazine content, and the amount of phenylhydrazine in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance is 20 ppm or less.

[0055] In this embodiment, the method may further include obtaining a first measurement value by measuring the phenylhydrazine content in a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, a first acidic water, and a first water-soluble organic solvent, and exhibiting a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL; obtaining a second measurement value by measuring the phenylhydrazine content in a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, a second acidic water, and a second water-soluble organic solvent; and detecting the phenylhydrazine content in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first and second measurement values, and determining that the phenylhydrazine amount is 20 ppm or less.

[0056] In one embodiment of the present invention, a method for treating amyotrophic lateral sclerosis or suppressing disease progression may include a drug holiday during a drug treatment period, with the drug treatment period and drug holiday period counted as one unit and repeated twice or more. For example, two or more 14-day drug holidays may be included during the drug treatment period, with the drug treatment period and drug holiday period counted as one unit and repeated twice or more. For example, when a drug treatment period and drug holiday are repeated twice, the result is "drug period, drug holiday, drug period, drug holiday," but this also includes "drug period, drug holiday, drug period" without a final drug holiday.

[0057] A drug holiday is a period during which no drug is administered, for example, for 7 or more consecutive days. The dosing period may be, for example, 14 days, or 10 days out of 14 days. "10 days out of 14 days" means any 10 days out of 14 consecutive days, and these 10 days of dosing may be 10 consecutive days, or may be 10 non-consecutive days separated by 1-4 days of no dosing. A preferred dosing period may be selected while observing the patient's condition. In one embodiment of the present invention, the drug holiday period is preferably 14 days.

[0058] When a 14-day dosing period and a 14-day drug-free period are repeated, the number of repetitions is not particularly limited as long as it is two or more times. When a 14-day initial dosing period is followed by a 14-day initial withdrawal period, followed by repeating a 10-day dosing period and a 14-day withdrawal period out of 14 days, the number of times the 10-day dosing period and the 14-day withdrawal period out of 14 days is repeated is not particularly limited as long as it is repeated at least once. In other embodiments, dosing may be repeated daily or nearly daily without a withdrawal period.

[0059] The daily dose of the active ingredient can be appropriately selected depending on conditions such as the age and condition of the patient. Generally, for an adult, the amount of 3-methyl-1-phenyl-2-pyrazolin-5-one (when the active ingredient is 3-methyl-1-phenyl-2-pyrazolin-5-one, the amount of 3-methyl-1-phenyl-2-pyrazolin-5-one; when the active ingredient is a physiologically acceptable salt of 3-methyl-1-phenyl-2-pyrazolin-5-one, the amount equivalent to 3-methyl-1-phenyl-2-pyrazolin-5-one; the same applies hereinafter) is preferably about 15 mg to about 240 mg, more preferably about 30 mg to about 180 mg, even more preferably about 60 mg to about 120 mg, and particularly preferably about 60 mg.

[0060] When administration is repeated daily or almost daily without a drug holiday, the amount of 3-methyl-1-phenyl-2-pyrazolin-5-one per day for adults is generally preferably about 60 mg, about 120 mg, or about 180 mg, and particularly preferably about 60 mg or about 120 mg.

[0061] There is no limit to the number of doses per day during the administration period, and a preferred number may be selected while observing the patient's condition. However, taking into consideration the burden on the patient, three, two, or one dose is preferred, with one dose being more preferred.

[0062] The route of administration of the active ingredient is not particularly limited, and may be oral or parenteral. Furthermore, bolus administration and continuous administration are possible, with continuous administration being preferred. Continuous administration includes intravenous administration by drip infusion, transdermal administration, oral administration using sublingual tablets, and oral and rectal administration using sustained-release preparations, with intravenous administration by drip infusion being preferred. For bolus administration by injection or intravenous administration by drip infusion, injections such as those described in Japanese Patent Application Laid-Open Nos. 63-132833 and 2011-62529 may be used. The disclosures of these publications are incorporated herein by reference.

[0063] When administered intravenously by infusion, the administration rate is desirably about 0.5 mg / min to about 1 mg / min of 3-methyl-1-phenyl-2-pyrazolin-5-one, which corresponds to a time period of about 15 minutes to about 480 minutes, preferably about 30 minutes to about 120 minutes, more preferably about 30 minutes to about 60 minutes, and even more preferably about 60 minutes.

[0064] Other administration forms include those that are substantially equivalent to intravenous administration by infusion at a dose of about 0.5 mg to about 1 mg of 3-methyl-1-phenyl-2-pyrazolin-5-one per minute. An administration form that is substantially equivalent to intravenous administration by infusion at a dose of about 0.5 mg to about 1 mg of 3-methyl-1-phenyl-2-pyrazolin-5-one per minute may be any administration form that is substantially equivalent in pharmacokinetics. Specific examples include administration forms that are recognized to result in substantially equivalent changes in plasma concentration of unchanged 3-methyl-1-phenyl-2-pyrazolin-5-one over time after administration of 3-methyl-1-phenyl-2-pyrazolin-5-one or a physiologically acceptable salt thereof. Examples of such administration forms include transdermal administration, oral administration using sublingual tablets, and oral and rectal administration using sustained-release formulations.

[0065] A method for analyzing the phenylhydrazine content in a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance according to one embodiment of the present invention may include preparing a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, a first acidic water, and a first water-soluble organic solvent; and / or preparing a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, a second acidic water, and a second water-soluble organic solvent.

[0066] In another embodiment of the method for analyzing the phenylhydrazine content in a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, the first measured value of phenylhydrazine is a value obtained by measuring a phenylhydrazine standard solution using high performance liquid chromatography, and / or the second measured value of phenylhydrazine is a value obtained by measuring a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution using high performance liquid chromatography.

[0067] In another embodiment of the present invention, a method for treating or inhibiting the progression of amyotrophic lateral sclerosis comprises administering to a patient in need thereof a medicament containing an effective amount of 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, wherein the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance has been analyzed and determined to contain 20 ppm or less of phenylhydrazine. Analyzing the amount of phenylhydrazine includes obtaining a first measurement value by measuring the phenylhydrazine content in a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, a first acidic water, and a first water-soluble organic solvent and exhibiting a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL; obtaining a second measurement value by measuring the phenylhydrazine content in a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, a second acidic water, and a second water-soluble organic solvent; and detecting the phenylhydrazine content in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first and second measurement values to determine that the amount of phenylhydrazine is 20 ppm or less.

[0068] In one embodiment of the present invention, a method for producing a drug for treating amyotrophic lateral sclerosis or suppressing the progression of the disease includes analyzing the amount of phenylhydrazine in a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, and mixing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance having a phenylhydrazine amount of 20 ppm or less with at least one pharmacologically and pharmaceutically acceptable additive. Here, the analysis of the amount of phenylhydrazine includes obtaining a first measurement value by measuring the phenylhydrazine content in a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, a first acidic water, and a first water-soluble organic solvent and exhibiting a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL; obtaining a second measurement value by measuring the phenylhydrazine content in a 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, a second acidic water, and a second water-soluble organic solvent; and detecting the phenylhydrazine content in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first and second measurement values to determine that the amount of phenylhydrazine is 20 ppm or less.

[0069] In yet another embodiment of the present invention, the drug is a drug for intravenous administration, which contains 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance having a phenylhydrazine content of 20 ppm or less. In one embodiment of the present invention, in a method for producing a medicament for treating amyotrophic lateral sclerosis or suppressing the progression of the disease, at least one pharmacologically and pharmaceutically acceptable additive comprises distilled water for injection or physiological saline. [Example]

[0070] Hereinafter, embodiments of the present invention will be described using examples, but the present invention is not limited to the following examples. In these examples, the drug substance 3-methyl-1-phenyl-2-pyrazolin-5-one will be referred to as edaravone drug substance (sometimes referred to as EDVP).

[0071] [Test Example 1] Study of dissolving solution The conditions under which edaravone drug substance and phenylhydrazine or its salts can be dissolved will be investigated by changing the concentration of acidic water, the ratio of acidic water to water-soluble organic solvent, etc. In addition, HPLC measurements will be performed to confirm the peak shape of phenylhydrazine, and the optimal concentration of acidic water and the ratio of acidic water to water-soluble organic solvent will be investigated.

[0072] Sample solutions were prepared using the following solutions (a) to (f), and the dissolution state of the edaravone drug substance was examined. Next, for the solutions in which the edaravone drug substance was soluble, phenylhydrazine solutions prepared with these solutions were measured using HPLC, and the peak shape of phenylhydrazine was examined.

[0073] Solution (a) 0.1 mol / L hydrochloric acid test solution (b) 0.1 mol / L hydrochloric acid / acetonitrile mixture (8:2) (c) 0.1 mol / L hydrochloric acid / acetonitrile mixture (6:4) (d) 0.1 mol / L hydrochloric acid / acetonitrile mixture (4:6) (e) 0.1 mol / L hydrochloric acid / acetonitrile mixture (2:8) (f) 1 mol / L hydrochloric acid / acetonitrile mixture (6:4)

[0074] Preparation of sample solutions Approximately 0.10 g of edaravone bulk drug was weighed and dissolved in the dissolving solutions (a) to (f) to make exactly 10 mL, which was used as a sample solution.

[0075] Preparation of phenylhydrazine solution 5 μL of phenylhydrazine was taken and 0.1 mol / L hydrochloric acid test solution was added to make exactly 100 mL. 0.1 mL of this solution was accurately measured, and solutions (a) to (f) were added to make exactly 10 mL to make a phenylhydrazine solution (0.5 μg / mL).

[0076] Test conditions The HPLC measurements were carried out under the following mobile phase flow conditions: Mobile phase A: pH 2.5 0.05 mol / L sodium perchlorate aqueous solution / acetonitrile mixture (90:10) Mobile phase B: pH 2.5 0.05 mol / L sodium perchlorate aqueous solution / acetonitrile mixture (10:90) Mobile phase delivery: The concentration gradient is controlled by changing the mixing ratio of mobile phase A and mobile phase B as follows:

[0077] [Table 1]

[0078] The results for each dissolution medium are shown in Table 2, and the chromatograms for the phenylhydrazine solution are shown in Figures 1 to 4. Judging from the solubility of EDVP and the peak shape of phenylhydrazine, (c) a 0.1 mol / L hydrochloric acid / acetonitrile mixture (6:4) is the most suitable, and (f) a 1 mol / L hydrochloric acid / acetonitrile mixture (6:4) can also be used.

[0079] [Table 2]

[0080] *5) Measured with pH test paper

[0081] In addition, this test example revealed that phenylhydrazine hydrochloride is less likely to decompose during storage and is easier to handle than phenylhydrazine, making it more suitable as a standard substance. Therefore, in this test example, a standard solution prepared using phenylhydrazine hydrochloride was measured. The number of theoretical plates and the symmetry factor can be confirmed from the phenylhydrazine peak. Furthermore, this test example revealed that, from the viewpoint of the solubility of EDVP and the peak shape of phenylhydrazine, it is not possible to prepare an optimal dissolution solution for use in the analysis of edaravone drug substance simply by adjusting the pH to 2.

[0082] [Test Example 2] Further investigation of the dissolving solution The results of Test Example 1 revealed that dissolving solution (c), a 0.1 mol / L hydrochloric acid / acetonitrile mixture (6:4), was the most suitable dissolving solution. However, in order to obtain a more preferable peak shape for the phenylhydrazine solution, a standard solution was prepared using dissolving solution (g), a 0.1 mol / L hydrochloric acid / acetonitrile mixture (7:3), and a comparison was made.

[0083] Standard solutions were prepared using the following dissolving solutions (c) and (g), and the peak shape of phenylhydrazine was further examined.

[0084] Solution (c) 0.1 mol / L hydrochloric acid / acetonitrile mixture (6:4) (g) 0.1 mol / L hydrochloric acid / acetonitrile mixture (7:3)

[0085] Preparation of standard solutions Approximately 13.4 mg of phenylhydrazine hydrochloride (approximately 10 mg as free form) was precisely weighed and dissolved in dissolving solution (c) and dissolving solution (g) respectively to make exactly 100 mL. 2 mL of this solution was accurately measured and dissolving solution (c) and dissolving solution (g) were added respectively to make exactly 20 mL. 2 mL of this solution was accurately measured and dissolving solution was added to make exactly 20 mL to use as the standard solution (phenylhydrazine: 1 μg / mL).

[0086] Test conditions The mobile phase was pumped under the following conditions: Mobile phase A: 0.05 mol / L sodium perchlorate solution at pH 2.5 / acetonitrile mixture (90:10) Mobile phase B: 0.05 mol / L sodium perchlorate solution at pH 2.5 / acetonitrile mixture (40:60) Mobile phase delivery: The concentration gradient is controlled by changing the mixing ratio of mobile phase A and mobile phase B as follows:

[0087] [Table 3]

[0088] When a sample solution was prepared using the dissolving solution (g) of 0.1 mol / L hydrochloric acid / acetonitrile mixture (7:3), the edaravone drug substance was found to be soluble. The measurement results of the standard solutions prepared with the above solution (c) and solution (g) are shown in Table 4 and Figures 1 and 5. This test revealed that the use of dissolving solution (g) can improve the peak shape of the phenylhydrazine solution.

[0089] [Table 4]

[0090] [Example 1] A first sample solution was prepared by dissolving 0.1020 g of edaravone bulk drug in a 0.1 mol / L hydrochloric acid / acetonitrile mixed solution (volume ratio 7:3; hereinafter referred to as the dissolving solution) to make exactly 10 mL. Separately, 13.693 mg of phenylhydrazine hydrochloride (approximately 10 mg of phenylhydrazine) was dissolved in a solvent to make exactly 100 mL. 2 mL of the resulting solution was accurately measured, and the solvent was added to make exactly 20 mL. 2 mL of the resulting solution was accurately measured, and the solvent was added to make exactly 20 mL to prepare a first standard solution (phenylhydrazine: 1 μg / mL). Separately, 5 μL of phenylhydrazine was dissolved in a solvent to make exactly 100 mL. 1 mL of the resulting solution was precisely measured, and the solvent was added to make exactly 10 mL. 1 mL of the resulting solution was precisely measured, mixed with 0.1010 g of edaravone drug substance, and the solvent was added to make exactly 10 mL to prepare a first additive sample solution. After a certain period of time had elapsed, 10 μL of each of the first standard solution, the first sample solution, and the first added sample solution was taken and tested by high performance liquid chromatography under the following conditions, and the peak area of the phenylhydrazine peak in each solution (measured value of phenylhydrazine) was measured by automatic integration. The amount of phenylhydrazine in the edaravone drug substance can be calculated by the following formula 3.

[0091] Test conditions Degassing / nitrogen bubbling of the solution: No Storage temperature for the prepared first sample solution, first standard solution, and first added sample solution: 5°C Containers (preparation and storage of the first sample solution, first standard solution, and first added sample solution): Brown glass bottles Detector: UV spectrophotometer (measurement wavelength: 225 nm) Column: Octadecylsilanized silica gel packed column (YMC-Pack Pro C18) Column temperature: constant temperature around 40°C Mobile phase: Start with 100% mobile phase A and control the concentration gradient up to 100% mobile phase B Mobile phase A: 0.05 mol / L sodium perchlorate aqueous solution at pH 2.5 / acetonitrile mixture (volume ratio 9:1) Mobile phase B: 0.05 mol / L sodium perchlorate aqueous solution at pH 2.5 / acetonitrile mixture (volume ratio 2:3) *0.05 mol / L sodium perchlorate aqueous solution with pH 2.5: 7.0 g of sodium perchlorate was dissolved in 1000 mL of water, and perchloric acid was added to adjust the pH to 2.5.

[0092] The results of the stability investigation of phenylhydrazine and / or 3-methyl-1-phenyl-2-pyrazolin-5-one in the first standard solution, the first sample solution, and the first spiked sample solution are shown in Table 5 and FIG.

[0093] [Table 5]

[0094] The elapsed time (h) was calculated from the time when the first standard solution, the first sample solution, or the first added sample solution was first injected into the HPLC within 30 minutes after preparation, which was defined as 0 hours.

[0095] Equation 3: Amount of phenylhydrazine in edaravone drug substance (ppm) = M S / M T ×AT / A S x1 x 0.748 where: M S : Amount of phenylhydrazine hydrochloride in the first standard solution (mg) M T : Amount of edaravone drug substance weighed in the first sample solution (g) A T : Peak area of phenylhydrazine in the first sample solution (μV sec) A S : Peak area of phenylhydrazine in the first standard solution at 0 hours (μV sec) 0.748: Conversion factor of phenylhydrazine hydrochloride to phenylhydrazine

[0096] The ratio of the phenylhydrazine peak area of the first standard solution to the initial value was 90% or more even after 28 hours, indicating that the phenylhydrazine in the solution was stable. In addition, the ratio of the phenylhydrazine peak area of the first added sample solution to the initial value was 90% or more even after 5 hours, indicating that the phenylhydrazine and 3-methyl-1-phenyl-2-pyrazolin-5-one in the solution were stable. Furthermore, in the first sample solution, the phenylhydrazine peak area did not change even after 5 hours, indicating that the 3-methyl-1-phenyl-2-pyrazolin-5-one in the solution was stable. Furthermore, by using a 0.1 mol / L hydrochloric acid / acetonitrile mixture (volume ratio 7:3) as a dissolving solution, it was possible to completely dissolve the edaravone drug substance and phenylhydrazine, and to accurately measure the phenylhydrazine content in the solution. Furthermore, when performing HPLC, as described above, the use of mobile phase A and mobile phase B enabled reliable separation of the edaravone drug substance contained in the solution. Furthermore, by setting the composition ratio of mobile phase A and mobile phase B to the above ratio, quantitative measurement was possible without the presence of other peaks near the retention time of phenylhydrazine, and the edaravone drug substance in the sample solution could be eluted in a short time, allowing efficient HPLC despite the use of two mobile phases.

[0097] From the above, it was revealed that this method enables accurate, quantitative, and simple measurement of the phenylhydrazine content in edaravone drug substance without performing complicated procedures such as degassing / nitrogen bubbling of the dissolution solution or rapid measurement after solution preparation.

[0098] [Example 2] Dissolve 13.517 mg of phenylhydrazine hydrochloride (approximately 10 mg of phenylhydrazine) in a 0.1 mol / L hydrochloric acid / acetonitrile mixture (volume ratio 7:3; hereafter referred to as the dissolving solution) to make exactly 100 mL. Measure 2 mL of the resulting solution accurately and add the dissolving solution to make exactly 20 mL. Measure 2.5 mL of the resulting solution accurately and add the dissolving solution to make exactly 100 mL to prepare the second standard solution (phenylhydrazine: 0.25 μg / mL). Take exactly 10 μL of the second standard solution and test it using high performance liquid chromatography under the following conditions, and measure the peak area of the phenylhydrazine peak in the solution (measured value of phenylhydrazine) using the automatic integration method.

[0099] Test conditions Degassing / nitrogen bubbling of the solution: No Storage temperature for the prepared second sample solution and second standard solution: 5°C Container (preparation and storage of the second sample solution and the second standard solution): Brown glass bottle Detector: UV spectrophotometer (measurement wavelength: 225 nm) Column: Octadecylsilanized silica gel packed column (YMC-Pack Pro C18 or equivalent) Column temperature: constant temperature around 40°C Mobile phase: Start with 100% mobile phase A and control the concentration gradient up to 100% mobile phase B Mobile phase A: pH 2.5 0.05 mol / L sodium perchlorate aqueous solution / acetonitrile mixture (volume ratio 9:1) Mobile phase B: pH 2.5 0.05 mol / L sodium perchlorate aqueous solution / acetonitrile mixture (volume ratio 2:3) *0.05 mol / L sodium perchlorate aqueous solution with pH 2.5: 7.0 g of sodium perchlorate was dissolved in 1000 mL of water, and perchloric acid was added to adjust the pH to 2.5.

[0100] The results of the stability study of phenylhydrazine in the second standard solution are shown in Table 6.

[0101] [Table 6]

[0102] The elapsed time (h) was calculated by setting the time when the second standard solution was first injected into HPLC within 30 minutes after preparation as 0 hours.

[0103] When the second standard solution is used, the amount of phenylhydrazine in the edaravone drug substance in the first sample solution can be calculated using Equation 4.

[0104] Equation 4: Amount of phenylhydrazine in edaravone drug substance (ppm) = M S / M T ×A T / A S x 0.25 x 0.748 where: M S : Amount of phenylhydrazine hydrochloride in the second standard solution (mg) M T : Amount of edaravone drug substance weighed in the first sample solution (g) A T : Peak area of phenylhydrazine in the first sample solution (μV sec) A S : Peak area of phenylhydrazine in the second standard solution at 0 hours (μV sec) 0.748: Conversion factor of phenylhydrazine hydrochloride to phenylhydrazine

[0105] The ratio of the phenylhydrazine peak area of the second standard solution to the initial value was 90% or more even after 12 hours, indicating that the phenylhydrazine in the solution was stable. Furthermore, it was revealed that the second standard solution prepared in this example can be used in the analytical method of the present invention, just like the first standard solution prepared in Example 1. This demonstrates that this method enables quantitative and simple measurement of the phenylhydrazine content in edaravone drug substance without the need for complicated procedures such as degassing / nitrogen bubbling of the solution or rapid measurement after solution preparation.

[0106] [Example 3] The organic solvent in the dissolution solution was changed from acetonitrile to methanol, and the stability of phenylhydrazine and / or 3-methyl-1-phenyl-2-pyrazolin-5-one in the solution was confirmed. A mixture of 0.1 mol / L hydrochloric acid and methanol was prepared, and the solvent was selected so that the organic solvent ratio was as low as possible within the range in which EDVP was soluble (previous studies had shown that the peak shape improved when the organic solvent ratio was closer to that of the mobile phase).The stability of phenylhydrazine and / or 3-methyl-1-phenyl-2-pyrazolin-5-one in the solution was investigated by changing the solvent and using the same conditions as in Example 1. Sample solutions were prepared using the following three types of dissolving solutions.

[0107] [1] 0.1 mol / L hydrochloric acid / methanol mixture (7:3) [2] 0.1 mol / L hydrochloric acid / methanol mixture (6:4) [3] 0.1 mol / L hydrochloric acid / methanol mixture (5: 5)

[0108] As a result, EDVP was found to be soluble in the dissolving solution [3]. Therefore, a spiked sample solution (edaravone drug substance: 0.10111 g, phenylhydrazine: 5 μL), a standard solution (phenylhydrazine hydrochloride: 13.154 mg), and a sample solution (edaravone drug substance: 0.10275 g) were prepared using a 0.1 mol / L hydrochloric acid / methanol mixture (5:5), and the stability of the solutions was investigated. The amount of phenylhydrazine in the edaravone drug substance can be calculated using a method based on the calculation method of Equation 3. The results of the stability study of phenylhydrazine in a 0.1 mol / L hydrochloric acid / methanol mixed solution (5:5) are shown in Table 7 and Figure 7. Compared to the results of Example 1 prepared using a 0.1 mol / L hydrochloric acid / acetonitrile mixed solution (7:3), the decomposition of phenylhydrazine tended to be faster.

[0109] [Table 7]

[0110] *15) The elapsed time (h) was calculated by setting the time when the spiked sample solution, standard solution, and sample solution were first injected into the HPLC within 30 minutes of preparation as 0 hours.

[0111] Phenylhydrazine is easily decomposed by light and oxygen. Therefore, to prevent decomposition of phenylhydrazine in solution, complicated procedures are usually required, such as degassing the solution and bubbling with nitrogen in advance, and starting measurement promptly after preparing the solution. However, in the embodiment of the present invention, it has been found that decomposition of phenylhydrazine in solution can be suppressed for a long period of time without the need for degassing and bubbling with nitrogen in advance.

[0112] The results of Examples 1 to 3 demonstrate that the analytical method according to the embodiment of the present invention, which uses a hydrochloric acid / acetonitrile mixed solution (dissolution solution) and a hydrochloric acid / methanol mixed solution (dissolution solution), can suppress the decomposition of phenylhydrazine and the decomposition of 3-methyl-1-phenyl-2-pyrazolin-5-one in the solution for a long period of time without degassing the solution or bubbling with nitrogen, and can quantitatively and simply measure the phenylhydrazine content in the edaravone drug substance.

[0113] Further modifications and variations of the present invention are possible in light of the above teachings, and it is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

1. 1. A method for producing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance having a phenylhydrazine content of 20 ppm or less, the method comprising: producing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance; measuring the phenylhydrazine content of a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, first acidic water, and a first water-soluble organic solvent, the phenylhydrazine standard solution exhibiting a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL to obtain a first measurement value; measuring the phenylhydrazine content of the obtained 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, the second acidic water, and the second water-soluble organic solvent to obtain a second measurement value; and detecting the content of phenylhydrazine in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first measurement value and the second measurement value; If the detection result shows that the concentration is 20 ppm or more, purifying the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance by a conventional method; wherein the first acidic water contains 0.1 mol / L to 1 mol / L of hydrochloric acid, the first water-soluble organic solvent is acetonitrile, the second acidic water contains 0.1 mol / L to 1 mol / L of hydrochloric acid, and the second water-soluble organic solvent is acetonitrile; and The volume ratio of the first acidic water to the first water-soluble organic solvent is in the range of 6:4 to 7:3, and the volume ratio of the second acidic water to the second water-soluble organic solvent is in the range of 6:4 to 7:

3.

2. The method according to claim 1, wherein the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance is produced by a conventional method.

3. 3. The method of claim 1, wherein the first acidic water contains 0.1 mol / L hydrochloric acid, and the second acidic water contains 0.1 mol / L hydrochloric acid.

4. 4. The method according to claim 1, wherein the volume ratio of the first acidic water to the first water-soluble organic solvent is 7:3, and the volume ratio of the second acidic water to the second water-soluble organic solvent is 7:

3.

5. 1. A method for producing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance having a phenylhydrazine content of 20 ppm or less, the method comprising: producing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance; measuring the phenylhydrazine content of a phenylhydrazine standard solution containing phenylhydrazine or a salt thereof, first acidic water, and a first water-soluble organic solvent, the phenylhydrazine standard solution exhibiting a phenylhydrazine concentration of 0.01 μg / mL to 10 μg / mL to obtain a first measurement value; measuring the phenylhydrazine content of the obtained 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, the second acidic water, and the second water-soluble organic solvent to obtain a second measurement value; and detecting the content of phenylhydrazine in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first measurement value and the second measurement value; If the detection result shows that the concentration is 20 ppm or more, purifying the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance by a conventional method; wherein the first acidic water contains 0.1 mol / L to 1 mol / L of hydrochloric acid, and the first water-soluble organic solvent is methanol; the second acidic water contains 0.1 mol / L to 1 mol / L of hydrochloric acid, and the second water-soluble organic solvent is methanol; The volume ratio of the first acidic water to the first water-soluble organic solvent is 5:5, and the volume ratio of the second acidic water to the second water-soluble organic solvent is 5:

5.

6. The method according to claim 5, wherein the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance is produced by a conventional method.

7. 7. The method of claim 5 or 6, wherein the first acidic water contains 0.1 mol / L hydrochloric acid, and the second acidic water contains 0.1 mol / L hydrochloric acid.

8. 8. The method of any one of claims 1 to 7, further comprising: Preparing the phenylhydrazine standard solution containing the phenylhydrazine or a salt thereof, the first acidic water, and the first water-soluble organic solvent; and preparing the 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution containing the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance, the second acidic water, and the second water-soluble organic solvent.

9. 9. The method according to claim 1, wherein the first measurement value is a value obtained by measuring the phenylhydrazine content of the phenylhydrazine standard solution using high performance liquid chromatography, and the second measurement value is a value obtained by measuring the phenylhydrazine content of the 3-methyl-1-phenyl-2-pyrazolin-5-one sample solution using high performance liquid chromatography.

10. The method according to any one of claims 1 to 9, for producing a 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance having a phenylhydrazine content of 20 ppm or less.

11. 11. The method of claim 10 further comprising: Detecting the phenylhydrazine content in the 3-methyl-1-phenyl-2-pyrazolin-5-one drug substance from the first measurement value and the second measurement value and determining that the phenylhydrazine amount is 20 ppm or less.

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