Method for purifying mitomycin C

Abstract

Provided are a method for purifying mitomycin C, comprising a step of recrystallizing crude mitomycin C crystals using a high-purity methanol; mitomycin C obtainable by the method; and the like.

Claims

1. A method for purifying mitomycin C, comprising a step of recrystallizing crude mitomycin C crystals using a high-purity methanol.

2. The method according to claim 1, wherein the high-purity methanol has a purity of 99.00 to 99.99%.

3. The method according to claim 1, wherein the high-purity methanol has a purity of 99.50 to 99.99%.

4. The method according to claim 1, wherein the high-purity methanol has a total metal concentration of 0.01 to 300 ppb.

5. The method according to claim 1, wherein the high-purity methanol has a total metal concentration of 0.01 to 100 ppb.

6. The method according to claim 1, wherein the high-purity methanol has a zinc concentration of 0.01 to 100 ppb.

7. The method according to claim 1, wherein the high-purity methanol has a zinc concentration of 0.01 to 10 ppb.

Description

DESCRIPTION OF EMBODIMENTS

(1) The method of the present invention for purifying crude mitomycin C crystals is described in detail.

(2) The crude mitomycin C crystals used in the present invention can be obtained by, for example, the methods described in JP-B 35-17897, JP-B 36-9094, JP-A 4-187092, etc. Specifically, the crude mitomycin C crystals can be obtained by subjecting the culture medium harvested after the culture of microorganisms to a treatment such as purification by adsorption to activated carbon or resin (e.g., reverse-phase adsorption resin etc.), alumina chromatography and countercurrent distribution; and shall meet the following requirements: i) the identification test is passed; ii) the mitomycin C content in 1 mg of the crude mitomycin C crystals is 950 g or more as measured based on 1 mg of a mitomycin C standard; and iii) the loss on drying is 1.0% or lower.

(3) The identification test and the measurement of the content and the loss on drying can be performed according to, for example, the methods described in the sections under the heading Identification, Assay and Loss on drying of Mitomycin C in the Japanese Pharmacopoeia, 16th edition.

(4) The unit v/w in the present invention represents the volume (mL) of a liquid per gram of a substance.

(5) Examples of the poor solvent in the present invention include ethyl ether, petroleum ether and the like.

(6) Examples of the metal in the present invention include organometallic compounds, elemental metals, metal salts and the like. Examples of the elemental metal include: nickel, lead, cadmium, iron, zinc and the like. Examples of the metal salt include nickel salts such as nickel chloride, nickel oxide and the like; lead salts such as lead chloride, lead oxide and the like; cadmium salts such as cadmium chloride, cadmium oxide and the like; iron salts such as iron chloride, iron oxide and the like; and zinc salts such as zinc chloride, zinc oxide and the like.

(7) The total metal concentration represents the sum of the content of all the metals detected:, for example;, as the respective metal ions by the metal analysis methods described in Inductively Coupled Plasma Atomic Emission Spectroscopy and Inductively Coupled Plasma Mass Spectrometry <2.63> in the Japanese Pharmacopoeia, 16th edition, and the like,

(8) Examples of the zinc in the present invention include organozinc compounds, metal zince, zinc salts and the like. Examples of the zinc salt include zinc chloride, zinc oxide and the like.

(9) Production Method

(10) To the crude mitomycin C crystals, 50 to 100 v/w, preferably 60 to 70 v/w of methanol is added, and the mixture is stirred at a temperature between 50 and 70 C., preferably between 55 and 67 C. for dissolution.

(11) If desired, the resulting solution can be filtered to remove insoluble matter etc.

(12) Examples of the methanol include a methanol having a low total metal concentration; and the like. Examples of the methanol having a low total metal concentration include distilled methanol prepared from a commercial methanol; a commercial high-purity methanol having a low total metal concentration; and the like.

(13) The commercial high-purity methanol having a low total metal concentration is, for example, a commercial purified methanol (manufactured by Toyo Gosei Co., Ltd.) or the like.

(14) The purity of the methanol is, for example, 99.00 to 99.99%, preferably 99.50 to 99.9941 more preferably 99.60 to 99.99%, and particularly preferably 99.80 to 99.99%. The total metal concentration in the methanol is preferably as low as possible. The total metal concentration is, for example, 0.01 to 300 ppb, preferably 0.01 to 100 ppb, more preferably 0.01 to 30 ppb, and particularly preferably 0.01 to 10 ppb. The zinc concentration in the methanol is preferably as low as possible. The zinc concentration is, for example, 0.01 to 100 ppb, preferably 0.01 to 10 ppb, more preferably 0101 to 7 ppb, and particularly preferably 0.01 to 6 ppb.

(15) The solution obtained above or the filtrate obtained above by filtration of the solution is cooled, 70 to 110 v/w, preferably 85 to 95 v/w of ethyl ether relative to the amount of the crude crystals is added, and 200 to 250 v/w, preferably 220 to 230 v/w of petroleum ether relative to the amount of the crude crystals is further added. The resulting mixture is allowed to stand with or without stirring at 10 to 15 C., preferably 5 to 10 C., for 0.5 to 2 hours, preferably 0.8 to 1.2 hours. The precipitated mitomycin C crystals are collected by filtration and dried to give a purified mitomycin C.

(16) Hereinafter, the present invention will he illustrated in more detail by examples, but the present invention is not limited thereto.

EXAMPLE 1

(17) To crude mitomycin C crystals (1.8 g), distilled methanol (117 mL) which had been prepared from a commercial methanol (Wako Pure Chemical Industries, Ltd., first grade) was added, and the mixture was heated at an outside temperature of 65 C. with stirring for 30 minutes. The resulting solution was filtered through a membrane filter with a pore size of 0.22 m, and the filtrate was harvested. The filtrate was gradually cooled down to 2 C., and then kept at this temperature for 2 hours. To the filtrate, ethyl ether (160 mL) cooled to 10 C. was added dropwise, and then petroleum ether (400 mL) cooled to 7 C. was added dropwise. After 1-hour aging at 2 C., the resulting crystals were collected by filtration under reduced pressure and then dried at room temperature under reduced pressure to give mitomycin C (1.61 g).

COMPARATIVE EXAMPLE 1

(18) Mitomycin C (1.51 g) was obtained, using crude Mitomycin C crystals (1.8 g) and the commercial methanol (Wako Pure Chemical Industries, Ltd., first grade), in the same manner as in Example 1.

COMPARATIVE EXAMPLE 2

(19) Mitomycin C (1.56 g) was obtained, using crude mitomycin C crystals (1.8 g) and 5-fold concentrated methanol prepared from the commercial methanol (Wako Pure Chemical Industries, Ltd., first grade) with a rotary evaporator, in the same manner as in Example 1.

TEST EXAMPLE 1

(20) Quantification of Zinc in Methanol and Mitomycin C

(21) 1) Methanol

(22) 500 mL of methanol was charged into an eggplant-shaped flask and concentrated to dryness with an evaporator. The eggplant-shaped flask was washed out with a 4% aqueous nitric acid solution prepared by dilution of special-grade nitric acid (Wako Pure Chemical. Industries, Ltd.), and the washout solution (final volume: 50 mL) was harvested and used as a sample solution.

(23) 2) Mitomycin C

(24) 0.2 g of each mitomycin C prepared in the above Example 1 and Comparative Examples 1 and 2 was separately weighed out in a vessel. To the vessel, 3 mL of special-grade nitric acid (Wako Pure Chemical Industries, Ltd.) and 3 mL of special-grade hydrogen peroxide solution (Junsei Chemical Go., Ltd.) were added, and water was added to make up the volume to 50 mL exactly. The resulting solution was used as a sample solution.

(25) The sample solutions were tested according to the calibration curve method of inductively coupled plasma atomic emission spectroscopy (<2.63> in the Japanese Pharmacopoeia) under the following conditions. Test conditions Wavelength; 213.856 nm for zinc High frequency power: 1.2 kW Carrier gas: Argon Carrier gas flow rate; 0.7 L/min Auxiliary gas flow rate: 0.6 L/min Coolant gas flow rate: 10.0 L/min

(26) The measured concentration of zinc in each methanol used for the purification of the crude mitomycin C crystals in the above Example 1 and Comparative Examples 1 and 2 is shown in Table 1.

(27) TABLE-US-00001 TABLE 1 Zinc concentration in solvents Zinc concentration in Methanol used methanol (ppb) Example 1 Distilled product 0 Comparative Example 1 Commercial product 628 Comparative Example 2 Concentrated product 2549 *The quantitative limit is 1.26 ppb.

(28) The measured concentration of zinc in each mitomycin C purified in the above Example 1 and Comparative Examples 1 and 2 is shown in Table 2.

(29) TABLE-US-00002 TABLE 2 Zinc concentration in mitomycin C Zinc concentration (ppb) Example 1 2223 Comparative Example 1 30394 Comparative Example 2 120337 The quantitative limit is 1250 ppb.

(30) The number of particles formed during the storage of each mitomycin C purified in Example 1 and Comparative Examples 1 and 2 was examined according to the method described in the following Test Example 2.

TEST EXAMPLE 2

(31) Change in the Number of Particles in Mitomycin C in a Stability Test under Severe Conditions

(32) 300 mg of each mitomycin C prepared in Example 1 and Comparative Examples 1 and 2 is separately placed into a vial, and stored at 50 C. under uncontrolled humidity. Sampling was performed after 0, 191 and 300 hours from the onset of the storage, and whether the number of particles would increase was examined according to the following section Quantification of Particles in Mitomycin C.

(33) Quantification of Particles in Mitomycin C

(34) 10 mg of mitomycin C was weighed out accurately, water for injection was added to make up the volume to 25 mL exactly, and the resulting solution was used as a sample solution.

(35) The particles in mitomycin C were analyzed with MFI DPA5200 (Protein Simple). Prior to the analysis of the sample solution, the passage in the analytical device was washed with water for injection to obtain the particle-free baseline. 1 mL of the sample solution was drawn into a pipette tip and then loaded on the sample holder. The sample solution was allowed to pass into the passage at a flow rate 0.1 mL/min to completely replace the liquid in the passage, and then the analysis of the sample; solution was started (volume for analysis: 0.61 mL).

(36) The obtained data was filtered on MVAS 1.3 software to extract particles having an aspect ratio of less than 0.85 for the purpose of excluding bubbles. The number of particles having a diameter of 10 m or more in 10 mg of mitomycin C and the number of particles having a diameter of 25 m or more in 10 mg of mitomycin C were separately counted using Microsoft Excel.

(37) The above procedure was repeated 3 times, and the average value of the triplicates was used as the quantitative value.

(38) TABLE-US-00003 TABLE 3 Time-dependent change in number of particles in 10 mg of mitomycin C under severe conditions Initial 191 hours 300 hours 10 m 25 m 10 m 25 m 10 m 25 m Example 1 3047 151 3624 273 5683 314 Comparative 2118 246 7063 861 22527 3005 Example 1 Comparative 3853 178 279617 51953 301967 36216 Example 2

(39) The results snow that the mitomycin C prepared in Example 1 showed almost no increase in either the number of particles having a diameter of 10 m or more or the number of particles having a diameter of 25 m or more even after 300-hour exposure to severe conditions. In contrast, the mitomycin C prepared in Comparative Example 1 or 2, which contained a larger amount of zinc than that in Example 1, showed a significant increase in both the number of particles having a diameter of 10 m or more and the number of particles having a diameter of 25 m or more after 300-hour exposure to severe conditions.

(40) Therefore, from the test results, it was found that the reduction of the zinc concentration in mitomycin C is effective for the reduction of the time-dependent increase in insoluble particulate matter in mitomycin C. To this end, the reduction of the residual, zinc concentration in mitomycin C by the mitomycin C purification method of the present invention using a high-purity methanol is considered effective.

INDUSTRIAL APPLICABILITY

(41) The present invention provides a method for purifying mitomycin C; and the like.