JP7148642B2 - Method for producing stable pharmaceutical composition containing azacitidine - Google Patents

Description

The present invention relates to a method of making stable azacitidine-containing pharmaceutical compositions using solvents and low temperature processes, including acetonitrile.

Azacytidine (AZA) having the following chemical structure is 4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-[1,3,5]triazin-2-one or 5-azacytidine, etc., and is currently marketed as the drug VIDAZA (registered trademark).

Azacytidine is a chemical analogue of cytidine, a nucleoside found in DNA and RNA. Azacytidine and its dioxy derivative, 5-aza-2'deoxycytidine, are nucleoside analogues designed to reduce DNA methylation and have been used clinically to treat acute myeloid leukemia and others. (Non-Patent Document 1). After being incorporated into replicating DNA, azacitidine forms a covalent complex with DNA methyltransferase, inhibits its activity, and induces DNA hypomethylation, thereby regulating normal cell cycle; Re-expression of genes involved in differentiation and death allows cells to restore normal function. The cytotoxic effects of azacitidine can also result in the death of rapidly dividing cells, including cancer cells, that are no longer responsive to the regulatory mechanisms of normal cell growth. Azacytidine has therefore been tested in clinical trials and has shown considerable anti-tumor activity, particularly in the treatment of myelodysplastic syndrome (MDS).

However, azacitidine has the problem of being difficult to synthesize, process and store on a large scale for commercial purposes due to its hydrolyzable properties. The s-triazine ring of azacitidine is prone to water degradation, but rapid hydration of the double bond of the 5,6-imine of azacitidine in aqueous solution at neutral pH followed by bond cleavage. , to give the formyl derivative N-(formylamidino)-N′-β-D-ribofuranosylurea (RGU-CHO), which is then deformylated to give 1-β- It irreversibly produces D-ribofuranosyl-3-guanylurea (RGU). Due to such instability of azacitidine in aqueous solution, it was not easy to process and store azacitidine in solvents containing water.

Therefore, there was a need for a stable, simple and industrially viable process for processing and storing azacitidine.

Molecules, v. 19, no. 3, pp. 3149-3159, 2014

To produce a lyophilized formulation of a compound such as azacitidine, the compound is prepared in solution, sterilized, lyophilized, and the solvent removed. In this process, when azacitidine is exposed to an aqueous solution for several hours, there is a problem that analogous substances (impurities) are generated due to hydrolysis.
Accordingly, an object of the present invention is to provide a method for producing a pharmaceutical composition containing azacitidine with minimal formation of related substances by finding temperature and solvent conditions that can suppress the hydrolysis of azacitidine. .

Unless explicitly stated otherwise, certain terms used throughout this specification may be defined as follows.

Throughout this specification, unless otherwise stated, "comprising" or "containing" means including without limitation any element (or component), other elements ( or components) are not construed as excluding the addition of

Also, "azacitidine" refers to azacitidine free base as well as salts, polymorphs, isomers, enantiomers, anhydrates, hemihydrates, solvates, prodrugs or any mixture thereof. There may be. As used herein, a "salt" of azacitidine means an acid addition salt of azacitidine derived from inorganic and/or organic acids and may be formed from hydrochloric, hydrobromic, boric, phosphoric, or sulfuric acid. . Alternatively, salts may be formed from acetic acid, citric acid, fumaric acid, maleic acid, malic acid, malonic acid, oxalic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, or trifluoroacetic acid. good. As used herein, a "polymorph" of azacitidine means a solid crystalline form of azacitidine or a salt or complex thereof. A "solvate" of azacitidine also means azacitidine or a salt thereof further comprising stoichiometric or non-stoichiometric amounts of solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate may be a hydrate, eg monohydrate, dihydrate, trihydrate or tetrahydrate. A "prodrug" of azacitidine is a functional derivative of azacitidine and means a compound that can be easily converted to azacitidine in vivo.

In order to solve the above problems, the present invention provides: 1) a step of dissolving azacitidine in a mixed solvent of acetonitrile and water maintained at a temperature of −8° C. to −1° C.; and 2) the solution of step 1) filling a container under conditions where the ambient temperature is maintained at 15° C. or lower; and a method for producing a pharmaceutical composition containing azacitidine.
The present invention will be described in detail below.

In one embodiment, in step 2), the ambient temperature may be maintained at 10° C. or less, but is not limited to this. Hydrolysis of azacitidine during the container filling process can be inhibited if the ambient temperature is maintained at or below this temperature. Even if the dissolution of azacitidine in step 1) is carried out at a low temperature, when the ambient temperature exceeds 15° C., for example, room temperature in the container filling step, azacitidine is rapidly hydrolyzed and the production of related substances is increased. It is important to maintain the ambient temperature in step 2) below a certain temperature. The ambient temperature may be 15° C. or lower or 10° C. or lower, and in one embodiment, the ambient temperature may be 0° C. or higher, 3° C. or higher, or 5° C. or higher.

In one embodiment, in step 2), the solution may be filled within 3 hours, preferably within 2 hours, but is not limited thereto.

During the industrial production of azacitidine-containing pharmaceutical compositions, the filling operation requires a certain amount of time. At this time, depending on the temperature and required time of the filling operation, azacitidine may be hydrolyzed to produce undesirable analogues. However, according to one embodiment of the present invention, if step 2) is performed at an ambient temperature of 15° C. or 10° C. or less within 3 hours or 2 hours and the freeze-drying is started, analogous Material production can be minimized.

In one embodiment, in step 1), the temperature of the mixed solvent is maintained at -8°C to -1°C, preferably -7°C to -1°C, more preferably -5°C to -1°C. Although there may be, it is not limited to this. When the temperature of the mixed solvent is within the above range, hydrolysis during the dissolving step of azacytidine can be further suppressed.

In one embodiment, in step 1), the volume ratio of acetonitrile and water (acetonitrile:water) may be from 5:95 to 30:70, but is not limited thereto. If the volume ratio of acetonitrile to water exceeds the above range, not only the stability of azacytidine is reduced, but also problems of subsequent removal of residual solvent may occur. When the volume ratio of acetonitrile to water is less than the above range, the stability of azacitidine may not be sufficiently ensured. The volume ratio of acetonitrile and water is preferably 10:90 to 25:75, more preferably 15:85 to 25:75, even more preferably 20:80, but is not limited thereto.

In one embodiment, the azacitidine concentration within said pharmaceutical composition can be about 0.001-50 mg/mL, preferably about 0.1-10 mg/mL, more preferably about 1-10 mg/mL, It may be about 2 to 7 mg/mL, or about 3 to 5 mg/mL, but is not limited thereto, and can be adjusted as appropriate according to the administration subject and purpose.

In one embodiment, the method of the present invention further comprises, but is not limited to, sterile filtering the solution of step 1) above. The sterilizing filtering step may be performed by, but not limited to, simple filtering, and the pore size of the filter can be selected according to the desired degree of sterilization. It can be carried out without particular limitation by a sterile filtration method commonly used in the field to which the invention belongs.

In one embodiment, the method of the present invention may further comprise, but is not limited to, the step of freeze-drying the filling of step 2). The freeze-drying step can be performed without particular limitation by a freeze-drying method commonly used in the field to which the present invention belongs.

In one embodiment, in step 1), the mixed solvent further contains a freeze-drying aid, but is not limited thereto.

In one embodiment, the lyophilization aid is mannitol, sodium dihydrogen phosphate, potassium dihydrogen phosphate, tartaric acid, gelatin, glycerin, dextrose, dextran, citric acid, ascorbic acid, sodium hydrogen tartrate phosphate, hydroxide It is selected from the group consisting of sodium and mixtures thereof, but is not limited thereto, and can be appropriately selected and used by a person skilled in the art from commonly used freeze-drying adjuvants.

For example, the freeze-drying adjuvant may be contained in the mixed solvent of step 1) at 1-10 mg/mL, preferably 2-8 mg/mL, more preferably 5 mg/mL, but is not limited thereto. Considering the concentration of the active ingredient, the type of freeze-drying aid, and the like, a person skilled in the art can adjust the amount appropriately.

In one embodiment, the step 1) comprises: 1-a) a step of dissolving a freeze-drying aid in water to obtain a solution; 1-b) adding acetonitrile to the solution to obtain a mixed solvent; -c) lowering the temperature of the mixed solvent to -8°C to -1°C; and 1-d) dissolving azacytidine in the mixed solvent at said temperature;

Pharmaceutical compositions according to the present invention can be used for treatment or amelioration of various cancers and tumor-related diseases, including myelodysplastic syndrome and acute myelogenous leukemia (AML). More specifically, the pharmaceutical composition can be used to treat or ameliorate diseases such as disorders associated with abnormal cell proliferation and blood disorders.

In one embodiment, the content of azacitidine-derived analogs contained in the pharmaceutical composition is less than 2.5%, less than 2%, less than 1.5%, less than 1.2%, less than 1%, 0.5% It may be less than 8%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1%. The content of such related substances varies depending on conditions such as criteria for determining related substances, time required for the process, process temperature and concentration of acetonitrile.

The administration route of the pharmaceutical composition is not particularly limited, and may be oral or parenteral administration. Parenteral routes of administration include, but are not limited to, various routes such as injection administration, transdermal, nasal, intraperitoneal, intramuscular, subcutaneous, and intravenous injection.

Therefore, the pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable carrier, and may further contain a buffering agent, a tonicity agent and/or an antibacterial agent.

In one embodiment, the azacitidine-containing pharmaceutical composition may be, but is not limited to, an injectable formulation.

For example, the pharmaceutical composition of the present invention can be formulated into an injectable formulation by a method known in the art, together with a pharmaceutically acceptable parenteral carrier. In this case, the injectable formulation must always be sterile and protected from contamination with microorganisms such as bacteria and fungi. Examples of suitable parenteral carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol), mixtures thereof and/or vegetable oils. may Preferably, suitable carriers include Hank's solution, intravenous solution, PBS with triethanolamine or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% dextrose, and the like. can do. Also, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may be further included to protect the injectable formulation from microbial contamination. Alternatively, the injectable formulation further comprises a tonicity agent such as a sugar or sodium chloride selected from the group consisting of mannitol, lactose, dextrose and trehalose. As other pharmaceutically acceptable carriers, reference can be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th Edition, 1995, Mack Publishing Company, Easton, PA). In addition, the injectable formulation can be filled into a pharmaceutically suitable container.

In one embodiment, the container in step 2) may be, but is not limited to, a glass vial.

According to one embodiment of the present invention, it is possible to manufacture a pharmaceutical composition containing azacitidine that minimizes the generation of analogues due to hydrolysis of azacitidine and meets the criteria for analogues under the strictest conditions (USP monograph), The fact that it is not complicated or requires no additional steps makes processing and storage of azacitidine viable on an industrial scale.
Also, stable pharmaceutical compositions containing azacitidine as described above can be used to treat various types of cancers and tumor-related diseases in mammals.

Production Examples 1 to 4: Production of low-temperature preparation using solvent (1) Production of low-temperature preparation using water for injection To 3000 mL of water for injection in a preparation tank, 15 g of mannitol was added and completely dissolved. The preparation tank was cooled sufficiently to maintain a temperature of 5±3°C. After confirming that the temperature of the prepared solution had dropped to the refrigeration temperature (5±3° C.), 15 g of azacytidine was added and stirred at 500±50 rpm for 20±10 minutes. After confirming complete dissolution, the preparation was sterile filtered.

(2) Preparation of low-temperature preparation liquid using organic solvent 15 g of mannitol was added to 2400 mL of water for injection in the preparation tank and dissolved completely. The preparation tank was cooled sufficiently to maintain -3±2°C. When mannitol was completely dissolved, 600 mL of organic solvents (acetonitrile, ethanol or tertiary butanol) as shown in Table 1 below were added and mixed well. After confirming that the temperature of the prepared solution had dropped to -3±2° C., 15 g of azacytidine was added and stirred at 500±50 rpm for 70±10 minutes. After confirming complete dissolution, the preparation was sterile filtered.
After putting 30 mL of each of the prepared solutions into vials, they were stored in a chamber at 5° C., 15° C. or 25° C., sampled at intervals of 1 hour, and related substances were measured for up to 4 hours.

Experimental Example 1: Evaluation of Related Substances by Storage Temperature and Type of Solvent Related substances in preparation solutions of Production Examples 1 to 4 were analyzed by the following method. The analytical standard solution and test solution were stored at 2 to 8°C.

1) Preparation of diluted solution Purified water was used to adjust the concentration of sodium hydrogen sulfite to 10.0 g/L, and diluted sulfuric acid was used to adjust the pH to 2.5.

2) Preparation of standard solution In a 50 mL volumetric flask, 5.0 mg of accurately quantified azacytidine was added, and the marked line was aligned with 20% acetonitrile. After that, the standard line was aligned with the diluent (concentration 0.01 mg/mL).

3) Preparation of system suitability solution 25 mg of accurately quantified azacitidine standard was placed in a 5 mL volumetric flask, dissolved by adding 20% acetonitrile, and aligned. 2 mL of this solution was taken and placed in a vial containing 3 mL of diluent and mixed well (concentration 2 mg/mL).

4) Preparation of test solution 2 mL of the well-dissolved solution (5 mg/mL) was taken, placed in a vial containing 3 mL of diluent, and mixed well to obtain a test solution (concentration: 2 mg/mL).

5) Liquid chromatography conditions a. Column: Orosil C18, 4.6 mm x 25 cm, 3 µm or similar column b. Detector: UV spectrophotometer (measurement wavelength: 210 nm)
c. Injection volume: 5 μL
d. Flow rate: 0.8 mL/min e. Automatic sample injector temperature: 5°C
g. Mobile Phase: Gradient Elution The running conditions are shown in Table 2 below.

Mobile phase A: 1.54 g/L ammonium acetate aqueous solution Mobile phase B: Acetonitrile:methanol:mobile phase A = 20:30:50

6) System suitability and method of operation Injecting the system suitability solution, the tailing of the azacitidine peak is 2.0 or less, and the standard solution is injected more than 6 times, and the partner standard deviation of the peak area is 10.0% or less. At that time, it was determined that the system was suitable, and the standard solution and the test solution were injected in that order to determine the peak area.

7) Calculation The concentration of each related substance was calculated using the following formula.
Each related substance (%) = (area of each related substance in test solution/peak area of azacitidine standard solution) × (concentration of azacitidine standard solution (mg/mL)/theoretical concentration of test solution (mg/mL)) × Purity of standard product (%)

Judgment criteria for azacitidine analogues are shown in Table 3 below (however, peaks of 0.04% or less are not reported). In addition, Tables 4 to 7 show the analysis results of related substances according to the azacitidine solvent used and temperature. Table 4 shows water for injection (Production Example 1), Table 5 shows acetonitrile (Production Example 2), Table 6 shows ethanol (Production Example 3), and Table 7 shows tertiary butanol (Production Example 4). It shows the results of analogue analysis after preparation of the prepared solution.

a. 1-β-D-ribofuranosyl-3-guanylurea b. N-(diaminoethylene)N'-(β-D-ribofuranosyl)carbamimidic acid c. 1-β-D-ribofuranosyl-3-aminocarbonylguanidine d. 1-β-D-ribofuranosyl-3-iminohydroxylmethylguanidine e. N-(formylamidino)-N'-β-D-ribofuranosyl urea (RGU-CHO)
f. Exclude N-(formylamidino)-N'-β-D-ribofuranosyl urea from total related substances

In the case of a composition prepared only with water without using an organic solvent, the prepared liquid was frozen when it was prepared at 0° C. or below, so there was a problem in producing it below the refrigeration temperature. Therefore, in the case of the composition prepared with water for injection, it was not possible to maintain below the standard for related substances for 1 hour at any temperature except storage at 5°C. On the other hand, in the case of the prepared liquid prepared using an organic solvent, it was possible to prepare at a low temperature of about -3°C. Acetonitrile showed the best stability among formulations made with three solvents, acetonitrile, ethanol and tertiary butanol. Next, ethanol and tertiary butanol showed superior stability in that order. In particular, acetonitrile was very effective in modulating the water and temperature sensitive name RGU-CH.

Production Examples 5-7: Production of preparation liquids by acetonitrile concentration 15 g of mannitol was added to 2100 mL of water for injection in the preparation tank and completely dissolved. The preparation tank was cooled sufficiently to maintain -3±2°C. When mannitol was completely dissolved, water for injection and/or acetonitrile was added and mixed well as shown in Table 8 below. After confirming that the temperature of the prepared solution had dropped to -3±2° C., 15 g of azacytidine was added, and the mixture was stirred at 500±50 rpm for 70±10 minutes. After confirming complete dissolution, the preparation was sterile filtered.
After putting 30 mL of this preparation into a vial, it was stored in a 5° C., 10° C. or 15° C. chamber, sampled at 1 hour intervals, and related substances were measured for up to 3 hours.

Experimental Example 2: Evaluation of Related Substances by Storage Temperature Associated with Acetonitrile Concentration Analogous substances in the preparation liquids of Production Examples 5 to 7 were analyzed in the same manner as in Experimental Example 1 above. Tables 9 to 11 below show the results of analysis of related substances after preparing preparations using 10%, 20% and 30% acetonitrile (ACN), respectively.

In the case of the preparation solution prepared using 10% acetonitrile (Preparation Example 5), the standard for related substances was exceeded after 2 hours at 10°C and after 1 hour at 15°C. In the case of the preparation solution prepared with a 30% acetonitrile solution (Production Example 7), the related substances exceeded the standard after 3 hours at 10 ° C., but in the case of the preparation solution prepared with a 20% acetonitrile solution (Production Example 6), 10 3 hours at 15°C and 2 hours at 15°C.

Example 1: Sample Preparation in a Cold Process with 20% Acetonitrile To 2400 mL of water for injection in a preparation tank was added 15 g of mannitol and completely dissolved. The preparation tank was cooled sufficiently to maintain -3±2°C. Once the mannitol was completely dissolved, 600 mL acetonitrile was added and mixed well. After confirming that the temperature of the prepared solution had dropped to -3±2° C., 15 g of azacytidine was added and stirred at 500±50 rpm for 70±10 minutes. After confirming complete dissolution, the preparation was sterile filtered. After filling the vial with the preparation solution under the condition of 10 ° C. or less, considering the time required for filling in the actual factory, it is further stored in a 10 ° C. chamber for 2 hours, placed in a freeze dryer, and freeze-dried. A sample was taken and analyzed.

Comparative Example 1: Sample Preparation at Room Temperature Process Using 20% Acetonitrile A prepared solution was prepared in the same manner as in Example 1 and then sterilized and filtered. After the preparation was filled into vials at room temperature, it was further stored at room temperature for 2 hours, considering the actual factory filling time, placed in a lyophilizer, freeze-dried, and then sampled for analysis. .

Comparative Example 2: Sample preparation in a low-temperature process using purified water A prepared solution was prepared in the same manner as in Example 1, except that purified water was used and the preparation temperature was maintained at 5 ± 3 ° C., and then sterilized. Filtered. After filling the vial with the preparation solution under the condition of 10 ° C. or less, considering the time required for filling in the actual factory, it is further stored in a 10 ° C. chamber for 2 hours, placed in a freeze dryer, and freeze-dried. A sample was taken and analyzed.

Comparative Example 3: Sample preparation at room temperature process using purified water A prepared solution was prepared in the same manner as in Example 1, except that purified water was used and the preparation temperature was maintained at 5 ± 3 ° C., and then sterilized. Filtered. After the preparation is filled into vials under room temperature conditions, it is further stored at room temperature for 2 hours, placed in a freeze dryer, freeze-dried, and sampled for analysis, considering the time it takes to fill in the actual factory. did.
The preparation conditions according to Comparative Examples 1 to 3 are compared and shown in Table 12 below.

Experimental Example 3: Evaluation of Related Substances for Optimal Temperature and Storage Conditions Similar substances in freeze-dried samples according to Example 1 and Comparative Examples 1-3 were analyzed in the same manner as in Experimental Example 1. At this time, about 10 mL of 20% acetonitrile was added to the freeze-dried sample using a syringe, well shaken to dissolve, and then transferred to a 20 mL volumetric flask. Vials were washed 1-2 times with a small amount of 20% acetonitrile, placed in a volumetric flask, and marked with 20% acetonitrile (5 mg/mL concentration). 2 mL of this well-dissolved solution was taken, placed in a vial containing 3 mL of diluent, and mixed well to obtain a test solution (concentration: 2 mg/mL). The results of analogue analysis of the freeze-dried samples are shown in Table 13 below.

Claims (5)

1) dissolving azacitidine in a mixed solvent of acetonitrile , water and a freeze-drying aid maintained at a temperature of -7 °C to -3 °C at 450-550 rpm within 60-80 minutes ;
2 ) filling the solution of step 1) into a container within 2 hours under conditions where the ambient temperature is maintained at 10 °C or less; and
3) freeze-drying the charge of step 2;
A method of manufacturing an azacitidine-containing pharmaceutical composition on an industrial scale of 3,000 mL or more of azacitidine solution, comprising :
In the step 1), the volume ratio of acetonitrile and water (acetonitrile:water) is 10:90 to 25:75,
The azacitidine concentration of the azacitidine-containing pharmaceutical composition is 3 to 5 mg/mL,
The freeze-drying adjuvant is mannitol, the concentration of mannitol in the mixed solvent is 2 to 8 mg / mL,
A method wherein said pharmaceutical composition containing azacitidine contains less than 0.8% analogues under conditions of lyophilization for at least 3 hours after the ambient temperature is maintained at 10°C or less . 2. The method for producing an azacitidine-containing pharmaceutical composition according to claim 1, further comprising the step of sterile filtering the solution of step 1). Step 1) is
1-a) a step of dissolving a freeze-drying aid in water to obtain a solution;
1-b) adding acetonitrile to the solution to obtain a mixed solvent;
1-c) a step of lowering the temperature of the mixed solvent to −7 ° C. to −3 ° C.; and 1-d) a step of dissolving azacitidine in the mixed solvent at the temperature at 450-550 rpm within 60-80 minutes ;
The method for producing the azacitidine-containing pharmaceutical composition according to claim 1, comprising 2. The method for producing an azacitidine-containing pharmaceutical composition according to claim 1, wherein the azacitidine-containing pharmaceutical composition is an injectable preparation. The method for producing a pharmaceutical composition containing azacitidine according to claim 1, wherein the container in step 2) is a glass vial.