JP2022500350A - Method for Producing Stable Azacitidine-Containing Pharmaceutical Composition - Google Patents

Abstract

The present invention relates to a method for producing a stable azacitidine-containing pharmaceutical composition using a solvent containing acetonitrile and a low temperature step.

Description

The present invention relates to a method for producing a stable azacitidine-containing pharmaceutical composition using a solvent containing acetonitrile and a low temperature step.

Azacitidine (AZA) having the following chemical structure is 4-amino-1- (3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl) -1H- [1,3,5] triazine-2-one. Alternatively, it is also known as 5-azacitidine and the like, and is currently marketed as a pharmaceutical product VIDAZA (registered trademark).

Azacitidine is a chemical analog of cytidine, a nucleoside present in DNA and RNA. Azacitidine and its dioxy derivative 5-aza-2'deoxycytidine are nucleoside analogs designed to reduce DNA methylation and have been clinically used to treat acute myeloid leukemia and the like. (Non-Patent Document 1). After being contaminated in replication DNA, azacitidine forms a covalent complex with DNA methyltransferase, suppresses its activity, and induces DNA hypomethylation to regulate the normal cell cycle. Re-expression of genes involved in differentiation and death allows cells to restore normal function. In addition, the cytotoxic effects of azacitidine can result in the death of rapidly dividing cells, including cancer cells, which no longer respond to the regulatory mechanisms of normal cell growth. Therefore, azacitidine has been tested in clinical trials and has shown considerable antitumor activity, especially in the treatment of myelodysplastic syndrome (MDS).

However, azacitidine has a problem that it is difficult to synthesize, process and store it on a large scale for commercial purposes because of its easily hydrolyzed property. The s-triazine ring of azacitidine tends to be degraded by water, but the bond is cleaved after rapid hydration of the azacitidine 5,6-imine double bond in an aqueous solution at neutral pH. , A formyl derivative N- (formylamidino) -N'-β-D-ribofuranosylurea (RGU-CHO), which is then deformylated by 1-β-. D-ribofuranosyl-3-guanylurea (RGU) is irreversibly produced. Due to the instability of azacitidine in such an aqueous solution, it was not easy to treat and store azacitidine in a solvent containing water.

Therefore, there was a need for a stable, simple and industrially viable method of treating and storing azacitidine.

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

In order to produce a lyophilized preparation of a compound such as azacitidine, a solution-state compound is prepared, sterilized, and then freeze-dried to remove the solvent. In such a process, when azacitidine is exposed to an aqueous solution for several hours, there is a problem that related substances (impurities, impurities) are produced by hydrolysis.
Therefore, an object of the present invention is to provide a method capable of producing a azacitidine-containing pharmaceutical composition by minimizing the formation of related substances by finding a temperature and solvent conditions capable of suppressing the hydrolysis of azacitidine. ..

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

In the whole of the present specification, unless otherwise specified, "containing" or "containing" means to include any component (or component) without any special limitation, and other components (including other components). Or it is not interpreted as excluding the addition of constituents).

In addition, "azacitidine" is not only a azacitidine free base, but also a salt of azacitidine, a polymorph, an isomer, an enantiomer, an anhydrate, a hemihydrate, a solvate, a prodrug, or any mixture thereof. There may be. Here, the "salt" of azacitidine means an acid addition salt of azacitidine derived from an inorganic acid and / or an organic acid, and may be formed from hydrochloric acid, hydrobromic acid, boric acid, phosphoric acid, or sulfuric acid. .. Alternatively, the salt may be formed from acetic acid, citric acid, fumaric acid, maleic acid, malic acid, malonic acid, oxalic acid, succinic acid, tartaric acid, p-toluene sulfonic acid, trifluoromethanesulfonic acid, or trifluoroacetic acid. good. Here, the "polymorph" of azacitidine means a solid crystalline form of azacitidine or a salt or complex thereof. Also, "solvate" of azacitidine means azacitidine or a salt thereof further comprising a stoichiometric or non-stoichiometric amount of solvent bonded by a non-covalent intermolecular force. When the solvent is water, the solvate may be a hydrate, for example a monohydrate, a dihydrate, a trihydrate or a tetrahydrate. Further, the "prodrug" of azacitidine means a compound that is a functional derivative of azacitidine and can be easily converted to azacitidine in vivo.

In order to solve the above problems, the present invention 1) dissolves azacitidine in a mixed solvent of acetonitrile and water whose temperature is maintained at -8 ° C to -1 ° C; and 2) the solution of step 1). Provided is a method for producing an azacitidine-containing pharmaceutical composition, which comprises a step of filling a container under the condition that the outside air temperature is maintained at 15 ° C. or lower.
Hereinafter, the present invention will be described in detail.

In one embodiment, the outside air temperature may be maintained at 10 ° C. or lower in the step 2), but the present invention is not limited to this. When the outside air temperature is maintained below the above temperature, hydrolysis of azacitidine during the container filling step can be suppressed. Even if the azacitidine is dissolved at a low temperature in the above step 1), the azacitidine is rapidly hydrolyzed and the production of related substances is increased when the outside air temperature is over 15 ° C., for example, at room temperature in the container filling step. It is important to maintain the outside air temperature in step 2) below a certain temperature. The outside air temperature may be 15 ° C. or lower or 10 ° C. or lower, and in one embodiment, the outside air 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, and at this time, the azacitidine is hydrolyzed according to the temperature and the required time of the filling operation, and an undesired related substance is produced. However, according to one embodiment of the present invention, when the filling operation is completed within 3 hours or 2 hours at an outside air temperature of 15 ° C. or 10 ° C. or lower and the freeze-drying is started when the step 2) is performed, it is similar. The production of substances can be minimized.

In one embodiment, in the 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. There may be, but it is not limited to this. When the temperature of the mixed solvent is within the above range, hydrolysis during the azacitidine dissolution step can be further suppressed.

In one embodiment, the volume ratio of acetonitrile to water (acetonitrile: water) may be 5:95 to 30:70 in the step 1), but is not limited thereto. If the volume ratio of acetonitrile to water exceeds the above range, not only the stability of azacitidine is reduced, but also the problem 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 to water may be, but is not limited to, preferably 10:90 to 25:75, more preferably 15:85 to 25:75, and even more preferably 20:80.

In one embodiment, the concentration of azacitidine in the pharmaceutical composition can be from about 0.001 to 50 mg / mL, preferably from about 0.1 to 10 mg / mL, more preferably from about 1 to 10 mg / mL. It may be about 2 to 7 mg / mL or about 3 to 5 mg / mL, but is not limited to this, and can be appropriately adjusted according to the administration subject, purpose and the like.

In one embodiment, the method of the present invention further includes, but is not limited to, the step of sterilizing and filtering the solution of the above step 1). The step of sterilizing and filtering may be performed by simple filtration, but is not limited to this, 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 the sterilization filtration method usually performed in the field to which the invention belongs.

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

In one embodiment, in step 1), the mixed solvent further comprises, but is not limited to, a lyophilization aid.

In one embodiment, the freeze-drying aid is mannitol, sodium dihydrogen phosphate, potassium dihydrogen phosphate, tartrate acid, gelatin, glycerin, dextrose, dextran, citric acid, ascorbic acid, sodium hydrogen tartrate, hydroxylation. It is selected from the group consisting of sodium and a mixture thereof, but is not limited to these, and can be appropriately selected and used by a conventional technician from commonly used freeze-drying aids.

For example, the freeze-drying aid may be contained in the mixed solvent of step 1) at a concentration of 1 to 10 mg / mL, preferably 2 to 8 mg / mL, and more preferably 5 mg / mL, but the present invention is not limited thereto. , The concentration of the active ingredient, the type of freeze-drying aid, and the like can be taken into consideration by an ordinary technician to appropriately adjust the amount before use.

In one embodiment, the step 1) is 1-a) a step of dissolving a freeze-drying aid in water to obtain a solution; 1-b) a step of adding acetonitrile to the solution to obtain a mixed solvent; 1 -C) A step of lowering the temperature of the mixed solvent to -8 ° C to -1 ° C; and 1-d) a step of dissolving azacitidine in the mixed solvent of the above temperature; but not limited to this.

The pharmaceutical composition according to the present invention can be used for the treatment or amelioration of various cancer and tumor-related diseases including myelodysplastic syndrome and acute myeloid 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 the azacitidine-derived relatives 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. 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 route of administration 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 of injection, such as, but not limited to, transdermal, nasal, abdominal, muscle, subcutaneous, and intravenous injections.

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

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

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

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

According to one embodiment of the present invention, it is possible to produce an azacitidine-containing pharmaceutical composition that minimizes the generation of related substances due to hydrolysis of azacitidine and meets the related substance standards under the strictest conditions (USP monograph). Due to its complexity or the lack of additional steps, it enables the processing and storage of azacitidine that is feasible on an industrial scale.
In addition, the stable azacitidine-containing pharmaceutical composition as described above can be used for the treatment of various types of cancer and tumor-related diseases in mammals.

Production Examples 1 to 4: Production of low-temperature preparation solution using solvent (1) Production of low-temperature preparation solution using water for injection 15 g of mannitol was added to 3000 mL of water for injection in the preparation tank and completely dissolved. The temperature of this preparation tank was lowered sufficiently so that it was maintained at 5 ± 3 ° C. After confirming that the temperature of the preparation solution had dropped to the refrigerating temperature (5 ± 3 ° C.), 15 g of azacitidine was added, and the mixture was stirred at 500 ± 50 rpm for 20 ± 10 minutes. After confirming that it was completely dissolved, this preparation was sterile filtered.

(2) Production of low-temperature preparation solution using organic solvent To 2400 mL of water for injection in the preparation tank, 15 g of mannitol was added and completely dissolved. The temperature of this preparation tank was sufficiently lowered to maintain it at -3 ± 2 ° C. When mannitol was completely dissolved, 600 mL of an organic solvent (acetonitrile, ethanol or tertiary butanol) of the type shown in Table 1 below was added and mixed thoroughly. After confirming that the temperature of the preparation solution had dropped to -3 ± 2 ° C., 15 g of azacitidine was added, and the mixture was stirred at 500 ± 50 rpm for 70 ± 10 minutes. After confirming that it was completely dissolved, this preparation was sterile filtered.
The prepared solution was placed in a vial of 30 mL each, stored in a chamber at 5 ° C., 15 ° C. or 25 ° C., and sampled at 1-hour intervals to measure related substances for up to 4 hours.

Experimental Example 1: Evaluation of related substances by storage temperature according to the type of solvent The related substances in the preparation liquid of Production Examples 1 to 4 were analyzed by the following method. The standard solution and the test solution at the time of analysis were stored at 2 to 8 ° C.

1) Preparation of diluted solution The concentration of sodium bisulfite was adjusted to 10.0 g / L using purified water, and the pH was adjusted to 2.5 using diluted sulfuric acid.

2) Preparation of standard solution In a 50 mL volumetric flask, 5.0 mg of precisely quantified azacitidine was placed, marked with 20% acetonitrile, 1 mL of this solution was placed in a 10 mL volumetric flask, and 3 mL of 20% acetonitrile was added thereto. After that, the marked lines were aligned with the diluted solution (concentration 0.01 mg / mL).

3) Preparation of system compatibility solution 25 mg of a precisely quantified azacitidine standard product was placed in a 5 mL volumetric flask, 20% acetonitrile was added to dissolve it, and the marked lines were aligned. 2 mL of this solution was taken, 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 diluted solution, and mixed well to prepare a test solution (concentration 2 mg / mL).

5) Liquid chromatography conditions a. Column: Orosil C18, 4.6 mm × 25 cm, 3 μm, or similar column b. Detector: Ultraviolet absorptiometer (measurement wavelength: 210 nm)
c. Injection volume: 5 μL
d. Flow rate: 0.8 mL / min e. Temperature of automatic sample injector: 5 ° C
g. Mobile phase: Gradient elution Execution 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 compatibility and operation method A system-compatible solution is injected, tailing of azacitidine peak is 2.0 or less, standard solution is injected 6 times or more, and the partner standard deviation of the peak area is 10.0% or less. At that time, it was judged that the system was suitable, and the standard solution and the test solution were injected in this order to determine the peak area.

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

The criteria for determining azacitidine-related substances are shown in Table 3 below (however, peaks of 0.04% or less have not been reported). The results of analysis of related substances by azacitidine solvent and temperature used are shown in Tables 4 to 7. Table 4 uses water for injection (Production Example 1), Table 5 uses acetonitrile (Production Example 2), Table 6 uses ethanol (Production Example 3), and Table 7 uses tertiary butanol (Production Example 4) as solvents. The results of analysis of related substances are shown after the preparation liquid is produced.

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

In the case of a composition produced only with water without using an organic solvent, the preparation solution freezes when produced at 0 ° C. or lower, so that there is a problem in producing at a refrigerating temperature or lower. Therefore, in the case of the composition produced with water for injection, it was not possible to maintain below the relative substance standard for 1 hour at all temperatures except storage at 5 ° C. On the other hand, in the case of the preparation liquid produced using an organic solvent, it was possible to produce at a low temperature of about -3 ° C. Acetonitrile showed the best stability in the preparations prepared using the three solvents of acetonitrile, ethanol and tertiary butanol. Next, the excellent stability was shown in the order of ethanol and tertiary butanol. In particular, in the case of acetonitrile, it was very effective in regulating the water and temperature sensitive name RGU-CH.

Production Examples 5 to 7: Preparation of preparation liquid according to acetonitrile concentration To 2100 mL of water for injection in the preparation tank, 15 g of mannitol was added and completely dissolved. The temperature of this preparation tank was sufficiently lowered to maintain it at -3 ± 2 ° C. When mannitol was completely dissolved, water for injection and / or acetonitrile was added as shown in Table 8 below, and the mixture was thoroughly mixed. After confirming that the temperature of the preparation solution had dropped to -3 ± 2 ° C., 15 g of azacitidine was added, and the mixture was stirred at 500 ± 50 rpm for 70 ± 10 minutes. After confirming that it was completely dissolved, this preparation was sterile filtered.
30 mL of this preparation was placed in vials and then 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 storage temperature-specific related substances according to acetonitrile concentration The related substances in the preparation liquid according to Production Examples 5 to 7 were analyzed by the same method as in Experimental Example 1. Tables 9 to 11 below show the results of analysis of related substances after the preparation liquid was prepared using 10%, 20% and 30% acetonitrile (ACN), respectively.

In the case of the preparation solution prepared using 10% acetonitrile (Production Example 5), the relative substance standard was exceeded after 2 hours at 10 ° C. and 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 It was confirmed that the stability was shown at 3 hours at ° C. and 2 hours at 15 ° C., and the best stability was shown at a ratio of acetonitrile of 20%.

Example 1: Preparation of a sample in a low temperature step using 20% acetonitrile To 2400 mL of water for injection in a preparation tank, 15 g of mannitol was added and completely dissolved. The temperature of this preparation tank was sufficiently lowered to maintain it at -3 ± 2 ° C. When mannitol was completely dissolved, 600 mL of acetonitrile was added and mixed well. After confirming that the temperature of the preparation solution had dropped to -3 ± 2 ° C., 15 g of azacitidine was added, and the mixture was stirred at 500 ± 50 rpm for 70 ± 10 minutes. After confirming that it was completely dissolved, this preparation was sterile filtered. After filling the vial under the condition of 10 ° C. or lower, the prepared solution is further stored in a 10 ° C. chamber for 2 hours in consideration of the time required for filling in an actual factory, placed in a freeze-dryer, and freeze-dried. Samples were taken and analyzed.

Comparative Example 1: Sample production in a room temperature step using 20% acetonitrile A preparation solution was produced in the same manner as in Example 1, and then sterilized and filtered. After filling the vial at room temperature, the preparation solution was further stored at room temperature for 2 hours in consideration of the time required for filling in the actual factory, placed in a lyophilizer, lyophilized, and then a sample was taken and analyzed. ..

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

Comparative Example 3: Sample production using purified water in a room temperature step After producing a preparation solution in the same manner as in Example 1 except that the preparation temperature was maintained at 5 ± 3 ° C. using purified water, it was sterilized. Filtered. After filling the vial under room temperature conditions, considering the time required for filling in the actual factory, the preparation solution is further stored at room temperature for 2 hours, placed in a lyophilizer, lyophilized, and then a sample is taken and analyzed. did.
The preparation conditions according to Comparative Examples 1 to 3 are shown in comparison with Table 12 below.

Experimental Example 3: Evaluation of related substances for optimum temperature and storage conditions The related substances in the lyophilized samples according to Example 1 and Comparative Examples 1 to 3 were analyzed by the same method as in Experimental Example 1. At this time, about 10 mL of 20% acetonitrile was added to the sample after freeze-drying using a syringe, and the sample was dissolved by shaking well 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 (concentration 5 mg / mL). 2 mL of this well-dissolved solution was taken, placed in a vial containing 3 mL of diluted solution, and mixed well to prepare a test solution (concentration 2 mg / mL). The results of analysis of related substances in the freeze-dried sample are shown in Table 13 below.

Claims (10)

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) Conditions under which the solution of step 1) is maintained at an outside air temperature of 15 ° C or lower. Below, the process of filling the container;
A method for producing a pharmaceutical composition containing azacitidine. The method for producing an azacitidine-containing pharmaceutical composition according to claim 1, wherein the solution is filled in step 2) within 3 hours. The method for producing an azacitidine-containing pharmaceutical composition according to claim 1, wherein in step 1), the volume ratio of acetonitrile to water (acetonitrile: water) is 5:95 to 30:70. The method for producing an azacitidine-containing pharmaceutical composition according to claim 1, further comprising a step of sterilizing and filtering the solution of step 1). The method for producing an azacitidine-containing pharmaceutical composition according to claim 1, further comprising a step of freeze-drying the filler in step 2). The method for producing an azacitidine-containing pharmaceutical composition according to claim 5, wherein in step 1), the mixed solvent further contains a lyophilization aid. Hydration aids include mannitol, sodium dihydrogen phosphate, potassium dihydrogen phosphate, tartaric acid, gelatin, glycerin, dextrose, dextran, citric acid, ascorbic acid, sodium hydrogen tartrate, sodium hydroxide, and mixtures thereof. The method for producing a azacitidine-containing pharmaceutical composition according to claim 6, which is selected from the group consisting of. Step 1)
1-a) A step of dissolving a freeze-drying aid in water to obtain a solution;
1-b) A step of adding acetonitrile to the solution to obtain a mixed solvent;
1-c) Step of lowering the temperature of the mixed solvent to -8 ° C to -1 ° C; and 1-d) Step of dissolving azacitidine in the mixed solvent of the temperature;
The method for producing an azacitidine-containing pharmaceutical composition according to claim 1. 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 an azacitidine-containing pharmaceutical composition according to claim 1, wherein the container in step 2) is a glass vial.