WO2019231225A1 - Method for preparing stable azacitidine-containing pharmaceutical composition - Google Patents

Abstract

The present invention relates to a method for preparing a stable azacitidine-containing pharmaceutical composition by means of a solvent comprising acetonitrile, and a low temperature process.

Description

Process for preparing stable azacytidine-containing pharmaceutical composition

The present invention relates to a process for preparing a stable azacytidine-containing pharmaceutical composition using a solvent comprising acetonitrile and a low temperature process.

Azazitidine (AZA) having the chemical structure shown below is 4-amino-1- (3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl) -1H- [1, 3,5] triazine-2-one or 5-azacytidine and the like, and are currently marketed as the drug VIDAZA ^™ .

Azatisitidine

Azacytidine is a chemical analog of cytidine, a nucleoside present in DNA and RNA, and azacytidine and its deoxy derivative 5-aza-2'deoxycytidine are designed to reduce DNA methylation. It has been used clinically for the treatment of acute myeloid leukemia and the like as a cleoside analog (Molecules, v.19, no.3, pp.3149-3159, 2014). Azacytidine, after incorporation into replicating DNA, forms covalent complexes with DNA methyltransferases and inhibits their activity to induce DNA hypomethylation, thereby re-expressing genes involved in normal cell cycle regulation, differentiation and death. Thereby allowing cells to restore normal function. The cytotoxic effect of azacytidine can also result in the death of rapidly dividing cells, including cancer cells, which no longer respond to normal cell growth control mechanisms. Thus, azacytidine has been tested in clinical trials and has shown significant antitumor activity, particularly in the treatment of myelodysplastic syndromes (MDS).

However, azacytidine has a problem in that it is difficult to synthesize, process, and store large scale for commercial purposes because of its easy hydrolysis property. The s -triazine ring of azacytidine tends to decompose in water, and after the hydration of the 5,6-imine double bond of azacytidine occurs in an aqueous solution at neutral pH, the bond is cleaved to form N- , a formyl derivative. (formyl amidino) - N '-β-D- ribo furanyl nosil urea (RGU-CHO) is obtained, which is then de-formylated-β-D- ribo furanyl by being 1-3 nosil not obtain urea (RGU ) Is irreversibly generated. Due to the instability of azacytidine in this aqueous solution, the treatment and storage of azacytidine in a solvent containing water was not easy.

Therefore, a need exists for a method for the treatment and storage of azacytidine that is stable, simple and can be performed on an industrial scale.

To prepare a lyophilized formulation of compounds such as azacytidine, a solution is prepared, sterilized, and lyophilized to remove the solvent. In the process, azacytidine is exposed to an aqueous solution for several hours. As a result, there was a problem in that a flexible material (impurity, impurities) is generated by hydrolysis.

Accordingly, an object of the present invention is to provide a method for preparing azacytidine-containing pharmaceutical compositions by minimizing the formation of analogues by searching for temperature and solvent conditions that can inhibit the hydrolysis of azacytidine. .

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

Unless specifically stated throughout this specification, "including" or "containing" refers to including any component (or component) without particular limitation, and excludes the addition of other components (or components). Not interpreted as

"Azacytidine" may also be azacytidine free base, as well as salts, polymorphs, isomers, enantiomers, anhydrides, hemihydrates, solvates, prodrugs, or any mixture thereof. have. “Salts” of azacytidine herein refer to acid addition salts of azacytidine derived from inorganic and / or organic acids 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-toluenesulfonic acid, trifluoromethanesulfonic acid, or trifluoroacetic acid. The “polymorph” of azacytidine herein refers to the solid crystalline form of azacytidine or salts or complexes thereof. "Solvate" of azacytidine also refers to azacytidine or a salt thereof, further comprising a stoichiometric or non-stoichiometric amount of solvent bound by noncovalent intermolecular forces. If the solvent is water, the solvate may be a hydrate, for example monohydrate, dihydrate, trihydrate or tetrahydrate. "Prodrug" of azacytidine also refers to compounds that are functional derivatives of azacytidine and are readily convertible to azacytidine in vivo.

In order to solve the above problems, the present invention comprises the steps of 1) dissolving azacytidine in a mixed solvent of acetonitrile and water, the temperature of which is maintained at -8 ℃ to -1 ℃; And 2) filling the solution of step 1) into a container under conditions where the outside temperature is maintained at 15 ° C. or lower.

Hereinafter, the present invention will be described in detail.

In one embodiment, the outside air temperature in step 2) may be maintained below 10 ° C, but may not be limited thereto. When the outside air temperature is kept below the above temperature, hydrolysis of azacytidine during the vessel filling process can be suppressed. Even if the azacytidine dissolution in step 1) is carried out at low temperature, when the outside air temperature is higher than 15 ° C., for example, room temperature, in the vessel filling step, the azacytidine is rapidly hydrolyzed to increase the formation of the flexible substance, It is important to keep the outside air temperature in 2) below a certain temperature. The outside air temperature may be 15 ° C. or less or 10 ° C. or less, and in one embodiment, the outside air temperature may be 0 ° C. or more, 3 ° C. or more, or 5 ° C. or more.

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

In the industrial preparation of the azacytidine-containing pharmaceutical composition, it takes a certain amount of time to fill. At this time, the azacytidine is hydrolyzed according to the temperature and the time required for the filling operation to produce an undesirable flexible substance. When performing step 2) 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 less and start lyophilization, generation of a flexible material may be minimized.

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

In one embodiment, the volume ratio (acetonitrile: water) of acetonitrile and water in step 1) may be 5:95 to 30:70, but may not be limited thereto. If the volume ratio of acetonitrile to water exceeds the above range, not only the stability of azacytidine is reduced, but also a problem of removing residual solvent may occur later. If the volume ratio of acetonitrile to water is below the above range, azacytidine May not be sufficiently secured. The volume ratio of acetonitrile to water may be preferably 10:90 to 25:75, more preferably 15:85 to 25:75, and more preferably 20:80, but is not limited thereto. Can be.

In one embodiment, the azacytidine concentration in the pharmaceutical composition may be about 0.001 to 50 mg / ml, preferably about 0.1 to 10 mg / ml, more preferably about 1 to 10 mg / ml, about 2 to 7 mg / ml, or about 3 to 5 mg / ml, but may not be limited thereto and may be appropriately adjusted depending on the administration target and purpose.

In one embodiment, the method of the present invention may further comprise the step of sterile filtration of the solution of step 1), but may not be limited thereto. The sterile filtration step may be performed by simple filtering, but may not be limited thereto, and may be selected by varying the pore size of the filter according to the degree of sterilization desired. It can be carried out without particular limitation by a sterile filtration method carried out.

In one embodiment, the method of the present invention may further include the step of lyophilizing the filler of step 2), but may not be limited thereto. The freeze-drying step may be carried out without particular limitation by a lyophilization method commonly performed in the field to which the present invention belongs.

In one embodiment, the mixed solvent in step 1) may further include a lyophilization aid, but may not be 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 tartaric acid hydrogen sulfite, sodium hydride It may be selected from the group consisting of a lockside, and mixtures thereof, but may not be limited thereto, and may be appropriately selected and used by those skilled in the art from commonly used lyophilization aids.

For example, the lyophilization aid may be included in the mixed solvent of step 1) 1 to 10 mg / ml, preferably 2 to 8 mg / ml, more preferably 5 mg / ml, but may not be limited thereto. In consideration of the concentration of the active ingredient and the type of lyophilization aid, a person skilled in the art can adjust the amount appropriately.

In one embodiment, the step 1) is 1-a) lyophilization aid in water to obtain a solution, 1-b) acetonitrile to the solution to obtain a mixed solvent, 1-c) of the mixed solvent Lowering the temperature to -8 ° C to -1 ° C, 1-d) may include dissolving azacytidine in the mixed solvent of the temperature, but may not be limited thereto.

The pharmaceutical compositions according to the invention can be used for the treatment or amelioration of various cancers and tumor related diseases, including myelodysplastic syndromes and acute myeloid leukemia (AML). More specifically, the pharmaceutical composition can be used for treating or ameliorating diseases such as disorders associated with abnormal cell proliferation and blood disorders.

In one embodiment, the content of azacytidine-derived analog contained in the pharmaceutical composition is less than 2.5%, less than 2%, less than 1.5%, less than 1.2%, less than 1%, less than 0.8%, less than 0.6%, It may be less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%, and the content of such flexible material may be determined by conditions such as the criteria for determining the flexible material, time spent in the process, process temperature and concentration of acetonitrile. Can vary.

The route of administration of the pharmaceutical composition is not limited thereto, and may be administered orally or parenterally. Parenteral routes of administration may include, but are not limited to, several routes of injection administration, for example, transdermal, nasal, abdominal, intramuscular, subcutaneous, intravenous, and the like.

Thus, the pharmaceutical composition according to the present invention may comprise a pharmaceutically acceptable carrier and may further comprise a buffer, an isotonic agent and / or an antimicrobial agent.

In one embodiment, the azacytidine-containing pharmaceutical composition may be an injection preparation, but may not be limited thereto.

For example, the pharmaceutical compositions of the present invention may be formulated according to methods known in the art in the form of injectable preparations with a pharmaceutically acceptable parenteral carrier. In this case, the preparation for injection must be sterile and protected from contamination of 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 (e.g., glycerol, propylene glycol and liquid polyethylene glycols, etc.), mixtures thereof, and / or vegetable oils. have. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Etc. can be used. In addition, it may further include various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like to protect the injectable preparations from microbial contamination. Alternatively, the injectable preparation may further comprise an isotonic agent such as sugar or sodium chloride selected from the group consisting of mannitol, lactose, dextrose and trihalose. Other pharmaceutically acceptable carriers, etc. may be referred to those described in the following literature (Remington's Pharmaceutical Sciences, 19th Edition, 1995, Mack Publishing Company, Easton, PA). In addition, the injectable preparation may be filled in a pharmaceutically suitable container.

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

According to one embodiment of the present invention, a stable azacytidine-containing pharmaceutical composition which can meet the conformation criteria of the most stringent conditions (USP monograph) by minimizing the generation of analogues due to hydrolysis of azacytidine It is possible to prepare and to handle and store azacytidine which can be performed on an industrial scale as it is not complicated or requires additional processing.

In addition, such stable azacytidine-containing pharmaceutical compositions can be used for the treatment of various types of cancer and tumor related diseases in mammals.

Preparation Examples 1 to 4 Preparation of Low Temperature Preparation Solution According to Solvent

(1) Preparation 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 to dissolve completely. The preparation tank was cooled down sufficiently to maintain 5 ± 3 ° C. After confirming that the temperature of the preparation liquid was lowered to the refrigerating temperature (5 ± 3 ° C.), 15 g of azacytidine was added thereto and stirred at 500 ± 50 rpm for 20 ± 10 minutes. It was confirmed that it was completely dissolved and the preparation was sterile filtered.

(2) Preparation of low temperature preparation liquid using organic solvent

15 g of mannitol was completely dissolved in 2400 ml of water for injection in the preparation tank. The preparation tank was cooled down sufficiently to maintain -3 ± 2 ° C. When mannitol was completely dissolved, 600 ml of organic solvents (acetonitrile, ethanol or tert-butanol) of the type shown in Table 1 below were added and mixed well. After confirming that the temperature of the preparation liquid was lowered to -3 ± 2 ° C., 15 g of azacytidine was added thereto, followed by stirring at 500 ± 50 rpm for 70 ± 10 minutes. It was confirmed that it was completely dissolved and the preparation was sterile filtered.

30 ml of the above preparations were added to the vial and then stored in a 5 ° C., 15 ° C. or 25 ° C. chamber, and sampled at 1 hour intervals to measure the flexible material up to 4 hours.

TABLE 1

[ Experimental Example  1] Storage temperature by solvent type Leading substance  evaluation

A flexible material in the preparation liquid of Preparation Examples 1 to 4 was analyzed by the following method. The standard solution and the sample solution were stored at 2 ~ 8 ℃ for analysis.

1) Preparation of diluent

The concentration of sodium hydrogen sulfite was adjusted to 10.0 g / L with purified water and adjusted to pH 2.5 with diluted sulfuric acid.

2) Standard Solution

Precipitate 5.0 mg of azacytidine in a 50 mL volumetric flask, mark it with 20% acetonitrile, add 1 mL of this solution to a 10 mL volumetric flask, add 3 mL of 20% acetonitrile, and dilute Mark was adjusted (concentration 0.01 mg / mL).

3) Preparation of system compatibility solution

Accurately weigh 25 mg of azacytidine standard into a 5 mL volumetric flask, add 20% acetonitrile to dissolve and mark. 2 mL of this solution was taken into 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 and placed in a vial containing 3 ml of diluent, mixed well to obtain a sample solution (concentration 2 mg / mL).

5) Liquid chromatograph conditions

a. Column: Orosil C18, 4.6 mm × 25 cm, 3 μm, or similar column

b. Detector: UV absorbance photometer (wavelength: 210 nm)

c. Injection volume: 5 μl

d. Flow rate: 0.8 mL / min

e. Autosampler temperature: 5 ℃

g. Mobile phase: gradient elution

Performance conditions are shown in Table 2 below.

TABLE 2

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 operation

When the system compatibility solution is injected, the azacytidine peak tailing is 2.0 or less and the standard solution is injected 6 times or more. If the relative standard deviation of the peak area is 10.0% or less, the system is considered suitable. Obtained.

7) Calculation

The concentration of each analog was calculated using the following formula.

% Of analogous substance =

The criteria for determining azacytidine analogs are shown in Table 3 below, except that peaks below 0.04% are not reported. In addition, the azacytidine solvent and the temperature of the used material analysis results are shown in Tables 4 to 7. Table 4 is water for injection (Preparation Example 1), Table 5 is acetonitrile (Preparation Example 2), Table 6 is ethanol (Preparation Example 3), and Table 7 is prepared using a tertiary butanol (Preparation Example 4) as a solvent It shows the result of analysis of lead substance after liquid preparation.

TABLE 3

a. 1-β-D-ribofuranosyl-3-guanylurea

b. N- (diaminoethylene) N '-(β-D-ribofuranosyl) carbamidic acid

c. 1-β-D-ribofuranosyl-3-aminocarbonyl guanidine

d. 1-β-D-ribofuranosyl-3-iminohydroxymethyl guanidine

e. N- (formyl amidino) -N'-β-D-ribofuranosylurea (RGU-CHO)

f. Excluding N- (formyl amidino) -N'-β-D-ribofuranosylurea from total flexible materials

TABLE 4

TABLE 5

TABLE 6

TABLE 7

In the case of a composition prepared only by water without using an organic solvent, when the preparation was made at 0 ° C. or lower, the preparation was frozen. Therefore, the composition prepared with water for injection did not maintain below the flexible material standards for 1 hour at all temperatures except 5 ℃ storage. On the other hand, the preparation prepared using the organic solvent was possible at a low temperature of about -3 ℃. Acetonitrile showed the highest stability in the preparation prepared using acetonitrile, ethanol and tertiary butanol three solvents, followed by ethanol and tertiary butanol. In particular, acetonitrile was very effective in regulating water and temperature sensitive RGU-CHO.

Preparation Examples 5 to 7 Preparation of Preparation Solutions for Each Acetonitrile Concentration

15 g of mannitol was completely dissolved in 2100 ml of water for injection in the preparation tank. The preparation tank was cooled down sufficiently to maintain -3 ± 2 ° C. When mannitol was completely dissolved, water for injection and / or acetonitrile were added and mixed well as shown in Table 8 below. It was confirmed that the temperature of the preparation liquid was lowered to −3 ± 2 ° C., and 15 g of azacytidine was added thereto, followed by stirring at 500 ± 50 rpm for 70 ± 10 minutes. It was confirmed that it was completely dissolved and the preparation was sterile filtered.

30 ml of this preparation solution was added to the vial, and then stored in a 5 ° C., 10 ° C. or 15 ° C. chamber, and sampled at 1 hour intervals to measure the flexible material up to 3 hours.

TABLE 8

[ Experimental Example  2] Acetonitrile  According to concentration By storage temperature Leading substance  evaluation

In the same manner as in Experiment 1, the flexible material in the preparation according to Preparation Examples 5 to 7 was analyzed. Tables 9 to 11 below show the analysis results of analogues after preparation of the preparation solution using 10%, 20% and 30% acetonitrile (ACN), respectively.

TABLE 9

TABLE 10

TABLE 11

In the case of the preparation (Preparation Example 5) prepared using 10% acetonitrile, the flexible substance standard was exceeded in 2 hours at 10 ° C. and 1 hour at 15 ° C. In the case of the preparation liquid prepared in 30% acetonitrile solution (preparation example 7), the flexible substance exceeded the standard in 10 hours at 10 ° C., but in the case of the preparation liquid prepared in 20% acetonitrile solution (preparation example 6) 10 ° C. In 3 hours, at 15 ℃ 2 hours stability was confirmed that the best stability in the acetonitrile ratio of 20%.

[ Example  1] 20% Acetonitrile  Sample preparation at low temperature process

15 g of mannitol was completely dissolved in 2400 ml of water for injection in the preparation tank. The preparation tank was cooled down sufficiently to maintain -3 ± 2 ° C. Once mannitol was completely dissolved, 600 ml acetonitrile was added and mixed well. After confirming that the temperature of the preparation liquid was lowered to -3 ± 2 ° C., 15 g of azacytidine was added thereto, followed by stirring at 500 ± 50 rpm for 70 ± 10 minutes. It was confirmed that it was completely dissolved and the preparation was sterile filtered. After the above preparation was filled in vials under 10 ° C. or less, the sample was stored for 2 hours in a 10 ° C. chamber and lyophilized in a lyophilizer in consideration of the time taken for filling in the factory.

[ Comparative example  1] 20% Acetonitrile  Sample preparation at room temperature

The preparation was prepared in the same manner as in Example 1, followed by sterile filtering. After filling the vial at room temperature with the vial, the sample was stored for 2 hours at room temperature and lyophilized in a lyophilizer in consideration of the time required for filling in the factory.

[ Comparative example  2] Purified water  Sample preparation at low temperature process

Purified water was used, and except that the preparation temperature was maintained at 5 ± 3 ° C., the preparation was prepared in the same manner as in Example 1, followed by sterile filtering. After the above preparation was filled in vials under 10 ° C. or less, the sample was stored for 2 hours in a 10 ° C. chamber and lyophilized in a lyophilizer in consideration of the time taken for filling in the factory.

[ Comparative example  3] Purified water  Sample preparation at room temperature

Purified water was used and the preparation was prepared in the same manner as in Example 1 except that the preparation temperature was maintained at 5 ± 3 ° C., followed by sterile filtering. After the above preparation was filled in vials at room temperature, the sample was stored for 2 hours at room temperature and lyophilized in a lyophilizer in consideration of the time taken for filling in the factory.

Preparation conditions according to Comparative Examples 1 to 3 are shown in comparison with Table 12 below.

TABLE 12

Experimental Example 3 Evaluation of Flexible Materials for Optimal Temperature and Storage Conditions

In the same manner as in Experiment 1, the flexible material in the lyophilized samples according to Example 1 and Comparative Examples 1 to 3 was analyzed. At this time, the lyophilized sample was added to about 10 mL of 20% acetonitrile using a syringe, shaken well, and then transferred to a 20 mL volumetric flask. The vial was rinsed once or twice with a small amount of 20% acetonitrile and placed in a volumetric flask and labeled with 20% acetonitrile (concentration 5 mg / mL). 2 mL of this well-dissolved solution was taken and placed in a vial containing 3 ml of diluent, mixed well to obtain a sample solution (concentration 2 mg / mL). The results of analogue analysis of lyophilized samples are shown in Table 13 below.

TABLE 13

Claims (10)

1) dissolving azacytidine in a mixed solvent of acetonitrile and water whose temperature is maintained at -8 ° C to -1 ° C; And

2) filling the container of step 1) in a container under conditions in which the outside temperature is maintained at 15 ° C. or lower.

A process for preparing azacytidine-containing pharmaceutical composition.

The method of claim 1, wherein the solution is filled within 3 hours in step 2).

The method of claim 1, wherein the volume ratio of acetonitrile to water (acetonitrile: water) in step 1) is from 5:95 to 30:70.

The method of claim 1, further comprising sterile filtration of the solution of step 1).

The method of claim 1, further comprising lyophilizing the fill of step 2).

The method of claim 5, wherein the mixed solvent in step 1) further comprises a lyophilization aid.

The lyophilization aid according to claim 6, wherein the lyophilization aid is mannitol, sodium dihydrogen phosphate, potassium dihydrogen phosphate, tartaric acid, gelatin, glycerin, dextrose, dextran, citric acid, ascorbic acid, sodium tartaric acid hydrogen sulfite, sodium hydride A method for preparing an azacytidine-containing pharmaceutical composition, which is selected from the group consisting of a lockside, and mixtures thereof.

The method of claim 1, wherein step 1)

1-a) dissolving the lyophilization aid in water to obtain a solution,

1-b) acetonitrile was added to the solution to obtain a mixed solvent,

1-c) lowering the temperature of the mixed solvent from -8 ° C to -1 ° C,

1-d) comprising the step of dissolving azacytidine in the mixed solvent of the temperature,

A process for preparing azacytidine-containing pharmaceutical composition.

The method of claim 1, wherein the azacytidine-containing pharmaceutical composition is an injectable preparation.

The method of claim 1, wherein the container of step 2) is a glass vial.