KR101286802B1 - Pre-freeze dried preparation of azacitidine, freeze dried preparation of azacitidine and method of manufacturing for the same - Google Patents

Abstract

The present invention relates to a pre-lyophilized formulation of azacytidine, a lyophilized formulation of azacytidine and a method for preparing the same, wherein the pre-lyophilized formulation of azacytidine according to the present invention has improved stability in aqueous solution and , Freeze-dried preparations of azacytidine with improved stability can be advantageously prepared by freeze-drying using the pre-lyophilized preparations of azacytidine under process conditions including a predetermined drying cycle.

Description

Pre-freeze-dried preparation of azacytidine, freeze-dried preparation of azacytidine and its manufacturing method{PRE-FREEZE DRIED PREPARATION OF AZACITIDINE, FREEZE DRIED PREPARATION OF AZACITIDINE AND METHOD OF MANUFACTURING FOR THE SAME}

The present invention relates to a pre-lyophilized formulation of azacytidine, a lyophilized formulation of azacytidine and a method for preparing the same.

[Advanced technical literature]

[Patent Document]

[Patent Document 1] Republic of Korea Patent Publication No. 10-2011-0026019, 2011.03.14, page 5, [0005] to [0007] paragraph

[Non-patent literature]

[Non-Patent Document 1] Product Information [on-line] for 5-Azacytidine from sigma, ( http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/a2385pis.Par.0001.File. tmp/a2385pis.pdf ), search date: 2011.04.06

Azacitidine is a compound represented by the following Chemical Formula 1, 4-amino-1-β-D-ribofuranosyl-1,3,5-triazine-2(1H)-one (4-amino-1- Also called β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one ([Patent Document 1] Korean Patent Publication No. 10-2011-0026019, 2011.03.14, 5 pages, [0005] To paragraph).

[Formula 1]

Azacytidine is an anti-malignant tumor agent that is active against leukemia, lymphoma and various solid tumors. Azacytidine is a drug of the DNA demethylating agent family, acts as an inhibitor of DNA methyltransferase, and is approved for the treatment of myelodysplastic syndrome, a bone marrow disease system. The compound is currently marketed under the trade name VIDAZA ^® by Celgene ([Patent Document 1] Korean Patent Publication No. 10-2011-0026019, 2011.03.14, page 5, [0005] to [0007] paragraph) .

Since azacytidine decomposes rapidly due to hydrolysis with water in aqueous solution conditions, it was not able to be maintained for a long time in the pre-lyophilized formulation state in aqueous solution ([Non-Patent Document 1] Product Information [on Sigma's 5-Azacytidine] -line], ( http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/a2385pis.Par.0001.File.tmp/a2385pis.pdf )). Therefore, there was a difficulty in mass-producing azacytidine lyophilized preparations for using azacytidine as a pharmaceutical preparation.

Accordingly, the present inventors studied the pre-freeze dried preparation of azacitidine to improve the retention time and stability of the pre-lyophilized preparation state of azacitidine in aqueous solution, and the azacitidine The present invention was completed by developing a manufacturing process capable of mass-producing azacytidine lyophilized formulations with improved stability using pre-lyophilized formulations.

An object of the present invention is to provide an azacytidine pre-lyophilized formulation with improved stability.

Another object of the present invention is to provide a method for preparing azacytidine lyophilized preparation having improved stability.

Another object of the present invention is to provide an azacytidine lyophilized formulation prepared by a method for preparing azacytidine lyophilized formulation having improved stability.

In order to achieve the above object, the present invention provides a pre-lyophilized formulation of azacitidine in which azacitidine is dissolved in an aqueous solution containing 40 to 60% (volume/volume) of tert-butanol.

The pre-lyophilized preparation of azacytidine of the present invention means a preparation in which azacytidine is stabilized in an aqueous solution as a preparation before lyophilization.

The pre-lyophilized preparation of azacytidine according to the present invention may stabilize the pre-lyophilized preparation of azacytidine as the content of tertiary butanol increases in an aqueous solution containing tertiary butanol as a solvent. On the other hand, when the content of tertiary butanol (70% (volume/volume) or higher) becomes high, azacytidine recrystallization may occur in the pre-lyophilized preparation of azacytidine or it may not be dissolved.

The preferred pre-lyophilized preparation of azacytidine according to the present invention may be an aqueous solution containing 40 to 60% (vol/vol) of tert-butanol.

In general, the azacytidine aqueous solution has low stability even at 5°C, but the pre-lyophilized formulation of azacytidine according to the present invention is preferably stable at 5 to 20°C, more preferably 5 to 15°C can do.

The pre-lyophilized preparation of azacytidine according to the present invention is preferably prepared at 5 to 20°C, more preferably 5 to 15°C, and even more preferably 5°C. Can be.

The pre-lyophilized formulation of azacitidine may have a residual ratio of azacitidine of 85% or more after 10 hours at 5 to 20°C.

The pre-lyophilized formulation of azacytidine may have a residual rate of azacytidine of 95% or more after 10 hours at 5°C. Preferably, the pre-lyophilized formulation of azacytidine may have a residual rate of azacytidine of 98% or more after 10 hours at 5°C.

The lyophilized formulation of azacytidine prepared using the pre-lyophilized formulation of azacytidine prepared according to the above composition is also stable.

In order to achieve another object according to the present invention, the present invention forms a pre-lyophilized formulation of azacitidine in which azacitidine is dissolved in an aqueous solution containing 40 to 60% (volume/volume) of tert-butanol. step; Adding an excipient to the pre-lyophilized formulation of azacytidine; And lyophilizing the pre-lyophilized preparation to which the excipients have been added.

The excipient is a substance used to blend the active pharmaceutical ingredient (azacytidine) into a pharmaceutical formulation. Preferably, the excipient is a compound that does not lower or interfere with the therapeutic effect of the active pharmaceutical ingredient (azacytidine). The excipient may include a carrier, diluent, solubilizer, stabilizer, and the like.

The excipient may be a polyol. Preferably, the polyol may be one or two or more selected from the group consisting of mannitol, sorbitol, and trehalose. More preferably, the polyol may be mannitol.

The concentration of azacytidine in the pre-lyophilized formulation of azacytidine may be 0.2 to 5 mg/ml.

In order to achieve another object according to the present invention, the present invention freeze-dried the pre-lyophilized preparation to which the excipient was added is frozen by freezing the pre-lyophilized preparation of azacytidine at a temperature of -55°C. Forming an isolated formulation; Maintaining the frozen formulation at a temperature of -55°C for 6 hours; Forming a dried formulation by heating the formulation maintained at a temperature of -55 DEG C or lower for 6 hours to a primary drying temperature of -55 to -20 DEG C for 3 hours; Maintaining the formulation dried at the primary drying temperature at -20°C for 6 hours; Forming the dried formulation by heating the formulation maintained at -20°C for 6 hours to a secondary drying temperature of -20 to -15°C for 1 hour; Maintaining the formulation dried at the secondary drying temperature at -15°C for 20 hours; Forming the dried formulation by heating the formulation maintained at -15°C for 20 hours to a third drying temperature of -15 to -12°C for 30 minutes; Maintaining the formulation dried at the tertiary drying temperature at -12°C for 15.5 hours; Heating the formulation maintained at -12°C for 15.5 hours to a fourth drying temperature of -12 to 35°C for 15 hours to form a dried formulation; Maintaining the formulation dried at the fourth drying temperature at 35° C. for 10 hours; Heating the formulation maintained at 35° C. for 10 hours to a fifth drying temperature of 35 to 40° C. for 1 hour to form a dried formulation; And maintaining the formulation dried at the fifth drying temperature for 10 to 15 hours.

The solution maintained at a temperature of -55°C for 6 hours is heated to a primary drying temperature of -55 to -20°C for 3 hours to form a dried formulation, and the solution dried at the 5th drying temperature is 10 to In the step of holding for 15 hours, the pressure may be maintained at 150 mTorr.

In order to achieve another object according to the present invention, the present invention provides azacytidine lyophilized preparation prepared by the method for preparing the azacytidine lyophilized preparation.

The azacytidine lyophilized formulation may have a moisture content of less than 3% and a tertiary butanol content of less than 5,000 ppm.

The criterion for the content of tertiary butanol in the lyophilized formulation of azacytidine is Class 3 of USP (US Pharmacopoeia) <467> in view of the toxicity of tertiary butanol and is determined to be less than 5,000 ppm.

The azacytidine pre-lyophilized formulation according to the present invention has improved stability in an aqueous solution, and the stability is improved when lyophilized using the azacytidine pre-lyophilized formulation under process conditions including a predetermined drying cycle and the like. Azacytidine lyophilized formulations can be usefully produced.

1 is a graph showing the temperature change per unit time of the manufacturing process for preparing the azacytidine lyophilized formulations of Examples 2-1, 2-3 and 2-4 of the present invention.
Figure 2 is a graph showing the temperature change per unit time of the manufacturing process for preparing the azacytidine lyophilized formulation of Example 2-2 of the present invention.
Figure 3 is a graph showing the temperature change per unit time of the manufacturing process for preparing the azacytidine lyophilized formulation of Comparative Example 2-2 of the present invention.

The normal pressure in the present specification is a pressure when the pressure is not particularly reduced or increased, that is, a pressure of about 1 atmosphere, such as normal atmospheric pressure.

Hereinafter, the present invention will be described in more detail by the following examples and experimental examples. However, the following examples are only for explaining the details of the present invention, and the present invention is not limited by the examples.

<Example 1. Pre-lyophilized formulation of azacytidine and preparation thereof>

Pre-lyophilized formulation of azacytidine of Examples 1-1 to 1-6 and preparation thereof

As shown in Table 1, Examples 1-1 to 1-3 are tert-butyl alcohol (tert-butyl alcohol, Daejung Gold, Korea), 40 ml, 50 ml, and 60 ml, respectively, and water for injection Prepared as a pre-lyophilized formulation of azacitidine by dissolving 20 mg of azacitidine (Chemagis, Israel) in 100 ml of a mixture of 60 ml, 50 ml and 40 ml.

In addition, as shown in Table 1, Examples 1-4 to 1-6 were tert-butyl alcohol (tert-butyl alcohol, Daejung Gold, Korea), 12 ml, 10 ml, and 8 ml, respectively. Prepared as a pre-lyophilized formulation of azacitidine by dissolving 100 mg of azacitidine (Chemagis, Israel) in 20 ml of a mixture of 8 ml, 10 ml and 12 ml water for injection.

Example 1-1 Example 1-2 Example 1-3 Example 1-4 Example 1-5 Example 1-6 Azacitidine 20mg 20mg 20mg 100mg 100mg 100mg tert-BuOH 40 ml 50 ml 60ml 12 ml 10ml 8 ml Water for injection 60ml 50 ml 40 ml 8 ml 10ml 12 ml

< Comparative example 1-1 to 1-5 Azacitidine Pre-lyophilized formulation and preparation thereof>

The ratio of tertiary butanol (tert-butyl alcohol) in the pre-lyophilized formulation of azacytidine of Example 1 and the stability increase according to temperature conditions were confirmed, and the ratio of tertiary butanol (tert-butyl alcohol) Comparative Examples 1-1 to 1-5 were prepared in order to confirm whether recrystallization was precipitated.

As shown in Table 2, Comparative Example 1-1 was prepared by dissolving 20 mg of azacitidine (Azacitidine, Chemagis, Israel) in 100 ml of water for injection as a pre-lyophilized formulation of azacitidine.

As shown in Table 2 below, Comparative Examples 1-2 to 1-3 are tert-butyl alcohol (tert-butyl alcohol, Daejung Gold, Korea) 20 ml and 80 ml, respectively, and tertiary butanol, 80 ml and water for injection 80 ml, 20 mg of azacitidine (Chemagis, Israel) was dissolved in 100 ml of a mixed solvent to prepare a pre-lyophilized formulation of azacitidine.

In addition, as shown in Table 2, Comparative Examples 1-4 to 1-5 are tert-butyl alcohols (tert-butyl alcohol, Daejung Gold, Korea), respectively, 16 ml and 14 ml, and water for injection 4 Prepared as a pre-lyophilized formulation of azacitidine by dissolving 100 mg of azacitidine (Azacitidine, Chemagis, Israel) in 20 ml of a mixed solvent of 6 ml and 6 ml.

Comparative Example 1-1 Comparative Example 1-2 Comparative Example 1-3 Comparative Example 1-4 Comparative Example 1-5 Azacitidine 20mg 20mg 20mg 100mg 100mg tert-BuOH 0 ml 20ml 80ml 16ml 14 ml Water for injection 100ml 80ml 20ml 4 ml 6 ml

< Experimental Example  One. Azacitidine  Stability test of pre-lyophilized formulation>

Stability test of the pre-lyophilized formulation of azacytidine to confirm the effect of increasing stability according to the ratio of tert-butanol (tert-butyl alcohol) and temperature conditions of the pre-lyophilized formulation of azacytidine of Example 1 above Was performed.

At room temperature (25°C), 20°C, 15°C, and 5°C, respectively, at the time of preparation of Examples 1-1 to 1-3 (initial), 5 hours after production (5 hours), and 10 hours after production (10 hours) Time), the content of azacitidine was measured. At this time, the content of azacitidine was measured by High Performance Liquid Chromatography (HPLC). Table 4 shows the measurement results.

The content of azacytidine contained in Comparative Examples 1-1 and 1-2 was also measured by performing a method substantially the same as the method of measuring the content of azacytidine contained in Examples 1-1 to 1-3. Table 5 shows the measurement results.

The conditions of high performance liquid chromatography for measuring the content of azacytidine are shown in Table 3 below.

column Phenomenex gemini C18 (5 microns, 250 * 4.6 mm) device
(Waters e2695 (Waters Co. Milford, MA,
USA)) Detection wavelength 230 nm Column temperature 25 ℃ Acquisition time 60 minutes Injection volume 3 μl flux 1.0 ml/min Sample temperature 5 ℃ diluent 30% 10mM ammonium acetate (Ammonium acetate) and 70% tetrahydrofuran (THF) (volume/volume) Buffer Ammonium acetate 2.832 g / 4 ℓ Mobile phase_A Buffer: methanol (MeOH) = 98: 2 (volume/volume) Mobile phase_B Buffer: methanol (MeOH) = 70: 30 (volume/volume) gradient Min A B 0 100 0 12 100 0 27 0 100 50 0 100 50.1 100 0 57 100 0

Example 1-1 (t-BuOH: water for injection = 40: 60 (volume/volume)) Example 1-2 (t-BuOH: water for injection = 50: 50 (volume/volume)) Example 1-3 (t-BuOH: water for injection = 60: 40 (volume/volume)) Early 5 hours 10 hours Early 5 hours 10 hours Early 5 hours 10 hours 25℃ 96.1 87.6 79.2 96.2 89.1 84.3 96.5 92.0 86.6 20℃ 97.1 91.9 85.2 97.3 93.2 88.4 97.9 94.3 89.2 15℃ 97.9 92.3 86.3 98.7 95.7 90.1 99.1 96.6 91.4 5℃ 99.3 98.8 98.4 99.4 98.9 98.7 99.3 99.2 99.2

Comparative Example 1-1 (t-BuOH: water for injection = 0: 100 (volume/volume)) Comparative Example 1-2 (t-BuOH: water for injection = 20: 80 (volume/volume)) Early 5 hours 10 hours Early 5 hours 10 hours 25℃ 94.3 65.7 57.4 95.2 78.2 71.3 20℃ 94.7 69.1 58.2 95.9 80.3 72.7 15℃ 95.3 74.3 64.1 96.3 82.3 73.4 5℃ 96.1 79.2 68.3 98.2 85.4 80.2

As shown in Table 4 to Table 5, it was confirmed that the pre-lyophilized preparation of azacytidine was stable in a mixture of water for injection with a high proportion of tertiary butanol.

In Examples 1-1 to 1-3, the content of azacytidine after 10 hours at a temperature of 5°C to 20°C was all about 85% or more, and it was confirmed that the stability of azacytidine at the temperature was high. In addition, the content of azacytidine after 10 hours at a temperature of 5°C to 15°C is about 90% or more (however, data excluded after 10 hours at 15°C in Example 1-1) is the temperature above From, it was confirmed that the stability of azacytidine was particularly high.

Therefore, it was confirmed that by mixing tertiary butanol with an appropriate ratio in the azacytidine pre-lyophilized formulation, a pre-lyophilized formulation of azacytidine can be more stably prepared at a relatively high temperature.

< Experimental Example 2. Azacitidine Class 3 of pre-lyophilized formulations Butanol Dissolution and recrystallization according to the content ratio Precipitation  Check whether experiment>

To confirm that Examples 1-4 to 1-6 may be conditions for preparing a high content of azacytidine pre-lyophilized preparation, Examples 1-4 to 1-6 and Comparative Examples 1-4 Experiments were conducted to confirm whether 1-5 was dissolved at 5°C and precipitated recrystallized. The experiment was judged by observing that the crystals were visually observed by leaving the pre-lyophilized formulation under 5°C refrigeration conditions.

Table 6 shows the results of the experiment.

3 hours 24 hours Example 1-4 Non-precipitation Precipitation Example 1-5 Non-precipitation Precipitation Example 1-6 Non-precipitation Precipitation Comparative Example 1-4 Insoluble Insoluble Comparative Example 1-5 Precipitation Precipitation

As in Comparative Examples 1-4 to 1-5, in an aqueous solution having a tertiary butanol content of 70% (volume/volume) or higher, azacytidine is recrystallized and precipitated as crystals after 3 hours after the start of the experiment to determine whether to dissolve and recrystallize. It was confirmed that azacytidine was not dissolved.

On the other hand, in Examples 1-4 to 1-6, azacytidine was recrystallized even after 3 hours, and a phenomenon such as precipitation as a crystal or inability to dissolve azacytidine was not observed.

Therefore, it was confirmed that Examples 1-4 to 1-6 could be conditions for preparing a high content of azacytidine pre-lyophilized formulation.

< Example 2. Azacitidine  Preparation of lyophilized formulation>

Example  2-1 Azacitidine Preparation of lyophilized formulations

The azacytidine lyophilized formulation of Example 2-1 was added with 100 mg of mannitol for injection to the pre-lyophilized formulation of azacytidine of Example 1-5, lyophilized according to the conditions in Table 7 below and FIG. 1. It was prepared by performing.

number step Explanation Hours Temperature (℃) Pressure (mTorr) Tilt (℃/min) One Pre-crystalline phase
(Prefreezing phase) Cooling -55 Atmospheric pressure 2 Pre-determination
(Prefreezing hold) maintain 6 -55 Atmospheric pressure 3 Phase 1 Gaon 3 -20 150 0.19 maintain 6 -20 4 Phase 2 Gaon One -15 0.08 maintain 20 -15 5 Phase 3 Gaon 0.5 -12 0.10 maintain 15.5 -12 6 Phase 4 Gaon 15 35 0.05 maintain 10 35 7 Phase 5 Gaon One 40 0.08 maintain 10 40 - - Total time 88 - - -

Example 2-2 Azacitidine  Preparation of lyophilized formulations

The azacytidine lyophilized formulation of Example 2-2 was added with 100 mg of mannitol for injection to the pre-lyophilized formulation of azacytidine of Example 1-5, lyophilized according to the conditions in Table 8 below and FIG. 2. It was prepared by performing. The manufacturing process of the azacytidine lyophilized preparation of Example 2-2 differs in that the retention time of phase 5 is increased among the process conditions of Example 2-1.

number step Explanation Hours Temperature (℃) Pressure (mTorr) Tilt (℃/min) One Pre-crystalline phase
(Prefreezing phase) Cooling -55 Atmospheric pressure 2 Pre-determination
(Prefreezing hold) maintain 6 -55 Atmospheric pressure 3 Phase 1 Gaon 3 -20 150 0.19 maintain 6 -20 4 Phase 2 Gaon One -15 0.08 maintain 20 -15 5 Phase 3 Gaon 0.5 -12 0.10 maintain 15.5 -12 6 Phase 4 Gaon 15 35 0.05 maintain 10 35 7 Phase 5 Gaon One 40 0.08 maintain 15 40 - - Total time 93 - - -

Example 2-3 Azacitidine  Preparation of lyophilized formulations

The azacytidine lyophilized formulation of Example 2-3 was lyophilized substantially the same as Example 2-1 by adding 100 mg of mannitol for injection to the pre-lyophilized formulation of azacytidine of Examples 1-6. It was prepared by performing lyophilization according to the conditions (see Table 7 and Figure 1). That is, the manufacturing process of the azacytidine freeze-dried preparation of Example 2-3 is the manufacturing process of the azacytidine freeze-dried preparation of Example 2-1, the pre-lyophilization of azacytidine having a different content of tertiary butanol. It is the same except that the formulation is used.

Example  2-4 of Azacitidine  Preparation of lyophilized formulations

The azacytidine lyophilized formulation of Example 2-4 was added to 100 mg of mannitol for injection into the pre-lyophilized formulation of azacytidine of Example 1-4, and was substantially freeze-dried as in Example 2-1. It was prepared by performing lyophilization according to the conditions (see Table 7 and Figure 1). That is, the manufacturing process of the azacytidine freeze-dried preparation of Example 2-4 is the manufacturing process of the azacytidine freeze-dried preparation of Example 2-1, the pre-lyophilization of azacytidine having a different content of tertiary butanol. It is the same except that the formulation is used.

< Comparative example 2-1 to 2-2 Azacitidine Preparation of pre-lyophilized formulation>

Comparative example 2-1 Azacitidine Preparation of lyophilized formulations

In the lyophilized formulation of azacytidine of Comparative Example 2-1, 100 mg of mannitol for injection was added to the pre-lyophilized formulation of azacytidine of Example 1-5 to perform lyophilization according to the conditions in Table 9 below. Was prepared.

number step Explanation Hours Temperature (℃) Pressure (mTorr) Tilt (℃/min) One Pre-crystalline phase
(Prefreezing phase) Cooling -55 Atmospheric pressure 2 Pre-determination
(Prefreezing hold) maintain 4 -55 Atmospheric pressure 3 Phase 1 Gaon 1.9 -10 35 0.40 maintain 33.7 -10 4 Phase 2 Gaon 1.4 -5 0.06 maintain 4 -5 5 Phase 3 Gaon 8.3 5 0.02 maintain 0.5 5 6 Phase 4 Gaon 3 25 0.11 maintain 7 25 7 Phase 5 Gaon One 40 0.25 maintain 4 40 - - Total time 68.8 - - -

As a result of preparing the method for preparing azacytidine using the pre-manufacturing method of azacytidine according to the embodiment of the present invention under the freezing conditions in Table 9, as the freeze-drying process started, the azacytidine freeze-dried products were out of the container. There was a phenomenon of eruption. It was understood that this phenomenon occurred because the freeze-drying was not sufficiently performed at a low pressure and a low temperature of the freezing method of Table 9 previously established. Therefore, it was confirmed that the manufacturing process conditions of the azacytidine production method of Comparative Example 2-1 are not suitable for mass production of azacytidine lyophilized formulations.

Comparative example 2-2 Azacitidine  Preparation of lyophilized formulations

The lyophilized formulation of azacytidine of Comparative Example 2-2 was added to 100 mg of mannitol for injection into the pre-lyophilized formulation of azacytidine of Example 1-5, and was frozen according to the conditions of Table 10 and Figure 3 below. It was prepared by drying.

number step Explanation Hours Temperature (℃) Pressure (mTorr) Tilt (℃/min) One Pre-crystalline phase
(Prefreezing phase) Cooling -55 Atmospheric pressure 2 Pre-determination
(Prefreezing hold) maintain 6 -55 Atmospheric pressure 3 Phase 1 Gaon 3 -20 150 0.19 maintain 6 -20 4 Phase 2 Gaon One -15 0.08 maintain 20 -15 5 Phase 3 Gaon 0.5 -12 0.10 maintain 15.5 -12 6 Phase 4 Gaon 15 35 0.05 maintain 10 35 7 Phase 5 Gaon One 40 0.08 maintain 5 40 - - Total time 83 - - -

In the manufacturing process of the azacytidine manufacturing method of Comparative Example 2-2, the process conditions were changed to increase the pressure and increase the low-temperature holding time in the manufacturing process of the azacytidine manufacturing method of Comparative Example 2-1.

< Experimental Example  3. Azacitidine Confirmation of manufacturing process of lyophilized formulation and suitability of formulation>

Example  2-1 to 2-4 and Comparative example  2-2 Azacitidine  Water content and residual level 3 of the lyophilized formulation Butanol Content measurement

Examples 2-1 to 2-4 and Comparative Examples 2-1 to 2-2 to confirm the suitability of mass production of azacytidine lyophilized preparations and the compatibility of azacytidine lyophilized preparations with pharmaceutical products The moisture content and residual tertiary butanol content of the azacytidine lyophilized formulation were measured. The moisture content and residual tertiary butanol content of the azacytidine lyophilized formulation were measured using a gas chromatography (7890A/Agilent) and a moisture meter (682/Metrohm). It was tested according to the following pharmacopoeia general test method gas chromatograph method with the following sample and standard solutions.

1) Preparation of sample solution

About 200 mg of this was precisely weighed and placed in a 10 ml volumetric flask, dissolved in 1 ml of internal standard solution and water, and accurately marked with water. Exactly 5 ml of this solution was placed in a headspace vial and mixed by closing the lid.

2) Preparation of standard solution

After precisely weighing 100 mg of tertiary butanol and placing it in a 100 ml volumetric flask, water was accurately aligned. Take exactly 1 ml of this solution and 1 ml of the internal standard solution, put it in a 10 ml volumetric flask, and align the mark accurately with water. Exactly 5 ml of this solution was placed in a headspace vial and mixed by closing the lid.

3) Preparation of internal standard liquid

50 mg of acetone was precisely weighed and placed in a 100 mL volumetric flask, followed by precisely aligning it with water.

4) Gas chromatography system

-Detector: Flame Ionization Detector (FID), 250℃

-Column: DB-624 (30m×0.53㎜×3.0㎜)

-Inlet: 140℃, Split ratio 1:10

-Oven: 40℃ (10 minutes)-80℃ minute rise-200℃ (5 minutes)

-Carrier gas: Helium

-Headspace Operating Parameters

Headspace Operating Parameter Sets Equilibration temperature (℃) 90 Equilibration time (minutes) 30 Transfer line temperature (℃) 110 Carrier gas Helium gas (He) Pressurization time (minutes) 0.5 Injection volume (mL) One

5) Mathematical formula for the amount of residual solvent

Where S _W is the standard concentration (mg/ml), T _W is the sample concentration (mg/ml), T _A is the integrated area of each residual solvent in the sample solution, S _A is the residual solvent standard product integration area of the standard solution, and S _IS is the standard solution concentration. Integral area of the internal standard, T _IS is the integral area of the internal standard of the test solution, RS _A is the content of the solvent standard solution (%), and 10000 is the unit conversion multiple.

The moisture content was tested according to the Karl Fischer method in the general pharmacopoeia test method.

The measurement results are shown in Table 12 below, together with whether or not lyophilisates are ejected out of the container. In the case of Comparative Example 2-1, as a phenomenon in which lyophilisates were ejected out of the container, the azacytidine lyophilized formulation could not be prepared, and the moisture content and residual tertiary butanol content of the azacytidine lyophilized formulation were not confirmed.

Eruption Moisture content (%) Standard: 3.0% Based on residual tertiary butanol content (ppm): 5,000ppm Comparative Example 2-1 has exist - - Comparative Example 2-2 none 0.64% (suitable) 9,659.0ppm (not suitable) Example 2-1 none 1.04% (suitable) 1884.8ppm (suitable) Example 2-2 none 0.97% (suitable) 1647.4ppm (suitable) Example 2-3 none 1.12% (suitable) 1756.2ppm (suitable) Example 2-4 none 0.92% (suitable) 1945.2ppm (suitable)

The moisture content of azacytidine freeze-dried preparations should satisfy 3.0% or less, and residual tertiary butanol should satisfy 5,000 ppm or less.

Example 2-1 is different from Comparative Example 2-1 in that the pressure is high in the lyophilization condition and that the low temperature holding time is long. In addition, in Example 2-1, when compared with Comparative Example 2-2, there is a difference in that the retention time of phase 5 was increased among the process conditions of Comparative Example 2-2. In Example 2-1, unlike Comparative Example 2-1, the phenomenon that lyophilisates were not ejected out of the container did not occur, and unlike Comparative Example 2-2, the lyophilized formulation of azacytidine had a moisture content and residual tertiary butanol. It was confirmed that all the content criteria were satisfied (suitability). The moisture content of the azacytidine lyophilized formulation of Comparative Example 2-2 was 0.64%, which is suitable for the moisture content standard, but the residual tertiary butanol content is 9,959 ppm, which is about twice the residual tertiary butanol content standard, exceeding the reference value. Residual tertiary butanol content was not suitable.

Example 2-2 differs from Example 2-1 in that it increases the retention time of phase 5 among the process conditions of Example 2-1. Example 2-2 is different from Comparative Example 2-1 in that the pressure is high in the freeze-drying condition and that the low-temperature holding time is long. In addition, Example 2-2 has a difference in that the retention time of phase 5 is increased among the process conditions of Comparative Example 2-2 when compared with Comparative Example 2-2. In Example 2-2, unlike Comparative Example 2-1, the phenomenon that lyophilisates were not ejected out of the container did not occur, and unlike Comparative Example 2-2, the lyophilized formulation of azacytidine had a moisture content and residual tertiary butanol. It was confirmed that all the content criteria were satisfied (suitability).

The azacytidine lyophilized formulations of Examples 2-3 and 2-4 freeze azacytidine of Example 2-1, except that the pre-lyophilized formulations of azacytidine with different tertiary butanol contents are used. It was manufactured by the same manufacturing process as that of the dry formulation. As in Example 2-1, Examples 2-3 and 2-4 also did not cause lyophilisates to be ejected out of the container, and the lyophilized formulation of azacitidine was based on moisture content and residual tertiary butanol content standards. It was confirmed that all were satisfied (conformity).

Through the above results, the manufacturing process of the azacitidine lyophilized formulations of Examples 2-1 to 2-4 is suitable for mass production of lyophilized formulations because there is no phenomenon in which lyophilisates are ejected out of the container during the lyophilization process. It was confirmed that the lyophilized formulation of azacytidine produced by the manufacturing process satisfies both the moisture content and the residual tertiary butanol content standards (suitability) and satisfies the suitability of the drug product.

Example  2-1 to 2-4 Azacitidine  Lyophilized Decomposition products  Confirm

The degradation products of the lyophilized formulations of azacitidine of Examples 2-1 to 2-4 were analyzed under substantially the same conditions as the HPLC conditions of Experimental Example 1 (see Table 3). Azacitidine (Azacitidine, Chemagis, Israel) was taken in an amount corresponding to 20 mg and dissolved in 10 ml of dilution. It was placed in an ultrasonic generator (5510/Branson) and sonicated for 2 minutes. The results of preparing the test solution by filtering the sonicated solution with a membrane filter of 0.5 μm or less are shown in Table 13 below.

Impurity A
(Imp-A) Impurity B
(Imp-B) Impurity C
(Imp-C) Azacitidine Impurity D
(Imp-D) Fit Example 2-1 Relative Retention Time (RTR) conditions 0.46 0.52 0.73 1.00 2.11 fitness Area value 12,496 2,478 42,451 8,686,415 8,255 content(%) 0.14 0.03 0.49 99.25 0.09 Example 2-2 Relative Retention Time (RTR) conditions 0.46 0.52 0.73 1.00 2.11 fitness Area value 12,478 2,484 39,458 8,678,436 8,342 content(%) 0.14 0.03 0.45 99.29 0.09 Example 2-3 Relative Retention Time (RTR) conditions 0.46 0.52 0.73 1.00 2.11 fitness Area value 36,368 5,425 96,453 8,083,254 9,324 content(%) 0.32 0.07 1.17 98.33 0.11 Example 2-4 Relative Retention Time (RTR) conditions 0.46 0.52 0.73 1.00 2.11 fitness Area value 8,324 1,532 23,756 9,145,312 6,144 content(%) 0.09 0.02 0.27 99.55 0.07

Conformity was judged to be suitable for the production of pharmaceuticals, and the azacytidine content of the decomposition products of the lyophilized formulation of azacitidine was judged as suitable when the content was 98% or more.

As shown in Table 13, the azacytidine contents of the decomposition products of the azacytidine lyophilized formulations 2-1 to 2-4 in the above Examples were 99.25%, 99.29%, 98.33% and 99.55%, respectively. All of the azacytidine lyophilized formulations of -1 to 2-4 obtained excellent content results suitable for pharmaceutical production. Through the above results, it was confirmed that the manufacturing process of the azacytidine lyophilized formulations of Examples 2-1 to 2-4 may be suitable for mass production of the azacytidine lyophilized formulation as a pharmaceutical product.

Claims (15)

Pre-lyophilized formulation of azacytidine in which azacytidine is dissolved in an aqueous solution containing 40-60% of tert-butanol (volume/volume). The pre-lyophilized formulation of azacytidine according to claim 1, wherein the azacytidine concentration is 0.2 to 5 mg/ml. The pre-lyophilized formulation of azacytidine according to claim 1, wherein the pre-lyophilized formulation of azacytidine is prepared at 5 to 20°C. The pre-lyophilized formulation of azacytidine according to claim 1, wherein the pre-lyophilized formulation of azacytidine is prepared at 5°C. The pre-lyophilized formulation of azacytidine according to claim 1, wherein the pre-lyophilized formulation of azacytidine has a residual ratio of azacytidine of 85% or more after 10 hours at 5 to 20°C. The pre-lyophilized formulation of azacytidine according to claim 1, wherein the pre-lyophilized formulation of azacytidine has a residual ratio of azacytidine of 95% or more after 10 hours at 5°C. Forming a pre-lyophilized preparation of azacytidine in which azacytidine is dissolved in an aqueous solution containing tertiary butanol 40 to 60% (volume/volume);
Adding a polyol to the pre-lyophilized formulation of azacytidine; And
A method for preparing a lyophilized formulation of azacytidine, comprising lyophilizing the pre-lyophilized formulation to which the polyol is added. delete The method of claim 7, wherein the polyol is one or two or more selected from the group consisting of mannitol, sorbitol and trehalose. The method of claim 7, wherein the polyol is mannitol, azacytidine. The method of claim 7, wherein the concentration of azacytidine in the pre-lyophilized formulation of azacytidine is 0.2 to 5 mg/ml. The method of claim 7, wherein the step of freeze-drying the pre-lyophilized formulation to which the polyol is added is
Freezing the pre-lyophilized formulation of azacytidine at a temperature of -55° C. to form a frozen formulation;
Maintaining the frozen formulation at a temperature of -55°C for 6 hours;
Forming a dried formulation by heating the formulation maintained at a temperature of -55 DEG C or lower for 6 hours to a primary drying temperature of -55 to -20 DEG C for 3 hours;
Maintaining the formulation dried at the primary drying temperature at -20°C for 6 hours;
Forming the dried formulation by heating the formulation maintained at -20°C for 6 hours to a secondary drying temperature of -20 to -15°C for 1 hour;
Maintaining the formulation dried at the secondary drying temperature at -15°C for 20 hours;
Forming the dried formulation by heating the formulation maintained at -15°C for 20 hours to a third drying temperature of -15 to -12°C for 30 minutes;
Maintaining the formulation dried at the tertiary drying temperature at -12°C for 15.5 hours;
Heating the formulation maintained at -12°C for 15.5 hours to a fourth drying temperature of -12 to 35°C for 15 hours to form a dried formulation;
Maintaining the formulation dried at the fourth drying temperature at 35° C. for 10 hours;
Heating the formulation maintained at 35° C. for 10 hours to a fifth drying temperature of 35 to 40° C. for 1 hour to form a dried formulation; And
A method for preparing a lyophilized formulation of azacytidine, comprising maintaining the formulation dried at the fifth drying temperature for 10 to 15 hours. The method of claim 12, wherein the solution maintained at a temperature of -55°C for 6 hours is heated to a primary drying temperature of -55 to -20°C for 3 hours to form a dried formulation to the 5th drying temperature. A method for preparing a lyophilized formulation of azacitidine where the pressure is maintained at 150 mTorr in the step of maintaining the dried solution for 10 to 15 hours. Claims 7 and 9 to 13 lyophilized formulation of azacitidine prepared by the method of any one of claims. The lyophilized formulation of azacytidine according to claim 14, wherein the azacytidine lyophilized formulation has a moisture content of less than 3% and a tertiary butanol content of less than 5,000 ppm.

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