FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
Complete Specification [See Sections 10 and rule 13]
Title: Process for preparation of Decitabine Injection
Applicant: (a) Astron Research Ltd
(b) Company Registered under Indian Company ACT
(c) 10th Floor, Premier House Bodakdev, Opp. Gurudwara Sarkhej - Gandhinagar Highway Ahmedabad 380054
Gujarat, India.
The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF THE INVENTION
This invention relates to an improved process to obtain a stable lyophilized Decitabine composition by allowing compressed air in lyophilizer for conversion of impurity to Decitabine.
BACKGROUND OF THE INVENTION
Decitabine, 5-aza-2'-deoxycytidine, is an antagonist of its related natural nucleoside, deoxycytidine. One of the major challenges with Decitabine is its chemical instability. It is well-known that Decitabine and related nucleoside analogues decompose within few hours at physiological temperature and pH.
Decitabine is degraded via hydrolytic opening of the triazine ring, deformylation, and anomerization. This result in reduced drug concentration and the degradation products may have pharmacological and toxic properties independent of the Decitabine. Hydrolytic ring-opening in Decitabine occurs at C-6 position in aqueous solutions under acidic conditions; its glycosidic bond is labile, releasing the free base azaC and deoxyribose.
Scheme for Decitabine decomposition in aqueous solution


Decitabine (I) is in equilibrium with its ring-open-formylated derivative (II), which undergoes irreversible deformylation and formation of the guanylurea derivative (III).
Decitabine, which is chemically unstable in aqueous solution, is commonly supplied as a sterile lyophilized powder for injection. In order to prepare lyophilized composition, bulk solution containing Decitabine is prepared and thereafter the solution is lyophilized in vials. However, it is observed by the inventors of the present invention that when lyophilized Decitabine injection is prepared by using normal lyophilization technique or by using nitrogen gas during lyophilization process, the amount of impurity present in the lyophilized product are high. Further, there is vial to vial variation in amount of impurity present in the lyophilized composition.
To overcome the above mentioned problems associated with lyophilized Decitabine injection composition, the inventors of the present invention have found out that by making a few incremental changes in the lyophilization process, a stable lyophilized Decitabine composition is obtained. Further, the process according to the present invention also minimizes vial to vial variation of impurity in lyophilized Decitabine injection composition.
OBJECTS OF THE INVENTION
The main object of the present invention is to prepare lyophilized Decitabine injection composition having reduced levels of impurity.

Another object of the present invention is to minimize vial to vial variation of impurity in lyophilized Decitabine injection composition.
Another object of the present invention is to minimize vial to vial variation of impurity at RRT 0.7 in lyophilized Decitabine injection composition.
Another object of the invention is to use compressed air in the lyophilizer after lyophilization process for the preparation of lyophilized Decitabine injection composition in order to convert impurity back to Decitabine.
SUMMARY OF THE INVENTION
The invention relates to process to obtain stable lyophilized Decitabine injection composition having reduced amount of impurity by allowing compressed air in lyophilizer for 1-24 hour after lyophilization cycle. During this time, the impurity formed during the bulk manufacturing process is reversed back to Decitabine.
Further the invention relates to the process for the preparation of lyophilized Decitabine injection composition wherein vial to vial variation of impurity is at minimum in the said lyophilized injection composition.
DETAILED DESCRIPTION
Lyophilization or freeze-drying is a process in which water is removed from a product after it is frozen and placed in a surrounding with vacuum, which allows the frozen water to sublimate directly from a solid to a vapor phase, without passing

through a liquid phase. The lyophilization process consists of three separate, unique, and interdependent processes; a freezing phase, a primary drying phase (sublimation), and a secondary drying phase (desorption). After the freeze-drying process is complete, the vacuum is usually broken with an inert gas, such as nitrogen, before the product is stoppered.
Lyophilization is sometimes employed to formulate injectable pharmaceuticals that exhibit poor active ingredient stability in aqueous solutions. It is common practice to sparge inert gas such as nitrogen during formulation of injectable preparation in order to minimize the degradation of active constituents.
It is observed by the inventors of the present inventors, that when lyophilized Decitabine injection composition is prepared by using normal lyophilization technique or by using nitrogen gas during lyophilization process, the amount of impurity present in the lyophilized injection is high. Also, there is vial to vial variation in amount of impurity present, compared to lyophilized injection prepared according to present invention i.e. allowing compressed air for about 18-24 hours in the lyophilizer after lyophilization cycle is complete. Allowing of compressed air in the lyophilizer after completion of lyophilization cycle helps reduce the impurity levels in the lyophilized product.
Presently, impurity in lyophilized Decitabine composition are divided into characterized / known impurity and unknown impurity; wherein known impurity is methyl-4-methyl benzoate, a anomer and mono ester impurity.
Single major unknown impurity is impurity at RRT 0.7; which when determined using HPLC equipped with DAD Detector (Make: Agilent 1100 series or equivalent) using Zorbax RX-SIL (250 x 4.6 mm) column and acetonitrile: water (19:1) as

mobile phase with 1.0 mL/min flow rate having sample temperature 2-8°C, having relative retention time of 0.7 with respect to Decitabine which is having retention time of about 12 min in described conditions.
Two isomeric forms of Decitabine are established as a anomer and β anomer; wherein a anomer is an inactive form and β anomer is the active form of Decitabine. In aqueous solution, Decitabine gets converted from β anomer to a anomer.
As used herein, the term "compressed air" refers to compressed air or atmospheric air or a synthetic mixture of 21% oxygen and 7.9% nitrogen.
Currently, Decitabine is available as Dacogen™, which contains 50 mg Decitabine, 68 mg monobasic potassium phosphate (potassium dihydrogen phosphate) and 11.6 mg sodium hydroxide.
The amount of impurity present in Decitabine bulk solution is gradually increased as time passes during the manufacture of bulk solution. Also there is variation in amount of impurity present in vial to vial when lyophilized Decitabine injection is prepared according to conventional preparation methods known i.e. either using nitrogen sparging or without using nitrogen sparging.
It is found by the inventors that when vials having high amount of impurity were allowed to remain open, then the amount of impurity is decreased significantly.
Below mentioned data suggests that when vials having high levels of impurity are exposed to air, then it would significantly reduce the levels of impurity.

Time intervals Level of Impurity at RRT 0.7
Initial 2.5%
After 17 Hrs 0.04%
After 22 Hrs Not Detected
Based on above observations, inventors of the present invention designed a novel process for the preparation of Decitabine injection using compressed air in the lyophilizer.
The presence of oxygen / environmental air is useful for fast, complete and uniform conversion of impurity to Decitabine once water is removed during lyophilization.
According to present invention, once the lyophilization process is completed, vacuum is broken with compressed air and compressed air is allowed in lyophilizer for about 1 -24 hour, preferably for about 10-24 hours more preferably 18-24 hours.
According to one embodiment of the present invention, lyophilized Decitabine injection composition is prepared without using inert gas during bulk manufacturing process. The vacuum was broken with compressed air after lyophilization cycle was completed and the vials were kept as such without stoppering in lyophilizer for 18-24 hours in presence of compressed air for conversion of impurity into Decitabine. After exposing vials to air for 18-24 hours at room temperature in lyophilizer, the vials were stoppered and unloaded. The headspace of the vials were taken and confirmed that the headspace is approximately 20% - 21%.
Further, according to another embodiment, the present invention provides a stable lyophilized Decitabine injection composition, wherein the impurity does not exceed 0.2% w/w in the said composition.

Also, according to another embodiment, the present invention provides an improved process for the preparation of stable lyophilized Decitabine injection composition comprising impurity not exceeding 0.2% w/w; wherein the improvisation of lyophilization comprises the step of allowing compressed air in the lyophilizer for a time period of 1 to 24 hours after the completion of lyophilization cycle. More specifically, once the lyophilization process is completed, vacuum is broken with compressed air and compressed air is allowed in lyophilizer for about 1-24 hour, preferably for about 10-24 hours more preferably 18-24 hours.
Further, according to another embodiment, the present invention provides a stable lyophilized Decitabine injection composition, wherein variation of impurity at RRT 0.7 from vial to vial in the said stable lyophilized Decitabine injection is not more than 0.1% w/w.
Further, according to another embodiment, the present invention provides an improved process for the preparation of stable lyophilized Decitabine injection composition wherein variation of impurity at RRT 0.7 from vial to vial in the said stable lyophilized Decitabine injection is not more than 0.1% w/w; wherein the improvisation of lyophilization comprises the step of allowing compressed air in the lyophilizer for a time period of 1 to 24 hours, after the completion of lyophilization cycle. More specifically, once the lyophilization process is completed, vacuum is broken with compressed air and compressed air is allowed in lyophilizer for about 1 -24 hour, preferably for about 10-24 hours more preferably 18-24 hours.
In the following section embodiments are described by a way of examples to illustrate the process of invention. However, these are not intended in any way to limit the scope of present invention. Several variants of these examples would be evident to present person ordinarily skilled in the art.

Example 1:
Lyophilized Decitabine injection composition
a) Bulk solution preparation

Sr. No. Ingredient Qty / per vial
1 Decitabine 50 mg
2 Monobasic potassium phosphate 68 mg
3 Sodium hydroxide 11.6 mg
4 Water for injection* q.s.
* Removed during lyophilization
Process for preparation:
1. Prepare bulk solution comprising Decitabine, monobasic potassium phosphate and sodium hydroxide in water for injection.
Stability studies for the above disclosed bulk solution is described in Table 1 Table 1: Stability Data of Decitabine for Injection Bulk solution

Data at 2°C - 8°C Data at 12°C-15°C
Initial 3 hrs 6 hrs 8 hrs 2 hrs 4 hrs
Assay 104.8% 102.2% 100.7% 100.9% 101.5% 98.4%
Chromatograp hic Purity
a anomer 0.07% 0.08% 0.09% 0.10% 0.08% 0.11%
Single unknown impurity at RRT0.7 0.46% 2.37% 3.69% 4.45% 3.02% 4.98%
Total impurity 0.58% 2.76% 4.18% 4.94% 3.21% 5.50%
pH 6.98 6.98 6.96 6.97 7.01 7.01
From table 1, it is observed that during preparation of bulk solution of Decitabine, impurity is steadily increased in solution as time progresses.

b) Lyophilization of the Decitabine bulk solution using nitrogen sparging
Bulk solution prepared above is further lyophilized using nitrogen sparging during process and head space of the vial.
Table 2: Stability data of Bulk Solution and Lyophilized product (FP) produced from the same Bulk Solution

Conditions Bulk solution held for 4 hrsatl5°C-20°C FP after Bulk hold for 4 hrsatl5°C-20°C
Description Clear, colourless solution White lyophilized cake
Assay 93.0% 99.6%
Chromatographic Purity
a anomer 0.051% 0.046%
Single unknown impurity at RRT 0.7 7.803% 0,306%
Total impurity 8.203% 0.428%o
From table 2, it is observed that when Decitabine bulk solution was lyophilized using nitrogen sparging during process and in headspace of the vial, impurity is reverted back to Decitabine during lyophilization.
However, during scale-up of lyophilized process, it is observed that there is vial to vial variation of impurity, which gives a clue that the conversion of impurity into Decitabine is not uniform during / after the lyophilization cycle. (See Table 3)
Table 3- Vial to vial variation of impurity after lyophilization

Impurity Vial-1 Vial-2 Vial-3 Vial-4 ViaI-5
Methyl 4-methyl benzoate ND ND ND 0.055% 0.056%)
a anomer 0.05% 0.06% 0.06% 0.063% 0.064%
Mono ester Impurity 0.06% 0.06% 0.11% — --
Single unknown impurity at RRT 0.7 2.98%o 0.27% 0.24% 0.072%. 0.183%
Total impurity 3.26% 0.41% 0.43%) 0.190%o 0.303%
* ND - Not detected

c) Forced degradation study
Forced degradation study for Decitabine formulation was carried out under various conditions such as normal, water hydrolysis, acidic conditions, basic conditions and in presence of oxidising agent such as KMNO4. Objective of performing forced degradation study was to understand the vial to vial variation of impurity.
For normal conditions Decitabine powder is used as sample, for water hydrolysis solution of Decitabine in water is prepared, for acidic conditions 5.0mL 0.05M HCL is added to solution of Decitabine in water, for basic conditions 2.0mL 0.05M NaOH is added to solution of Decitabine in water, for oxidising conditions 1 mL 0.01M KMNO4 is added to solution of Decitabine in water.
Results obtained are described in table below: Table 4- Result of forced degradation study

Sample As such Water Hydrolysis Acid 1 Alkali 1 Oxidation
Condition As such S.OmL of Water 6Hr atRT 5.0mL of 0.05MHCL 6Hr at RT 2.0mL of 0.05M NaOH at RT for 30 min 1 mL of 0.01M KMN04 at
RT for 2 Hr
a anomer 0.01% 0.60% 0.07% 0.09% 0.01%
Impurity at RRT 0.09 ND ND ND ND 0.66%
Impurity at RRT 0.22 0.01% 0.01% 0.01% ND 5.06%
Impurity at RRT 0.48 0.42% 0.43% 0.40% 0.03% 0.21%
Impurity at RRT 0.68 ND 1.00% 0.04% ND ND
Impurity at RRT 0.7 ND 7.07% 0.06% 0.17% ND
Total Impurity 0.59% 9.47% 0.77% 1.31% 6.23%

It is observed from the forced degradation study that impurity at RRT 0.7 is either not getting formed or it is getting reversed to Decitabine in presence of KMnCU which is very strong oxidizing agent.
Example 2: Lyophilized Decitabine injection composition prepared according to the present invention
Composition of Decitabine Bulk solution

Sr. No. Ingredient Qty / per vial
1 Decitabine 50 mg
2 Monobasic potassium phosphate 68 mg
3 Sodium hydroxide 11.6 mg
4 Water for injection* q.s.
* Removed during lyophilization Process for preparation:
1. Prepare bulk solution comprising Decitabine, monobasic potassium phosphate and sodium hydroxide in water for injection with or without using any gas during manufacturing process.
2. Prepared bulk solution is subjected to lyophilization process using compressed air.
3. Vacuum was broken with compressed air after cycle was over and the vials were kept as such without stoppering in lyophilizer for 18-24 hours in presence of air.
4. After exposing vials to compressed air for 18-24 hours at room temperature in lyophilizer, the vials were stoppered and unloaded.
5. The headspace of the vials were taken and confirmed that the headspace is approximately 20% - 21%.
Analytical results of product obtained by present example: Table 5 - Stability data of Lyophilized product (FP)

Conditions
Initial 40 ± 2°C, 75 ± 5 % RH (Upright) 25 ± 2°C, 60 ± 5 % RH (Upright)

1M 2M 3M 6M 3M 6M 9M 12M
Description White lyophiHzed cake
Assay 98.4% 98.9% 99.9% 99.7% 97.3% 99.7% 99.3% 101.91% 100.0%
Chromatograf )hic Purity
Methyl 4-
methyl
benzoate 0.02% ND ND ND ND ND ND ND 0.02%
Mono ester impurity 0.02% 0.01% 0.02% 0.03% BQL 0.03% BQL BQL BQL
a anomer 0.04% 0.03% 0.03% 0.01% BQL 0.04 BQL BQL BQL
Single unknown impurity at RRT0.7 0.06% 0.01% 0.02% 0.01% BQL 0.02% ND BQL ND
Total impurity 0.10% 0.05% 0.07% 0.05% BQL 0.10% BQL BQL 0.02%
* BQL - Below the levels of quantification M - month
ND - Not detected
From the stability data according to table 5, it can be inferred that the lyophilized product obtained with respect to the process for preparation according to the present invention are stable and have reduced impurity levels.
Table 6 - Vial to vial variation of impurity after using compressed air

Impurity Vial-1 Vial-2 ViaL3 Vial-4 Vial-5
Methyl 4-methyl benzoate ND ND ND ND ND
Mono ester Impurity 0.046% 0.033% 0.038% 0.039% 0.034%
a anomer 0.081% 0.072% 0.082% 0.079% 0.077%
Single unknown Impurity at RRT 0.7 0.020% ND ND ND ND
Total impurity 0.147% 0.105% 0.120% 0.118% 0.111%
Water by KF 1.9% 2.4% 2.0% 2.0% 1.3%
* ND - Not detected

According to the data of table 6 for vial to vial variation of impurity with regards to lyophilized product obtained according to the present invention, it can be inferred that vial to vial variation of impurity is reduced and is uniform as compared to the lyophilized product obtained with conventional lyophilization process.

We claim,
1) An improved process to reduce impurity in lyophilized Decitabine injection
composition, wherein the improvisation comprises the step of
- allowing compressed air in lyophilizer after the completion of lyophilization cycle for a time period of 1 to 24 hours.
2) The improved process according to claim 1, wherein the impurity is impurity at RRT 0.7.
3) The improved process according to claim 2, wherein the impurity at RRT 0.7 does not exceed 0.2% w/w in the said stable lyophilized Decitabine injection composition.
4) The improved process according to claim 1, wherein variation of impurity at RRT 0.7 from vial to vial in the said stable lyophilized Decitabine injection composition is reduced as compared to lyophilized Decitabine injection composition obtained with respect to conventional lyophilization process.
5) The improved process according to claim 4, wherein variation of impurity at RRT 0.7 from vial to vial in the said stable lyophilized Decitabine injection composition is not more than 0.1 % w/w.
6) The improved process according to claim 1, wherein the time period for allowing compressed air in lyophilizer is preferably between 10 to 24 hours.
7) The improved process according to claim 6, wherein the time period for allowing compressed air in lyophilizer is more preferably between 18 to 24 hours.

8) A stable lyophilized Decitabine injection composition, wherein the impurity at
RRT 0,7 does not exceed 0.2% w/w in the said composition.
9) A stable lyophilized Decitabine injection composition as obtained by the
improvised lyophilization process as described here within the description.