FORM 2
THE PATENTS ACT, 1970 (39 of1 970)
&
THE PATENTS RULES. 2003
Complete Specification
[See Sections 10 and rule 13]
Title: Process for preparation of Azacitidine Composition.
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 describes the invention and the manner in which this is to be performed:

FIELD OF THE INVENTION
This invention relates to an improved method of preparation to obtain a stable lyophiiized Azacitidine composition with reduced levels of impurity.
BACKGROUND OF THE INVENTION
Azacitidine is a pyrimidine nucleoside analog of cytidinc and its chemical name is 4-amino-l-p-D-ribofuranosyl-s-triazin-2(lH)-one. Us structural formula is as follows:

The empirical formula of Azacitidine is C8H12N4O5 and its molecular weight is 244. The marketed product, VIDAZA" is indicated for treatment of patients with the myelodysplasia syndromes and related disorders.
VIDAZA® (azacitidine for injection) is supplied in a sterile form for reconstitution as a suspension for subcutaneous injection or reconstitution as a solution with further dilution for intravenous infusion. Vials of VIDAZA contain 100 mg of azacitidine and 100 mg mannitol as a sterile lyophiiized powder.

The major challenge with Azacitidine is its chemical instability. It is known from several studies that Azacitidine and related nucleoside analogues such as Decitabine. decompose quickly in hours in water at physiological temperature and pH.
The rapid in vitro chemical decomposition of azacitidine in aqueous solutions was described by Pithova et al. (1965; cited by von hoff and Slavik. 1977). In neutral and basic media, azacitidine is hydrolyzed to l-B-ribofuranosyl-3-guanylurea (probably the same compound as guanylurea ribonuclcoside) via oxidative degradation of the bond between the carbon atom at the 6 position of the cytosinc ring and the nitrogen atom at position 1 accompanied by loss of that carbon atom as formaldehyde or formate, further degradation was reported to give alpha-D-ribofuro (1' 2 4.5)-2-azolidone. guanidine. and D-ribose. At 37°C and pH 7.2. the concentration of azacitidine underwent an initial rapid decline, but approximately 25% of the azacitidine was unchanged after 24h.


In acidic media, azacitidine underwent deglycosylalion (loss of the ribose moiety) to give 5-Azacytosine. Oxidative deamination of 5-Azacytosine gave 5-Azauracil. Deamination occurring before deglycosylation led to 5-Azauridine. (Pithova et al. (1965; cited by von hoff and Slavik. 1977).
There exists a need to develop a method to obtain a stable lyophilized Azacitidine composition. To overcome the above mentioned instability problems associated with Azacitidine composition, the inventors of the present invention have found an improved method of preparation to obtain a stable lyophilized Azacitidine composition. Further, the process according to the present invention also minimizes vial-to-vial variation of impurity in lyophilized Azacitidine injection composition.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide an improved method of preparation to obtain a stable lyophilized Azacitidine composition with reduced levels of impurity.
Another object of the invention is use of heat for a particular temperature and duration to control impurity.
Another object of the invention is heating of finished product after the lyophilisation cycle, so as to reduce the level of impurity.

SUMMARY OF THE INVENTION
One embodiment of the present invention to provide an improved method of preparation to. obtain a stable lyophilized Azacitidine composition with reduced levels of impurity,
Another embodiment of the present invention is to minimize vial-to-vial variation of impurity in lyophilized Azacitidine injection composition.
Another embodiment of the present invention is use of heat for a particular temperature and duration to control impurity.
Another embodiment of the present invention is heating of finished product after the lyophilisation cycle, so as to reduce the level of impurity.
DETAILED DESCRIPTION
Unless otherwise indicated, terms in this specification are intended to have their ordinary meaning in the relevant art.
The present invention refers to an improved method of preparation to obtain a stable lyophilized Azacitidine composition with reduced levels of impurity.
The marketed product. VIDAZA' (azacitidine for injection) is supplied in a sterile form for reconstitution as a suspension for subcutaneous injection or reconstitution as a solution with-further dilution for intravenous infusion.

The major challenge with Azacitidine is its chemical instability. It is known from several studies that Azacitidine and related nucleoside analogues such as Decitabine. decompose quickly in hours in water at physiological temperature and pH.
The below mentioned bulk hold stability data suggests that the Azacitidine is very sensitive in aqueous solvents.
Table 1: Bulk Hold Stability Data of Azacitidine Injection

Bulk Hold Stability Data at 2°C - 8°C without any gas s parging
Initial 90 min 180 min 270 min 360 min 650 min
Assay 97.2 94.5 92.4 91.2 90.3 86.9
Related substances (in %)
SPT1222SM1 0.05 0.05 0.06 0.O6 0.07 0.14
SPT1222SM2 ND ND ND ND ND ND
Max. single unknown impurity 7.90 14.04 20.38 25.52 30.71 40.86
Total impurity 8.14 14.34 20.68 25.87 31.16 41.46
To overcome the above mentioned instability problems associated with Azacitidine composition, there exists a need to develop a method to obtain a stable lyophilized Azacitidine composition.
It is observed by the inventors of the present invention that when lyophilized Azacitidine injection composition is prepared by using normal lyophilization technique, the amount of impurities present in the lyophilized injection is high. Moreover, the impurities, which are formed in aqueous solution gets reverted to Azacitidine once the water is removed during lyophilization.

Surprisingly the inventors found that, after the completion of lyophilization cycle, when the finished product is heated at 40-90 °C for 6 hrs to 45 days, and preferably heated at 60-80 °C for 12 hrs to 30 days for reaction in a closed condition or sterile condition, the reversal of impurities is upto acceptable level.
Following data indicates that the single unknown impurity which is formed in bulk solution gets reverted back to Azacitidine and this single unknown impurity is reduced in the final lyophilized product. For this, bulk solution was prepared without any gas sparging and after lyophilization: samples were kept open in lyophilizer at room temperature in presence of compressed air for 24 hrs and then stopper the vials with headspace air.
Table 2: Chemical Analytical Results of Lyophilized product (Untreated and Treated)

Condition Without air treated , sample Compressed air
treatment for 24
hrs@25°C 1M/ 40°C 2M/ 40°C 3M/ 40°C 3M/ 25°C 6M/ 40°C 6M/
25°C
Related substa nces (in %)
SPT1222SM1 0.306 0.512 0.789 0.609 0.407 0.618 0.362 0.581
SPTI222SM2 0 0 o ND ND ND ND ND
Max. single
unknown
impurity 15.439 9.239 0.641 0.427 0.185 4.655 0,157 2.268
Total Impurity 16.388 10.249 1.721 1.415 0.817 5.573 0.764 2.997
Assay 92.8 92.6 92.8 91.1 91.7 92.6 91.2 91.9
Based on above data it was observed that the single impurity is decreased in the presence of air and temperature.
To demonstrate the effect of oxygen following bulk hold was carried out.
Bulk solution was prepared by adding 1 % w/v of H2O2 at 2-8°C and 25°C.

Table 3: Bulk hold at 2-8°C

Condition SPTI222SM1 SPT1222SM2 Max. single unknown impurity Total Imp. Assay
Initial 0.07% ND 2.58% 2.88% 107.7%
180 min 2-8°C 0.42% ND 1.49% ! .96% 106.9%
360 min 2-8°C 0.72% ND 1.43% 2.41% 104.5%
540 min 2-8cC 0.95% ND 1.57% 2.84% 102.3
Table 4: Bulk hold at 25°C

Condition SPT1222SM1 SPTI222SM2 Max. single unknown impurity Total imp. Assay
Initial 0.07% ND 2.58% 2.88% 107.7%
90 min 25°C 0.62% ND 2.25% 3.13% 104.2%
270 min 25°C 0.70% ND 2.59% 4.10% 87.7%
450min25°C 1.03% ND 2.27% 4.74% 74.8%
Based on above studies, it was observed that the oxygen may play a role to control the impurity during bulk preparation and further to prove the role of oxygen and temperature on impurity, following trial was taken.
Bulk solution was prepared by sparging the oxygen in bulk solution and hold the bulk solution for 3 hrs at 2-8°C and then lyophilized the product, after completion of the lyophilization cycle the vials were kept open in lyophilizer at 45°C in presence of oxygen for 24 hrs and then stopper the vials with headspace oxygen and further stability was carried out.

Table 5:

Condition: Oxygen treatment for 24 hrs@ 45°C Initial filling IM/40°C 2M/40 °C 3M/40 °C 3M/25°C
Related substances (in %)
SPTI222SM1 0.206 0,387 0.74 0.703 0.531
SPT1222SM2 NO NO ND ND ND
Max. single unknown impurity 13.498 3.871 2.374 0.698 9.896
Total Imp. 14.521 4.647 3.500 1.725 10.765
Assay 104.6 104,7 103,3 104.3 104.9
Based on above data it was observed that impurity is decreased in presence of oxygen with temperature.
Further trials were taken to prove this fact and samples were treated at different conditions mentioned below.
Table 6:

Oxygen Oxygen
Oxygen treatment for 24 hrs
@. 45°C treatment for 24 hrs treatment for 24 hrs Oxygen
treatme Oxygen treatment for 72 hrs
@ 45°C Oxygen trcatme
Condition Untreated
@45°Cand
FP sample
hold @60°C @45°Cand
FP sample
hold @S0°C ntfor48 hrs@
45°C
nt for 96
hrs @ 45°C
/24hr /24hr
SPT1222SM1 0.435% 0.666% 1.130% 0.307% 1.008% 1.187% 1,336%
SPT1222SM2 ND ND ND ND ND ND ND
Max. single -
unknown 20.800% 14.782% 1.106% 0.234% 8.308% 2.178% 1.049%
impurity
Total Impurity 22.435% 16,459% 2.837% 0,838% 9.971% 3.988% 2.863%
Assay 99.0% NA NA NA 99.0% NA 98.7

Based on above data it was observed that single unknown impurity is decreased in presence of oxygen with temperature. But in presence of oxygen known impurity SPT1222SM1 was increased.
Further trials were taken to see the effect of temperature to stabilize the product impurity level in presence of oxygen.
Bulk solution was prepared by sparging the oxygen in bulk solution and hold the bulk solution for 6 hrs at 2-8°C and then lyophilizcd the product, after completion of the lyophilization cycle the half of the total vials were kept open in lyophiiizer at 45°C in presence of Oxygen for 24 hrs and then stopper the vials and remaining vials stopper directly without any treatment with headspace oxygen.
Table 7: Effect of temperature on untreated samples

Condition SPT1222SM1 SPTI222SM2 Max. single unknown impurity Total Impurity % Assay
Initial 0.031% ND 11.796% 12.743% 102.1
80°C.24Hrs 0.042% ND 0.308% 0.752% 102.6
80°C,48Hrs 0.043% ND 0,194% 0.589% 102.8
80°C.96Hrs 0.050% ND 0.204% 0.551% 102.1
60°C,72Hrs 0.033% ND 1.015% 1.955% 103.0
60°C.96Hrs 0.036% ND 0.611% 1.385% 102.6

Tabic 8: Effect of temperature on Treated samples:

Condition SPT1222SM1 SPTI222SM2 Max. single unknown impurity Total Imp. % Assay
Initial 0.032% ND 12.93% 13.851% 100.2
80°C.24Hrs 0.039% ND 0.246% 0,712% 101.1
80°C,48Hrs 0.046% ND 0.203% 0.630% 101.6
80°C,96Hrs 0.044% ND 0.164% 0.430% 99.5
60°C,72Hrs 0.021% ND 0.495% 1.140% 102.4
60°C ,96Hrs 0.017% ND 0.497% 1.135% 101.5
90°C.36Hrs 0.042% . ND 0.164% 0.464% NA
Based on above data it was observed that there was no any significant impact of oxygen treatment but there was significant impact of temperature on finished product in presence of oxygen. Also the known impurity SPT1222SM1 is controlled. So maybe there was because of temperature effect., there may not be role of oxygen so to prove this fact further following trial was taken.
Bulk solution was prepared without sparging any gas in bulk solution and hold the bulk solution for 6 hrs at 2-8°C and then lyophilized the product, after completion of the lyophilization cycle the half of the total vials were stopper using nitrogen with vacuum and then stopper the vials and remaining vials stopper directly without any gas.

Table 9: Trial with Head Space Nitrogen with Vacuum

Condition SPT1222SMI SPTI222SM2 Max. single unknown impurity Total Imp. %Assay
Initial ND ND 21.622% 23.106% 99.9
80°C.12HTS ND ND 0.671% 1.421% NA
80°C,24Hrs ND ND 0.415% 0.916% NA
80°C.48Hrs ND ND 0.278% 0.651% NA
60°C7 Days ND ND 0.679% 1.621% NA
60oC, 10 Days ND ND 0.614% 1.498% NA
60°C,l4Days ND ND 0.552% 1.286% NA
Table 10: Trial with Head Space air without Vacuum

Condition SPT1222SMI SPTI222SM2 Max. single unknown impurity Total Imp. % Assay
Initial ND ND 21.622% 23.106% NA
80oC,12Hrs ND ND 0.607% 1.316% NA
80°C,24Hrs ND ND 0.321% 0.799% NA
80°C,48Hrs ND ND 0.241% 0.601% NA
60°C,7 Days ND ND 0.623% 1.455% NA
60°C,10Days ND ND 0.553% 1.395% NA
60°C,14 Days ND ND 0.414% 1.174% NA
Based on above data it was observed that there no significant impact of any inert gas sparging on product stability but there is significant role of temperature to control the impurity level in finished drug product.

We claim:
1. A method for preparation of a stable lyophilized azacitidine composition with reduced levels of impurity.
2. The method for preparation according to claim 1. wherein (he stable lyophilized azacitidine composition is prepared by providing heat for a particular temperature and duration to control impurity.
3. The method for preparation according to claim 2. wherein the stable lyophilized azacitidine composition is prepared by heating of the finished product after the lyophilization cycle.
4. The method for preparation according to claim 1. wherein the stable lyophilized azacitidine composition is healed at 40-90 °C for 6 hrs to 45 days in a sterile condition.
5. The method for preparation according to claim 4. wherein the stable lyophilized azacitidine composition is heated at 60-80 °C for 12 hrs to 30 days in a sterile condition.
6. The method for preparation according to claim 1, wherein the said method minimizes vial-to-vial variation of lyophilized azacitidine injection.
7. A stable lyophilized azacitidine composition with reduced levels of impurity prepared by any of (he preceding claims.