FORM 2
THE PATENT ACT 1970 & THE PATENTS RULES, 2003
COMPLETE SPECIFICATION (See Section 10 and Rule 13)
1. TITLE OF THE INVENTION
Preparation of formulations of oxaliplatin.
2. APPLICANT(S)
(a) NAME: Emcure Pharmaceuticals Ltd.
(b) NATIONALITY: an Indian Company
(b) ADDRESS: P-l,IT-BT Park
MIDC Phase-2, Hinjwadi, Pune-411057, INDIA
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

4. DESCRIPTION
Field of the invention
The present invention relates to an improved process for the preparation of formulations of oxaliplatin using carbon dioxide as a principal acidity maintaining agent that form the inorganic acid in solution. And formulations so prepared having significantly improved impurities profile and stability for clinical applications.
Background of the invention

Oxaliplatin is chemically known as cis-[(-lR,2R)-cyclohexanediamine-N,N'] [oxalato(2-)-0,0'] platinum and has the structural formula [1]. It is a fine white powder is slightly soluble in water and insoluble in ethanol and acetone. It is used in combination with 5-fluorouracil and leucovorin, and is indicated for the treatment of metastatic carcinoma of the colon or rectum as a second-line therapy agent. Its mechanism of action is mediated by its formation of platinum complexes with DNA bases in the rapidly growing cancerous cells and limiting proliferation of these cells. The formulations of oxaliplatin in water are stabilized with different organic acids as the parent compound is unstable and forms many impurities on decomposition that are

undesired in clinical applications. Oxaliplatin is especially unstable in alkaline conditions. It is stable in acidic conditions; however, strong acidic conditions of parenteral solutions are not recommended as they cause severe reactions in patients due to local irritation at the site of injection.
The major impurities of present in oxaliplatin formulations are - impurity A:
oxalic acid; impurity B: (SP-4-2)-diaqua[1R,2R)-cydohexane-l,2-diamine-
KN, KN']platinum (diaquodiaminocyclohexane platinum); impurity C: (OC-
6-33)-[(lR,2R)-cyclohexane-l,2-diamine-KN, KN'][ethanedioato(2-)-K01,
K02]dihydroxyplatinum; impurity D: (SP-4-2)-[(lS,2S)-cyclohexane-l,2-diamine-KN, KN'][ethanedioato(2-)- KOI, K02]platinum (S,S-enantiomer of oxaliplatin); and impurity E: (SP-4-2)-di-μ-oxobis[{lR,2R)-cyclohexane-l,2-diamine-kN, KN']diplatinum-(diaquodiaminocyclohexaneplatinum dimer). These impurities are formed in the several preparations of oxaliplatin and are important as they get developed in the storage conditions to high levels.
In the prior art, there are disclosed several methods of preparation of formulations of oxaliplatin using organic acids such as tartaric acid, lactic acid, etc. US6306902 discloses oxaliplatin compositions comprising a therapeutically effective amount of oxaliplatin, an effective stabilizing amount oi oxalic acid or its alkali metal salt as a buffering agent. US6476068 discloses oxaliplatin compositions comprising oxaliplatin, and a stabilizing amount of lactic acid or its salt. US20060063833 discloses oxaliplatin compositions of oxaliplatin in water with an acid selected from the group consisting of phosphoric acid, sulfuric acid, methane sulfonic acid, ethane sulfonic acid, para-toluene sulfonic acid, and mixtures thereof and carbohydrates such as lactose, glucose, maltose, fructose, galactose, and/or dextrans. US20060264501 discloses oxaliplatin compositions comprising a

therapeutically effective amount of oxaliplatin in water with an acid selected from the group consisting of group consisting of citric acid, maleic acid, saccharic acid, succinic acid, malic acid, tartaric acid, and mixtures thereof. US5716988 discloses oxaliplatin compositions using nitrogen before sealing the containers hermetically for long-term stability of the formulations. EP1466600 discloses various organic and inorganic acids as stabilizing agents for oxaliplatin compositions, except used of carbon dioxide as a stabilizing agent. WO2009087660 discloses use of carbon dioxide as a stabilizing agent, but fails to disclose the effective concentration of carbon dioxide required to achieve clinically useful compositions of the oxaliplatin. Besides, this application discloses the method that use CO2 sparging of the compositions for longer period of time of about 60 min, which is not practical in production plants and also lead to undesired product conditions such as high pH of the solutions [see WO2009087660]. These compositions also have pH range outside compendial requirements rendering them of no use as clinical compositions. Besides, the above compositions/ formulations have limitations with respect to the guidelines for pharmaceutical products such as ICH and USP monographs on oxaliplatin formulations.
The foregoing processes do not involve use of carbon dioxide as disclosed herein in solution to form the inorganic carbonic acid for the preparation of formulations of oxaliplatin that have significantly improved stability and impurities profile compared with the known formulations used in clinical applications. Besides, the foregoing processes suffer from serious disadvantages such as low stability of the formulations and development of undesired impurities in the formulations due to the coordinate nature of oxaliplatin and the stabilizing agents not sufficient in constancy

maintenance in common storage conditions of the liquid formulation. Consequently, it would be a significant contribution to the art to provide an improved process for the preparation of formulations of oxaliplatin for clinical applications, which would be scalable, cost effective, and environment friendly.
Objects of the invention
An object of the present invention is to provide a novel route for the preparation of oxaliplatin formulations of higher stability and improved impurity profile for clinical -applications. Another object of the present invention is to use carbon dioxide as an acidity and stability maintaining agent in the preparation of the formulations of oxaliplatin. Yet another object of the present invention is to prepare the formulations of oxaliplatin using carbonic acid as the acidity and stability agent and the carbonic acid prepared in situ using carbon dioxide.
Detail description
In an embodiment of the present invention, there is provided an improved process for the preparation of formulations of oxaliplatin with good stability and improved purity profile by using carbon dioxide as an acidity and stability maintaining agent in the preparation of the formulations. In the first part, the oxaliplatin API of at least 98% purity is prepared or obtained and a solution of about 5 mg/mL is made is high purity water like water for injection. In the second part, this solution is treated (sparged or bubbled through the solution of oxaliplatin) with carbon dioxide gas of at least 99% purity ,so that the pH of the solution becomes acidic till about 5.0. At this point the concentration of carbon dioxide in the solution is kept between 300

- 600 ppm. In the third part, this acidified solution of oxaliplatin in packed in air tight containers in the presence of CO2, so that the gas content remains stable in the formulation containers. To test the effect of the concentration oi 100 -1000 ppm of CO2 on the impurities formed on storage in the oxaliplatin formulations, the impurity profile of the samples at different time points were analysed by HPLC for the impurities B and E as shown in Table 1. The CO2 content was analysed using a carbonation meter in-line at the time of preparation, packaging and after storage of the formulations of oxaliplatin.
As shown in Table 1 different batches of formulations of oxaliplatin were subjected to the conditions as numerated. in the case of batches of formulations sparged with nitrogen, oxygen or air, the impurities B and E were not stable compared with the batches that were sparged with CO2 at about 450 ppm of concentration. At an elevated temperature over a period of three months impurity E and B increased in the batches that were not sparged with CO2. The pH of batches treated with CO2 for 5 min were also stable compared to other batches and were having lowest amount of impurity E compared to the batches not treated with CO2 gas as acidifying agent. This data showed that the CO2 concentration in the range of 300 - 600 ppm in the formulations of oxaliplatin, specially improved its stability and impurities profile vis-a-vis impurities E and B. These formulations also had stable pH conditions over the period of storage of the formulations at elevated temperature for a longer period of time. These characteristics of the formulation of oxaliplatin disclosed herein are advantageous and there are fewer impurities formed as well as pH remains stable, as lower pH of prior art- formulations may cause adverse reactions such as phlebitis, phlebothombosis and local irritation in the patients.

] Table 1: The CO2 content and formation of impurities on storage in the oxaliplatin formulations.

No. Sample Type* Conditions [temperature/ humidity/ months] Gas
Sparged [5 min] CO2
Content [ppm] pH Impurity E [%] Impurity
B[%] Total Impurities ,[%] (. Assay [%]
1 RLD 40/75/0 - <50 6.2 0.077 0.052 0.182 1 100.20
2 RLD 40/75/3 - <50 5.2 0.067 0.043 0.207 101.10
3 Batch A 40/75/0 N2 <50 6.6 0.262 0.052 0.460 101.10
4 Batch A 40/75/3 N2 <50 6.6 0.322 0.055 0.875 98.90
5 Batch B 40/75/0 O2 <50 6.6 0.041 0.075 0.221 100 30
6 Batch B 40/75/3 O2 <50 6.7 0.286 0.056 1.154 97.30
'/ Batch C 40/75/0 Air <50 6.6 0.078 0.065 0.243 100.70
8 Batch C 40/75/3 Air <50 6.7 0.288 0.056 0.804 ., 9790
9 Batch D 40/75/0 COz -450 5.8 0.017 0.031 0.089 100.60
10 Batch D 40/75/3 CO2 -450 5.3 0.184 0.045 0.737 97.30
*RLD - Reference standard; different batches of formulations were used for the above experiments.

In the present invention is disclosed the use of carbon dioxide as an acidity and stability agent in the formulations of oxaliplatin that are stable for longer period of time with improve impurity profile. The presence of a critical impurity, impurity E, in the stored formulations of oxaliplatin is the problem and the present method eliminates the formation of this impurity, along with other impurities as disclosed. The process of the present invention is simple, improved, eco-friendly, cost-effective, commercially significant, robust and reproducible on an industrial scale. Having thus described the invention with reference to particular preferred embodiments and illustrative examples, those in the art may appreciate modification to the invention as described and illustrated that do not depart from the spirit and scope of the invention as disclosed in the specification. The examples are set to aid in understanding the invention but are not intended to, and should not be construed to; limit its scope in any way. The examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinarily skill in the art. All references mentioned herein are incorporated in their entirety.
Example 1: Preparation of Oxaliplatin
The oxaliplatin API was produced with known processes or obtained as required. The process for the preparation of oxaliplatin API was: reacting of potassium tetrachloroplatinate with (1R, 2R)-(-)-l,2-diamino-cyclohexane in aqueous medium to obtain 1,2-cyclohexanediamine-dichloroplatinate. Next, this compound was reacted with silver oxalate in aqueous medium leading to the formation of the coordinate compound of oxaliplatin with total impurities of less than 0.5% in the final batches.

Example 2: Preparation of Formulations of Oxaliplatin Using Carbon Dioxide as Acidity and Stability Maintaining Agent
In a vessel 95 mL water for injection was taken at temperature-between 15 to 30 °C and pH around 6.5. To this 500 mg oxaliplatin API [of at least 98% purity] was added. Solution was stirred well to clarity and made up to 100 mL of water, to obtain the oxaliplatin solution of 5 mg/mL. This was followed by the sparging of CO2 gas [of at least 99% purity] for various time periods. The content of CO2 in these solutions was measured with a CO2 analyser to determine the amount of gas in solution. These solutions were then filtered through 0.44 micron and 0.22 micron filters, respectively, to remove particulate and microbial contaminants. The bulk solutions were then dispensed into vial as required, while CO2 or other gases were purged through the vials before and after the dispensing and the filled vials closed with rubber stoppers and sealed.
Example 3: Quantification of CO2 in the Formulations
The CO2 content of the formulations of oxaliplatin was measured using a carbonation meter [CarboQC, ANTON PAAR GmbH]. The resolution limit of the meter was 0.001%/L. To measure CO2 in the samples the probe of the meter was dipped in the solution for about 90 sec. and reading recorded. The amount of CO2 and its relation to the acidity of solution for a representative batch [Batch D] is shown in Table 2. As the time of expose of CO2 to the solution increased, the pH decreased. The pH 4.0 at room temperature was achieved at about 1050 ppm of CO2 concentration in the composition in about 15 min.

Table 2: The CCh content and pH of the oxaliplatin formulations.

CCh sparging time [minute] pHat RT CO2
Content
[ppm]
1 0 5.8 <50
2 <1 4.3 250
3 1-1.5 4.3 290
4 5 4.2 450
5 15 4.0 1050
Table 3 gives the ratios of CCh to that of oxaliplatin base in the formulation at different CO2 concentrations. Table 3:

No. CO2 content (ppm) CO2 to oxaliplatin ratios
1 300 5.4:50000
2 500 9:50000
3 600 10.8:50000
Example 4: Analysis of the Oxaliplatin Formulations
The parameters of HPLC method for the detection of impurities B and E in the formulations of oxaliplatin were as given in Table 3. The relative retention time for impurity B was 1.0 min and for impurity E was 1.4 min.

Table 3: HPLC method parameters
Column L1, Hyerpsil BDS C18 (4.6 mm x 100 mm), 3 μrn
Flow rate l.0mL/min
Elution Retention time for about 18 minutes
Wavelength 210 ran
Injection volume 20 μL
Column temperature 10 0C
. Runtime 55 min.
Diluent 0.01N nitric acid
Sample concentration 5 mg/mL
Solution A Monobasic potassium phosphate [1.36 g], 1-heptane sulphonic acid sodium salt [0.55 g] in 1 L water, pH adjusted to 3.0 with phosphoric acid.
Mobile phase Mobile phase A: Solution A and methanol
81:19
Mobile phase B: Solution A and methanol
49.5:50.5
Gradient program

Time (min) Mobile phase-A % Mobile phase-B %

0 100 0

45 0 100

45.5 100 0

53 100 0

5. CLAIMS
We claim:
1. A process for the preparation of a stable formulation of oxaliplatin
having pH between 4.5 to 7, comprising the steps of:
a) dissolving oxaliplatin of at least 98% purity in water,
b) sparging the said solution with carbon dioxide to reduce the pH, and
c) packing the said solution in air tight container for long term storage.

2. A process of claim 1, wherein the purity of carbon dioxide used is at least 99%.
3. A process of claim 1, wherein ratio of carbon dioxide to oxaliplatin is between 5:50000 to 12:50000.
4. A process of claim 1, wherein temperature applied is between 15 to 30 °C
5. A formulation of oxaliplatin according to claim 1, having carbon dioxide concentration in the range of 100 to 1000 ppm, preferably in the rage of 300 to 600 ppm.
6. A formulation of oxaliplatin according to claim 1, having the impurity E not more than 0.5%.
7. A pharmaceutical composition according to claim 1 in a unit dosage form comprising an effective amount of oxaliplatin.
8. A pharmaceutical composition according to claim 7 comprising a formulation of oxaliplatin in association with a pharmaceutically

acceptable carrier and/or an inactive compound and/or another active compound.
9. A process for preparing a formulation of oxaliplatin substantially as described with reference to the examples.