FORM 2
THEi PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
Provisional Specification
[See Sections 10 and rule 13]
Title: A stable composition of ready-to-use Gemcitabine injection

Applicant: (a) Astron Research Limited
(b) Nationality: Indian

(c) 10,h Floor, Premier House

Bodakdev, Opp. Gurudwara
Sarkhej -- Gandhinagar Highway
Ahmedabad 380054

Gujarat, India.



The following specification describes the invention:


Field of the invention
The present invention relates to pharmaceutical composition containing Gemcitabine or its pharmaceutically acceptable salts in the form of ready-to-use solutions and processes for preparing such compositions.
Background
Chemically, Gemcitabine is l-(2-oxo-4-amino-l, 2-dihydropyrimidin-l-yl)-2-deoxy-2, 2-difluororibose having the following formula:

Gemcitabine was earlier disclosed in US patent 4808614. Gemcitabine is currently been marketed as a hydrochloride salt in a lyophilized formulation (Gemzar®) by Eli Lily and Company.
Gemcitabine is a pro-drug (pyrimidine antimetabolite) which is metabolised intracellularly to active diphosphate and triphosphate nucleosides. It inhibits DNA synthesis by inhibiting DNA polymerase and ribonucleotide reductase. It also induces apoptosis and is primarily active against cells in the S-phase of DNA synthesis.
The Journal of Pharmaceutical Sciences, vol. 89, No.7, Pg. No. 885-891, discloses Gemcitabine aqueous solution. The pH of the solution was adjusted to 3.2 by using sodium acetate. The degradation of Gemcitabine in aqueous solution at pH 3.2 indicates that development of formulation was feasible when stored at refrigerated temperature. However at thermally stressed conditions four significant degradation products were obtained.
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EP1479388 discloses Gemcitabine ready-to-use preparation having Gemcitabine dissolved in mixture of water and physiologically acceptable solvent or solubilising agent.
The disclosed concentration according to this patent is 16mg/ml to 1 lOmg/ml and having pH of 3.5 to 10. The disclosed examples suggest that the preparation were aqueous in nature and contains a mixture of water and solvents which are water soluble or insoluble in nature.
EPl479389 discloses Gemcitabine inorganic acid addition salt composition for ready-to-use solution which was not reconstituted from a solid prior to administration, having pH of the solution above 3.5 and Gemcitabine in at least 0.05mg/ml. According to a preferred form of execution, the pH of the invention solutions is in a range from 3.5 to 10.0 and Gemcitabine concentration of 0.05 mg/ml to 16.0 mg/ml of solvent. The stability data of disclosed examples in the patent demonstrate that compositions with higher concentration of Gemcitabine (more than 16.0 mg/ml) cause precipitation when stored at lower temperatures.
The solvent used in this invention was selected from the ethyl alcohol, polyethylene glycol 200-600, propylene glycol and mixture thereof but their use was restricted to preparations wherein the concentration of Gemcitabine was 0.05 mg/ml to 16.0 mg/ml.
US20060154891 discloses, ready-to-use Gemcitabine aqueous preparation in glass container having specified dimensional relationships to demonstrate shelf life of over wide range of solution pH values. The ratio of surface area wetted by the composition to the volume of the solution contained in the container, expressed in cm /cm is less than 3.4.
WO2007143895 discloses, stable supersaturated solution of Gemcitabine hydrochloride, which is prepared by dissolving completely Gemcitabine
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hydrochloride in a medium by heating it at pH 4-8 to give a supersaturated solution of Gemcitabine having a concentrate of 15-45g/L.
Thus to overcome the disadvantages of precipitation of ready-to-use Gemcitabine composition at lower temperature (2° - 8° C) for concentration above 16mg/ml in aqueous solution, there is need to develop a stable ready-to-use preparation containing Gemcitabine or its pharmaceutically acceptable salts using non-aqueous solvents.
The present invention relates to non-aqueous pharmaceutical preparations containing Gemcitabine or its pharmaceutically acceptable salts in the form of ready-to-use solutions wherein the concentration of Gemcitabine is above 16 mg/ml and having a pH of about 3.5 to 10.0.
The present invention meets these objectives by providing Gemcitabine or its pharmaceutically acceptable salts compositions, with superior stability even at higher concentration of Gemcitabine or its pharmaceutically acceptable salts as compared with preparations known in background references. It has been found that the non-aqueous solvents used for ready-to-use compositions of Gemcitabine or its pharmaceutically acceptable salts are suitable for intravenous (i.v.) administration in humans and provide better stability to the composition.
Objects of the invention
The main object of the invention is to provide non-aqueous pharmaceutical preparation containing Gemcitabine or its pharmaceutically acceptable salts as a ready-to-use preparation wherein the concentration of Gemcitabine is above 16 mg/ml and having a pH of about 3.5 to 10.0.
Another object of the present invention is to provide stable non-aqueous pharmaceutical preparation containing Gemcitabine or its pharmaceutically acceptable salts as a ready-to-use preparation wherein the concentration of Gemcitabine is above 16 mg/ml and having a pH of about 3.5 to 10.0.
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Still another object of this invention is to provide a process for preparation of non-aqueous pharmaceutical preparation containing Gemcitabine or its pharmaceutically acceptable salts as a ready-to-use preparation wherein the concentration of Gemcitabine is above 16 mg/ml and having a pH of about 3.5 to 10.0.
Summary of the Invention
This invention relates to a stable non-aqueous pharmaceutical preparation of Gemcitabine or its pharmaceutically acceptable salts in the form of ready-to-use preparation wherein the concentration of Gemcitabine is above 16 mg/ml and having a pH of about 3.5 to 10.0.
Further this invention also relates to processes for preparation of a stable non¬aqueous pharmaceutical preparation containing Gemcitabine hydrochloride or its pharmaceutically acceptable salts in the form of ready-to-use preparation wherein the concentration of Gemcitabine is above 16 mg/ml and having a pH of about 3.5 to 10.0.
Detailed Description of the Invention
The primary object of the present invention is to provide a stable non-aqueous pharmaceutical preparation containing Gemcitabine hydrochloride or its pharmaceutically acceptable salts in the form of ready-to-use formulation.
This invention is directed towards a stable non-aqueous pharmaceutical preparation containing Gemcitabine or its pharmaceutically acceptable salts in concentration of above 16 mg/ml and a pH of about 3.5 to 10.0. Preferably Gemcitabine or its pharmaceutically acceptable salts are in a concentration of about 16 mg/ml to about 200 mg/ml and the pH of the composition is about 5.0 to 8.0. More preferably the Gemcitabine or its pharmaceutically acceptable salts are in concentration of about 60 mg/ml to about 140 mg/ml and the pH of the composition is about 5.0 to 8.0.
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Further the present invention also provides stable ready-to-use composition containing Gemcitabine or its pharmaceutically acceptable salts comprising of physiologically acceptable non-aqueous solvent wherein the concentration of Gemcitabine or its pharmaceutically acceptable salt is above 16 mg/ml and the pH of the composition is about 3.5 to 10.0.
The physiologically acceptable non-aqueous solvent according to present invention comprises propylene glycol, polyethylene glycols, ethanol or the like thereof either alone or in combination thereof.
Another embodiment of the inventions is directed towards process for the preparation of non-aqueous ready-to-use pharmaceutical preparation containing Gemcitabine or its pharmaceutically acceptable salts wherein the process comprises of dissolving Gemcitabine or its pharmaceutically acceptable salt in a non-aqueous solvent or a mixture of non-aqueous solvents, adjusting the pH of the composition to 3.5 to 10.0 using sodium hydroxide to completely solubilize Gemcitabine or its pharmaceutically acceptable salt and filing the obtained solution in vials.
Further, the non-aqueous ready-to-use pharmaceutical preparation of Gemcitabine or its pharmaceutically acceptable salts of the present invention are stable at room temperature as well as at lower temperatures.
Examples
The present invention has been described by way of example only, and it is to be recognized that modifications thereto which fall within the scope and spirit of the appended claims, and which would be obvious to a skilled person based upon the disclosure herein, are also considered to be included within the invention.
The above said invention of non-aqueous preparation containing Gemcitabine or its pharmaceutically acceptable salts in the form of ready-to-use solutions can be illustrated by but not limited to following examples.
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Example 1: Gemcitabine injection (RTU), lOOmg/ml, pH: 7.8

Sr. No. Ingredients Qty.Jml
1 Gemcitabine Hydrochloride 113.85 mg
2 PEG-400 180.00 mg
3 Sodium Hydroxide / cone. HC1 q.s. topH 7.5
4 Propylene Glycol q.s. to 1ml
Procedure:
1. Take 18 gm of PEG-400 & 62 gm of propylene glycol and stir to form uniform mixture.
2. Add 1.5 gm of sodium hydroxide palettes and stir to get clear solution.
3. Sparge nitrogen for 30 minutes and add Gemcitabine hydrochloride and stir to get clear solution.
4. Adjust the pH to 7.5 (7.5 -7.8) using sodium hydroxide and /or concentrated hydrochloric acid.
5. Make up volume with propylene glycol, stir for 20 minutes with nitrogen sparging.
6. Filter with 0.22". PVDF filter & fill in vial and seal.
Stability studies:

Parameters Initial Storage conditions

25tt C ± 2°C & 60% ± 5%RH 40°C±2°C&75% ±5%RH

1 Month ,2 Month 1 Month 2 Month
Assay 101.3 100.8 110.3 101.2 100.1
PH 7.8 7.8 7.6 7.8 7.5
Related substances
Cytosine BQL BQL BQL BQL BQL
Alfa anomer 0.01 0.01 0.01 0.01 0.01
unknown 0.04 0.04 0.03 0.04 0.04
Total 0.09 0.09 0.11 0.09 0.08
BQL-Below quantification limit
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Results of the stability studies performed for stable ready-to-use non-aqueous Gemcitabine composition mentioned according to example 1 demonstrates that the pH of the composition, assay for Gemcitabine and the amount of impurities formed after long time and accelerated studies conducted for 2 months were within the acceptable limits.
Example 2: Gemcitabine injection (RTU), lOOmg/ml, pH: 7.8

Sr. No. Ingredients Qty./ml
1 Gemcitabine Hydrochloride 113.85 mg
2 PEG-400 180.00 mg
3 Sodium Hydroxide / cone. HC1 q.s. topH 7.5
4 Propylene Glycol q.s. to 1ml
Procedure:
1. Take 18 gm of PEG-400 & 62 gm of propylene glycol and stir to form uniform mixture.
2. Sparge nitrogen for 30 minutes and add Gemcitabine hydrochloride and stir to get uniform suspension.
3. Solubilize the Gemcitabine hydrochloride using sodium hydroxide solution,
4. Adjust the pH to 7.5 (7.5 -7.8) using sodium hydroxide solution and /or concentrated hydrochloric acid.
5. Make up volume with propylene glycol, stir for 20 minutes with nitrogen.
6. Filter with 0.22|i PVDF filter & fill in vial and seal.
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Stability studies:

Parameters Initial Storage conditions

25° C ± 2°C & 60% ±5% RH 40°C±2°C&75% ±5%RH

1 Month 2 Month 1 Month 2 Month
Assay 102.5 100.5 100.3 100.5 100.2
PH 7.5 7.5 7.5 7.5
Related substances
Cytosine BQL BQL BQL BQL BQL
Alfa anomer 0.01 0.0 i 0.01 0.01 0.01
unknown 0.04 0.04 0.03 0.04 0.04
Total 0.1 0.09 0.08 0.09 0.1
BQL-Below quantification limit
Results of the stability studies performed for Gemcitabine composition mentioned according to example 2 demonstrates that the pH, assay for Gemcitabine and the amount of impurities formed after long time and accelerated studies conducted for 2 months were within the acceptable limits.
Example 3: Gemcitabine injection (RTU) Batch size: 200ml, pH: 7.5

Sr. No. Ingredients Qty./ml
1 Gemcitabine Hydrochloride 113.85 mg
2 PEG-300 180.00 mg
3 Dehydrated Alcohol 100.0 mg
4 Sodium Hydroxide / cone. HC1 q.s. to pH 7.5
5 Propylene Glycol q.s. to I ml
Procedure:
1. Take 36 gm of PEG-300 & 120 gm of Propylene Glycol and stir to form uniform mixture.
2. Sparge nitrogen for 30 minutes and add API and stir to get uniform suspension.
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3. Solubilize the API using Alcoholic Sodium Hydroxide solution.
4. Adjust pH 7.5 (7.5 -7.8) using Alcoholic Sodium Hydroxide solution and / or concentrated hydrochloric acid.
5. Make up volume with Propylene Glycol, stir for 20 minutes with nitrogen sparging.
6. Filter with 0.22U. PVDF filter & fill in vial and seal.
Example 4: Gemcitabine injection (RTU) Batch size: 200ml, pH: 5.5

Sr. No. Ingredients Qty./ml
1 Gemcitabine Hydrochloride 113.85 mg
2 PEG-300 180.00 mg
3 Dehydrated Alcohol 100.0 mg
4 Sodium Hydroxide solution in alcohol / cone. HC1 q.s. to pH 5.5
5 Propylene Glycol q.s. to 1ml
Procedure:
1. Take 36 gm of PEG-300 & 120 gm of Propylene Glycol and stir to form uniform mixture.
2. Sparge nitrogen for 30 minutes and add API and stir to get uniform suspension.
3. Solubilize the API using alcoholic sodium hydroxide solution.
4. Adjust pH to 5.5 (5.0-6.0) using alcoholic sodium hydroxide solution and / or concentrated hydrochloric acid.
5. Make up volume with propylene glycol, stir for 20 minutes with nitrogen sparging.
6. Filter with 0.22u. PVDF filter & fill in vial and seal.
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Example 5: Gemcitabine injection (RTU), 200ml, pH: 5.5

Sr. No. Ingredients Qty./ml
1 Gemcitabine Hydrochloride 113.85 mg
2 PEG-300 180.00 mg
3 Sodium Hydroxide / cone. HC1 q.s. to pH 5.5
4 Propylene Glycol q.s. to 1ml
Procedure:
1. Take 36 gm of PEG-300 & 120 gm of propylene glycol and stir to form uniform mixture.
2. Sparge nitrogen for 30 minutes and add API and stir to get uniform suspension.
3. Solubilize the API using 50% sodium hydroxide solution.
4. Adjust pH to 5.5 (5.0 -6.0) using sodium hydroxide solution and /or concentrated hydrochloric acid.
5. Make up volume with propylene glycol, stir for 20 minutes with nitrogen sparging.
6. Filter with 0.22u PVDF filter & fill in vial and seal.
The results obtained from the stability studies performed on non-aqueous Gemcitabine compositions according to example 1 and example 2 demonstrate that preparation is stable in normal and accelerated storage conditions.

.
Ketana Laljibhai Babaria
For and on behalf of the applicant To,
The Controller of Patents, The Patent Office, At Mumbai.
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