FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patent Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rule 13)
PHARMACEUTICAL COMPOSITION


SUN PHARMA ADVANCED RESEARCH COMPANY LTD.
A company incorporated under the laws of India having their office at 17/B, MAHAL INDUSTRIAL ESTATE, MAHAKALI CAVES ROAD, ANDHERI (E), MUMBAI-400093, MAHARASHTRA, INDIA.
The following specification describes the invention.





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The present invention also provides lyophilized pharmaceutical composition comprising bortezomib in therapeutically effective amount and tromethamine wherein the pH of the composition is in the range of 7.6
to 8.4, preferably 7.9 to 8.1.
When the composition is a lyophilized composition, a bulking agent may be added in the composition. As used herein, the term "bulking agent" is intended to mean a compound used to add bulk to the lyophilized product and/or assist in the control of the properties of the formulatiori during lyophilization. Examples of bulking agents that may be used include, but are not limited to, dextran, trehalose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol, albumin, calcium lactobionate, and others known to those of ordinary skill in the art.
When the pharmaceutical composition is in the form of a lyophilized product, the composition may include apart from tonicity adjusting agents, bulking agent, cryoprotectants, The term 'cryoprotectant' used is intended to mean a compound used to protect an active therapeutic agent from physical or chemical degradation during lyophilization. Examples of cryoprotectants that may be used include, but are not limited to, carbohydrates such as monosaccharides, disaccharides and sugar alcohols. Examples of the carbohydrates that may be used include, but are not limited to, mannito], sucrose and others known to those of ordinary skill in the art.
The pharmaceutical composition of the present invention may be prepared by simply mixing the required amounts of bortezomib with the tromethamine and adding water for injection with continuous stirring. The solution may be warmed at 45 °C to 50 °C to form a clear solution. The solution may then be filtered through 0.2 to 0.8 micron syringe filter, preferably 0.45 micron filter and then the pH is determined. The pH of the clear solution is further adjusted with hydrochloric acid in the range of 7.6 to 8.4, preferably in the range of 7.9 to 8.1. According to one embodiment, the pH adjusted clear solution may be subjected to lyophilization or freeze drying.
Generally, the lyophilization involves two steps namely, thermal treatment step wherein no vacuum is applied and the actual primary drying step wherein vacuum is applied. The solution subjected to lyophilization is filled into vials with specialized stoppers. The vials filled with the solution to be dried are placed in the lyophilizer. In the thermal treatment step, temperature of shelf of lyophilizer where the vials of solution filled is stored, is gradually decreased from 20 ° C to - 40 ° C. Then the frozen solution is subjected to drying step. For example, in this instance, the temperature is set frcrn - 40 ° C to - 15 ° C for the time cycle of about 5 to 6 hours at a vacuum of about 100 to 2C0 mTorr. At this time and temperature, the ice is dried. Then the temperature of the material is raised to + 10 ° C to about + 25 ° C at a vacuum of about 50 mTorr in which the residual solvent if any is removed. The lyophilized composition or commonly referred to as lyophilized cake in the vials may then be subjected to reconstitution at the time of administration.
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It was found that the reconstitution of the Iyophilized cake of the pharmaceutical composition of the present invention takes less than 90 seconds without any need of bath sonication. Also, the reconstituted solution was found to be stable in terms of particle formation for at least 24 hours i.e no particulates were observed and also was chemically stable.
It will be understood by those of skill in the art that numerous modifications can be made without departing from the spirit of the present invention. Therefore, it should be clearly understood that the following examples are illustrative only and should not to be construed to limit the scope of the present invention.
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Table 1: Comparative examples I-IX

Comparative Example Ingredients per vial Observation conclusion
1 Bortezomib (Img) + polysorbate (100 mg) The solution was stirred at 50 to 60 °C., water for injection quantity sufficient to 0.5 ml Solution was stored at RT Precipitation was observed within
2 hours Not soluble in polysorbate 80
II Bortezomib (1 mg) + polysorbate (100 mg). water for injection quantity sufficient to 1 ml -sonication applied Fibrous particles observed Not soluble in polysorbate 80
III Bortezornr'b (Img) + etfianof (0.1 ml) + polysorbate (100 mg) water for injection quantity sufficient to 1 ml Fibrous particles observed when stored at 2-8°C Not soluble in polysorbate 80 and ethanol
IV Bortezom ib (1 mg) + polyethylene glycol 400 (600 mg), water for injection quantity sufficient to 5 ml Clear solution initially but fibrous particles observed on storage Not soluble on storage
V Bortezomib (5 mg) + polyethylene glycol 400 (600 mg) +polysorbate 80 water for injection quantity sufficient to 5 ml Hazy solution particles observed on storage at RT after 2 hours Not soluble on storage
VI Bortezomib (3.5 mg) + n-butanol 0.3 ml, 30 mg sodium chloride water for injection sufficient to 0.7 ml Bortezomib solubilizers in 30 % t-butanol, after reconstitution with water for injection, particles observed Not soluble on storage
VII Bortezomib (3.5 mg) + n-butanol 0.4 ml + L-Cysteine hydrochloride hydrate 4.78 mg + sodium chloride 30 mg, water for injection sufficient to 0.6 ml -final solution filtered using 0.45 micron filter and lyophilized Clear solution on bath sonication Reconstitution of lyophilized cake takes more than 2 minutes Reconstitution of cake not easy and takes long time
VIII Bortezomib (3.5 mg) -^-polyethylene glycol 400 (420 mg)+ hydroxypropyl beta cyclodextrin 175 mg, water for injection sufficient to make 3.5 ml Particulate matter observed Drug not solubilized
IX Bortezomib (3.5 mg) + sodium hydroxide (2.4 mg) + L-Cysteine hydrochloride.hydrate (478 mg) + sodium chloride (2.4 mg) water for injection sufficient to make 1.0 ml- final solution filtered using 0.45 micron filter and lyophilized Clear solution was obtained
Reconstitution of lyophilized cake took long time (more than 2 minutes) Reconstitution of cake not easy and takes long time
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Comparative Example X
Bortezomib (3.5 mg) was accurately weighed and taken in a 5 ml vial. Tromethamine (3.31 mg) was weighed and mixed in the vial. Water for injection (quantity sufficient to make 1 ml) was added with continuous stirring. The solution was warmed at 45 to 50 °C to form a clear solution. The solution was cooled and 30 mg of sodium chloride was added to the cooled solution. The solution was filtered through 0.45 micron syringe filter and the pH was determined. The pH of the clear solution was found to be 8.51. The clear solution was lyophilized. The solid mass when subjected to reconstitution it was found that it took more than 120 seconds. A bath sonication was required to break the lyophilized cake to get a solution. After reconstitution, the solution was found to be stable in terms of particle formation for 24 hours.
Comparative Example XI
Bortezomib (3.5 mg) was accurately weighed and taken in a 5 ml vial. Tromethamine (3.31 mg) was weighed and mixed in the vial. Water for injection (quantity sufficient to make 1 ml) was added with continuous stirring. The solution was warmed at 45 to 50 °C to form a clear solution. The solution was cooled and 30 mg of sodium chloride was added to the cooled solution. The solution was filtered through 0.45 micron syringe filter and the pH was determined. The pH of the clear solution was 8.63 which was adjusted with I % hydrochloric acid to 7.28. The clear solution was lyophilized. The reconstitution of the lyophilized cake took more than 120 seconds. After reconstitution, the solution was found to be unstable in terms of particle formation for 24 hours i.e particulates were observed.
Comparative Example X
Bortezomib (3.5 mg) was accurately weighed and taken in a 5 ml vial. Tromethamine (3.31 mg) was weighed and mixed in the vial. Water for injection (quantity sufficient to make 1 ml) was added with continuous stirring. The solution was warmed at 45 to 50 °C to form a clear solution. The solution was filtered through 0.45 micron syringe filter and the pH was determined. The pH of the clear solution was 8.63 which was further adjusted with 1 % hydrochloric acid to 7.5. The clear solution was lyophilized. The reconstitution of the lyophilized cake took more than 120 seconds. After reconstitution, the solution was found to be unstable in terms of particle formation for 24 hours i.e particulates were observed.
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Example I
Borlezomib (3.5 mg) was accurately weighed and taken in a 5 ml vial. Tromethamine (3.31 mg) was weighed and mixed in the vial. Water for injection (quantity sufficient to make 1 ml) was added with continuous stirring. The solution was warmed at 45 to 50 °C to form a clear solution. The solution was cooled and 30 mg of sodium chloride was added to the cooled solution. The solution was filtered through 0.45 micron syringe filter and the pH was determined. The pH of the clear solution was 8.68 which was adjusted with 5 % hydrochloric acid to 8.01. The clear solution was lyophilized. The reconstitution of the lyophilized cake took less than 30 seconds without any need of bath sonication. After reconstitution, the solution was found to be stable in terms of particle formation for 24 hours i.e no particulates were observed. The 1R spectrum of the lyophilized composition was recorded. The 1R spectrum is given in Figure 1. The IR spectroscopy of the formula indicates formation of tromethamine salt having a strong B-N bond.
Example II
Bortezomib (3.5 mg) was accurately weighed and taken in a 5 ml vial. Tromethamine (3.31 mg) was weighed and mixed in the vial. Water for injection (quantity sufficient to make 1 ml) was added with continuous stirring. The solution was warmed at 45 to 50 °C to form a clear solution. The solution was filtered through 0.45 micron syringe filter and the pH was determined. The pH of the clear solution was 8.75 which was adjusted with 5 % hydrochloric acid to 7.89. The clear solution was lyophilized. The reconstitution of the lyophilized cake took less than 30 seconds without any need of bath sonication. After reconstitution, the solution was found to be stable in terms of particle formation for 24 hours i.e no particulates were observed. The IR spectrum of the lyophilized composition was recorded. The IR spectrum is given in Figure 2. The IR spectroscopy of the formula indicates formation of tromethamine salt having a sirona B-N bond.
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Example 111
Bortezomib (3.5 mg) was accurately weighed and taken in a 5 m| vial. Tromethamine (3.31 mg) was
weighed and mixed in the vial. Water for injection was added with continuous stirring. The mixture was warmed at 35 ^C to 40 °C. The mixture was stirred and subjected to bath sonication till clear solution was obtained. Accurately weighed quantity of sodium chloride (30 mg) was added in the vial and dissolved in the solution. The pH of the clear solution was adjusted with 5 % hydrochloric acid to 8.0. The volume was made up and then the solution was filtered through 0.2-0.8 micron syringe filter. The clear solution was lyophilized in the vials. The lyophilized cake in the vials was reconstituted with 3.5 ml of water for injection. The reconstitution was quick and took less than 90 seconds. The final reconstituted solution was clear. This reconstituted solution was stored at 2 °C to 8 °C for a period of 48 hours. The reconstituted solution was also subjected to storage temperatures of 20 °C to 25 °C. The stored solution was analyzed for related substances i.e unknown impurities and total impurities and the bortezomib content. The assay and the impurities were determined by HPLC. The solutions were found to be stable over a period of 48 hours.
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Dated this 9lh Day of January 2009