FORM 2
THE PATENTS ACT. 1970
(39 of 1970)
&
THE PATENTS RULES. 2003
Complete Specification
[See Sections 10 and rule 13]
TITLE: A Process for Preparing a Stable Lyophilized Composition
Applicant: (a) ASTRON Research Limited
(b) A Company incorporated 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 a process to obtain a stable lyophilized composition by lowering the pH during solution phase before lyophilization without addition of an acid or buffer during preparation of composition.
Background of the Invention & Prior Art:
It is well known fact that degradation of compounds takes place in water either by oxidation or by hydrolysis and that results into generation of impurities, e.g. anthracycline glycoside like doxorubicin undergoes hydrolysis during process for preparation of its composition and there by hydrolysis related impurities are generated. This type of problem is solved in the present invention.
Anthracycline glycoside is an antineoplastic antibiotic, which is obtained from the fermentation of slreptomyces paucities. Anthracycline glycosides are DNA interacting drugs that are widely used in chemotherapy for treatment of various types of cancer including breast, ovarian, bladder and lung cancer as well as non-Hodgkin's lymphoma. Hodgkin's disease and sarcoma. These drugs bind up the DNA of the cancer cells and stop the growth and multiplication of the cells.
Anthracycline glycosides bind to nucleic acids, presumably by specific intercalation of the planar anthracycline nucleus with the DNA double helix. The anthracycline ring is lipophilic, but the saturated end of the ring system contains abundant hydroxy 1 groups adjacent to the amino sugar, producing a hydrophilic center. The molecule is amphoteric, containing acidic functions in the ring phenolic groups and a basic function in the sugar amino group. It binds to cell membranes as well as plasma proteins.
Anthracycline glycosides have been used successfully to produce regression in disseminated neoplastic conditions such as acute lymphoblastic leukemia, acute myeloblastic leukemia. Wilms tumor, neuroblastoma, soft tissue and bone sarcomas.

breast carcinoma, ovarian carcinoma, transitional cell bladder carcinoma, thyroid carcinoma, gastric carcinoma. Hodgkin's disease, malignant lymphoma and bronchogenic carcinoma in which the small cell histologic type is the most responsive compared to other cell types.
It is known that solubility problem encountered in reconstitution of anthracycline glycoside containing lyophilized composition, because the lyophilized cake dissolves somewhat slowly and complete dissolution requires prolonged shaking. This problem is solved in US 4840938 that claims for method of lyophilized preparation containing an anthracycline glycoside or a salt with co-solubilizing agent and an inert excipient followed by freeze drying the resulting combination. The co-solublising agents are used to enhance solubility of lyophilized cake at the time of reconstitution.
WO 2006122309 disclose stable lyophilized composition of anthracycline glycoside salt and process of preparations where in one of the process is for inhibition of the generation of aglycon and bis anhydro impurities and thereby stability of the composition is achieved by using a buffer and appropriate solvent and in another process the same is achieved by using resin and a solvent.
US 20070004653 disclose stable lyophilized composition of anthracycline glycoside salt, wherein anthracycline glycoside is combined with a solvent between 50-70° C followed by an addition of an acid to adjust pH between 3.2 to 3.8.
US 4946831. US 5124318. US 5124317. US 6107285. US 5977082. US 6284738, US 6087340, US 6632799. US 6596697. US 2003013666. US 2004077559 and US 2004077560 disclose ready to use pharmaceutical compositions of an anthracycline glycoside in a suitable aqueous solvent having pH in the range between 2.5 to 6.5 wherein pH adjustment is made by using physiologically acceptable acid or buffer.

Further WO 2006085336 discloses ready to use composition of anthracycline glycosides in aqueous solvent with one or more excipients and with or without use of inert gas to maintain the pH of solution. Here the inert gas is used to inhibit the generation of aglycon and bis anhydro impurities in the composition. The inventive step in this patent specifically relates to adjustment of pH of the solution with physiologically acceptable use of inert gas wherein the said composition is for ready to use.
One critical characteristic of the anthracycline glycosides is their tendency to get hydrolyzed in aqueous solution when pH reaches above 4. This critical characteristic originate during the preparation of lyophilized composition when pH of the solution reaches above 4, being responsible for generation of hydrolysis impurities and thereby inhibit long-term stability of composition as impurities resides in the lyophilized cake. Using either organic or inorganic acid or buffer that maintain the pH of the solution less than 4 solves this critical characteristic. Although the purpose to maintain the pH of the solution as well as inhibition of hydrolysis impurities is solved by an addition of either acid or buffer, this addition results in the generation of impurities other than hydrolysis impurities and also results in the pH of the lyophilized composition after reconstitution below 4 which is not acceptable as per pharmacopeial specifications of doxorubicin lyophilized injection (Limit: 4.5-6.5).
Hence to achieve stable, readily soluble lyophilized composition of anthracycline glycosides, the inventors of the present invention have tried to solve above said problems by providing a process that includes the continuous sparge of carbon dioxide in solution before lyophilization process commences.
The term "Anthracycline glycosides" above and hereinafter refers to as "Anthracycline glycosides or its pharmaceutically acceptable salts". The preferred anthracycline glycosides of the present invention are doxorubicin or its pharmaceutically acceptable salts. The present invention is a step forward in the direction to provide a stable, lyophilized pharmaceutical composition

Object of the Invention
The main object of the present invention is to maintain an acidic pH of solution during the preparation of composition without addition of acid or buffer and hence preclude the generation of undesired impurities.
Another object of the invention is to prevent hydrolysis of ingredient(s) in aqueous solution to inhibit generation of impurities during preparation.
Another object of the invention is to obtain lyophilized cake having the pH range 4.5-6.5 that is acceptable range under pharmacopeias specifications.
Another object of the invention is to provide a process that maintain the pH of the anthracycline glycoside solution less than 4 to produce stable, readily soluble, lyophilized composition of anthracycline glycosides preferably doxorubicin.
One more object of the invention is to inhibit the development of hydrolysis impurities by maintaining the pH less than 4 during the process of preparation of lyophilized formulation doxorubicin.
Summary Of The Invention
This invention relates to process to obtain stable lyophilized composition of an active ingredient showing stability at acidic pH by maintaining pH of solution wherein the process does not include addition of acid or buffer during the preparation. This process is useful to obtain stable lyophilized composition without addition of acid or buffer.
Further the present invention relates to a process for preparing stable, readily soluble, lyophilized composition of anthracycline glycosides in which pH of solution containing

anthracycline glycoside and co-solublizing agent is maintained less than 4 by continuous sparge of carbon dioxide till the lyophilisation process commences.
Detailed Description of The Invention
It is well known fact that degradation of compounds takes place in water either by oxidation or by hydrolysis and thereby generating impurities. Anthracycline glycosides and more preferably doxorubicin degrade at pH below 4.0 generating hydrolysis impurities. In the present invention, inventors have tried to stabilize the composition by maintaining an acidic pH of solution without addition of acid or buffer during the preparation.
This process of the present invention prevents hydrolysis of ingredient(s) by keeping the pH of the composition below 4 and thereby generation of impurities in the composition / solution wherein the process includes continuous sparging of carbon dioxide.
Generally the term "sparging" refers to a technique, which involves bubbling a chemically inert gas through a liquid with connotation to remove dissolved gases (e.g. oxygen) from the liquid and thereby prevent the oxidation or hydrolysis of active ingredient in a composition. In the present invention the term "sparging" involves bubbling of a carbon dioxide gas through a solution not only remove dissolved gases but also to maintain an acidic pH.
This invention relates to process of maintaining acidic pH of solution for stabilizing the active ingredient without addition of an acid or buffer. Therefore, to maintain pH of the solution and by that means inhibit the generation of impurities, this process includes continuous sparge of carbon dioxide till lyophilisation commences. Further sparged carbon dioxide gas is removed during lyophilization by applying vacuum and hence lyophilized cake attains pH between 4.5-6.5 that is acceptable as per pharmacopeial specification.

An active ingredient showing pH dependency are selected from the group comprising of anthracycline glycoside derivatives and the like.
The present invention provides a stable, readily soluble, lyophilized composition of anthracycline glycosides by a process involving sparge of carbon dioxide wherein carbon dioxide is sparged till lyophilization process commences.
Our experimental study shows, on combining anthracycline glycosides with suitable co-solublizers and water, PH of the solution reaches above 4. At this pH 1 hydrolysis impurities start to generate and till the lyophilisation process commences, percentage of these impurities cross ICH limits and the same is maintained in the lyophilized cake. Hence it is necessary to keep the pH of the solution less than 4 to avoid generation of hydrolysis impurities. This necessity is fulfilled in present invention by sparge of carbon dioxide in a solution.
Accordingly, the present invention describes the process related to an improvement in stability of lyophilized composition including dissolution of co-solublizer and one or more inert excipients in water followed by sparge of carbon dioxide to maintain pH less than 4. Now anthracycline glycoside is dissolved in the solution with continuous stirring during which sparging of carbon dioxide also continues until clear solution is obtained. Now carbon dioxide sparged water is added to the solution to achieve the desired volume wherein pH of the solution is maintained less than 4. The resulting solution is clarified and filtered with the help of carbon dioxide gas and distributed in sterile vials with partial stoppering. Partially stoppered vials are placed in lyophilizer wherein lyophilization is carried out in a conventional way. Generally the conventional way of lyophilisation includes freezing of the solution at 40 to 50° C followed with primary drying below eutectic point and secondary drying at final temperature of 35-40° C and the vials are sealed under sterile conditions by conventional procedure.

The content of dry unopened vials arc completely stable for at least 6 months at 40° C. The reconstitution of lyophilized composition is made by conventional way. In one of the method physiological saline is used wherein volume of saline may vary and it depends on the kind and amount of active ingredient present in lyophilized composition.
The main components of lyophilized composition prepared by the above process are anthracycline glycoside as active drug substance, pharmaceutically acceptable co-solublizers to enhance solubility during reconstitution and optionally inert excipient(s).
The said anthracycline glycoside can be doxorubicin, epirubicin. esorubicin. daunorubicin. idarubicin or its pharmaceutically acceptable salts, wherein the active drug substance is in the range from 1 to 100 mg/mL.
The co-solublising agent is in the range of 0.1-10 mg/ml. The co-solublising agent used to enhance solubility during reconstitution of lyophilized composition is selected from the group comprising of p-hydroxybenzoic acid and the methyl ester thereof, p-amino benzoic acid and the methyl ester thereof-, o-hydroxy-benzoic acid and the methyl ester thereof 3-methyl-4-chlorophenol and the 3.5-dimethyl-4-chlorophenol, or a combination of two or more of said compounds. A particularly preferred co-solubiliser in the present invention is p-hydroxy-benzoic acid methyl ester.
The inert excipient is in the range of 5 50 mg/ml. can be selected from but not limited to lactose, mannitol. sorbitol, or maltose, preferably lactose monohydrate in suitable amount.
Throughout this specification it is to be understood that the words "comprise" and 'include" and variations such as "comprises", "comprising", "includes", "including" are to be interpreted inclusively, unless the context requires otherwise. That is, the use of these words may imply the inclusion of an element or elements not specifically recited.
Example

The present invention has been described by way of example only, and it is to be recognized that modifications thereto falling within the scope and spirit of this specification and which would be obvious to a person skilled in the art based upon the disclosure herein, are also considered to be included within the scope of this invention. The proportion of the ingredients in the composition can be in the range as below:
Example

1. Take 5 % w/w water for injection of the proposed batch size & cosolublize p-hydroxy-benzoic acid methyl ester at a temperature of 80-90° C
2. lake remaining water for injection at 8-15° C and sparge of carbon dioxide for approximately 30 minutes.
3. Mix the solution of step 1 & step 2 and add Doxorubicin hydrochloride in resulting solution with constant stirring.
4. Stir the solution continuously with sparge of carbon dioxide until a clear solution is obtained.
5. Add Lactose monohydrate in the solution & make up the volume upto the required batch size with carbon dioxide sparged water.
6. Filter and till the final bulk solution in vial
7. Load the partially stoppered vials in the lyophilizer
8. Lyophilize the solution in conventional way

The present invention can be illustrated by but not limited to following example(s) Example 1

1. Take 5 % w/w water for injection of the proposed batch size & cosolublize p-hydroxy-benzoic acid methyl ester at a temperature of 80-90° C
2. Take remaining water for injection at 8-15° (' and sparge of carbon dioxide for approximately 30 minutes.
3. Mix the solution of step 1 & step 2 and add Doxorubicin hydrochloride in resulting solution with constant stirring.
4. Stir the solution continuously with sparge of carbon dioxide until a clear solution is obtained.
5. Add Lactose monohydrate in the solution & make up the volume upto the required batch size with carbon dioxide sparged water.
6. Filter and fill the final bulk solution in vial
7. Load the partially stoppered vials in the lyophilizer
8. Lyophilize the solution in conventional way

We Claim,
1. A process to maintain acidic pH of the solution without addition of acid or buffer during preparation.
2. A process to maintain acidic pH as claimed in claim 1. includes continuous sparging of carbon dioxide during preparation.
3. A process for preparing stable. Iyophilized composition of an active ingredient showing stability at acidic pH. wherein the process includes continuous sparge of carbon dioxide in the solution before lyophilisation commences.
4. A process as claimed in claim 3. wherein the active ingredient is selected from the group comprising anthracy clinc glycosidc derivatives and the like.
5 An anthracycline glycoside as claimed in claim 4 is selected from the group comprising doxorubicin, epirubicin. esorubicin. daunorubicin and idarubicin or its pharmaceutically acceptable salt.
6. A process for preparing stable, readily soluble, Iyophilized composition of doxorubicin or its pharmaceutically acceptable salt comprising continuous sparge of carbon dioxide during the preparation of solution wherein pH of the solution is maintained less than 4.
7. A process as claimed in claim 6. containing a doxorubicin or its pharmaceutically acceptable salt, said process-comprising steps;
a. Take 5 % vv/w water for injection of the proposed batch size & cosolublize p-hydroxy-benzoie acid methyl ester at a temperature of 80-90° C

c. Mix the solution of step 1 & step 2 and add Doxorubicin hydrochloride in
resulting solution with constant stirring.
d. Stir the solution continuously with sparge of carbon dioxide until a clear
solution is obtained.
e. Add lactose monohydrate in the solution & make up the volume upto the
required batch size with carbon dioxide sparged water.
f. Filter and fill the final bulk solution in vial
g. Lyophilize the solution in conventional way
8. A lyophilized composition prepared by a process as claimed in claim 6 & 7, comprising doxorubicin or its pharmaceutically acceptable salt, co-solublisers to enhance solubility during reconstitution and an inert excipient.
9. A co-solublizing agent as claimed in claim 7 and 8 is selected from p-hydroxybenzoic acid and the methyl ester thereof, p-amino benzoic acid and the methyl ester thereof-, o-hydroxy-benzoic acid and the methyl ester thereof 3-methyl-4-chlorophenol and the 3.5-dimethyl-4-chlorophenol. or a combination of two or more of said compounds.

10. A process as claimed in claim 7. wherein said process also includes an inert

Abstract
This invention relates to process to obtain stable lyophilized composition by lowering the pH during solution phase before lyophilization without addition of an acid or buffer during preparation of composition.