FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
Complete Specification
[See Sections 10 and rule 13]
Title: A stable parenteral composition of Topotecan
Applicant: (a) INTAS Pharmaceuticals Limited
(b) Company Registered Under Indian Company ACT
(c) 2nd Floor, Chinubhai Centre, Ashram Road Ahmedabad - 380009 Gujarat. India.
The following specification particularly describes the invention and the manner in which it is to be performed:

Field of the invention
The present invention relates to a stable injectable composition of Topotecan or its pharmaceutically acceptable salts and methods for preparing the same.
Background
The chemical name for Topotecan is (S)-10-[(dimethyIamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indoHzino[ ] ,2-fo]quinoline-3,14-(4H, 12H) -dione having the following formula:

Topotecan is a semi-synthetic water-soluble analog of camptothecin which is an inhibitor of topoisomerase-1. Topotecan, like other camptothecin analogs, stabilizes the covalent complex between topoisomerase-1 and DNA, resulting in enzyme-Jinked DNA cleavage and single-strand breaks. The cytotoxicity of Topotecan is thought to be due to double strand DNA damage produced during DNA synthesis, when replication enzymes interact with the ternary complex formed by Topotecan, topoisomerase-1, and DNA. Mammalian cells cannot efficiently repair these double strand breaks.

Topotecan was first disclosed in European patent EP 321122. Topotecan HC1 for Injection (Hycamtin, GlaxoSmithKIine) has been approved for metastatic carcinoma of the ovary after failure of initial or subsequent chemotherapy and small cell lung cancer sensitive disease after failure of first-line chemotherapy by the United States Food and Drug Administration. HYCAMTIN for Injection is supplied as a sterile lyophilized powder available in single-dose vials. Each vial contains Topotecan hydrochloride equivalent to 4 mg of Topotecan as free base. Therapy for Hycamtin (Topotecan hydrochloride) comprises administration of a dose of 1.5 mg/m2 of Topotecan by intravenous infusion over 30 minutes daily for 5 consecutive days, starting on day 1 of a 21-day course.
EP1643972 discloses a reconstituted lyophilized Topotecan liposomal composition comprising Topotecan, liposomes and cryoprotectant, wherein Topotecan substantially entrapped in the liposome interior. When Topotecan is entrapped within liposomes it may improve the stability, but it does not prevent other degradation of the Topotecan in aqueous media. Such other approaches have been attempted to stable composition of Topotecan or salt thereof by liposomal/ lyophilizing method are mentioned below.
CN101283983 describes a stable liposome composition comprises camptothecin or derivatives thereof, phospholipid, cholesterol, organic acids and the buffer salts of the camptothecins, and pharmaceutically-acceptable excipient.

KR2008076099 discloses the title drug delivery system comprises negative charged drug preparation, and injectable biodegradable temperature and pH-sensitive (hydrophilic and hydrophobic) block copolymer hydrogel.
WO2007076117 discloses a liposomal composition comprising at least one therapeutic agent having a protonable amino group and a neutrally buffered secondary or tertiary amine.
WO9508986 disclose pharmaceutical composition comprising a water-soluble analog of Camptothecin with a lipid in the form of multilamellar or unilamellar vesicles.
JP2003342167 discloses a water-soluble preparation in which the pharmaceutical composition contains a drug-containing polymer micelle produced by sealing a camptothecin derivative in a polymer micelle containing a hydrophilic segment and a hydrophobic segment.
Another problem with camptothecin and its analogues (including Topotecan) is that camptothecin and its analogues get reversibly hydrolyzed in a pH-dependent reaction in aqueous solutions to a ring-open hydroxy acid, a form that exhibits little activity against topoisomerase-1.

Kearney et al. (Int. J. Pharm. (1996) 127:229-237) report that Topotecan in aqueous media undergoes a pH and temperature dependent degradation. It further reveals that desirable solubilities (i.e. 2.5 mg/mL in free base equivalents of Topotecan) are achievable at pH < 4, whereas desirable stabilities (i.e. < 2% degradation of Topotecan over 2 years) are achievable at pH < 3. The stability results are consistent with a degradation mechanism involving deamination of Topotecan, resulting in the formation of 10-hydroxy camptothecin and a camptothecin dimer. In the experiment to maintain stability, pH of the storage medium is adjusted to less than 3 by addition of hydrochloric acid or tartaric acid, which overcomes the degradation of the Topotecan in the composition.
DE2O2O07016060 discloses stable ready (o use Topotecan based solution for injection by adjusting the pH of the formulation below 3.0 by addition of a pharmaceutically acceptable acid and/or its salt or their mixtures thereof. Further the patent application discloses that the pH of the formulation is adjusted below 3.0 by addition of organic or inorganic acids.
Hence to attain a stable Topotecan aqueous solution the pH of the formulation is adjusted using pharmaceutically acceptable acids, salts of the pharmaceutically acceptable acids or mixtures thereof.
The purpose of the present invention is to develop a stable Topotecan composition for parenteral use by overcoming

1. Problems disclosed in the background references relating to stability and
2. Addition of pH adjusting agents to the ready-to-use aqueous Topotecan
injectable composition to obtain stable composition.
Objects of the invention
The main object of the invention is to provide an aqueous stable ready-to-use composition of Topotecan salt for parenteral administration by preventing the hydrolysis of Topotecan without adjusting the pH of the formulation by addition of an acid, acid addition salt, pH adjusting agent or stabilizer.
Another object of the invention is to provide an aqueous ready-to-use composition of Topotecan salt for parenteral administration which is stable at higher concentration than 2.5 mg/mL of equivalent Topotecan free base.
Another object of the invention is to provide a process for the preparation of an aqueous stable ready-to-use composition of Topotecan salt for parenteral administration without adjusting the pH of the composition by addition of an acid, acid addition salt, pH adjusting agent or stabilizer.
Summary of the Invention
Present invention relates to an aqueous ready-to-use composition of Topotecan salt wherein the pH of the formulation is not adjusted using an acid, acid addition salt, pH adjusting agent or stabilizer resulting in stable aqueous solution.

Further invention relates to provide a process for preparing a stable injectable ready-to-use parenteral solution of Topotecan salt.
Detailed Description of the Invention
It is desirable not to add any pharmaceutically acceptable excipient in an injectable composition, however if the active ingredient is unstable then maintaining the pH of an injectable composition is a method used as one of the . strategies to obtain a stable injectable composition. A desirable pH of the composition is attained by addition of pH adjusting agents like an acid, acid addition salt or pH stabilizers.
It has been found by the inventors of the present invention that a stable ready-to-use Topotecan injectable composition can be attained by increasing the concentration of Topotecan salt in the injectable composition. The stability of the ready-to-use aqueous Topotecan composition is attained without the addition of pH adjusting agents.
Present invention involves the use of Topotecan salt in injectable composition where stability of the composition increases as the concentration of Topotecan salt increases. Topotecan in water gets hydrolyzed to 10-hydroxy camptothecin derivatives. When higher concentration of Topotecan salt is added in water to make a solution, the ratio of Topotecan to water molecules in the composition

decreases, which further reduces the hydrolysis of Topotecan in the solution. Further, increase in topotecan strength also helps to bring down the pH of the bulk solution, which in turn reduces the rate of hydrolytic reaction. No additional pharmaceutically acceptable excipients are needed to be added in the Topotecan composition to adjust the pH of the composition.
The concentration of Topotecan salt used in the formulation may be between 2.5 mg/mL to 32 mg/mL. Preferably, the concentration of Topotecan salt in the composition is 4 mg/mL to 16 mg/mL.
Topotecan salt used in the ready-to-use injectable composition comprise of pharmaceutically acceptable salt like hydrochloride salt, phosphate, sulfate, lactate, tartrate, maleate, citrate, benzoate, acetate, tosylate, mesylate and the likes thereof, wherein the preferable salt of Topotecan is hydrochloride salt.
The pH of the ready-to-use Topotecan injectable composition of the present invention is between pH 1 0 to pH 4.0. Preferably, the pH of the composition is between pH 1.5 to pH 3.5.
Another embodiment of the inventions is directed towards process for the preparation of aqueous ready-to-use pharmaceutical preparation containing Topotecan salt wherein the process comprises of dissolving Topotecan salt in

inert gas sparged water for injection, making volume with water for injection, checking pH of the solution and filling fiJtered solution in to dear glass vials.
It is recommended to produce the solution under an inert gas, for example nitrogen or carbon dioxide.
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 aqueous preparation containing Topotecan Hydrochloride ready-to-use injection can be illustrated by but not limited to following examples.
Example 1: Topotecan HC1 (RTU); 4 mg/mL, pH 2-2.9 Composition:
Sr. No. Ingredients Qty./ml
1 Topotecan HC1 eq. to Topotecan base 4.0
2 Water for Injection q.s.to 1 ml
3 Carbon dioxide q.s.
Procedure for manufacturing:
1. 95 % of water for injection is taken in pharmaceutical compounding vessel. Sparge with carbon dioxide for minimum of 30 minutes.
2. Add Topotecan HCI in to solution (stepl) and stir for 5-10 min.

3. Make up the volume to required batch size. Check the pH of solution.
4. Filter the solution through 0.2 u filter.
5. Fill the filtered solution in to clear glass vials. Plug the vials using rubber stopper with pre and post carbon dioxide sparging & seal with aluminum flip off seal.
Stability:
Topotecan Hydrochloride Inj. (Ready to use)
4 mg/mI(Co2 Sparge)
Initial 2-8°C 25 'c
1M 2M 1M 2M
Description yellow color clear liquid soln yellow
color clear liquid soln yellow color clear liquid soln yellow color clear liquid soln yellow color clear liquid soln
pH 2.7 2.8 2.7 . - 2.8 2.7 ,
Assay 101.4 100.0 99.9 100.0 100.2
Related substances
10-Hydroxy Camptothecin 0.01 0.03 0.03 0.13 0.22
Camptothecin ND ND ND ND ND
Single Max 0.08 0.08 0.08 0.08 0.08
Total imp 0.1 0.12 0.13 0.24 0.35
Example 2: Topotecan HC1 (RTU), 8 mg/ml, pH 2-2.9
Composition:
Sr. No. Ingredients Qty./ml
1 Topotecan HC1 eq. to Topotecan base 8.0
2 Water for Injection q.s.to 1 ml
3 Carbon dioxide q.s. to sparge

Procedure for manufacturing:
1. 95 % of water for injection is taken in pharmaceutical compounding vessel. Sparge with carbon dioxide for minimum of 30 minutes.
2. Add Topotecan HC1 in to solution (step]) and stir for 5-10 min.
3. Make up the volume to required batch size. Check the pH of solution.
4. Filter the solution through 0.2μ filter.
5. Fill the filtered solution in to clear glass vials. Plug the vials using rubber stopper with pre and post carbon dioxide sparging & seal with aluminum flip off seal.
Stability:
Topotecan Hydrochloride Inj. (Ready to use)
8 mg/ml (C02 Sparge)
Initial 2-8°C 25°c
1M 2M 1M 2M
Description yellow color clear liquid soln yellow color clear liquid soln yellow color clear liquid soln yellow color clear liquid soln yellow color clear liquid soln
pH 2.4 2.4 2.4 2.4 2.4
Assay 103.8 102.0 103.0 100.4 102.8
Related substances
10-Hydroxy Camptothecin 0.01 '' 0.02 0.02 0.06 0.11
Camptothecin ND ND ND ND
Single Max 0.08 0.08 0.08 0.08 0.08
Total imp 0.1 0.12 0.12 0.16 0.22

Example 3: Topotecan HCl (RTU), 16mg/ml,pH 1.5-2.5
Composition:
Sr. No. Ingredients Qty./ml
1 Topotecan HC1 eq. to Topotecan base 16.0
2 Water for Injection q.s.to l ml
3 Carbon dioxide q.s. to sparge
Procedure for manufacturing:
1. 95 % of water for injection is taken in pharmaceutical compounding vessel. Sparge wjth carbon dioxide for minimum of 30 minutes.
2. Add Topotecan HCl in to solution (stepl) and stir for 5-10 min.
3. Make up the volume to required batch size. Check the pH of solution.
4. Filter the solution through 0.2 μ filter.
5. Fill the filtered solution in to clear glass vials, Plug the vials using rubber stopper with pre and post carbon dioxide sparging & seal with aluminum flip off seal.
Stability:
Topotecan Hydrochloride Inj. (Ready to use )
16 mg/ml(C02 Sparge)
Initial 2-8°C 25 ;°c
1M 2M 1M 2M
Description yellow color clear liquid soln yellow color clear liquid soln yellow color clear liquid soln yellow color clear liquid soln yellow color clear liquid soln
pH 2.1 2.1 2.1 2.2 2.2
Assay 100.3 100.4 101.2 100.4 101.3
Related substances
10-Hydroxy Camptothecin 0.01 0.02 0.02 0.04 0.06
Camptothecin ND 0.01 ND ND ND
Single Max 0.08 0.08 0.08 0.08 0.08
Total imp 0.11 0.12 0.12 0.13 0.17

Results of the stability studies performed for stable ready-to-use aqueous Topotecan hydrochloride composition mentioned according to above examples demonstrate that the pH of the composition, assay for Topotecan HCl, the amount of impurities and single max study conducted for 1 month, 2 months and 3 months were within the acceptable limits.
Example 4: Topotecan HCl (RTU), 16mg/mL, pH 1.5-2.5 (no sparging)
Composition: *
Sr.No. Ingredients Qty./ml
1 Topotecan HCl eq. to Topotecan base 16.0
2 Water for Injection q.s.to l ml
Procedure for manufacturing:
1. 95 % of water for injection is taken in pharmaceutical compounding vessel.
2. Add Topotecan HCl in to solution (step1) and stir for 5-10 min.
3. Make up the volume to required batch size. Check the pH of solution.
4. Filter the solution through 0.2 μ filter.
5. Fill the filtered solution in to clear glass vials. Plug the vials using rubber stopper & seal with aluminum flip off seal.

We claim:
1. An aqueous stable ready-to-use injectable composition of Topotecan salt, having a pH below 3.0; wherein the pH of the composition is attained and maintained without the addition of pH adjusting agent,
2. The composition as disclosed in claim I, wherein the concentration of Topotecan salt is between 2.5 mg/ml and 35 mg/ml.

3. The composition as disclosed in claim 1 and'2, wherein the concentration of Topotecan salt is preferably between 4mg/ml and 16mg/ml.
4. The composition as disclosed in any of the preceding claims, wherein Topotecan salt used in the said composition comprise of pharmaceutically acceptable salts of Topotecan.
5. The composition as disclosed in claim 4, wherein pharmaceutically acceptable salts of Topotecan comprise of hydrochloride salt, phosphate salt, sulphate salt, lactate salt, tartarate salt, maleate salt, citrate salt, benzoate salt, acetate salt, tosylate salt, mesylate salt and the likes thereof.
6. The composition as disclosed in claim 5, wherein the Topotecan salt is preferably hydrochloride salt.

7. An aqueous ready-to-use injectable composition of Topotecan salt, having the
following formula:
Topotecan HC1 in a range between 2.5 - 35 mg/ml
Water for injection - q.s. for I ml
Optional use of Carbon dioxide - q.s. for sparging (at any time during the
process)
8. An aqueous stable ready-to-use injectable composition of Topotecan salt
comprising a solution of Topotecan salt in water as herein described with
foregoing description and examples.