DESC:Field of invention:
The present invention relates to stable aqueous, pharmaceutical composition of Cetrorelix acetate along with a suitable stabilizing agent and tonicity adjusting agent for parenteral administration. The pharmaceutical composition provides sufficient stabilization of Cetrorelix acetate thus improving the shelf life and free from aggregate formation during storage. The invention further relates to a process of preparation thereof.

Background of the invention:
Cetrorelix acetate injection is a synthetic decapeptide with gonadotropin-releasing hormone (GnRH) antagonistic activity. Cetrorelix acetate is an analogue of native GnRHwith substitutions of amino acids at positions 1, 2, 3, 6, and 10. The molecular formula is Acetyl-D-3-(2´-naphtyl)-alanine-D-4-chlorophenylalanine-D-3-(3´-pyridyl)-alanine-L-serine-L-tyrosine-D-citruline-L-leucine-L-arginine-L-proline-D-alanine-amide, and the molecular weight is 1431.06, calculated as the anhydrous free base. The structural formula is as follows:
Cetrorelix acetate
.X ACOH

(Ac-D-Nal1-D-Cpa2-D-Pal3-Ser4-Tyr5-D-Cit6-Leu7-Arg8-Pro9-D-Ala10-NH2)

Cetrorelix acetate is marketed as lyophilized product for Subcutaneous use by EMD Serono US, Inc. under the trade name Cetrotide® 0.25 mg. Cetrorelix acetate is indicated for the treatment of the inhibition of premature luteinizing hormone(LH) surges in women undergoing controlled ovarian stimulation.
CA2115943 discloses that bulking agent such as Mannitol is necessary in the lyophilised composition of Cetrorelix to obtain a stable cake. CA2115943 also discloses that during sterile filtration of the bulk solution, formation of gels in the sterile filter would increase the viscosity of the solution and hence hinder the filtration step. To overcome the gel formation, use of acetic acid is suggested.
US7718599 claims a pharmaceutical composition for parenteral administration, which contains Cetrorelix acetate in a concentration of 2.5mg/ml and comprises a pharmaceutically acceptable acid selected from a group of gluconic acid, glucaric acid or galactouronic acid which is capable of imparting a pH between 2.5 to 4.5 to the composition which helps in suppressing aggregation of Cetrorelix acetate.
US7214662 discloses an aqueous solution of LHRH antagonist (Cetrorelix), wherein the composition comprises of an acid selected from a group of carboxylic acid, gluconic acid, hydrocarboxylic acid and gluconic acid deltalactone in combination with a surfactant Tween 80; to improve the solubility of the LHRH antagonist and to reduce the tendency of LHRH substances to aggregate.

Cetrorelix acetate is marketed as ready to use solution for subcutaneous use by Intas Pharmaceutical limited India, under the Brand name Cetrolix-PFS. Cetrorelix acetate is indicated for the treatment of the inhibition of premature LH surges in women undergoing controlled ovarian stimulation.

Currently available ready to use solution formulations in pre filled syringe contains Glacial acetic acid as stabilizing agent. Also, the formulation containing Glacial acetic acid 4.5 mg/1.0ml, as mentioned in Indian patent application no.3306/MUM/2010.

Significant drawbacks exist with the use of acetic acid as published in “ Acetic acid sensitivity as a cause of cold urticaria” by Richard D. Wiseman, M.D., Daniel K. Adler, M.D in The journal of Allergey and clinical immunology,January 1956 Volume 27, Issue 1, Pages 50–56© 1956 Published by Elsevier Inc. Many people have acetic acid sensitivity especially when given as subcutaneous / parenteral route of administration.

Indian patent Application number 3306/MUM/2010 discloses a pharmaceutical composition of Cetrorelix which comprises Glacial acetic acid as stabilizing agent to prevent gel formation.

Developing a stable aqueous solution of Cetrorelix acetate is hindered by the known fact that Cetrorelix being an oligopeptide particularly having a terminal acidamide group is prone to gel formation.

Further, Cetrorelix acetate is generally unstable in aqueous solution, it is necessary to develop a pharmaceutical composition by stabilizing the compound and the salts thereof for parenteral administration.

In view of the instability of Cetrorelix acetate in preparing the ready to use solution for parenteral administration as mentioned above, the present inventors felt a need to provide a stable Cetrorelix acetate injectable composition which is free from Acetic acid with good shelf life and to avoid acetic acid intolerance.

Therefore, it is an object of the present invention to provide a pharmaceutical compositions containing Cetrorelix acetate which is devoid of acetic acid and surfactant.

While screening for suitable agents for stabilizing the formulation, the inventors have unexpectedly found that stabilized pharmaceutical composition of Cetrorelix can be prepared for parenteral administration, without employing acetic acid.

Summary of the invention:
In accordance with above object, in an aspect, the present invention provides aqueous pharmaceutical composition comprising Cetrorelix or pharmaceutically acceptable salt thereof along with a suitable stabilizing agent, tonicity adjusting agent and optionally other pharmaceutically acceptable excipients along with water as an aqueous vehicle, wherein, the composition is free from acetic acid and surfactant. In a preferred aspect, pharmaceutically acceptable salt of Cetrorelix is Cetrorelix acetate.

The Cetrorelix acetate aqueous pharmaceutical composition is stabilized by adding stabilizing agents selected from the group consisting of a lactic acid, propionic acid, aspartic acid, and mixture thereof in aqueous vehicle to obtain formulation. In a preferred aspect, the stabilizing agent is Propionic acid.

In another aspect, aqueous pharmaceutical composition of Cetrorelix acetate and stabilizing agent is prepared and provided as a drug concentrate, suitable for parenteral administration.

According to the invention, the aqueous composition comprises an effective amount of stabilizer in the range of 1.0 mg to 10 mg per 0.5 to 1.0ml.

The pH of the aqueous injectable composition is maintained in the range of 2.5 to 4.

In another aspect, the present invention provides a method for stabilization of Cetrorelix acetate in an aqueous solution which comprises combining Cetrorelix acetate in an aqueous vehicle with a stabilizing agent selected from the group consisting of lactic acid, propionic acid, aspartic acid and mixture thereof; preferably Propionic acid.

Detailed description of the invention:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be fully understood and appreciated.

Cetrorelix acetate is generally unstable in aqueous form, It is therefore necessary to develop a pharmaceutical composition for stabilizing the compound and the salts thereof for parenteral administration.

Accordingly, the present invention provides stable pharmaceutically acceptable composition for parenteral administration comprising of Cetrorelix acetate as the active using a suitable stabilizer and maintaining appropriate pH of the formulation.

According to the present invention, the pharmaceutical preparation comprise of tonicity adjusting agents. Tonicity adjusting agents decrease the hemolysis of blood cells and reduce pain and irritation at the injection site. Tonicity adjusting agents that can be included in the said pharmaceutical preparation comprise of Sucrose, lactose, trehalose, sulfobutyl ether betacyclodextrin sodium and the like. Preferred tonicity adjusting agent for the said pharmaceutical preparation is the combination of sucrose and sulfobutyl ether betacyclodextrin sodium.

According to the present invention, the amount of tonicity adjusting agent used in the said preparation is adjusted to obtain osmolality of the said preparation in the range of 250 to 340mOsm/kg. An osmometer can be used to check and adjust the amount of tonicity adjusting agent to be added to obtain the desired osmolality.

In an embodiment, the present invention provides aqueous pharmaceutical composition comprising Cetrorelix acetate; a pharmaceutically acceptable stabilizing agent selected from the group consisting of lactic acid, propionic acid, aspartic acid and mixture thereof; in an aqueous vehicle and combination of sucrose and sulfobutyl ether betacyclodextrin sodium as tonicity adjusting agent, wherein, pH of said composition is in the range of 2.5 to 4.5 and wherein, the aqueous composition is devoid of acetic acid and surfactant. The aqueous vehicle according to the invention is water for injection.

In an embodiment, Cetrorelix is present in the aqueous composition in an amount ranging from 0.25mg to 1.0 mg per 0.5 ml; more preferably 0.25 mg per 0.5 ml.
Accordingly, in another embodiment, Cetrorelix is present in the aqueous composition in an amount of 0.50 mg per 1 ml.

Cetrorelix acetate is stabilized using a suitable stabilizing agent where Cetrorelix acetate can be maintained in a dissolved state in the aqueous solution thereby preventing gel formation of Cetrorelix acetate. The stabilizing agent is selected from the group consisting of lactic acid, propionic acid, aspartic acid and mixture thereof. In one preferred embodiment, the stabilizing agent is Propionic acid.

In a preferred embodiment, the aqueous pharmaceutical composition comprises Cetrorelix in an amount ranging from 0.25 mg to 1.0 mg per 0.5 to 1 ml; an effective amount of Propionic acid in the range of 1.0 mg to 10mg per 0.5 to 1 ml as a stabilizing agent in aqueous vehicle and a combination of Sucrose and sulfobutyl ether betacyclodextrin sodium in an amount of 25to 120 mg per 0.5 to 1ml as tonicity adjusting agents .The aqueous composition thus provided in aqueous vehicle is filtered to remove particulate matter, filled into vials to provide pre filled syringes.

The present inventors have observed during experimentation that there is an increase in total impurities on storage of the aqueous composition at 25?C for 6 months. Since, cetrorelix is not stable to pH above 4.0 it was necessary to reduce pH to the desirable extent to obtain the stable aqueous product with reduced total impurities and increased stability.

Accordingly, in an embodiment, the present invention provides aqueous composition of Cetrorelix with pH limit 2.5 to 4.0, preferably in the range of 2.5 to 3.5; most preferably the pH is in range of about 2.9 to 3.3.
In another embodiment, the present invention provides stable, aqueous, pharmaceutical composition for parenteral administration, which is free from acetic acid, comprising;
a) Cetrorelix acetate equivalent to Cetrorelix in an amount of 0.25 mg to 1.0 mg per 0.5 to 1.0 ml;
b) a pharmaceutically acceptable stabilizing agent selected from the group consisting of a lactic acid, propionic acid, aspartic acid and mixture thereof in an amount of 1.0 mg to.10 mg per 0.5 ml to 1.0 ml; and
c) combination of sucrose and sulfobutyl ether betacyclodextrin sodium in an amount of 25 to 120 mg per 0.5ml to 1.0ml as Tonicity adjusting agent(s), wherein, pH of said composition is in the range of 2.5 to 4.0.

The stabilizing agent is preferably Propionic acid used in an amount of 1.0 to 5.0 mg/ 0.5 ml. Alternately, Propionic acid may be used in an amount of 5 to 10 mg per 1 ml.

In one preferred embodiment, the stable aqueous pharmaceutical composition for parenteral administration, comprising;
i. Cetrorelix acetate equivalent to Cetrorelix in an amount of 0.25 mg per 0.5 ml;
ii. 3.0 mg of Propionic acid in aqueous vehicle as a stabilizing agent per 0.5ml;
iii. 35.0 mg Sucrose and 20.0 mg Sulfobutylether betacyclodextrin sodium as tonicity adjusting agents per 0.5 ml; wherein pH of said composition is in the range of 2.5 to 4.0.
In another preferred embodiment, the stable aqueous pharmaceutical composition for parenteral administration comprising;
i. Cetrorelix acetate equivalent to Cetrorelix in an amount of 0.5 mg per 1.0 ml;
ii. 6.0 mg of Propionic acid in aqueous vehicle as a stabilizing agent per 1.0 ml;
iii. 70.0 mg Sucrose and 40.0 mg Sulfobutylether betacyclodextrin sodium as tonicity adjusting agents per 1.0ml; wherein pH of said composition is in the range of 2.5 to 4.0.
The aqueous composition of Cetrorelix containing Propionic acid in aqueous vehicle as a stabilizing agent with pH range of 2.5 to 4.0 have total impurities less than the prior art formulations as mentioned in Indian patent Application number 3306/MUM/2010. These compositions are chemically and physically stable over an extended period of time and are suitable for intended pharmaceutical use.

In yet another embodiment, the aqueous composition of the present invention is provided as prefilled syringe, as ready to use composition for parenteral administration.

In another embodiment, the invention provides process for preparation of aqueous pharmaceutical composition of the invention which comprises; providing aqueous composition consisting of Cetrorelix acetate dissolved in aqueous vehicle containing stabilizing agent and tonicity adjusting agent(s).
Accordingly, the process for preparation of aqueous pharmaceutical composition which comprises;
a) dissolving cetrorelix in an aqueous solution containing Propionic acid; Sulfobutyl ether betacyclodextrin sodium and Sucrose in water for injection; and
b) adjusting the pH of the composition in the range of 2.5 to 4.0 using Propionic acid solution;
c) filtering the solution and filling into the syringes.
The pharmaceutical compositions of the present invention can be administered to a patient according to a dosing regimen. It should be understood that the specific dosing regimen for any particular patient will depend on a variety of factors, including age, body weight, general health, sex, diet, time of administration, specific disease being treated, and the severity of the condition among other factors and the judgment of the treating physician.

Thus, the present invention provides aqueous pharmaceutical composition comprising Cetrorelix acetate which is free from acetic acid to avoid acetic acid sensitivity/intolerance in patients without compromising the stability of drug and its solution composition with a pH range of 2.5 to 4.0, with reduced amount of total impurities which is stable for the entire period of the shelf life.

Other features and embodiments of the invention will become apparent by the following examples which are given for illustration of the invention rather than limiting its intended scope. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art.

Experimental:
Example: 1
Aqueous pharmaceutical composition
Sr. No Ingredient Concentration Each 0.5 ml contains in Prefilled syringe
1 Cetrorelix acetate 0.270 gm 0.27 mg
2 Propionic acid 3.0 gm 3.0 mg
3 Sulfobutyl ether betacyclodextrin sodium 20.0 gm 20.0 mg
4. Sucrose 35.0 gm 35.0 mg
5 Water for Injection q.s 0.5 ml

Manufacturing process:
0.270 gm Cetrorelix acetate (active) was added to an aqueous solution containing 3.0 gm of Propionic acid, 20.0 gm Sulfobutyl ether betacyclodextrin sodium and 35.0 gm Sucrose in water for injection having temperature below 25°C and stirred for some time to get clear solution. The pH of the solution limit (2.5 to 4.5) was checked adjusted the pH to about 3.15 with Propionic acid solution and made up the volume. Filtered the solution and the resulting solution was filled into 0.5 ml prefilled syringes with each containing 0.5 ml solution. The resulting preparation (prefilled syringe) was stored at 2-8°C and 25°C and 60 % RH, and the residue of Cetrorelix was tested at initial, after 3 months and 6 months and the results are provided in below table.

Parameters Initial After 3 months After 6 months
2-8°C 25°C and 60 % RH 2-8°C 25°C and 60 % RH
Description Clear colorless solution Clear colorless solution Clear colorless solution Clear colorless solution Clear colorless solution
Aggregation No No No No No
pH 3.15 3.16 3.15 3.17 3.14
Assay 101.07 100.54 100.11 99.54 99.18
Related Substances
Single Max.
Total Impurities
0.12
0.45
0.20
0.61
0.41
0.91
0.25
1.25
0.81
1.75
Osmolality 308 - - - -

Example: 2
Aqueous pharmaceutical composition
Sr. No Ingredient Concentration Each 0.5 ml contains in Prefilled syringe
1 Cetrorelix acetate 0.270 gm 0.27 mg
2 Propionic acid 1.0 gm 1.0 mg
3 Sulfobutyl ether betacyclodextrin sodium 20.0 gm 20.0 mg
4. Sucrose 35.0 gm 35.0 mg
5 Water for Injection q.s 0.5 ml

0.270gmCetrorelix acetate (active) was added to an aqueous solution containing 1.0gm of Propionic acid, 20.0 gm Sulfobutyl ether betacyclodextrin sodium and 35.0 gm Sucrose in water for injection having temperature below 25°C and stirred for some time to get clear solution. The pH of the solution limit (2.5 to 4.5) was checked and adjusted the pH to about 4.05 with Propionic acid solution and made up the volume. Filtered the solution and the resulting solution was filled into 0.5 ml prefilled syringes with each containing 0.5 ml solution. The resulting preparation (prefilled syringes) was stored at 2-8°C and 25°C and 60 % RH, and the residue of Cetrorelix was tested at initial, after 3 months and 6 months and the results are provided in below table.
Parameters Initial After 3 months After 6 months
2-8°C 25°C and 60 % RH 2-8°C 25°C and 60 % RH
Description Clear colorless solution Clear colorless solution Clear colorless solution Clear colorless solution Clear colorless solution
Aggregation No yes yes yes yes

pH, assay and related substances test were not performed due to aggregation found in sample.

Example: 3
Aqueous pharmaceutical composition
Sr. No Ingredient Concentration Each 0.5 ml contains in Prefilled syringe
1 Cetrorelix acetate 0.270 gm 0.27 mg
2 Propionic acid 3.0 gm 3.0 mg
3 Sulfobutyl ether betacyclodextrin sodium 20.0 gm 20.0 mg
4. Sucrose 10 gm 10.0 mg
5 Water for Injection q.s 0.5 ml

Manufacturing process:
0.270 gm Cetrorelix acetate (active) was added to an aqueous solution containing 3.0 gm of Propionic acid, 20.0 gm Sulfobutyl ether betacyclodextrin sodium and 10 gm Sucrose in water for injection having temperature below 25°C and stirred for some time to get clear solution. The pH of the solution limit (2.5 to 4.5) was checked adjusted the pH to about 3.02 with Propionic acid solution and made up the volume. Filtered the solution and the resulting solution was filled into 0.5 ml prefilled syringes with each containing 0.5 ml solution. The resulting preparation (prefilled syringe) was stored at 2-8°C and 25°C and 60 % RH, and the residue of Cetrorelix was tested at initial, after 3 months and 6 months and the results are provided in below table.

Parameters Initial After 3 months After 6 months
2-8°C 25°C and 60 % RH 2-8°C 25°C and 60 % RH
Description Clear colorless solution Clear colorless solution Clear colorless solution Clear colorless solution Clear colorless solution
Aggregation No No No No No
pH 3.09 3.11 3.02 3.15 3.12
Assay 101.89 101.25 100.57 100.69 99.83
Related Substances
Single Max.
Total Impurities
0.10
0.39
0.15
0.54
0.35
0.81
0.21
1.09
0.71
1.62
Osmolality 273 - - - -

Conclusion:
The results of the studies performed for Example1, 2 and 3 according to the present invention, confirms that the aqueous pharmaceutical preparations of Cetrorelix are stable within the pH range of 2.5 to 4 and hence can be administered to a person in need thereof.
,CLAIMS:1. A stable aqueous pharmaceutical composition for parenteral administration comprising;

a) Cetrorelix acetate equivalent to Cetrorelix in an amount of 0.25 mg to 1.0 mg per 0.5 to 1 ml;
b) propionic acid as stabilizing agent in an amount of 1.0 mg to 10 mg per 0.5 to 1ml; and
c) combination of Sucrose and sulfobutyl ether betacyclodextrin sodium in an amount of 25 to 120 mg per 0.5 to 1ml as tonicity adjusting agent(s),
wherein, pH of said composition is in the range of 2.5 to 4.0 and wherein, the composition is free from acetic acid.

2. The composition as claimed in claim 1, wherein, the Propionic acid used in an amount of 1.0 to 5 mg per 0.5 ml.

3. The composition as claimed in claim 1, wherein, the Propionic acid used in an amount of 5 to 10 mg per 1 ml.

4. The composition as claimed in claim 1, wherein, the composition comprises;
a) Cetrorelix acetate equivalent to Cetrorelix in an amount of 0.25 mg per 0.5 ml;
b) 3.0 mg of Propionic acid in aqueous vehicle as a stabilizing agent;
c) 10 mg Sucrose and 20.0 mg Sulfobutylether betacyclodextrin sodium as tonicity adjusting agents;
Wherein, pH of said composition is in the range of 2.5 to 4.0.

5. The composition as claimed in claim 1, wherein, the composition comprises;
a) Cetrorelix acetate equivalent to Cetrorelix in an amount of 0.5 mg per 1.0 ml;
b) 6.0 mg of Propionic acid in aqueous vehicle as a stabilizing agent per 1.0 ml;
c) 20 mg Sucrose and 40.0 mg Sulfobutylether betacyclodextrin sodium as tonicity adjusting agents per 1.0 ml;
wherein pH of said composition is in the range of 2.5 to 4.0.

6. A process for preparation of stable aqueous pharmaceutical composition comprising,
a) dissolving cetrorelix in an aqueous solution containing Propionic acid; Sulfobutyl ether betacyclodextrin sodium and Sucrose in water for injection; and
b) adjusting the pH of the composition in the range of 2.5 to 4.0 using Propionic acid solution;
c) filtering the solution and filling into the syringes.