Title of Invention	A FREEZE DRIED PARENTERAL COMPOSITION OF TIGECYCLIN AND PROCESS FOR PREPARATION THEREOF
Abstract	“A Freeze Dried Parenteral Composition of Tigecycline and Process for Preparation Thereof” Abstract: A stable, freeze dried pharmaceutical composition comprising Tigecycline along with sulfobutyl ether betacyclodextrin sodium as stabilizing agent for parenteral administration. The pharmaceutical composition provides stabilization of Tigecycline thereby improving the shelf life during storage. The invention further discloses a process of preparation of said composition.

Title of Invention

A FREEZE DRIED PARENTERAL COMPOSITION OF TIGECYCLIN AND PROCESS FOR PREPARATION THEREOF

Abstract

“A Freeze Dried Parenteral Composition of Tigecycline and Process for Preparation Thereof” Abstract: A stable, freeze dried pharmaceutical composition comprising Tigecycline along with sulfobutyl ether betacyclodextrin sodium as stabilizing agent for parenteral administration. The pharmaceutical composition provides stabilization of Tigecycline thereby improving the shelf life during storage. The invention further discloses a process of preparation of said composition.

Full Text	Claims:1. A stable freeze-dried pharmaceutical composition for parenteral administration comprising, a) Tigecycline or pharmaceutically acceptable salts thereof in an amount of 1mg to 200mg/vial; and b) Stabilizing agent such as sulfobutyl ether betacyclodextrin sodium in an amount of 100mg to 4000mg. 2. The stable freeze-dried pharmaceutical composition according to claim 1, wherein stabilizing agent is present in an amount of 100mg to 2000mg. 3. The stable freeze-dried pharmaceutical composition according to claim 1 and 2, wherein stabilizing agent is present in an amount of 100mg to 1500mg. 4. The stable freeze-dried pharmaceutical composition according to claim 1 comprising Tigecycline and sulfobutyl ether betacyclodextrin sodium; wherein pH of said composition is adjusted between 4.5-7.0 using acidifying agent such as dilute hydrochloric acid. 5. The stable freeze-dried pharmaceutical composition according to any of the preceding claims, wherein the composition comprises; a) Tigecycline or pharmaceutically acceptable salts thereof in an amount of 50mg/vial; b) sulfobutyl ether betacyclodextrin sodium in an amount of 1000mg and c) dilute hydrochloric acid for adjusting the pH in the range of 4.5 to 7.0. 6. The stable freeze-dried pharmaceutical composition according to any of the preceding claims, wherein the composition comprises; a) Tigecycline or pharmaceutically acceptable salts thereof in an amount of 150mg/vial; b) sulfobutyl ether betacyclodextrin sodium in an amount of 3000mg and c) dilute hydrochloric acid for adjusting the pH in the range of 4.5 to 7.0. 7. A process for increasing the stability of freeze-dried pharmaceutical composition of Tigecycline according to any of the preceding claims, in an aqueous solution comprising a step of combining Tigecycline or a pharmaceutically acceptable salts thereof with sulfobutyl ether betacyclodextrin sodium. , Description: FORM 2 THE PATENTS ACT, 1970 (39 of 1970) AND The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10 and rule 13) 1. TITLE OF THE INVENTION: “A FREEZE DRIED PARENTERAL COMPOSITION OF TIGECYCLIN AND PROCESS FOR PREPARATION THEREOF” 2. APPLICANT: (a) Name: GUFIC BIOSCIENCES LIMITED (b) Nationality: Indian Company incorporated under the Companies Act, 1956 (c) Address: N.H.No.8, Near Grid, Kabilpore 396 424, Navsari, Gujarat, India. 3. PREAMBLE TO THE DESCRIPTION: The following specification particularly describes the invention and the manner in which it is to be performed. Technical Field of the Invention: The present invention relates to a stable, freeze dried pharmaceutical composition of Tigecycline along with a suitable stabilizing agent and acidifying agent for parenteral administration. The pharmaceutical composition provides stabilization of Tigecycline thereby improving the shelf life during storage. The invention further relates to a process for preparation of said composition. Background of the Invention: Tigecycline, a derivative of minocycline that is modified to overcome tetracycline resistance, is the first of a novel class of glycylcyclines with expanded-spectrum properties. It is active in-vitro against a broad range of Gram-positive and Gram-negative bacteria, anaerobes, ‘atypical’ bacteria as well as against many species of drug-resistant strains [e.g. vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae and multidrug resistant (MDR) Acinetobacter baumannii]. Similar to tetracyclines, Tigecycline inhibits protein translation by reversibly binding to the 30S subunit of the bacterial ribosome, which impedes amino acid synthesis. Although the ribosomal binding sites of Tigecycline are similar to those of tetracycline, Tigecycline binds five times more effectively than tetracyclines. This allows Tigecycline to evade the common ribosomal protection mechanisms associated with resistance to tetracyclines. Because of its long side chain that blocks binding to most efflux proteins and transporters, Tigecycline also overcomes the efflux mechanisms of tetracycline resistance. In particular, since New Delhi Metallo-Lactamase-1 (NDM-1) was found among Gram-negative bacteria, which were highly resistant to all antibiotics except Tigecycline and colistin, Tigecycline has received high attention and has been regarded as the last resort to treat pan drug-resistant-bacteria (International Journal of Antimicrobial Agents 41 (2013) 110– 116). The first tetracycline antibiotics were discovered more than 50 years ago, and represented a significant advance in the treatment of many Gram-positive and Gram-negative bacterial infections. However, following their initial widespread use, a high incidence of tetracycline resistance among many bacteria has led to tetracyclines being relegated to second- or third-line therapy. In an attempt to restore the potential of tetracyclines as broad-spectrum antibiotics, systematic searches for tetracycline analogues with activity against both tetracycline-susceptible and tetracycline-resistant organisms were performed in the early 1990s. These efforts led to the identification of the glycylcyclines, including Tigecycline (Nature Reviews Drug Discovery 4, 809-810 (October 2005). Tigecycline is a tetracycline derivative (a glycylcycline) for intravenous infusion. The chemical name of Tigecycline is (4S,4aS,5aR,12aS)-9-[2-(tert-butylamino)acetamido]-4,7-bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,10,12,12a-tetrahydroxy-1,11-dioxo-2-naphthacenecarboxamide. The empirical formula is C29H39N5O8 and the molecular weight is 585.65gm/mol. Tigecycline is a light-sensitive, hygroscopic orange powder that is freely soluble in water and isotonic sodium chloride solution (Daily Med). The chemical structure of Tigecycline is given below: Tigecycline is marketed as lyophilized product for intravenous infusion by Wyeth Pharmaceuticals Inc. a subsidiary of Pfizer Inc. under the trade name Tygacil®. Tigecycline is indicated for the treatment of patients 18 years of age and older for: Complicated skin and skin structure infections, complicated intra-abdominal infections and Community-acquired bacterial pneumonia. Currently available formulations of Tigecycline is in lyophilized vial containing lactose monohydrate as stabilizing agent which does not provide desirable stability due to the sensitivity of Tigecycline against environmental stress. Further, Tigecycline is generally unstable to light, heat, humidity, acid, and the like and forms degradation product Tigecycline epimer, hence, it is necessary to develop a pharmaceutical composition which stabilizes the active compound and salt thereof for parenteral administration. In view of unstable nature of Tigecycline and the resultant challenges in preparing the lyophilized product for parenteral administration as mentioned above, the present inventors felt a need to develop a stable Tigecycline injectable composition using a suitable stabilizing agent which lowers the degradation of the active ingredient and improves shelf life of the composition. Indian Patent Number 268331 has disclosed a stable pharmaceutical composition of Tigecycline in lyophilized form comprising lactose as stabilizing agent. It is further disclosed in IN’331 that suitable carbohydrates stabilize Tigecycline against epimer formation at acidic pH. Examples of suitable carbohydrates include anhydrous, hydrated and solvated forms of mono and disaccharides selected from aldose monosaccharide or a disaccharide such as lactose, mannose, sucrose and glucose; preferably, a disaccharide such as lactose and sucrose. Lactose is most preferred. Tigecycline marketed products and the compositions known in the art contain higher percentage of degradation impurities which includes Tigecycline epimer impurity. In pursuit to circumvent the above problem, there is a need in the art to provide improved/stable Tigecycline freeze-dried composition which can reduce Tigecycline epimer formation and other oxidative degradation impurities thereby making the product stable with longer shelf-life. The above objective is realised in the present invention by providing a stable, freeze-dried pharmaceutical composition comprising Tigecycline and a suitable stabilizing agent which reduces the Tigecycline epimer formation and other degradation impurities with improved shelf life of the composition. Summary of the Invention: In accordance with above, the present invention provides a stable, freeze dried pharmaceutical composition comprising Tigecycline or pharmaceutically acceptable salt thereof along with a suitable stabilizing agent and acidifying agent for parenteral administration. In the present invention, Tigecycline is stabilized by adding stabilizing agents selected from cyclodextrin derivatives, specially, Sulfobutyl ether betacyclodextrin sodium. In an aspect, the pharmaceutical composition of Tigecycline and stabilizing agent is freeze dried and is provided as a drug concentrate. In another aspect, the present invention provides a method for stabilization of Tigecycline in an aqueous solution. 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. The present invention discloses a stable, freeze-dried pharmaceutical composition for parenteral administration comprising Tigecycline or pharmaceutically acceptable salt thereof as an active ingredient along with stabilizing agent and acidifying agent. The composition provides stabilization of Tigecycline with low degradation of the active ingredient thereby improving shelf life of the composition during storage and equally reducing Tigecycline epimer formation. Accordingly, in a preferred embodiment, the present invention provides a stable, freeze dried pharmaceutical composition for parenteral administration comprising; i. Tigecycline or a pharmaceutically acceptable salt thereof, and ii. Stabilizing agent selected from cyclodextrin derivatives, wherein the pH of said composition is maintained within the range of 4.5 to 7.0 using acidifying agent. Tigecycline or a pharmaceutically acceptable salt is present in the composition in an amount of 1mg to 200mg/vial; more preferably, 50mg/vial and 150mg/vial. Tigecycline is stabilized using a suitable stabilizing agent where Tigecycline can be maintained in a dissolved state in the aqueous solution thereby preventing crystallization or crystalline growth of Tigecycline. The stabilizing agent is selected from the cyclodextrin derivatives preferably, Sulfobutyl ether betacyclodextrin sodium. The composition of the present invention comprises Sulfobutyl ether betacyclodextrin sodium as stabilizing agent present in an amount of 100mg to 4000mg, preferably, 100mg to 2000mg, more preferably, 100mg to1500mg. The pH of the composition is maintained within the range of 4.5 to 7 using acidifying agent, such as dilute hydrochloric acid solution. In an embodiment, the present invention discloses a stable, freeze-dried pharmaceutical composition for parenteral administration comprising; a) Tigecycline or a pharmaceutically acceptable salts thereof in an amount of 1mg to 200mg/vial; b) stabilizing agent such as Sulfobutyl ether betacyclodextrin sodium in an amount of 100mg to 4000mg and c) acidifying agent such as dilute hydrochloric acid solution for adjusting the pH in the range of 4.5 to 7.0. In another embodiment, the present invention discloses a stable, freeze-dried pharmaceutical composition comprising; a) Tigecycline or a pharmaceutically acceptable salts thereof in an amount of 50mg/vial; b) Sulfobutyl ether betacyclodextrin sodium in an amount of 1000mg and c) Dilute hydrochloric acid solution for adjusting the pH in the range of 4.5 to 7.0. In yet another embodiment, the present invention discloses a stable, freeze-dried pharmaceutical composition comprising; a) Tigecycline or a pharmaceutically acceptable salts thereof in an amount of 150mg/vial; b) Sulfobutyl ether betacyclodextrin sodium in an amount of 3000mg and c) Dilute hydrochloric acid solution for adjusting the pH in the range of 4.5 to 7.0. The composition of the present invention after freeze drying is chemically and physically stable over an extended period of time and is suitable for intended pharmaceutical use after reconstitution with sodium chloride injection or 5% dextrose injection. Freeze drying process involves cooling of the desired composition at suitable temperature not less than -45°C, raising temperature to 0°C at suitable pressure of 150 mtorr to 100 mtorr in 35 hours, then at 50mtorr, further raising temperature to +35°C in 15 hrs. The freeze dried Tigecycline when reconstituted with 5 ml of suitable vehicle contains final drug concentrate of 10 mg/ml. In another embodiment, the pharmaceutical composition of the invention described herein is freeze dried composition, which may also be prepared by dissolving Tigecycline first in aqueous vehicle containing stabilizing agent then filter the solution and fill in to 10 ml glass vial. Developing freeze drying process for such composition, the freeze dried drug may be diluted with suitable diluents before administration as IV injection. The final concentration of solution may be reduced to further desired level using 5% Dextrose infusion prior to administration to a patient. The pharmaceutical composition of the present invention is useful in the treatment of various gram-positive and gram-negative bacterial infections such as complicated skin and skin structure infections, complicated intra-abdominal infections and community-acquired bacterial pneumonia. The pharmaceutical compositions of the present invention are 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. Industrial Advantages: 1. The pharmaceutical composition comprising Tigecycline as active with Sulfobutyl ether betacyclodextrin sodium without compromising stability of drug and its solution before Lyophilisation has a pH between 4.5 to 7.0. 2. The composition 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. Example 1: Sr. No. Ingredient Function Amount 1. Tigecycline or pharmaceutically acceptable salts thereof Active ingredient 1mg to 200mg 2. Sulfobutyl ether betacyclodextrin sodium Stabilizing agent 100mg to 4000mg 3. Dilute hydrochloric acid pH adjuster QS (To adjust pH between 4.5-7.0) 4. Water for Injection Vehicle QS Example 2: Sr. No. Ingredient Function Amount 1. Tigecycline or pharmaceutically acceptable salts thereof Active ingredient 50mg 2. Sulfobutyl ether betacyclodextrin sodium Stabilizing agent 1000mg 3. Dilute hydrochloric acid pH adjuster QS (To adjust pH between 4.5-7.0) 4. Water for Injection Vehicle QS Example 3: Sr. No. Ingredient Function Amount 1. Tigecycline or pharmaceutically acceptable salts thereof Active ingredient 150mg 2. Sulfobutyl ether betacyclodextrin sodium Stabilizing agent 3000mg 3. Dilute hydrochloric acid pH adjuster QS (To adjust pH between 4.5-7.0) 4. Water for Injection Vehicle QS Process for preparation of compositions of Examples 1 to 3: a) Dissolving Tigecycline or pharmaceutically acceptable salts thereof in aqueous vehicle containing Sulfobutyl ether betacyclodextrin sodium and b) adjusting the pH of the composition using dilute hydrochloric acid in the range of 4.5 to 7.0, followed by freeze drying. Experimental: Different trials were conducted and tested before narrowing down to the present composition. These trials are discussed in brief below to emphasize the inventiveness of the current invention and analysis was carried out as per USP 38. Example 4 1.0 gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 2.0 gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (10 ml) with each containing 2.5 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 50 mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested. The result is given in below Table 1. Table 1: Period Assay (%) pH Tigecycline epimer (%) Total impurities (%) Storage temp. Color Initial Bulk solution 99.07 5.45 0.61 0.79 2-8°C Clear orange color solution Lyophilized 99.01 5.36 0.62 0.88 25°C Orange color lyophilized cake After 24 hours 98.65 - 0.92 1.23 70°C Orange color lyophilized cake Reconstituted with 5 ml 0.9% Sodium chloride Example 5 1.0 gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 5.0 gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (10 ml) with each containing 2.5 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 50 mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested. The result is given in below Table 2. Table 2: Period Assay (%) pH Tigecycline epimer (%) Total impurities (%) Storage temp. Color Initial Bulk solution 99.17 5.45 0.61 0.89 2-8°C Clear orange color solution Lyophilized 99.08 5.31 0.60 0.92 25°C Orange color lyophilized cake After 24 hours 98.85 - 0.77 1.09 70°C Orange color lyophilized cake Reconstituted with 5 ml 0.9% Sodium chloride Example 6 1.0 gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 10.0 gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (10 ml) with each containing 5.0 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 50 mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested. The result is given in below Table 3. Table 3: Period Assay (%) pH Tigecycline epimer (%) Total impurities (%) Storage temp. Color Initial Bulk solution 99.31 5.45 0.53 0.65 2-8°C Clear orange color solution Lyophilized 99.23 5.35 0.53 0.69 25°C Orange color lyophilized cake After 24 hours 99.14 - 0.60 0.80 70°C Orange color lyophilized cake Reconstituted with 5 ml 0.9% Sodium chloride Example 7 1.0 gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 15.0 gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (10 ml) with each containing 5.0 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 50 mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested. The result is given in below Table 4. Table 4: Period Assay (%) pH Tigecycline epimer (%) Total impurities (%) Storage temp. Color Initial Bulk solution 99.34 5.45 0.54 0.72 2-8°C Clear orange color solution Lyophilized 99.21 5.34 0.57 0.75 25°C Orange color lyophilized cake After 24 hours 99.16 - 0.62 0.79 70°C Orange color lyophilized cake Reconstituted with 5 ml 0.9% Sodium chloride Example 8 1.0 gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 20.0 gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (10 ml) with each containing 5.0 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 50 mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested. The result is given in below Table 5. Table 5: Tests Initial After 24 hours (Store at 70°C) After 15 days (Store at 40°C/75% RH) After 40 days (Store at 40°C/75% RH) Bulk solution (Store at 2-8°C) Lyophilized (Store at 25°C) Colour Clear orange color solution Orange color lyophilized cake Orange color lyophilized cake Orange color lyophilized cake Orange color lyophilized cake pH(Limit: 4.5 to 5.5) 5.45 5.26 - - - % Assay (Limit: 96.0% to 116.0%) 99.53 99.48 99.42 99.31 98.87 Impurities Tigecycline open ring (Limit: NMT 0.15%) - - - - - Tigecycline 12-oxo-11-hydroxy (Limit: NMT 0.5%) - - - 0.02 0.04 Tigecycline related compound B (Limit: NMT 0.7%) - - - 0.02 0.05 Tigecycline epimer (Limit: NMT 2.0%) 0.48 0.49 0.51 0.55 0.79 Tigecycline quinone analog (Limit: NMT 0.3%) - - - - - Minocycline - - - 0.06 0.09 Tigecycline tricyclic analog (Limit: NMT 0.5%) - - - - - Any individual unspecified degradation product (Limit: NMT 0.2%) - 0.02 0.02 0.03 0.04 Total degradation product (Limit: NMT 6.0%) 0.48 0.51 0.53 0.69 1.01 Reconstituted with 5 ml 0.9% Sodium chloride It is observed from the above examples that, the composition comprising Tigecycline and Sulfobutyl ether betacyclodextrin sodium exhibits good stability with less impurity after 40 days (Example 8). Example 9 3.0gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 60.0gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (30 ml) with each containing 15.0 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 150mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested.

Full Text

Claims:1. A stable freeze-dried pharmaceutical composition for parenteral administration comprising,
a) Tigecycline or pharmaceutically acceptable salts thereof in an amount of 1mg to 200mg/vial; and
b) Stabilizing agent such as sulfobutyl ether betacyclodextrin sodium in an amount of 100mg to 4000mg.

2. The stable freeze-dried pharmaceutical composition according to claim 1, wherein stabilizing agent is present in an amount of 100mg to 2000mg.

3. The stable freeze-dried pharmaceutical composition according to claim 1 and 2, wherein stabilizing agent is present in an amount of 100mg to 1500mg.

4. The stable freeze-dried pharmaceutical composition according to claim 1 comprising Tigecycline and sulfobutyl ether betacyclodextrin sodium; wherein pH of said composition is adjusted between 4.5-7.0 using acidifying agent such as dilute hydrochloric acid.

5. The stable freeze-dried pharmaceutical composition according to any of the preceding claims, wherein the composition comprises;
a) Tigecycline or pharmaceutically acceptable salts thereof in an amount of 50mg/vial;
b) sulfobutyl ether betacyclodextrin sodium in an amount of 1000mg and
c) dilute hydrochloric acid for adjusting the pH in the range of 4.5 to 7.0.

6. The stable freeze-dried pharmaceutical composition according to any of the preceding claims, wherein the composition comprises;
a) Tigecycline or pharmaceutically acceptable salts thereof in an amount of 150mg/vial;
b) sulfobutyl ether betacyclodextrin sodium in an amount of 3000mg and
c) dilute hydrochloric acid for adjusting the pH in the range of 4.5 to 7.0.
7. A process for increasing the stability of freeze-dried pharmaceutical composition of Tigecycline according to any of the preceding claims, in an aqueous solution comprising a step of combining Tigecycline or a pharmaceutically acceptable salts thereof with sulfobutyl ether betacyclodextrin sodium.
, Description:
FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:

“A FREEZE DRIED PARENTERAL COMPOSITION OF TIGECYCLIN AND PROCESS FOR PREPARATION THEREOF”

2. APPLICANT:

(a) Name: GUFIC BIOSCIENCES LIMITED

(b) Nationality: Indian Company incorporated under the Companies Act, 1956

(c) Address: N.H.No.8, Near Grid, Kabilpore 396 424, Navsari, Gujarat, India.
3. PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the invention and the manner in which it is to be performed.

Technical Field of the Invention:
The present invention relates to a stable, freeze dried pharmaceutical composition of Tigecycline along with a suitable stabilizing agent and acidifying agent for parenteral administration. The pharmaceutical composition provides stabilization of Tigecycline thereby improving the shelf life during storage. The invention further relates to a process for preparation of said composition.

Background of the Invention:
Tigecycline, a derivative of minocycline that is modified to overcome tetracycline resistance, is the first of a novel class of glycylcyclines with expanded-spectrum properties. It is active in-vitro against a broad range of Gram-positive and Gram-negative bacteria, anaerobes, ‘atypical’ bacteria as well as against many species of drug-resistant strains [e.g. vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae and multidrug resistant (MDR) Acinetobacter baumannii]. Similar to tetracyclines, Tigecycline inhibits protein translation by reversibly binding to the 30S subunit of the bacterial ribosome, which impedes amino acid synthesis. Although the ribosomal binding sites of Tigecycline are similar to those of tetracycline, Tigecycline binds five times more effectively than tetracyclines. This allows Tigecycline to evade the common ribosomal protection mechanisms associated with resistance to tetracyclines. Because of its long side chain that blocks binding to most efflux proteins and transporters, Tigecycline also overcomes the efflux mechanisms of tetracycline resistance. In particular, since New Delhi Metallo-Lactamase-1 (NDM-1) was found among Gram-negative bacteria, which were highly resistant to all antibiotics except Tigecycline and colistin, Tigecycline has received high attention and has been regarded as the last resort to treat pan drug-resistant-bacteria (International Journal of Antimicrobial Agents 41 (2013) 110– 116).

The first tetracycline antibiotics were discovered more than 50 years ago, and represented a significant advance in the treatment of many Gram-positive and Gram-negative bacterial infections. However, following their initial widespread use, a high incidence of tetracycline resistance among many bacteria has led to tetracyclines being relegated to second- or third-line therapy.

In an attempt to restore the potential of tetracyclines as broad-spectrum antibiotics, systematic searches for tetracycline analogues with activity against both tetracycline-susceptible and tetracycline-resistant organisms were performed in the early 1990s. These efforts led to the identification of the glycylcyclines, including Tigecycline (Nature Reviews Drug Discovery 4, 809-810 (October 2005).

Tigecycline is a tetracycline derivative (a glycylcycline) for intravenous infusion. The chemical name of Tigecycline is (4S,4aS,5aR,12aS)-9-[2-(tert-butylamino)acetamido]-4,7-bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,10,12,12a-tetrahydroxy-1,11-dioxo-2-naphthacenecarboxamide. The empirical formula is C29H39N5O8 and the molecular weight is 585.65gm/mol. Tigecycline is a light-sensitive, hygroscopic orange powder that is freely soluble in water and isotonic sodium chloride solution (Daily Med). The chemical structure of Tigecycline is given below:

Tigecycline is marketed as lyophilized product for intravenous infusion by Wyeth Pharmaceuticals Inc. a subsidiary of Pfizer Inc. under the trade name Tygacil®. Tigecycline is indicated for the treatment of patients 18 years of age and older for: Complicated skin and skin structure infections, complicated intra-abdominal infections and Community-acquired bacterial pneumonia.

Currently available formulations of Tigecycline is in lyophilized vial containing lactose monohydrate as stabilizing agent which does not provide desirable stability due to the sensitivity of Tigecycline against environmental stress.

Further, Tigecycline is generally unstable to light, heat, humidity, acid, and the like and forms degradation product Tigecycline epimer, hence, it is necessary to develop a pharmaceutical composition which stabilizes the active compound and salt thereof for parenteral administration.
In view of unstable nature of Tigecycline and the resultant challenges in preparing the lyophilized product for parenteral administration as mentioned above, the present inventors felt a need to develop a stable Tigecycline injectable composition using a suitable stabilizing agent which lowers the degradation of the active ingredient and improves shelf life of the composition.

Indian Patent Number 268331 has disclosed a stable pharmaceutical composition of Tigecycline in lyophilized form comprising lactose as stabilizing agent. It is further disclosed in IN’331 that suitable carbohydrates stabilize Tigecycline against epimer formation at acidic pH. Examples of suitable carbohydrates include anhydrous, hydrated and solvated forms of mono and disaccharides selected from aldose monosaccharide or a disaccharide such as lactose, mannose, sucrose and glucose; preferably, a disaccharide such as lactose and sucrose. Lactose is most preferred.

Tigecycline marketed products and the compositions known in the art contain higher percentage of degradation impurities which includes Tigecycline epimer impurity.

In pursuit to circumvent the above problem, there is a need in the art to provide improved/stable Tigecycline freeze-dried composition which can reduce Tigecycline epimer formation and other oxidative degradation impurities thereby making the product stable with longer shelf-life.

The above objective is realised in the present invention by providing a stable, freeze-dried pharmaceutical composition comprising Tigecycline and a suitable stabilizing agent which reduces the Tigecycline epimer formation and other degradation impurities with improved shelf life of the composition.

Summary of the Invention:
In accordance with above, the present invention provides a stable, freeze dried pharmaceutical composition comprising Tigecycline or pharmaceutically acceptable salt thereof along with a suitable stabilizing agent and acidifying agent for parenteral administration.

In the present invention, Tigecycline is stabilized by adding stabilizing agents selected from cyclodextrin derivatives, specially, Sulfobutyl ether betacyclodextrin sodium.

In an aspect, the pharmaceutical composition of Tigecycline and stabilizing agent is freeze dried and is provided as a drug concentrate.

In another aspect, the present invention provides a method for stabilization of Tigecycline in an aqueous solution.

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.

The present invention discloses a stable, freeze-dried pharmaceutical composition for parenteral administration comprising Tigecycline or pharmaceutically acceptable salt thereof as an active ingredient along with stabilizing agent and acidifying agent. The composition provides stabilization of Tigecycline with low degradation of the active ingredient thereby improving shelf life of the composition during storage and equally reducing Tigecycline epimer formation.

Accordingly, in a preferred embodiment, the present invention provides a stable, freeze dried pharmaceutical composition for parenteral administration comprising;
i. Tigecycline or a pharmaceutically acceptable salt thereof, and
ii. Stabilizing agent selected from cyclodextrin derivatives,
wherein the pH of said composition is maintained within the range of 4.5 to 7.0 using acidifying agent.

Tigecycline or a pharmaceutically acceptable salt is present in the composition in an amount of 1mg to 200mg/vial; more preferably, 50mg/vial and 150mg/vial.

Tigecycline is stabilized using a suitable stabilizing agent where Tigecycline can be maintained in a dissolved state in the aqueous solution thereby preventing crystallization or crystalline growth of Tigecycline. The stabilizing agent is selected from the cyclodextrin derivatives preferably, Sulfobutyl ether betacyclodextrin sodium.

The composition of the present invention comprises Sulfobutyl ether betacyclodextrin sodium as stabilizing agent present in an amount of 100mg to 4000mg, preferably, 100mg to 2000mg, more preferably, 100mg to1500mg.

The pH of the composition is maintained within the range of 4.5 to 7 using acidifying agent, such as dilute hydrochloric acid solution.

In an embodiment, the present invention discloses a stable, freeze-dried pharmaceutical composition for parenteral administration comprising;
a) Tigecycline or a pharmaceutically acceptable salts thereof in an amount of 1mg to 200mg/vial;
b) stabilizing agent such as Sulfobutyl ether betacyclodextrin sodium in an amount of 100mg to 4000mg and
c) acidifying agent such as dilute hydrochloric acid solution for adjusting the pH in the range of 4.5 to 7.0.

In another embodiment, the present invention discloses a stable, freeze-dried pharmaceutical composition comprising;
a) Tigecycline or a pharmaceutically acceptable salts thereof in an amount of 50mg/vial;
b) Sulfobutyl ether betacyclodextrin sodium in an amount of 1000mg and
c) Dilute hydrochloric acid solution for adjusting the pH in the range of 4.5 to 7.0.

In yet another embodiment, the present invention discloses a stable, freeze-dried pharmaceutical composition comprising;
a) Tigecycline or a pharmaceutically acceptable salts thereof in an amount of 150mg/vial;
b) Sulfobutyl ether betacyclodextrin sodium in an amount of 3000mg and
c) Dilute hydrochloric acid solution for adjusting the pH in the range of 4.5 to 7.0.

The composition of the present invention after freeze drying is chemically and physically stable over an extended period of time and is suitable for intended pharmaceutical use after reconstitution with sodium chloride injection or 5% dextrose injection.

Freeze drying process involves cooling of the desired composition at suitable temperature not less than -45°C, raising temperature to 0°C at suitable pressure of 150 mtorr to 100 mtorr in 35 hours, then at 50mtorr, further raising temperature to +35°C in 15 hrs.

The freeze dried Tigecycline when reconstituted with 5 ml of suitable vehicle contains final drug concentrate of 10 mg/ml.

In another embodiment, the pharmaceutical composition of the invention described herein is freeze dried composition, which may also be prepared by dissolving Tigecycline first in aqueous vehicle containing stabilizing agent then filter the solution and fill in to 10 ml glass vial.

Developing freeze drying process for such composition, the freeze dried drug may be diluted with suitable diluents before administration as IV injection. The final concentration of solution may be reduced to further desired level using 5% Dextrose infusion prior to administration to a patient.

The pharmaceutical composition of the present invention is useful in the treatment of various gram-positive and gram-negative bacterial infections such as complicated skin and skin structure infections, complicated intra-abdominal infections and community-acquired bacterial pneumonia.

The pharmaceutical compositions of the present invention are 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.

Industrial Advantages:
1. The pharmaceutical composition comprising Tigecycline as active with Sulfobutyl ether betacyclodextrin sodium without compromising stability of drug and its solution before Lyophilisation has a pH between 4.5 to 7.0.
2. The composition 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.

Example 1:

Sr. No. Ingredient Function Amount
1. Tigecycline or pharmaceutically acceptable salts thereof Active ingredient 1mg to 200mg
2. Sulfobutyl ether betacyclodextrin sodium Stabilizing agent 100mg to 4000mg
3. Dilute hydrochloric acid pH adjuster QS (To adjust pH between 4.5-7.0)
4. Water for Injection Vehicle QS

Example 2:

Sr. No. Ingredient Function Amount
1. Tigecycline or pharmaceutically acceptable salts thereof Active ingredient 50mg
2. Sulfobutyl ether betacyclodextrin sodium Stabilizing agent 1000mg
3. Dilute hydrochloric acid pH adjuster QS (To adjust pH between 4.5-7.0)
4. Water for Injection Vehicle QS

Example 3:

Sr. No. Ingredient Function Amount
1. Tigecycline or pharmaceutically acceptable salts thereof Active ingredient 150mg
2. Sulfobutyl ether betacyclodextrin sodium Stabilizing agent 3000mg
3. Dilute hydrochloric acid pH adjuster QS (To adjust pH between 4.5-7.0)
4. Water for Injection Vehicle QS

Process for preparation of compositions of Examples 1 to 3:
a) Dissolving Tigecycline or pharmaceutically acceptable salts thereof in aqueous vehicle containing Sulfobutyl ether betacyclodextrin sodium and
b) adjusting the pH of the composition using dilute hydrochloric acid in the range of 4.5 to 7.0, followed by freeze drying.

Experimental:
Different trials were conducted and tested before narrowing down to the present composition. These trials are discussed in brief below to emphasize the inventiveness of the current invention and analysis was carried out as per USP 38.

Example 4
1.0 gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 2.0 gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (10 ml) with each containing 2.5 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 50 mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested. The result is given in below Table 1.

Table 1:
Period Assay (%) pH Tigecycline epimer (%) Total impurities (%) Storage temp. Color
Initial Bulk solution 99.07 5.45 0.61 0.79 2-8°C Clear orange color solution
Lyophilized
99.01 *5.36 0.62 0.88 25°C Orange color lyophilized cake
After 24 hours 98.65 - 0.92 1.23 70°C Orange color lyophilized cake

*Reconstituted with 5 ml 0.9% Sodium chloride

Example 5
1.0 gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 5.0 gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (10 ml) with each containing 2.5 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 50 mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested. The result is given in below Table 2.
Table 2:
Period Assay (%) pH Tigecycline epimer (%) Total impurities (%) Storage temp. Color
Initial Bulk solution 99.17 5.45 0.61 0.89 2-8°C Clear orange color solution
Lyophilized
99.08 *5.31 0.60 0.92 25°C Orange color lyophilized cake
After 24 hours 98.85 - 0.77 1.09 70°C Orange color lyophilized cake

*Reconstituted with 5 ml 0.9% Sodium chloride
Example 6
1.0 gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 10.0 gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (10 ml) with each containing 5.0 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 50 mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested. The result is given in below Table 3.
Table 3:
Period Assay (%) pH Tigecycline epimer (%) Total impurities (%) Storage temp. Color
Initial Bulk solution 99.31 5.45 0.53 0.65 2-8°C Clear orange color solution
Lyophilized
99.23 *5.35 0.53 0.69 25°C Orange color lyophilized cake
After 24 hours 99.14 - 0.60 0.80 70°C Orange color lyophilized cake

*Reconstituted with 5 ml 0.9% Sodium chloride

Example 7
1.0 gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 15.0 gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (10 ml) with each containing 5.0 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 50 mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested. The result is given in below Table 4.

Table 4:
Period Assay (%) pH Tigecycline epimer (%) Total impurities (%) Storage temp. Color
Initial Bulk solution 99.34 5.45 0.54 0.72 2-8°C Clear orange color solution
Lyophilized
99.21 *5.34 0.57 0.75 25°C Orange color lyophilized cake
After 24 hours 99.16 - 0.62 0.79 70°C Orange color lyophilized cake

*Reconstituted with 5 ml 0.9% Sodium chloride

Example 8
1.0 gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 20.0 gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (10 ml) with each containing 5.0 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 50 mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested. The result is given in below Table 5.
Table 5:
Tests Initial After 24 hours (Store at 70°C) After 15 days
(Store at 40°C/75% RH) After 40 days
(Store at 40°C/75% RH)
Bulk solution
(Store at 2-8°C) Lyophilized
(Store at 25°C)
Colour Clear orange color solution Orange color lyophilized cake Orange color lyophilized cake Orange color lyophilized cake Orange color lyophilized cake
pH(Limit: 4.5 to 5.5) 5.45 *5.26 - - -
% Assay (Limit: 96.0% to 116.0%) 99.53 99.48 99.42 99.31 98.87
Impurities
Tigecycline open ring (Limit: NMT 0.15%) - - - - -
Tigecycline 12-oxo-11-hydroxy (Limit: NMT 0.5%) - - - 0.02 0.04
Tigecycline related compound B (Limit: NMT 0.7%) - - - 0.02 0.05
Tigecycline epimer (Limit: NMT 2.0%) 0.48 0.49 0.51 0.55 0.79
Tigecycline quinone analog (Limit: NMT 0.3%) - - - - -
Minocycline - - - 0.06 0.09
Tigecycline tricyclic analog (Limit: NMT 0.5%) - - - - -
Any individual unspecified degradation product (Limit: NMT 0.2%) - 0.02 0.02 0.03 0.04
Total degradation product (Limit: NMT 6.0%) 0.48 0.51 0.53 0.69 1.01

*Reconstituted with 5 ml 0.9% Sodium chloride

It is observed from the above examples that, the composition comprising Tigecycline and Sulfobutyl ether betacyclodextrin sodium exhibits good stability with less impurity after 40 days (Example 8).

Example 9
3.0gm Tigecycline (active) or pharmaceutically acceptable salt was added to an aqueous solution containing 60.0gm of Sulfobutyl ether betacyclodextrin sodium and stirred for some time. Adjust the pH about 5.45 with 1.0 N HCL solution (pH limit 4.5 to 7.0). The solution was filtered and the resulting solution was loaded into 20 vials (30 ml) with each containing 15.0 ml solution. The solution in each vial was lyophilized using freeze dryer according to conventional method, thereby obtaining the lyophilized compositions each comprising 150mg of Tigecycline. The resulting lyophilized preparation was stored at different storage temperatures at different time intervals and the content of Tigecycline was tested.

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Patent Number

279965

Indian Patent Application Number

201621000791

PG Journal Number

06/2017

Publication Date

10-Feb-2017

Grant Date

06-Feb-2017

Date of Filing

08-Jan-2016

Name of Patentee

GUFIC BIOSCIENCES LIMITED

Applicant Address

N.H.No.8, Near Grid, Kabilpore 396 424, Navsari, Gujarat India.

Inventors:

#	Inventor's Name	Inventor's Address
1	PATEL, Mitesh Natavarlal	Gufic Biosciences Limited N.H.No.8, Near Grid, Kabilpore 396 424, Navsari , Gujarat India.
2	DAVE, Mafatlal Tribhovandas	Gufic Biosciences Limited N.H.No.8, Near Grid, Kabilpore 396 424 Navsari , Gujarat India.
3	CHOKSI, Pranavkumar Jayesh	Gufic Biosciences Limited N.H.No.8, Near Grid, Kabilpore 396 424 Navsari , Gujarat India.

PCT International Classification Number

C07C231/21

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA