DESC:FORM 2

THE PATENTS ACT 1970
(SECTION 39 OF 1970)

&
THE PATENT RULES, 2003

COMPLETE SPECIFICATION
(Section 10 and Rule 13)

A STABLE READY-TO-USE INJECTABLE COMPOSITION OF GEMCITABINE OR ITS SALT AND METHODS FOR PRODUCING SAME

We, HETERO HEALTHCARE LIMITED,
a company incorporated under the companies act, 1956 having address at Sy. No. 80-84, Melange Towers, 4th Floor, C-wing, Patrikanagar, Madhapur, Hyderabad-500081, Telangana State, India.

The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF INVENTION
The present invention relates to stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent and other pharmaceutically acceptable excipients which can be infused without any prior dilutions before administration.

The present invention also relates to stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent and tonicity adjusting agent, pH adjusting agents, and aqueous vehicle as other pharmaceutically acceptable excipients.

The present invention specifically relates to method for the preparation of stable ready-to-use injectable infusion formulation of Gemcitabine or pharmaceutically acceptable salt which involves sterilization by aseptic filtration followed by heat treatment.

The present invention more specifically relates to method for the preparation of stable ready-to-use injectable infusion container formulation of Gemcitabine or pharmaceutically acceptable salt comprising the steps of adding, dissolving, pH adjusting, diluting, sterilization by aseptic filtration followed by heat treatment which avoids terminal sterilization and filled into the infusion container which does not contain aluminium over-pouch as secondary packaging.

BACKGROUND OF INVENTION
Lyophilization or freeze-drying is often used to stabilize various pharmaceutical products, including virus vaccines, protein and peptide formulations, liposome and small-chemical drug formulation. Lyophilized formulations have to be reconstituted, usually by injecting diluent through the septum into the vial. The solution is drawn up into a new syringe, the needle changed before finally being injected into the patient. These multiple steps make the lyophilized formulation inconvenient for use and provide risk of microbiological contamination during aseptic handling.

It is particularly desirable if the stable pharmaceutical composition can be prepared without the use of freeze drying techniques. A stable ready-to-use injectable solution could be stored at room temperature and the composition provides easier, safer handling, storage and distribution.

Compounds administered by infusion must be in a form ready to be dissolved (if solid) or diluted (if liquid) in an infusion liquid, or be provided as ready for infusion. Solid formulations must be such that the entire contents are readily soluble leaving essentially no solid particles and providing the entire active ingredient as homogeneously dissolved compound.

Gemcitabine is known as difluorodeoxycytidine, an analogue of cytosine arabinoside (ara-C) is a pyrimidine antimetabolite and it works by blocking the creation of new DNA, which results in cell death. Gemcitabine is a nucleoside metabolic inhibitor that exhibits antitumor activity which is used as chemotherapy medication to treat a number of types of cancers which include breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, and bladder cancer.

Chemically, Gemcitabine is 4-amino-1-(2-deoxy-2,2-difluoro-b-D-erythro-pentofuranosyl)pyrimidin-2(1H)-one, and has the following structural formula:

Gemcitabine free base has a molecular formula of C9H11F2N3O4/ 263.20 g/mol and its hydrochloride salt having molecular formula of C9H11F2N3O4.HCl/ 299.66 g/mol. Gemcitabine is a white to off-white crystalline powder which melts at 287-292°C and is soluble in water, slightly soluble in methanol, and practically insoluble in ethanol and polar organic solvents. The aqueous solubility of gemcitabine hydrochloride in purified water is 70.2 mg/mL.

Conventional formulations of Gemcitabine hydrochloride consists of sodium chloride as tonicity adjuster and pH adjusting agents. This formulation is clear, colourless, sterile solution in a single-dose, premixed intravenous large volume infusion bags with an aluminium over-pouch as secondary packaging and terminally sterilized by autoclave process at 121°C for 15 minutes.

The recommended dose for Gemcitabine is 1 g/m2 of body surface area and gemcitabine can be used cytostatically in the therapeutic treatment of various types of cancer. The administration of Gemcitabine to treat different types of cancer conditions is effected intravenously, in which case the active substance must be in the form of a solution.

Some patent literature describes the stable ready-to-use formulations of Gemcitabine or its pharmaceutically acceptable salt comprising pH modifying agents and tonicity adjusting agents such as sodium chloride and/or solubilizer such as propylene glycol, wherein the solution composition is sterilized by filtration sterilization.

Chinese patent No. CN 1302782 C discloses Gemcitabine hydrochloride injection solution comprising Gemcitabine hydrochloride, a solvent for injection, a stabilizer and an isotonic agent, wherein the injection pH range is in between 3.6-7.5.

European Pat. No. EP 1 479 388 B1 discloses pharmaceutical composition including gemcitabine in the form of a ready-to-use solution concentrate, wherein the gemcitabine is dissolved in a mixture of water and at least one other physiologically acceptable solvent or solubilising agent, wherein solubilising agents are ethanol, polyethylene glycol 200 - 600 and/or 1,2-propanediol (propylene glycol). The pH of the composition is in the range of 3.5 to 10.0 and gemcitabine concentration is 50 mg/ml or 80 mg/ml and solution sterilized by filtration.

European Pat. No. EP 1 479 389 B1 discloses Gemcitabine ready-to-use solution comprising 0.05 mg/ml to 16.0 mg/ml of Gemcitabine having pH of above 3.5. The stability data of examples disclosed in this patent demonstrate that compositions with higher concentration of Gemcitabine (more than 16.0 mg/ml) cause precipitation when stored at lower temperatures. The solvent was selected from the ethyl alcohol, polyethylene glycol 200-600, propylene glycol and mixture thereof. Solution is sterilized by filtration and the filtered solution is filled into vials.

U.S Pat. No. 9,241,948 B2 discloses large volume infusion dosage form of gemcitabine, comprising a stable large volume solution of gemcitabine or its pharmaceutically acceptable salt in an aqueous vehicle filled in a large volume infusion polymeric container covered with a secondary packaging system of aluminium over-pouch, wherein the solution is ready-to-be-infused. The injection dosage form is obtained by subjecting the solution filled in infusion container to terminal sterilization by autoclave at 121°C for 15 minutes.

U.S Pub No. US 2006/0089328 A1 discloses an injectable pharmaceutical composition comprising a solution of gemcitabine or its salt in a solvent having a pH of about 3.5 to about 10 and a gemcitabine concentration of about 0.5 mg/ml to about 16 mg/ml, wherein solvent is selected from the group consisting of water, ethyl alcohol, polyethylene glycol 200-600, and propylene glycol and mixtures thereof. Solution was sterilized by filtration and filled in stoppered vials.
U.S Pub No. US 2006/0154891 A1 discloses ready-to-use Gemcitabine aqueous preparation in glass container having specified dimensional relationships to demonstrate shelf life of over wide range of solution pH values. The ratio of surface area wetted by the composition to the volume of the solution contained in the container, expressed in cm2/cm3 is less than 3.4. Gemcitabine concentration is between 0.05 mg/mL and 110 mg/mL and pH of the aqueous solution is within a range of about 5.0 to about 10.0.

U.S Pub No. US 2012/0129799 A1 discloses stable non-aqueous pharmaceutical preparation comprising Gemcitabine or its pharmaceutically acceptable salts in a ready-to-use form comprising non-aqueous solvent, wherein non-aqueous solvent includes propylene glycol, polyethylene glycols, ethanol or the like thereof either alone or in combination thereof and the pH is between about 3.5 and about 10.0.

PCT Pub No. WO 2007/143895 A1 discloses, stable supersaturated solution of Gemcitabine hydrochloride, which is prepared by dissolving completely Gemcitabine hydrochloride in a medium by heating it at pH 4-8 to give a supersaturated solution of Gemcitabine having a concentrate of 15-45 g/L and the hot supersaturated solution was sterilized by filtration and dispensed in ampoules.

Patrick J. Jansen et al., The Journal of Pharmaceutical Sciences, 2000, vol. 89, No.7, Pg. No. 885-891, discloses the Gemcitabine aqueous solution. Sodium acetate was used to adjust pH of solution to 3.2. However degradation of Gemcitabine in aqueous solution at pH 3.2 indicates that development of formulation was feasible when stored at refrigerated temperature. However, at thermally stressed conditions caused significant degradation of products.

The commercially available product contain Gemcitabine or its pharmaceutically salt and is marketed under the trade name Gemzar® by Eli Lilly & Co which is supplied as a sterile lyophilised powder in single-dose vials for reconstitution which has to be dissolved for intravenous infusion. The product formulation contains, in addition to the active ingredient, mannitol, sodium acetate and hydrochloric acid and/ sodium hydroxide may have been added for pH adjustment. Gemzar® upon reconstitution, concentration is 40 mg/mL. It is reported that at concentrations greater than 40 mg/mL may result in incomplete dissolution, and therefore should be avoided.

It was discovered that a simple, isotonic saline solution of gemcitabine is not pharmaceutically acceptable for commercial purposes due to degradation of the solution to form unacceptable related substances. The chemical instability of gemcitabine is mainly attributed to their hydrolytic degradation. Hence, the main challenge lies in formulating a stable pharmaceutical composition of gemcitabine that has the minimum concentration of hydrolytic degradation impurities.

A ready-to-use solution can be filled in vial or glass container and supplied as single dose vial. Sterilization of injectable solution filled in the glass container plays a major role in maintaining stability and shelf life of the drug product. Sterilization of glass container can be achieved by many ways either by filtration sterilization, terminal sterilization by moist heat and dry heat sterilization. Hence, the use of stabilizer in the formulation and sterilization of infusion container are the key elements in developing a stable pharmaceutical composition of Gemcitabine or its salt.

Thus to overcome drawbacks associated with currently available formulation like precipitation issues including stability and solubility of ready-to-use Gemcitabine solution, there is a need to develop a stable ready-to-be-infused preparation containing Gemcitabine or its pharmaceutically acceptable salts. The inventors of the present invention have developed a stable ready-to-use injectable Gemcitabine composition comprising tromethamine as stabilizing and pH modifying agent, tonicity adjusting agents, pH modifying agents as other pharmaceutically acceptable excipients which provide better effect for the treatment of several forms of cancer including leukemias, lymphomas and breast cancer. The inventors of the present invention also provide method for the preparation of stable ready-to-use injectable infusion formulation of Gemcitabine or pharmaceutically acceptable salt comprising the steps of adding, dissolving, adjusting pH, diluting, sterilization by aseptic filtration followed by heat treatment.

OBJECTIVE OF INVENTION
The main objective of the present invention is to provide stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent and optionally other pharmaceutically acceptable excipients which can be infused without any prior dilutions before administration.

Another objective of the present invention is to provide stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent and other pharmaceutically acceptable excipients, wherein the solution can be infused without any prior dilutions before administration.

Another objective of the present invention is to provide stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, tonicity adjusting agents, pH modifying agents and aqueous vehicle as other pharmaceutically acceptable excipients.

Another objective of the present invention is to provide stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, sodium chloride or glycerine as tonicity adjusting agents, sodium hydroxide and hydrochloric acid as pH adjusting agents and water for injection as aqueous vehicle.

Another objective of the present invention is to provide stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, tonicity adjusting agents, pH modifying agents and aqueous vehicle as other pharmaceutically acceptable excipients, wherein the concentration of Gemcitabine or its pharmaceutically acceptable salts is 10 mg/ml and solution having a pH of about 8.0 to 9.0.

Another objective of the present invention is to provide stable ready-to-use aqueous composition of Gemcitabine or its pharmaceutically acceptable salts thereof which is physically stable at a temperature between 2° C to room temperatures and suitable to be given by intravenous route without any intervening step of reconstitution or dilution or mixing.

Still another objective of the present invention is to provide method for the preparation of stable ready-to-use injectable infusion formulation of Gemcitabine or pharmaceutically acceptable salt which involves sterilization by aseptic filtration followed by heat treatment.

Still another objective of the present invention is to provide method for the preparation of stable ready-to-use injectable infusion container formulation of Gemcitabine or pharmaceutically acceptable salt which does not contain aluminium over-pouch as secondary packaging comprising the steps of adding, dissolving, adjusting pH, diluting, sterilization by aseptic filtration followed by heat treatment which avoids terminal sterilization.

Still another objective of the present invention is to provide method for the preparation of stable ready-to-use injectable formulation of Gemcitabine or pharmaceutically acceptable salt, wherein the composition is filled in infusion container followed by heat treatment.

In yet another objective of the present invention is to provide better effect for the treatment of several forms of cancer including leukemias, lymphomas and breast cancer by administering stable ready-to-use injectable formulation of Gemcitabine or pharmaceutically acceptable salt.

SUMMARY OF INVENTION
Accordingly, the present invention provides stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent and optionally other pharmaceutically acceptable excipients.

Another embodiment of the present invention provides stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent and other pharmaceutically acceptable excipients, wherein the solution can be infused without any prior dilutions before administration.

Another embodiment of the present invention provides stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, tonicity adjusting agents, pH modifying agents, aqueous vehicle as other pharmaceutically acceptable excipients.

Another embodiment of the present invention provides stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, tonicity adjusting agents, pH modifying agents, aqueous vehicle as other pharmaceutically acceptable excipients, wherein the concentration of Gemcitabine or its pharmaceutically acceptable salts is 10 mg/ml and solution having a pH of about 8.0 to 9.0.

Another embodiment of the present invention provides stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, sodium chloride or glycerine as tonicity adjusting agents, sodium hydroxide and hydrochloric acid as pH adjusting agents and water for injection as aqueous vehicle.

Another embodiment of the present invention provides stable ready-to-use injectable formulation of Gemcitabine or its pharmaceutically acceptable salts thereof which is physically stable at a temperature between 2°C to room temperature and which can be administered through intravenous route without any intervening step of reconstitution or dilution or mixing.

Another embodiment of the present invention provides method for the preparation of stable ready-to-use injectable infusion container formulation comprising Gemcitabine or pharmaceutically acceptable salt, wherein sterilization involves aseptic filtration process followed by heat treatment which avoids terminal sterilization and the infusion container does not contain an aluminium over-pouch as secondary packaging.

Another embodiment of the present invention provides method for the preparation of stable ready-to-use injectable infusion formulation of Gemcitabine or pharmaceutically acceptable salt which involves sterilization by aseptic filtration, followed by heat treatment.

Another embodiment of the present invention provides method for the preparation of stable ready-to-use injectable infusion formulation of Gemcitabine or pharmaceutically acceptable salt comprising the steps of adding, dissolving, adjusting pH, diluting, sterilization by aseptic filtration followed by heat treatment which avoids terminal sterilization.

In another embodiment, the present invention provides method for the preparation of stable ready-to-use injectable formulation comprising of Gemcitabine or its pharmaceutically acceptable salts as active ingredient, tromethamine as stabilizing and pH modifying agent, tonicity adjusting agents, pH modifying agents, aqueous vehicle as pharmaceutically acceptable excipients, wherein the composition is filled in infusion container followed by heat treatment.

In another embodiment, the present invention provides a stable ready-to-use injectable formulation comprising:
(a) 0.1% to 1% (w/v) of Gemcitabine or its salt,
(b) 0.001% to 1.21% (w/v) of tromethamine,
(c) 0.25% to 3% (w/v) of tonicity adjusting agents,
(d) pH adjusting agents to adjust pH, and
(e) aqueous vehicle.

In yet another embodiment, the present invention provides a stable ready-to-use injectable formulation comprising:
(a) 0.1% to 1% (w/v) of Gemcitabine or its salt,
(b) 0.001% to 1.21% (w/v) of tromethamine,
(c) 0.25% to 3% (w/v) of sodium chloride or glycerine,
(d) sodium hydroxide and Hydrochloric acid as pH adjusting agents to adjust pH to 8.0 to 9.0, and
(e) water for injection.

Another embodiment of the present invention provides method for the preparation of stable ready-to-use injectable formulation comprising of Gemcitabine or its pharmaceutically acceptable salts comprising adding tromethamine, tonicity modifying agents in water, adding and dissolving Gemcitabine hydrochloride, adjusting pH to 8.0 to 9.0 using pH adjusting agents, diluting with remaining amount of water, adjusting the pH, sterilization by aseptic filtration followed by heat treatment.

In yet another embodiment, the present invention provides method for the preparation of stable ready-to-use injectable formulation comprising:
(a) adding tromethamine and tonicity modifying agents to 80% of water and stirred to get a clear solution,
(b) adding Gemcitabine hydrochloride to obtained solution of step (a) under stirring until it dissolves,
(c) adjusting pH of obtained solution of step (b) to 8.0 to 9.0 using pH adjusting agents,
(d) diluting the solution with remaining amount of water to get a stable solution,
(e) adjusting pH of stable solution of step (d) to 8.0 to 9.0 with pH adjusting agents, if required,
(f) filtering the solution aseptically through Nylon 66 Filter (0.2 micron) and filled into the stoppered glass container, and
(g) heat treatment at a temperature of 100°C to 105°C for a period of time ranging from 30 to 60 minutes after final packaging.

DETAILED DESCRIPTION OF THE INVENTION
The term "comprising", which is synonymous with "including", "containing", or "characterized by" here is defined as being inclusive or open-ended, and does not exclude additional, unrecited elements or method steps, unless the context clearly requires otherwise.
The present invention provides stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, optionally other pharmaceutically acceptable excipients.

The present invention provides stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, tonicity adjusting agents, pH modifying agents, aqueous vehicle as other pharmaceutically acceptable excipients.

The present invention provides stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, tonicity adjusting agents, pH modifying agents, aqueous vehicle as other pharmaceutically acceptable excipients, wherein the concentration of Gemcitabine or its pharmaceutically acceptable salts is 10 mg/ml and solution having a pH of about 8.0 to 9.0.

The present invention provides stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, sodium chloride or glycerine as tonicity adjusting agents, sodium hydroxide and hydrochloric acid as pH adjusting agents and water for injection as aqueous vehicle.

"Stable ready-to-use formulation” of the present invention includes injectable formulation suitable for administration to patient. In particular, a ‘stable aqueous composition’ is intended to refer to a solution which when stored at a temperature in the range of 2°C to room temperature, for a period of time, is physically stable i.e. it does not show the appearance of any visible particulates, crystals and is also chemically stable.

The term ‘chemically stable’ as used herein means that the impurities those resulting from chemical reaction in solution remains within acceptable limits during storage period at a temperature in the range of 2°C to room temperature.

Preferably used stabilizing and pH modifying agent is Tris(hydroxymethyl)aminomethane shown in formula (I) which is also referred to as TRIS, tromethamine and trisamine. It is a primary amine and a weak base with a pKa of 8.07 at 25°C.

Formula (I)

The concentration of tromethamine used in stable ready-to-use injectable formulation is from 0.001 to 1.21 % (w/v) and most preferably used concentration of tromethamine is from 0.003 to 0.12 % (w/v).

The term "tonicity agents" as used herein is to modulate the tonicity of the formulation. The formulation can be hypotonic, isotonic or hypertonic. An isotonic solution can have an osmolality of about 250-350 mOsmol/kg.

The term “tonicity adjusting agents” as used in the present invention includes but not limited to sodium chloride, calcium chloride, magnesium chloride, sodium lactate, glucose, fructose, sorbitol, mannitol, galactose, inositol, maltitol, lactose, trehalose, maltose, sucrose, dextran or organic solvents such as ethanol, glycerine, sorbitol, propylene glycol or combinations thereof. Preferred used tonicity adjusting agent is sodium chloride or glycerine.

The concentration of tonicity adjusting agents used in stable ready-to-use injectable formulation is from 0.25 to 3.0% (w/v).

The term “pH modifying agents” used in the present invention includes but not limited to inorganic acids and bases, such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, phosphorous acid, carbonic acid, sulfurous acid, nitrous acid, ascorbic acid, propionic acid, lactic acid, citric acid, formic acid, oxalic acid, benzoic acid, acetic acid, tartaric acid, malonic acid, maleic acid, pyruvic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, sodium hydroxide, calcium hydroxide, potassium hydroxide and magnesium hydroxide, sodium dihydrogen phosphate and its hydrates, sodium phosphate and its hydrates, disodium hydrogen phosphate and its hydrates, disodium sulfate, sodium hydrogen sulfate, sodium sulfite, calcium sulfite, magnesium sulfite, sodium carbonate, sodium hydrogen carbonate, calcium hydrogen carbonate, sodium nitrite, sodium nitrate, magnesium nitrate, calcium nitrite and magnesium nitrite, 1-deoxy-(methylamino)-D-glucitol (N-methylglucamine, meglumine).

Preferably, the pH of the solution is adjusted with hydrochloric acid, phosphoric acid, sulfuric acid, sodium hydroxide, sodium phosphate and their hydrates, sodium hydrogen phosphate and its hydrates, disodium hydrogen phosphate and its hydrates, acetic acid, lactic acid, citric acid. Most preferably used pH modifying agents are sodium hydroxide and hydrochloric acid. Sufficient quantity of pH adjusting agents can be used to adjust pH to 8.0 to 9.0

According to the present invention ready to be infused solution of Gemcitabine or its pharmaceutically acceptable salts can be filled in suitable large volume single compartment container. Suitable compartment containers include, for example, glass or polymeric vials which is devoid of aluminium over-pouch, ampoules, syringes, infusion bottles with sizes ranging from 20 ml to 500 ml.

The present invention provides a ready to be infused sterile solution which undergoes sterilization by heat treatment. Particularly, the present invention provides the use of aseptic filtration followed by heat treatment to destroy all viable microorganisms within the final, sealed container.

The present invention provides method for the preparation of stable ready-to-use injectable infusion formulation of Gemcitabine or pharmaceutically acceptable salt which involves sterilization by aseptic filtration followed by heat treatment.

The present invention provides method for the preparation of stable ready-to-use injectable infusion formulation of Gemcitabine or pharmaceutically acceptable salt comprising the steps of adding, dissolving, adjusting pH, diluting, sterilizing by aseptic filtration followed by heat treatment which avoids terminal sterilization.

The gemcitabine or its pharmaceutically acceptable salts solution composition of the present invention undergo Heat treatment at a temperature ranging from 95° C to 105° C and more preferably from 100° C to 105°C for a period of time ranging from 45 to 90 minutes, preferably 50 to 80 minutes, and more preferably 60 minutes. In particular, Hear treatment is preferably carried out in the temperature range of 100° C. to 105°C for a period of time 60 minutes.

The following examples describes the nature of the invention and are given only for the purpose of illustrating the present invention in more detail and are not limitative and relate to solutions which have been particularly effective on a bench scale and prepared by the process of the present invention.

Example 1
Excipients Quantity
mg/ml % w/v
Gemcitabine hydrochloride (expressed as free base) 10 1
Tromethamine 1.2 0.12
Sodium Chloride 5 0.5
Sodium Hydroxide qs Qs
Hydrochloric Acid qs Qs
WFI 1 100
pH 8.4

Example 2
Excipients Quantity
mg/ml % w/v
Gemcitabine hydrochloride (expressed as free base) 10 1
Tromethamine 0.8 0.08
Sodium Chloride 5 0.5
Sodium Hydroxide qs Qs
Hydrochloric Acid qs Qs
WFI 1 100
pH 8.4

Example 3
Excipients Quantity
mg/ml % w/v
Gemcitabine hydrochloride (expressed as free base) 10 1
Tromethamine 1.2 0.12
Glycerine 25 2.5
Sodium Hydroxide qs Qs
Hydrochloric Acid qs Qs
WFI 1 100
pH 8.4

Example 4
Excipients Quantity
mg/ml % w/v
Gemcitabine hydrochloride (expressed as free base) 10 1
Tromethamine 0.8 0.08
Glycerine 25 2.5
Sodium Hydroxide qs Qs
Hydrochloric Acid qs Qs
WFI 1 100
pH 8.4

Example 5
Excipients Quantity
mg/ml % w/v
Gemcitabine base 10 1
Tromethamine 0.3 0.03
Sodium Chloride 8 0.8
Sodium Hydroxide Qs Qs
Hydrochloric Acid Qs Qs
WFI 1 100
pH 8.4

Example 6
Excipients Quantity
mg/ml % w/v
Gemcitabine base 10 1
Tromethamine 1.2 0.12
Sodium Chloride 8 0.8
Sodium Hydroxide qs Qs
Hydrochloric Acid qs Qs
WFI 1 100
pH 8.4

Example 7
Excipients Quantity
mg/ml % w/v
Gemcitabine base 10 1
Tromethamine 0.3 0.03
Glycerine 25 2.5
Sodium Hydroxide Qs Qs
Hydrochloric Acid Qs Qs
WFI 1 100
pH 8.4

Example 8
Excipients Quantity
mg/ml % w/v
Gemcitabine base 10 1
Tromethamine 1.2 0.12
Glycerine 25 2.5
Sodium Hydroxide Qs Qs
Hydrochloric Acid Qs Qs
WFI 1 100
pH 8.4

Manufacturing process for preparation of ready-to-use solution of Gemcitabine Hydrochloride:

Tromethamine and tonicity modifying agents were added to 80% of water and stirred to get a clear solution. Gemcitabine hydrochloride is added and stirred until the mixture dissolves. pH was adjusted to 8.0 to 9.0 with pH adjusting agents like sodium hydroxide and hydrochloric acid. The solution was further diluted with remaining amount of water to get a stable solution. pH was adjusted, if required. The solution was filtered aseptically through Nylon 66 Filter (0.2 micron) and undergone heat treatment at a temperature of 100°C to 105°C for a period of time ranging from 30 to 60 minutes and obtained solution of Gemcitabine filled into the stoppered glass containers and final packaging.

The ready to be infused solution of all examples was sterilized by aseptic filtration (without Heat treatment) as well as by aseptic filtration followed by Heat treatment. The filtered solution was filled in infusion bottle. The filled bottles were stored at 2-8° C and 20-25°C. The solutions were evaluated for the physical stability i.e. presence of crystals or particulate matter. The solutions were observed regularly over a period of 6 months. Results of physical stability are given below table 1:

Reference Example

Excipients Quantity
mg/ml % w/v
Gemcitabine hydrochloride (expressed as free base) 10 1
Sodium Chloride 9 0.9
Sodium Hydroxide qs Qs
Hydrochloric Acid qs Qs
WFI 1 100
pH 8.4

Manufacturing process for preparation of conventional ready-to-use solution of Gemcitabine Hydrochloride:

Tonicity modifying agents were added to 80% of water and stirred to get a clear solution. Gemcitabine hydrochloride is added and stirred until the mixture dissolves. pH was adjusted to 8.0 to 9.0 with pH adjusting agents like sodium hydroxide and hydrochloric acid. The solution was further diluted with remaining amount of water to get a stable solution. pH was adjusted, if required. The solution was sterilized by aseptic filtration (without heat treatment) using Nylon 66 Filter (0.2 micron) and filled into the stoppered glass container.

Table 1: Physical stability of Ready to use Gemcitabine Injection of the present invention and of Reference Example product after 6 months of storage:
Example
Number 2-8° C 25° C (Room Temperature)
Without Heat treatment With Heat treatment Without Heat treatment With Heat treatment
1 Clear Solution Clear Solution Clear Solution Clear Solution
2 Clear Solution Clear Solution Clear Solution Clear Solution
3 Clear Solution Clear Solution Clear Solution Clear Solution
4 Clear Solution Clear Solution Clear Solution Clear Solution
5 Clear Solution Clear Solution Clear Solution Clear Solution
6 Clear Solution Clear Solution Clear Solution Clear Solution
7 Clear Solution Clear Solution Clear Solution Clear Solution
8 Clear Solution Clear Solution Clear Solution Clear Solution
Reference Example Crystals observed after 1 Month - Clear Solution -

The above data shows that the ready to use solution of Gemcitabine or its pharmaceutically acceptable salts are chemically and physically stable at 2-8°C as well as at room temperature. There was no observation of crystals formation or precipitation of gemcitabine ready-to-use solution which were prepared by using tromethamine as stabilizing and pH modifying agent, whereas conventional formulation prepared by tonicity modifying agents showed presence of crystals or particulate matter after 1 month when stored at 2-8°C. Thus, the use of stabilizing agent has played an important role in prevention of crystallization of Gemcitabine or its pharmaceutically acceptable salts and depict its need in formulation to prevent crystallization of Gemcitabine or its pharmaceutically acceptable salts during storage period, particularly at refrigerated condition. The combination of Heat treatment and aseptic filtration will increase the sterility assurance level of the product. The inventors of present invention believe that the stabilizing and pH modifying agent Tromethamine will improve solubility Gemcitabine or its pharmaceutically acceptable salts and prevent its crystallization during storage period.

Finished product was stored at different stability conditions as per ICH guideline. The initial sample of ready to use solution and samples stored at different stability conditions were analysed for assay, related substance (%), pH, absorbance at 420nm, % transmittance at 650 nm and osmolality. The solution is chemically stable having impurity levels within the acceptable pharmacopoeial and ICH limits. The results of the representative examples are given in Table 2 and Table 3.

Table 2: Stability results of Ready to use Gemcitabine Injection, 10 mg/mL, 100 mL Fill volume & Pack: 100 mL Vial (Example 1)

Conations/Tests Description Assay of Gemcitabine Related substances Absorbance at 420 nm % transmittance at 650 nm pH Osmolality Particulate matter
Cytosine
Gemcitabine a- anomer 2'-Deoxy-2',2' difluorouridine Highest unknown Impurity Total Impurities
Limits Clear colourless solution
NLT 95.0 % and NMT 105.0 % NMT 0.1 % NMT 0.1 % NMT 1.5 % NMT 0.2 % NMT 2.0 % NMT 0.1 AU NLT 95 % Between 8.0 to 9.0 250- 350 mOsm/L =10 µm: max 6000 per container
=25 µm: max 600 per container
Initial Clear colourless solution
99.8 BDL 0.006 0.178 0.029 0.218 0.0005 100.003 8.50 276 Complies as per USP
40°C± 2°C /75% ±5% RH
1M Clear colourless solution
100.4 BDL 0.005 0.397 0.007 0.420 0.0020 99.941 8.32 272 Complies as per USP
2M Clear colourless solution 100.1 0.001 0.008 0.552 0.006 0.572 0.0050 99.920 8.54 272 Complies as per USP
3M Clear colourless solution 99.0 0.001 0.009 0.627 0.006 0.652 0.0022 99.257 8.51 276 Complies as per USP
6M Clear colourless solution 98.2 BDL 0.014 1.185 0.006 1.205 0.0059 100.124 8.53 275 Complies as per USP
25°C± 2°C /60% ±5% RH
3M Clear colourless solution 99.8 BDL 0.005 0.271 0.006 0.290 0.0030 100.339 8.49 274 Complies as per USP
6M Clear colourless solution 99.7 BDL 0.006 0.369 0.005 0.381 0.0015 99.927 8.52 276 Complies as per USP
9 M Clear colourless solution 98.3 BDL 0.006 0.455 0.006 0.472 0.0021
99.881 8.48 274 Complies as per USP
12 M Clear colourless solution 98.3 BDL 0.009 0.445 0.036 0.516 0.0001 99.956 8.51 275 Complies as per USP
30°C± 2°C /75% ±5% RH
3M Clear colourless solution 100.1 0.001 0.007 0.371 0.006 0.388 0.0066 99.409 8.51 276 Complies as per USP
6M Clear colourless solution 99.6 BDL 0.008 0.574 0.005 0.587 0.0043 100.120 8.56 276 Complies as per USP
9 M Clear colourless solution 98.5 BDL 0.008 0.727 0.097 0.846 0.0135 99.767 8.47 275 Complies as per USP
12 M Clear colourless solution 98.3 BDL 0.011 0.764 0.006 0.784 0.0028 99.911 8.55 270 Complies as per USP

NLT: Not Less Than, NMT: Not More Than, BDL: Below Detection Limit

Table 3: Stability results of Ready to use Gemcitabine Injection, 10 mg/mL, 100 mL Fill volume & Pack: 100 mL Vial (Example 5)

Conations/Tests Description Assay of Gemcitabine Related substances Absorbance at 420 nm % transmittance at 650 nm pH Osmolality Particulate matter
Cytosine
Gemcitabine a- anomer 2'-Deoxy-2',2' difluorouridine Highest unknown Impurity Total Impurities
Limits Clear solution NLT 95.0 % and NMT 105.0 % NMT 0.1 % NMT 0.1 % NMT 1.5 % NMT 0.2 % NMT 2.0 % NMT 0.1 AU NLT 95 % Between 8.0 to 9.0 250- 350 mOsm/L =10 µm: max 6000 per container
=25 µm: max 600 per container
Initial Clear colourless solution 98.5 0.006 0.012 0.252 0.151 0.452 0.0064 99.628 8.34 295 Complies as per USP
40°C± 2°C /75% ±5% RH
1M Clear colourless solution 98.0 0.008 0.011 0.419 0.007 0.454 0.0005 99.409 8.21 295 Complies as per USP
2M Clear colourless solution 96.1 0.007 0.015 0.550 0.011 0.603 0.0013 100.256 8.23 294 Complies as per USP
3M Clear colourless solution 96.3 0.007 0.012 0.737 0.008 0.770 0.0060 100.200 8.26 295 Complies as per USP
6M Clear colourless solution 96.1 BDL 0.017 1.021 0.013 1.082 0.0099 100.018 8.39 295 Complies as per USP
25°C± 2°C /60% ±5% RH
3M Clear colourless solution 97.5 0.006 0.01 0.346 0.006 0.375 0.0005 99.409 8.21 295 Complies as per USP
6M Clear colourless solution 96.0 BDL 0.011 0.362 0.010 0.418 0.0086 99.864 8.31 295 Complies as per USP
9M Clear colourless solution 97.9 0.006 0.009 0.434 0.01 0.471 0.0032 99.949 8.21 291 Complies as per USP
12M Clear colourless solution 99.5 0.006 0.012 0.521 0.01 0.551 0.0022 99.276 8.20 290 Complies as per USP
30°C± 2°C /75% ±5% RH
3M Clear colourless solution 96.9 0.006 0.011 0.423 0.01 0.464 0.0005 99.409 8.21 295 Complies as per USP
6M Clear colourless solution 97.2 BDL 0.012 0.500 0.017 0.572 0.0099 100.016 8.34 295 Complies as per USP
9M Clear colourless solution 98.9 0.007 0.011 0.653 0.009 0.691 0.0032 99.873 8.26 293 Complies as per USP
12M Clear colourless solution 97.6 0.007 0.014 0.857 0.008 0.893 0.0007 98.945 8.24 290 Complies as per USP

NLT: Not Less Than, NMT: Not More Than, BDL: Below Detection Limit

,CLAIMS:We Claim:

1. A stable ready-to-use injectable formulation comprising Gemcitabine or pharmaceutically acceptable salt, tromethamine as stabilizing and pH modifying agent and optionally other pharmaceutically acceptable excipients.

2. The stable ready-to-use injectable formulation as claimed in claim 1, wherein the solution can be infused without any prior dilutions before administration.

3. The stable ready-to-use injectable formulation as claimed in claims 1 and 2 which is stable at a temperature between 2°C to room temperature.

4. The stable ready-to-use injectable formulation as claimed in claims 1 to 3, wherein concentration of tromethamine used in stable ready-to-use injectable formulation is from 0.001 to 1.21 % (w/v) and most preferably used concentration of tromethamine is from 0.003 to 0.12 % (w/v).

5. The stable ready-to-use injectable formulation as claimed in claims 1 to 4, wherein the formulation comprise tonicity adjusting agents, pH modifying agents, aqueous vehicle as other pharmaceutically acceptable excipients.

6. The stable ready-to-use injectable formulation as claimed in claim 5, wherein the tonicity adjusting agent is selected from sodium chloride, calcium chloride, magnesium chloride, sodium lactate, glucose, fructose, sorbitol, mannitol, galactose, inositol, maltitol, lactose, trehalose, maltose, sucrose, dextran or organic solvents such as ethanol, glycerine, sorbitol, propylene glycol or combinations thereof.

7. The stable ready-to-use injectable formulation as claimed in claim 6, wherein the concentration of tonicity adjusting agents used in stable ready-to-use injectable formulation is from 0.25 to 3.0% (w/v).

8. The stable ready-to-use injectable formulation as claimed in claim 3, wherein the pH modifying agent is selected from inorganic acids and bases, such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, phosphorous acid, carbonic acid, sulfurous acid, nitrous acid, ascorbic acid, propionic acid, lactic acid, citric acid, formic acid, oxalic acid, benzoic acid, acetic acid, tartaric acid, malonic acid, maleic acid, pyruvic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, sodium hydroxide, calcium hydroxide, potassium hydroxide and magnesium hydroxide, sodium dihydrogen phosphate and its hydrates, sodium phosphate and its hydrates, disodium hydrogen phosphate and its hydrates, disodium sulfate, sodium hydrogen sulfate, sodium sulfite, calcium sulfite, magnesium sulfite, sodium carbonate, sodium hydrogen carbonate, calcium hydrogen carbonate, sodium nitrite, sodium nitrate, magnesium nitrate, calcium nitrite and magnesium nitrite, 1-deoxy-(methylamino)-D-glucitol (N-methylglucamine, meglumine).

9. The stable ready-to-use injectable formulation as claimed in claims 1 to 8, wherein the concentration of Gemcitabine or its pharmaceutically acceptable salts is 10 mg/ml and the solution having a pH of about 8.0 to 9.0.

10. A stable ready-to-use injectable formulation as claimed in claims 1 to 9, wherein the formulation comprising:
(a) 0.1% to 1% (w/v) of Gemcitabine or its salt,
(b) 0.001% to 1.21% (w/v) of tromethamine as stabilizing and pH modifying agent,
(c) 0.25% to 3% (w/v) of tonicity adjusting agents,
(d) quantity sufficient of pH adjusting agents, and
(e) aqueous vehicle.

11. The stable ready-to-use injectable formulation as claimed in claims 1 to 10, wherein the formulation comprising Gemcitabine or pharmaceutically acceptable salt as active ingredient, tromethamine as stabilizing and pH modifying agent, sodium chloride or glycerine as tonicity adjusting agents, sodium hydroxide and hydrochloric acid as pH adjusting agents and water for injection as aqueous vehicle.

12. The stable ready-to-use injectable formulation as claimed in claims 1 to 10, wherein the infusion container does not contain an aluminium over-pouch as secondary packaging.

13. A method for the preparation of stable ready-to-use injectable infusion container formulation comprising Gemcitabine or pharmaceutically acceptable salt, wherein sterilization involves aseptic filtration process followed by heat treatment which avoids terminal sterilization.

14. The method for the preparation of stable ready-to-use injectable formulation as claimed in claim 13, wherein the method comprise:
(a) adding tromethamine and tonicity modifying agents to 80% of water and stirred to get a clear solution,
(b) adding Gemcitabine hydrochloride to obtained solution of step (a) under stirring until it dissolves,
(c) adjusting pH of obtained solution of step (b) to 8.0 to 9.0 using pH adjusting agents,
(d) diluting the solution with remaining amount of water to get a stable solution,
(e) adjusting pH of stable solution of step (d) to 8.0 to 9.0 with pH adjusting agents, if required,
(f) filtering the solution aseptically through Nylon 66 Filter (0.2 micron) and filled into the stoppered glass container, and
(g) heat treatment at a temperature of 100°C to 105°C for a period of time ranging from 30 to 60 minutes after final packaging.

Dated this Thirteenth (13th) day of August, 2020

__________________________________
Dr. S. Padmaja
Agent for the Applicant
IN/PA/883