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

FIELD OF THE INVENTION

Also provided in the present application, a stable lyophilized formulation of 5-Azacytidine Form I, and methods of preparing said lyophilized formulation.

BACK GROUND OF THE INVENTION

5-azacytidine (also known as 4-amino-l-P-D-ribofuranosyl-l, 3, 5-triazin-2(lH)-one) has the structure (I) as mentioned below - It is reported to be insoluble in acetone, ethanol, and methyl ethyl ketone, slightly soluble in ethanol and water (50:50), propylene glycol, and polyethylene glycol, sparingly soluble in water, water saturated octanol, 5% dextrose in water, N-methylpyrrolidone, normal saline, and 5% Tween™ 80 in water, and soluble in dimethylsulfoxide (DMSO). Azacitidine is used in the treatment of myelodysplastic syndrome.

5-azacytidine is believed to exert its antineoplastic effects by causing hypomethylation of DNA and direct cytotoxicity on abnormal hematopoietic cells in the bone marrow. Hypomethylation may restore normal functions to genes that are critical for differentiation and proliferation. The cytotoxic effects of azacitidine cause the death of rapidly dividing cells, including cancer cells that are no longer responsive to normal growth control mechanism. Non-proliferating cells are relatively insensitive to azacitidine.

5-Azacitidine (or Azacitidine) underwent NCI-sponsored clinical trials for the treatment of myelodysplastic syndromes (MDS). See Komblith et al., J. Clin. Oncol. 20(10): 2441-2452 (2002) and Silverman et al., J. Clin. Oncol. 20(10): 2429-2440 (2002).

Now, it is commercially available as product containing Azacitidine and is sold as VIDAZA , 5-azacytidine for injection, by Celgene. The VIDAZA® product received marketing approval in the USA in 2004 and is supplied in sterile lyophilized form for reconstitution as a suspension for subcutaneous injection, or reconstitution as a solution with further dilution for intravenous infusion. Vials of the VIDAZA® product contain 100 mg of azacitidine and 100 mg of mannitol, as a sterile lyophilized powder.

5-Azacytidine is approved for subcutaneous (SC) or intravenous (IV) administration to treat various proliferative disorders. It has been also reported that s-triazine ring of 5-azacytidine has a particular sensitivity towards water as Azacitidine rapidly degrades in aqueous solution via hydrolysis. See, e.g., Beisler, J. Med. Chem., 1978, 21(2), 204-08; Chan, et al., J. Pharm. Sci., 1979, 68(7), 807-12. In an aqueous environment both in vivo and in vitro, 5-azacytidine underwent a spontaneous hydrolysis and resulted in an equilibration with a labile product, n-formylguanyl-ribosylurea, and finally the irreversible formation of guanyl-ribosylurea. Due to this instability, an aqueous formulation was not a viable option to be explored as formulation. Hence, a lyophilized dosage form was developed to minimize water activity in the medicinal product. To minimize azacitidine degradation during product manufacturing, the manufacturing process was developed such that compounding, filtration and filling operations are performed as a continuous process at reduced temperatures.

Dumitru et al in US 6, 887,855 disclosed that 5-azacytidine exists in at least eight different polymorphic and pseudopolymorphic crystalline forms (Forms I-VIII), in addition to an amorphous form. Form I is a polymorph found in prior art retained samples of 5-azacytidine drug substance. Form II is a polymorph found in some prior art retained samples of the 5-azacytidine drug substance; in those samples, Form II is always found in mixed phase with Form I. Form III is a hydrate, and is formed when prior art retained and current samples of the drug product are reconstituted with water to form a "slurry" prior to administration to the patient. Form VI is found in prior art retained samples of the 5-azacytidine drug product, either substantially free of other polymorphs, or in mixed phase with Form I. The invention provides novel crystalline forms referred to as Form IV, Form V, Form VII and Form VIII. Forms I-VIII each has characteristic X-ray power diffraction (XRPD) patterns and is easily distinguished from one another using XRPD.

Dumitru et al in US 7,078,518 disclosed 5-azacytidine Forms IV, V, VI, and mixtures of form I and VII, are prepared by recrystallization processes that include dissolving 5-azacytidine in dimethylsulfoxide, and at least one co solvent is added to the solution of 5-azacytidine facilitating the crystallization; wherein the co solvents is toluene, methanol or chloroform.

Alexander et al in WO2008/088779A2 describes pure crystalline form V of 5-azacytidine is prepared by a process comprising lyophilizing a solution of 5-azacytidine in dimethylsulfoxide. The described process involves 5-azacytidine and dimethylsulfoxide, and heating the combination. Preferably, the heating is to a temperature of about 50°C to about 130°C, more preferably at about 70°C to about 80°C. Preferably, freezing the solution is done gradually. First, cooling to a temperature of about 30°C is done, and then cooling to a temperature of about is -18°C to about 30°C, is performed, providing a frozen solution. Typically, the evaporation of the solvents is done at about -18°C to about 30°C. Preferably, evaporation of the solvent is done under reduced pressure (less than one atmosphere). Preferably, the reduced pressure is used in the range of about 0.01 to 100 mBar, more preferably at about 0.1 to about 3 mBar.

Dumitru et al in US 6,943,249 disclosed methods for isolating crystalline Form I of 5-azacytidine substantially free of other forms, and also pharmaceutical compositions comprising Form I of 5-azacytidine. The method includes isolating crystalline Form I of 5-azacytidine substantially free of other forms, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising at least one primary solvent and at least one co-solvent selected from the group consisting of C2 - C5 alcohols, aliphatic ketones, and alkyl cyanides, by cooling said solvent mixture from a temperature selected to allow said 5-azacytidine to dissolve completely to about ambient temperature; and isolating the recrystallized 5-azacytidine.

Arnold et al in US 4,684,630 discloses a method of intravenously injecting an aqueous unstable anticancer agent into a warm-blooded mammal, the agent being selected from the group consisting of 5-azacytosine arabinoside and 5-azacytidine, comprising in combination the following steps:

(a) aqueously diluting a stable, anhydrous organic solution to form an organic-aqueous solution, the organic solution consisting of the agent and a highly water soluble organic solvent selected from the group consisting of dimethylsulfoxide and dimethylacetamide, the organic-aqueous solution having the organic solvent present in a concentration of less than about 5% based on the total weight of the organic-aqueous solution, the organic-aqueous solution being physiologically suitable for intravenous injection into the warm-blooded mammal, the agent being present in the organic-aqueous solution in an effective dosage concentration per unit volume of approximately 1 mg./ml.; and

(b) intravenously injecting the organic-aqueous solution into the warm-blooded mammal; the dilution step occuring immediately prior to the intravenous injection step.

U.S. Publication No.2011/0042247 disclose a pharmaceutical formulation of azacitidine, prepared by a process comprising preparing an aqueous solution containing azacitidine at about -3°C to about -1°C; and lyophilizing the solution.

Anthony et al in U.S. Publication No. 2012/0196823 disclose a liquid pharmaceutical composition comprising a cytidine analog selected from 5-azacytidine and Decitabine, and cold sterile water, which is substantially free of impurities.

Kim, Je Hak et al in WO2013/012135 disclose a pre-freeze dried azacitidine preparation, in which azacitidine is dissolved in an aqueous solution comprising 40 to 60 (v/v) % of tertiary butanol, and a method of manufacturing the freeze dried azacitidine preparation. The pre-freeze dried azacitidine preparation of the present invention has improved stability in an aqueous solution. When the pre-freeze dried azacitidine preparation is frozen and dried under predetermined process conditions including a freeze drying cycle, a stability-improved freeze dried azacitidine preparation can be usefully produced.

Khattar, Dhiraj et al in WO2013/117969 disclose a process of preparing a stable pharmaceutical composition of compounds which are susceptible to hydrolysis comprising a) addition of required quantity of pharmaceutically acceptable lyophilization excipients optionally in Water for Injection in a formulation vessel; b) addition of organic solvent to form a appropriate proportion of aqueous and organic solvent; c) maintaining the temperature of the formulation vessel from the range -5±1°C to - 5±3°C; d) addition of required quantity of compound susceptible to hydrolysis to form a solution and lyophilizing the solution.

Despite various disclosures, there still a need remains for a simple process of preparing a stable lyophilized formulation of 5-Azacytidine, avoiding low temperature settings (like less than 0°C Eg: -5±1°C to - 5±3°C), for liquid filling step before lyophilization, as low temperature maintenance is cumbersome and known to be a complicated procedure & requires expensive facility establishments, especially, for higher batch-sizes; further, a need remains too for a simple process which avoids the low temperature settings of liquid filling, as well as rule out the chances of degradation of 5-Azacytidine due to hydrolysis, during the process of preparing a lyophilized formulation of 5-Azacytidine.

The need remains for a simple process of preparing a chemically stable lyophilized formulation of 5-Azacytidine which potentially permit, inter alia, a convenient and hassle-free processing, during the manufacture of lyophilized formulation of 5-Azacytidine, and consistently ensure ease manufacturing of lower/higher batch-size (like lab-scale/exhibit/commercial batch scale) ranges of lyophilized Azacytidine powder for injection, with high standards of product quality and purity, having suitable impurity profile to minimize potential toxicity, and ultimately provides an accurate delivery of intended dose of cytidine analogs for treating new diseases or disorders or new patient populations; and/or other potential advantageous benefits.

SUMMARY OF THE INVENTION

Surprisingly, the present invention is directed to a pharmaceutical composition comprising 5-Azacytidine, mannitol, and dimethyl sulfoxide.

The present invention is directed to a pre-lyophilized composition comprising 5-Azacytidine, mannitol, and dimethyl sulfoxide.

The present invention is directed to a stable lyophilized composition comprising 5-Azacytidine and mannitol, derived from a pre-lyophilized composition comprising 5-Azacytidine, mannitol, and dimethyl sulfoxide.

Aspects of the present invention relates to a process of preparing a stable pharmaceutical composition comprising crystalline 5-Azacytidine Form I, characterized by the steps:

i) dissolving mannitol and 5-azacytidine in dimethyl sulfoxide.

ii) filtering the step i) solution.

iii) filling the filtered liquid into vials at temperature ranging between 10- 30°C, and

iv) lyophilizing the solution obtained from step (iii); wherein the pharmaceutical composition having not more than 0.2%w/w of known impurities and not more than l%w/w of total impurities, at release.

Aspects of the present invention relates to a process of preparing a stable pharmaceutical composition comprising crystalline 5-Azacytidine Form I, characterized by the steps:

i) dissolving mannitol and 5-azacytidine Form I, in the range of about 5 to 100 mg/ml, in dimethyl sulfoxide.

ii) filtering the step i) solution.

iii) filling the filtered liquid into vials at temperature ranging between 10-30°C, and

iv) lyophilizing the solution obtained from step (iii); wherein the pharmaceutical composition having not more than 0.2%w/w of known impurities and not more than l%w/w of total impurities, at release.

Aspects of the present invention relates to a process of preparing a stable pharmaceutical composition comprising crystalline 5-Azacytidine Form I, characterized by the steps:

i) dissolving mannitol and 5-azacytidine Form I, in the range of about 5 to 100 mg/ml, in dimethyl sulfoxide.

ii) filtering the step i) solution.

iii) filling the filtered liquid into vials at temperature ranging between 10- 30°C, and

iv) lyophilizing the solution obtained from step (iii); wherein the pharmaceutical composition having not more than 0.2%w/w of known impurities and not more than l%w/w of total impurities, when stored atleast for 40°C/75%RH for 3 months.

Aspects of the present invention relates to a stable lyophilized composition of 5-Azacytidine, having water content of the not more than l%w/w; derived from dimethyl sulfoxide solution comprising 5-Azacytidine & mannitol.

Aspects of the present invention, relates to a stable lyophilized composition comprising Crystalline 5-Azacytidine Form I for parenteral administration, in a sterile vessel is provided, for administration to a subject in need thereof.

Aspects of the present invention, herein relates to methods of using a stable lyophilized formulation comprising Crystalline 5-Azacytidine Form I for parenteral administration, provided herein to treat diseases or disorders.

BRIEF DESCRIPTION OF THE DRAWING

Fig.l is Illustration of X-ray powder diffraction (XRPD) pattern ,of Crystalline 5-Azacytydine Form I.

Fig.2 is Illustration of X-ray powder diffraction (XRPD) pattern of 5-Azacytidine lyophilized formulation of example 1.

Fig.3 is Illustration of X-ray powder diffraction (XRPD) pattern of placebo lyophilized formulation of example 2.

Comparative X-Ray powder diffraction patterns of: Crystalline 5-Azacytydine Form I, Example-1 5-Azacytydine lyophilized formulation & Example-2 placebo lyophilized formulation, provided hereunder the table below:

DETAILED DESCRIPTION OF THE INVENTION

In embodiment of the present invention, provides a pharmaceutical composition comprising 5-Azacytidine, mannitol, and dimethyl sulfoxide.

In embodiment of the present invention, provides a pre-lyophilized composition comprising 5-Azacytidine, mannitol, and dimethyl sulfoxide.

In embodiment of the present invention, provides a stable lyophilized composition comprising 5-Azacytidine and mannitol, derived from a pre-lyophilized composition comprising 5-Azacytidine, mannitol, and dimethyl sulfoxide.

In embodiment of the present invention, provides a process of preparing a stable pharmaceutical composition comprising crystalline 5-Azacytidine Form I, characterized by the steps:

i) dissolving mannitol and 5-azacytidine in dimethyl sulfoxide.

ii) filtering the step i) solution.

iii) filling the filtered liquid into vials at temperature ranging between 10-30°C, and

iv) lyophilizing the solution obtained from step (iii); wherein the pharmaceutical composition having not more than 0.2%w/w of known impurities and not more than l%w/w of total impurities, at release.

In embodiment of the present invention, provides a process of preparing a stable pharmaceutical composition comprising crystalline 5-Azacytidine Form I, characterized by the steps:

i) dissolving mannitol and 5-azacytidine in dimethyl sulfoxide.

ii) filtering the step i) solution.

iii) filling the filtered liquid into vials at about 25°C, and

iv) lyophilizing the solution obtained from step (iii);

wherein the pharmaceutical composition having not more than 0.2%w/w of known impurities and not more than l%w/w of total impurities, at release.

In embodiment of the present invention, provides a process of preparing a stable pharmaceutical composition comprising crystalline 5-Azacytidine Form I, characterized by the steps:

i) dissolving mannitol and 5-azacytidine Form I, in the range of about 5 to 100 mg/ml, in dimethyl sulfoxide.

ii) filtering the step i) solution.

iii) filling the filtered liquid into vials at temperature ranging between 10-30°C, and

iv) lyophilizing the solution obtained from step (iii); wherein the pharmaceutical composition having not more than 0.2%w/w of known impurities and not more than l%w/w of total impurities, at release.

In embodiment of the present invention, provides a process of preparing a stable pharmaceutical composition comprising crystalline 5-Azacytidine Form I, characterized by the steps:

i) dissolving mannitol and 5-azacytidine Form I, in the range of about 5 to 100 mg/ml, in dimethyl sulfoxide.

ii) filtering the step i) solution.

iii) filling the filtered liquid into vials at temperature ranging between 10-30°C, and

iv) lyophilizing the solution obtained from step (iii); wherein the pharmaceutical composition having not more than 0.2%w/w of known impurities and not more than l%w/w of total impurities, when stored atleast for 40°C/75%RH for 3 months.

In embodiment of the present invention, provides a process of preparing a stable pharmaceutical composition comprising crystalline 5-Azacytidine Form I, characterized by the steps:

i) dissolving mannitol and 5-azacytidine Form I, in the range of about 5 to 100 mg/ml, in dimethyl sulfoxide.

ii) filtering the step i) solution through 0.2|i filter.

iii) filling the filtered liquid into vials at temperature ranging between 10-30°C, and

iv) lyophilizing the solution obtained from step (iii);

wherein the pharmaceutical composition having not more than 0.2%w/w of known impurities and not more than l%w/w of total impurities, at release.

In embodiment of the present invention, provides a stable lyophilized composition of 5-Azacytidine, having water content of the not more than l%w/w; derived from dimethyl sulfoxide solution comprising 5-Azacytidine & mannitol.

The term "release" or "at release" means the finished drug product has met the release specifications and can be used for its intended pharmaceutical purpose.

The term "pharmaceutical composition" as used herein shall mean a composition that is made under conditions such that it is suitable for administration to humans, e.g., it is made under GMP conditions and contains pharmaceutically acceptable excipients, e.g., without limitation, stabilizers, bulking agents, buffers, carriers, diluents, vehicles, solubilizers, and binders. As used herein pharmaceutical composition includes but is not limited to a pre-lyophilization solution or dispersion as well as a liquid form ready for injection or infusion after reconstitution of a lyophilized preparation.

By "stable pharmaceutical composition" is meant any pharmaceutical composition having sufficient stability to have utility as a pharmaceutical product. Preferably, a stable pharmaceutical composition has sufficient stability to allow storage at a convenient temperature, preferably between -20°C. and 40°C, more preferably about 2°C to about 30°C, for a reasonable period of time, e.g., the shelf-life of the product which can be as short as one month but is typically six months or longer, more preferably one year or longer even more preferably twenty-four months or longer, and even more preferably thirty-six months or longer. The shelf-life or expiration can be that amount of time where the active ingredient degrades to a point below 90% purity. For purposes of the present invention stable pharmaceutical composition includes reference to pharmaceutical compositions with specific ranges of impurities as described herein. Preferably, a stable pharmaceutical composition is one which has minimal degradation of the active ingredient, e.g., it retains at least about 85% of un-degraded active, preferably at least about 90%, and more preferably at least about 95%, after storage at 2-30°C for a 2-3 year period of time.

The term "formulation" as used in the context of the present invention refers to any of various dosage forms suitable for administration of a drug, such as parenterally, intraperitoneally, intravenously, intraarterially, intramuscularly, subcutaneously, etc.

"Known impurities" refer to any of impurity-1, impurity-2, impurity-3, RGU & RGU-CHO.

"Total impurities" refer to sum of all known impurities like "Impurity-1", "Impurity-2", "Impurity-3" and RGU; but excluding RGU-CHO.

The term "chemical stability" or "chemically stable" as referred in the specification relates to maintaining an original drug purity of the formulation, in terms of drug-related impurities or drug-related substances.

In embodiment of the present invention, provides methods of using a stable lyophilized composition comprising Crystalline 5-Azacytidine Form I for parenteral administration, provided herein to treat diseases or disorders.

In embodiment of the present invention, provides a stable lyophilized formulation comprising Crystalline 5-Azacytidine Form I for parenteral administration, in a sterile vessel is provided, for administration to a subject in need thereof. The sterile vessel comprising a pharmaceutical formulation according to the present invention; for example, may be a vial, syringe, or ampoule.

In embodiment of the present invention provides methods of using a stable lyophilized formulation comprising Crystalline 5-Azacytidine Form I for parenteral administration, provided herein to treat diseases or disorders including, e.g., cancer, disorders related to abnormal cell proliferation, hematologic disorders, or immune disorders, among others. In certain embodiments, the pharmaceutical compositions of 5-azacytidine which are parenterally administered to subjects in need thereof to treat a cancer or a hematological disorder, such as, for example, Myelodysplastic syndromes (MDS), acute myelogenous leukemia (AML).

In another embodiment of the present invention, provides a stable lyophilized composition of 5-azacytidine, which is reconstituted with 4ml or 10ml of sterile water for injection.

In another embodiment of the present invention, provides a method of administering therapeutically effective amount of 5-azacytidine, from reconstituted 5-azacytidine solution, for treating medical conditions selected from Myelodysplastic syndromes, Chronic myelomonocytic leukaemia and Acute myeloid leukaemia.

In another embodiment of the present invention, provides a stable lyophilized formulation of 5-Azacytidine Form I, wherein the assay value of 5-Azacytidine is NLT 90%w/w and NMT 110%w/w.

In the context and the description of the present invention, the term "DMSO" referred to as Dimethyl sulfoxide.

In the context and the description of the present invention, the terms "NLT" means not less than, and "NMT" means not more than, the specified values.

5-azacytidine hydrolyzes quickly in water, and this is dependent on pH and temperature. It has been observed that, due to hydrolysis, around nine solid state forms have been identified: five polymorphic forms, three psueodpolymorphic forms and an amorphous form. Polymorphism could be of importance since speed of dissolution of azacitidine could affect its degradation. Azacitidine rapidly degrades in aqueous solutions via hydrolysis, and due to this instability a lyophilized dosage form was developed to minimize water activity in the dosage form. Hence, the water content of a formulation may impact the stability of the product.

Two major degradants have been observed due to hydrolysis. The hydrolytic pathway leads to the formation of an initial N-formyl compound hydrolysis product "RGU-CHO," which is a reversible reaction and the compounds are in equilibrium with each other. This is followed by ring opening and loss of formic acid which results in formation of an amine compound "RGU," which is an irreversible reaction. RGU-CHO is N-(formylamidino) N'-P-D-ribofuranosylurea ("N-formyl compound" below) and RGU is l-p-D-ribofuranosyl-3-guanylurea ("amine compound" below).

The term "Impurity-1" as referred in the specification relates to 4-amino-l, 3, 5-triazine-2-(lH)-one or also known as 5-Azacytosine.

The term "Impurity-2" as referred in the specification relates to l-O-Acetyl-2, 3, 5-Tri-O-benzoyl- P -D-ribofuranose.

The term "Impurity-3" as referred in the specification relates to l-(2, 3, 5-Tri-O-benzoyl-P-D-ribifuranosyl)-4-amino-l, 2-dihydro-l, 3, 5-triazin-2-one.

RGU = 1- P -D-ribofuranosyl- 3-guanylurea.

RGU-CHO = N-(formylamidino) N'-P-D-ribofuranosylurea.

The term "Known impurities" refer to any of impurity-1, impurity-2, impurity-3, RGU & RGU-CHO.

The term "Total impurities" as referred in the specification relates to sum of all known impurities like "Impurity-1", "Impurity-2", "Impurity-3" and RGU; but excluding RGU-CHO.

The term "water for injections or WFI" as referred in the specification relates to distilled or sterile water for injection.

Injectable formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solubilization or suspending in liquid prior to injection, or as emulsions. Sterile injectable formulations can be prepared according to techniques known in the art using suitable carriers, dispersing or wetting agents, and/or suspending agents. The injectable formulations may be sterile injectable solutions or suspensions in a nontoxic, parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media.

The technique known as lyophilization is sometimes employed to process injectable pharmaceuticals that exhibit poor active ingredient stability in aqueous solutions. Lyophilization processing is suitable for injectables because it can be conducted under sterile conditions, which is a primary requirement for parenteral dosage forms. Cryoprotectants are excipients whose primary function is to protect the active constituent during a freezing process. Cryoprotectants in the present invention include bulking agents that may be used in the invention are: mannitol, sorbitol, lactose, sucrose, or any mixtures of two or more thereof.

Lyophilization or freeze-drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from a solid to a vapor, without passing through a liquid phase. The process consists of three separate, unique, and interdependent processes; a freezing phase, a primary drying phase (sublimation), and a secondary drying phase (desorption). These processes may be optimized to enhance the product stability as well as decrease the manufacturing costs.

The appropriate vial sizes for filling the filtered bulk solution is 30mL/20mm flint moulded vial, and fill volume ranges from about 1ml to 25ml.

Freezing Phase:

A primary function of the freezing phase is to ensure that the entire container having the complex solution is completely frozen, prior to proceeding to a subsequent phase. Additionally, it is usually desired that these containers freeze in a uniform manner. While there are different ways that this can be accomplished, one option is to chill the containers after they are loaded onto the Iyophilizer shelves and holding for 30-60 minutes prior to initiation of the freezing cycle. It is generally not practical to equilibrate the shelves to a freezing temperature, because of frost accumulation during the filling and loading of the containers.

Primary Drying Phase:

Once the formulation is brought to the desired frozen state, primary drying via sublimation can proceed. The primary drying phase involves the removal of bulk water or solvent, at a product temperature below the ice transition temperature under a vacuum (pressures typically between 50-300 mTorr). This phase can be a critical one for stabilizing an active. The goal is to identify the glass transition temperature (Tg') for the formulation. The Tg' is the temperature at which there is a reversible change of state between a viscous liquid and a rigid, amorphous glassy state. One can measure the Tg' of candidate formulations using a differential scanning calorimeter (DSC), in particular with modulated DSC. Generally, the collapse temperature is observed to be about 2-5°C greater than the Tg'. Hence, the shelf temperature is set such that the target product temperature is maintained near or below the Tg' of the formulation throughout the removal of solvent during the primary dry phase.

As the solvent is progressively removed from the formulation containers, the product temperature will approach and reach the shelf temperature since it is no longer cooled by water sublimation. To optimize the duration of the primary dry phase, the removal of solvent vapor can be tracked using a moisture detector, or by monitoring the decrease in pressure difference between a capacitance manometer and a thermocouple pressure gauge or by a pressure drop measurement. The optimization of the primary dry cycle involves a removal of solvent as quickly as possible without causing cake collapse and subsequent product instability.

Secondary Drying Phase:

The secondary drying phase is the final segment of the lyophilization cycle, where residual moisture is removed from a formulation's interstitial matrix by desorption with elevated temperatures and/or reduced pressures. The final moisture content of a lyophilized formulation, which can be measured by Karl Fischer or other methods, is important because if the solid cake contains too much residual moisture, the stability of the active can be compromised. Hence, it is imperative that one achieves a moisture level as low as possible.

To accomplish a low residual moisture, the shelf temperature is typically elevated to accelerate desorption of water molecules. The duration of the secondary drying phase is usually short. When microstructure collapse occurs, the residual moisture is generally significantly greater than desired. One alternative is to purge the sample chamber of the lyophilizer with alternating cycles of an inert gas such as nitrogen, to facilitate displacement of bound water. However, another solution is to properly formulate the drug product and run an optimal lyophilization cycle.

The advantages of lyophilization include: ease of processing a liquid, which simplifies aseptic handling; enhanced stability of a dry powder; removal of water without excessive heating of the product; enhanced product stability in a dry state; and rapid and easy dissolution of reconstituted product. The product is dried without elevated temperatures, thereby eliminating adverse thermal effects, and then stored in the dry state in which there are relatively few stability problems.

Additionally, freeze dried products are often more soluble, dispersions are stabilized, and products subject to degradation by oxidation or hydrolysis are protected.

Pharmaceuticals to be freeze dried are frequently in aqueous solutions, ranging from about 0.01 to 40% by weight concentrations of total solids. Usually, an improvement in stability of the lyophilizate, compared to a solution, is due to the absence of water in the lyophilizate.

A pharmacologically active constituent of many pharmaceutical products is present in such small quantities that, if freeze dried alone, it may not give a composition of suitable mass, and in some cases its presence would be hard to detect visually. Therefore, excipients are often added to increase the amount of solids present. In most applications it is desirable for a dried product cake to occupy essentially the same volume as that of the original solution. To achieve this, the total solids content of the original solution is frequently about 10 to 25% by weight.

Bulking substances that are useful for this purpose, often in combination, include, but are not limited to, sodium or potassium phosphates (monobasic potassium phosphate, potassium dihydrogen phosphate, etc.), citric acid, tartaric acid, gelatin, lactose and other carbohydrates such as dextrose, mannitol and dextran, and occasionally preservatives. Various excipients contribute appearance characteristics to the cake, such as dull and spongy, sparkling and crystalline, firm or friable, expanded or shrunken, and uniform or striated. Therefore formulations of a composition to be freeze dried should be a result of consideration not only of the nature and stability characteristics required during the liquid state, both freshly prepared and when reconstituted before use, but also the characteristics desired in the final lyophilized cake.

In aspects the invention includes kits provided for delivery of the azacitidine or its salts. A kit according to the present invention comprises a container holding a stable lyophilized formulation comprising Crystalline 5-Azacytidine Form I, a sterile reconstitution vehicle, and a sterile syringe.

Certain specific aspects and embodiments of the invention will be further described in the following examples, which are provided only for purposes of illustration and are not intended to limit the scope of the invention in any manner.
 
Example 1:

Preparation of 5-Azacytidine lyophilized powder for injection lOOmg/vial

Brief method of preparation:

1. 80% batch quantity of DMSO was collected in a clean beaker or Duran Bottle.

2. Weighed batch quantity of Mannitol was added to step 1 and stirred to get a clear solution.

3. Weighted batch quantity of 5-Azacitidine Form I was added to step 2 and stirred to get a clear solution.

4. The final volume of the bulk solution is made up with remaining quantity of DMSO and the solution stirred for 5 mins.

5. The final bulk solution of step 4 is filtered through 0.2\i filter.

6. The vials are half stoppered and loaded into precooled Lyophilizer (temperature of shelf was -5°C).

Lyophilizer was runned as per below mentioned Recipe:

7. After completion of secondary drying, vacuum was breakdown with nitrogen then stoppering and sealing was done. After unloading, a good lyophilized cake is observed.

Example 2:

Preparation of Placebo lyophilized powder for injection:

A placebo formulation of example 2 is prepared with the same formula and manufacturing process of example 1 excluding 5-azacytidine Form I.

Brief method of preparation:

1. 80% batch quantity of DMSO was collected in a clean beaker or Duran Bottle.

2. Weighed batch quantity of Mannitol was added to step 1 and stirred to get a clear solution.

3. The final volume of the bulk solution is made up with remaining quantity of DMSO and the solution stirred for 5 mins.

4. The final bulk solution of step 3 is filtered through 0.2^1 filter.

5. The vials are half stoppered and loaded into precooled Lyophilizer (temperature of shelf was -5°C).

Lyophilizer was runned as per identical lyophilization recipe, as mentioned under example 1.

6. After completion of secondary drying, vacuum was breakdown with nitrogen then stoppering and sealing was done. After unloading, a good lyophilized cake is observed.

The input Crystalline 5-Azacytydine Form I (of example 1), Example-1 5-Azacytydine lyophilized formulation & Example-2 placebo lyophilized formulation are submitted for XRD study.

Comparative interpretations of X-Ray powder diffraction patterns of: Crystalline 5-Azacytydine Form I, Example-1 5-Azacytydine lyophilized formulation & Example-2 placebo lyophilized formulation, indicate 5-Azacytydine Form I got retained in the finished drug product.

Assay & Related substances are performed for example 1 formulation, by HPLC analytical methods discussed hereunder:

ASSAY METHOD:

Reagent preparation: Mobile phase-A: HPLC Grade Water & Mobile Phase-B: Acetonitrile.
Chromatographic conditions:

Column : Inertsil ODS 3V, C18, 250 cm X 4.6mm, 5m

Wavelength : 242nm

Flow rate : 2.0ml / minute

Injection volume : 5 uL

Run Time : 20 minute

Column Temperature: 20°C

Sampler Temperature: 25°C

Diluent : DMSO

Mode : Gradient

Gradient Programme:

Standard Solution: Weighed and transferred 5.0 mg of Azacitidine working Standard in to 10 ml volumetric flask and diluted to volume with Diluent.

Sample preparation:

For Bulk Solution: Transferred 1ml of given bulk solution into a 10ml volumetric flask containing 5ml of diluent. Make the volume 10ml with diluent and mixed well.

For Finished product:

Sample stock-1:

Open one vial of Azacitidine for injection lOOmg/vial (before open the vial pierce the stopper with needle to release the vacuum) add about 15ml of DMSO to dissolve the contents and carefully transfer the solution in to 50ml volumetric flask. Again rinse the vial with diluent 2-3 times and transfer the contents into 50ml volumetric flask. Made the volume 50ml with diluent and mix well.

Sample stock-2 (500PPM):

Transfer 5ml of sample stock-1 in to 20ml volumetric flask containing 10ml of diluent. Make the volume 20ml with diluent and mixed well.

Procedure: Separately inject Blank (diluent) one injection and standard solution five injections into chromatographic system.

RELATED SUBSTANCES METHOD:

lOOmM Ammonium Acetate Preparation: Weigh and dissolve 7.72g of Ammonium acetate in to 1000ml water. Mix well sonicate lOminutes it for degas.

Reagent preparation:

Mobile phase-A: lOOmM Ammonium acetate. (7.7g Ammonium acetate in 1000ml water).

Mobile Phase-B: Mix buffer and Acetonitrile in the ratio 5:95 (v/v).

Chromatographic conditions:

Column : Inertsil ODS 2, CI 8, 250 cm X 4.6mm, 5^;

Wavelength : 242nm

Flow rate : 1.0ml / minute

Injection volume : 5uL

Run Time : 65 minute
 
Column Temperature : 20°C
 
Sampler Temperature : 25°C
 
Diluent-1 : DMSO

Diluent-2 : Cold water

Mode : Gradient

Gradient Programme:
 
Standard Stock Solution (500PPM): Weigh and transfer 5.0 mg of Azacitidine working Standard in to 10 ml volumetric flask and add 5ml to dissolve and make final volume with Diluent-1 and mix well.

Standard Solution (4 PPM): Transferred 2.0ml of Azacitidine Standard Stock solution in to 25ml volumetric flask and diluted to volume with Diluent-1. Further transferred 2ml of this solution to 20ml with diluent-1 and mixed well.

Sample preparation:

For Bulk Solution:

Transferred 2ml of given bulk solution into a 5ml volumetric flask containing 2ml of diluent.

Make the volume 5ml with diluent and mixed well.

Note: Sample preparation with cold water should inject immediately and fallowed by DMSO sample.

For Finished product:

Sample preparation with DMSO: Open one vial and add 10ml of DMSO to reconstitute. Transfer 2ml of reconstituted solution in to 10ml volumetric flask and make the volume 10ml with DMSO and inject into HPLC.

Sample preparation with Cold water: Open one vial and add 10 ml of cold water to reconstitute and transfer contents carefully into 50ml volumetric flask and rinse 2-3 times with cold water again transfer into volumetric flask and made the volume 50ml with cold water and inject into HPLC.

Procedure: Separately inject Blank DMSO, water injected and followed by standard solution six injections into chromatographic system fallowed by sample preparations with water and later sample preparation with DMSO.

Assay & Related substances results of example 1 formulation, at initial and after storage of lyophilized vials under 40°C/75% RH for 3M, and 25°C/75%RH for 6M; are discussed under table below. The expressed percentages are of the label 5-azacytidine content.

Water content (by Karl fisher) test is performed before storage ("Initial") and after storage, at 40°C/75% RH for 3M, and 25°C/75%RH 3M.

While the foregoing provides a detailed description of the preferred embodiments of the invention, it is to be understood that the descriptions are illustrative only of the principles of the invention and not limiting. Furthermore, as many changes can be made to the invention •without departing from the scope of the invention, it is intended that all material contained herein be interpreted as illustrative of the invention and not in a limiting sense.

Claims:

1. A pharmaceutical composition comprising 5-Azacytidine, mannitol, and dimethyl sulfoxide.

2. A pharmaceutical composition according to claim-1 comprising a pre-lyophlized or a lyophilized composition.

3. A process of preparing a stable pharmaceutical composition comprising crystalline 5-Azacytidine Form I, characterized by the steps:

i) dissolving mannitol and 5-azacytidine in dimethyl sulfoxide.

ii) filtering the step i) solution.
 
iii) filling the filtered liquid into vials at temperature ranging between 10- 30°C, and
 
iv) lyophilizing the solution obtained from step (iii); wherein the pharmaceutical composition having not more than 0.2%w/w of known impurities and not more than l%w/w of total impurities, at release.

4. A process of preparing a stable lyophilized composition according to claim-3, wherein crystalline 5-azacytidine utilized in step i) is Form-I.

5. A stable pharmaceutical composition prepared according to claim-3, wherein 5-azacytidine is used in the range of about 5 to 100 mg/ml and mannitol is in the range of about 5 to 100 mg/ml.

6. A stable lyophilized composition according to claim-3, having water content of not more than 1% w/w.

7. A stable lyophilized composition of 5-azacytidine according to claims 2-6, contains not more than 0.2%w/w of known impurities and not more than l%w/w of total impurities, when stored atleast for 40°C/75%RH for 3 months.

8. A stable lyophilized composition of 5-azacytidine according to claims 2-7, reconstituted with 4ml or 10ml of sterile water for injection.

9. A method of administering therapeutically effective amount of 5-azacytidine, from reconstituted 5-azacytidine solution of claim-8, used for treating medical conditions selected from Myelodysplastic syndromes, Chronic myelomonocytic leukaemia and Acute myeloid leukaemia.