The present invention relates to stable injectable formulation comprising Cyclophosphamide as active ingredient and pharmaceutically acceptable excipients which is devoid of mannitol. The present invention also relates to methods for the preparation of pharmaceutical compositions of Cyclophosphamide by lyophilization process using solvent and optionally solubilizer wherein lyophilization process does not involve rehydration step. The present invention specifically relates to preparation of lyophilized pharmaceutical compositions of Cyclophosphamide with improved solubility and stability wherein the lyophilization process does not involve rehydration step and the composition is devoid of mannitol. The present invention specifically relates to preparation of stable injectable lyophilized formulation comprising Cyclophosphamide as active ingredient which is devoid of solubilizer as well as pharmaceutically acceptable excipients such as pH modifying agent and mannitol. BACKGROUND OF INVENTION

Cyclophosphamide is an alkylating agent used in the treatment of several forms of cancer including leukemias, lymphomas and breast cancer. Cyclophosphamide is example of a group of novel cyclic phosphoric acid ester amides and synthetic antineoplastic drug, chemically related to the nitrogen mustards. Cyclophosphamide, chemically is 2-[bis(2-chloroethyl)amino]tetrahydro-2H- 1,3,2-oxazaphosphorine 2-oxide, and has the following structural formula:

Cyclophosphamide has a molecular formula of C ₇ H ₁₅ Cl ₂ N ₂ O ₂ P and a molecular weight of 261.08 gm/mol. Cyclophosphamide is soluble in water, saline, or ethanol. Cyclophosphamide exhibits polymorphism and available in two forms i.e. monohydrate form and anhydrous form. Cyclophosphamide monohydrate has a molecular formula of C ₇ H ₁₅ Cl ₂ N ₂ O ₂ P.H ₂ O and a molecular weight of 279.1 gm/mol. The anhydrous form is less stable than monohydrate form, it easily picks up water when exposed to a relative humidity more than 20% and converted into the monohydrate form at room temperature. The monohydrate form is more stable than anhydrous form and it is preferably used for pharmaceutical processing. Cyclophosphamide easily interconvert between anhydrous and monohydrate form based on the storage condition, so it is recommended to store Cyclophosphamide at temperature not exceeding 30°C, preferably at about 25°C, under dry conditions where relative humidity is 20% or less, it start to lose the water which can create problems in manufacture. Some patent literature describes the lyophilized formulations using mannitol and sodium chloride by using the two stage freeze drying process with aqueous and non-aqueous solvents. Use of non-aqueous (organic) solvents like ethanol, isopropanol are used to prepare the lyophilized formulations with low residual moisture content product is in the critical range i.e. 2-7% by weight have also been reported in literature. U.S Pat. No. 4,537,883 A discloses a hydrated lyophilizate composition with improved stability, superior solubility characteristics and enhanced appearance comprising about 20 parts by weight of Cyclophosphamide anhydride, water and mannitol. U.S Pat. No. 4,659,699 A discloses the two stage freeze drying process for Cyclophosphamide to yield its hydrate form. The two stage freeze drying process involves in the freeze drying of aqueous solution of Cyclophosphamide with an excipient to reduce moisture content less than 2% by weight followed by rehydration of the freeze dried material until the moisture content of the product reach the range of 2 to 7% by weight. U.S Pat. No. 5,413,995 A discloses the process for producing a lyophilized Cyclophosphamide pharmaceutical solid composition which involves (a) dissolving about two parts by weight of Cyclophosphamide monohydrate and from about 1-8 parts by weight of excipient comprising mannitol in sufficient water to give about a 1.67 to 4% (w/v) concentrated Cyclophosphamide solution; (b) filling a container with said solution to a desired Cyclophosphamide content; (c) lyophilizing solution of desired Cyclophosphamide content in a container to a dryness of about 2% or less; and (d) humidifying the lyophilizate by means of exposure to water vapor at about room temperature and a pressure of from about 100 millitorr to atmospheric pressure for an effective time of at least 2 hours until about 1 equivalent of water per equivalent of anhydrous Cyclophosphamide is taken up. U.S Pat. No. 5,336,669 A discloses lyophilization of Cyclophosphamide. The lyophilized cake was prepared by the process of freezing Cyclophosphamide, lactose and water, lyophilizing part of the water, obtaining and melting a supersaturated Cyclophosphamide-lactose solution, precipitating Cyclophosphamide as a hydrated polymorph, refreezing the solution and lyophilizing the water not bound to Cyclophosphamide or lactose. PCT Pub. No. WO 1994/008592 A1 discloses lyophilized Cyclophosphamide compositions with excipient mainly dextran. The lyophilized formulations are prepared by lyophilizing a solution of Cyclophosphamide to obtain an anhydrous crystalline form. PCT Pub. No. WO 2014/068585 A1 discloses composition comprising lyophilized Cyclophosphamide monohydrate wherein lyophilization process is carried out without a rehydration step in presence of solvent or mixtures of solvents. Currently available formulation of Cyclophosphamide consists of sterile dry powder blend mixtures of Cyclophosphamide in monohydrate form and sodium chloride. This formulation can be used for oral or parenteral administration by dissolving the dry powder blend mixture in water prior to administration. However, due to low shelf life of the dissolved aqueous solution it is recommended to administer it immediately after reconstitution of formulation. However, this premixed product prone to a glassiness and or stickiness during manufacturing and storage results in unacceptable product with low potency as well as reduced solubility. Lyophilized product has several advantages over the dry powder premix product. First in lyophilization drying is performed at lower temperatures thus avoid temperature related degradation or stability problems. However, the lyophilization process currently used is critical and it requires rehydration steps to control moisture level, high energy and the skilled technicians to control process. It is well known that monohydrate form of Cyclophosphamide is more stable than anhydrous form of Cyclophosphamide. During lyophilization process, the water of crystallization is removed from the Cyclophosphamide which results in relative low stable product. However, to regain the hydrate form rehydration step is included in lyophilization process but it is difficult to achieve uniform water content across all the vials during rehydration step. Thus to overcome drawbacks associated with currently available formulations, the inventors of the present invention have developed a lyophilized injectable Cyclophosphamide composition comprising water as solvent and optionally ethanol as solubilizer which provide better effect for the treatment of several forms of cancer including leukemias, lymphomas and breast cancer. The goal of the present invention is achieving the lyophilization without rehydration step. The investigators found that by controlling vehicle system and process parameters, monohydrate form of Cyclophosphamide can be produced without requirement of any rehydration step. The lyophilized composition comprising Cyclophosphamide for reconstitution with water with improved storage stability, improved solubility, uniform hydrate integrity between the vials and enhanced appearance compared with currently available lyophilized and dry powder pre-mix compositions of Cyclophosphamide. OBJECTIVE OF INVENTION

The main objective of the present invention is to provide stable injectable formulation comprising Cyclophosphamide as active ingredient which is devoid of mannitol. Another objective of the present invention is to provide stable injectable formulation comprising Cyclophosphamide as active ingredient optionally containing pharmaceutically acceptable excipients which is devoid of mannitol. Another objective of the present invention is to provide methods for preparation of pharmaceutical compositions of Cyclophosphamide by lyophilization process using solvent and optionally solubilizer wherein lyophilization process does not involve rehydration step. Another objective of the present invention is to provide preparation of lyophilized pharmaceutical compositions of Cyclophosphamide with improved solubility and stability wherein the lyophilization process does not involve rehydration step and the composition is devoid of mannitol. Another objective of the present invention is to provide stable injectable lyophilized formulation comprising Cyclophosphamide as active ingredient which is devoid of solubilizer as well as pharmaceutically acceptable excipients such as pH modifying agent and mannitol. SUMMARY OF INVENTION

One embodiment of the present invention provides stable injectable formulation comprising Cyclophosphamide as active ingredient which is devoid of mannitol. One embodiment of the present invention provides stable injectable formulation comprising Cyclophosphamide as active ingredient optionally pharmaceutically acceptable excipients which is devoid of mannitol. Another embodiment, the present invention provides process for preparation of pharmaceutical compositions of Cyclophosphamide by lyophilization process using solvent and optionally solubilizer wherein lyophilization process does not involve rehydration step. Another embodiment, the present invention provides the preparation of lyophilized pharmaceutical compositions of Cyclophosphamide with improved solubility and stability wherein the lyophilization process does not involve rehydration step and the composition is devoid of mannitol. In another embodiment, the present invention provides the preparation of lyophilized pharmaceutical compositions of Cyclophosphamide comprising a vehicle system which includes water as solvent, optionally ethanol as solubilizer and pH modifying agents. In another embodiment, the present invention is to provide stable injectable lyophilized formulation comprising Cyclophosphamide as active ingredient which is devoid of solubilizer as well as pharmaceutically acceptable excipients such as pH modifying agent and mannitol. In another embodiment, the present invention provides process for preparation of Cyclophosphamide injection formulations comprising steps in lyophilization process which optionally involves annealing step. In another embodiment, the present invention provides process for preparation of Cyclophosphamide injection formulations comprising the steps of:

a) adding Cyclophosphamide to solvent and optionally added solubilizer, and optionally adjusting the pH of solution to 3.0 to 9.0 using pH modifying agents,

b) sterilizing the bulk solution by Filtration through 0.2 m sterile filter, c) filling vials with the solution and half stoppering of the vials,

d) loading the vials filled with the solution into a lyophilizer having a shelf temperature of 0°C and maintaining the shelf temperature to achieve thermal equilibrium,

e) freezing of the product solution at a shelf temperature of -45°C, at a ramp rate of 5°C per hour to 90°C per hour, and maintaining the shelf temperature to achieve uniform frozen mass of the product,

f) chilling condenser of the lyophilizer to a temperature -50°C or below, g) evacuating chamber of the lyophilizer to a pressure of less than 1200 mTorr, preferably 200-700 mTorr, and controlling the chamber pressure at the pressure of less than 700 mTorr, preferably 300-650 mTorr,

h) sublimation of frozen mass achieved by warming the lyophilizer to shelf temperature in range of -10°C to -2°C, and maintaining shelf temperature and the chamber pressure at the pressure below 650 mTorr, to obtain a dried product with uniform water content between the vials, i) this process includes optionally at least one annealing step with temperature ranging between 0°C and -10°C during freezing steps, and

j) the process of lyophilization wherein drying may optionally achieved by heating the product to negative temperature. BRIEF DESCRIPTION OF THE FIGURES

Figure 1: DSC thermograms of Cyclophosphamide monohydrate API and Example 6 lyophilizate.

Figure 2: TGA thermograms of Cyclophosphamide monohydrate API and Example 6 lyophilizate.

Figure 3: XRD Diffractograms of Cyclophosphamide monohydrate API and Example 6 lyophilizate.

Figure 4: IR spectra of Cyclophosphamide monohydrate API and Example 6 lyophilizate. The IR spectra of both Cyclophosphamide monohydrate API and lyophilizate are similar confirming that lyophilizate is a monohydrate form. 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 a stable injectable formulation comprising Cyclophosphamide as active ingredient and pharmaceutically acceptable excipients which is devoid of mannitol. The present invention provides the preparation of lyophilized pharmaceutical compositions of Cyclophosphamide with improved solubility and stability wherein the lyophilization process does not involve rehydration step and the composition is devoid of mannitol. The term“active ingredients” of the present invention is used to treat various types of cancer. Preferably used active ingredient is Cyclophosphamide, is nitrogen mustard which is bio transformed principally in the liver to active alkylating metabolites by a mixed function microsomal oxidase system. These metabolites interfere with the growth of susceptible rapidly proliferating malignant cells. The present invention relates to an innovative lyophilization process for manufacturing of a Cyclophosphamide monohydrate form that can be used for oral or parenteral administration. The said pharmaceutical composition of comprises Cyclophosphamide, pH modifying agents and suitable vehicle system to dissolve the Cyclophosphamide. The vehicle system contains water as solvent and optionally ethanol as solubilizer. The solubilizer present in the formulation is less than 15% v/v. The vehicle system is later removed during lyophilization process. The water content in formulation is controlled during lyophilization in such manner that it facilitates the formation of the stable Cyclophosphamide monohydrate form. As used herein the term "vehicle system" refers to the liquid phase of a composition which is capable to dissolve or suspend the solutes. The term "solubilizer" can refer to a vehicle that is miscible with water and enhances the solubilization of the solutes. The solubilizer used in the composition is ethanol which is less than 8 % v/v. The vehicle system contains more than 85 % v/v of water along with suitable ratio of solubilizing vehicle to obtain a lyophilized product with desirable characteristics. Preferably water is 100% v/v. The present invention involves innovative lyophilization process that leads to “in situ” formation of stable Cyclophosphamide monohydrate form without requirement of any rehydration step. The formation of stable Cyclophosphamide monohydrate form is achieved by using vehicle system and by optimizing different steps of the lyophilization process. Suitable pH modifying agents used in the compositions of the present invention to adjust the pH of the solution include, but are not limited to acids, bases or salt form of one or more, 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, tromethamine, 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, sodium carbonate, sodium hydrogen carbonate, calcium hydrogen carbonate, sodium nitrite, sodium nitrate, magnesium nitrate, calcium nitrite and magnesium nitrite; Tris hydrochloride or combinations thereof. Preferably, the pH modifying agent comprises at least one of tromethamine, citric acid, ascorbic acid, hydrochloric acid, lactic acid, tartaric acid, malonic acid, maleic acid, sulfuric acid, and sodium hydroxide, and more preferably mixture of these compounds. Lyophilization is also known as Freeze drying or cryodesiccation is a low temperature dehydration process which involves freezing the product, lowering pressure, then removing the ice by sublimation. This is in contrast to dehydration by most conventional methods that evaporate water using heat. Lyophilization or Freeze drying is a process in which solution is allowed to freeze followed by drying under low temperature and vacuum by removal of vehicle system from a frozen mass first by sublimation and then by desorption. The unique advantage of lyophilization is that drying of sample is performed at low temperature where water is removed from frozen mass without having to pass through the liquid phase by sublimation which restrict any chemical reactions thus reduce the degradation of product and improve their stability. The term“Annealing” used here shall be defined as process where product temperature raised above the final freezing temperature and then bringing the product temperature back to final freezing temperature. Annealing results in generation of larger crystal during freeze drying step thereby help in shorting drying times. The term“Frozen mass” refer to a product where a liquid product when stored at temperature below zero degrees Celsius converted to solid mass from liquid state. The term“Prelyophilizate” refer to a product composition where solute dissolved or suspended in vehicle system prior to freeze drying. The term "freeze-dried formulation" or "Lyophilizate" refer to a dried product obtained from lyophilization process. Lyophilization Instrument consists of condenser, vacuum system, a chamber solvent trap and software program to control lyophilization process parameters. Lyophilization of Cyclophosphamide:

The investigators found that by controlling vehicle system and process parameters, monohydrate form of Cyclophosphamide can be produced without requirement of any rehydration step. Different percentage of solubilizer (ethanol solution) (less than 8 % v/v) was evaluated for lyophilization of Cyclophosphamide for injection. Lyophilization Process:

The solid Cyclophosphamide composition of the invention can be obtained by an inventive lyophilization process. The lyophilization processes according to embodiments of the invention does not involve rehydration step which comprising the steps of: a) adding Cyclophosphamide to solvent and optionally added solubilizer, and optionally adjusting the pH of solution to 3.0 to 9.0 using pH modifying agents, b) sterilizing the bulk solution by filtration through 0.2 m sterile filter,

c) filling vials with the solution and half stoppering of the vials,

d) loading the vials filled with the solution into a lyophilizer having a shelf temperature of 0°C and maintaining the shelf temperature to achieve thermal equilibrium,

e) freezing of the product solution at a shelf temperature of -45°C, at a ramp rate of 5°C per hour to 90°C per hour, and maintaining the shelf temperature to achieve uniform frozen mass of the product,

f) chilling condenser of the lyophilizer to a temperature -50°C or below, g) evacuating chamber of the lyophilizer to a pressure of less than 1200 mTorr, preferably 200-700 mTorr, and controlling the chamber pressure at the pressure of less than 700 mTorr, preferably 300-650 mTorr,

h) sublimation of frozen mass achieved by warming the lyophilizer to shelf temperature in range of -10°C to -2°C, and maintaining shelf temperature and the chamber pressure at the pressure below 650 mTorr, to obtain a dried product with uniform water content between the vials,

i) this process includes optionally at least one annealing step with temperature ranging between 0°C and -10°C during freezing steps, and

j) the process of lyophilization wherein drying may optionally achieved by heating the product to negative temperature. Cyclophosphamide solution was prepared by dissolving Cyclophosphamide monohydrate in water and optionally ethanol, then optionally pH of solution was adjusted to 3.0 to 9.0 using suitable pH modifying agents. Then bulk solution of Cyclophosphamide was sterilized by filtration through a 0.2 m sterilizing filter followed by an aseptic filling in vials. Solution of drug was prepared in various percentage of ethanol solution (4% v/v to 8% v/v). Precipitation of drug was observed while filling and loading of vials in lyophilizer in a composition containing less than 7% v/v ethanol, while composition containing 8 % ethanol solution was stable so it was used for further development purpose. Unit dose of Cyclophosphamide varied from 100 mg to 2000 mg. Based on required dose Tubular or Molded vials with varied sizes are selected but not limited to 10 mL, 15 mL, 30 mL, 50 mL, and 100 mL. Based on the solution concentration each vial is filled aseptically with Cyclophosphamide solutions to achieve desired unit dose in vial. In a preferred embodiment of the invention, aseptic filling operation performed to achieve desired concentration of Cyclophosphamide monohydrate per unit vial. In a preferred embodiment of the invention, the proportion in vehicle system (water as solvent and optionally ethanol as solubilizer) were maintained in such way to eliminate or minimize precipitation of the product from the solution, allowing for a clear, homogeneous solution. In another preferred embodiment of the invention, the processing temperature is preferably maintained at 2°C to 10°C, more preferably at 5°C± 3°C, during the stirring and aseptic filling. However, as noted above, other methods of preparing the Cyclophosphamide solution is also within the scope of the invention. According to an embodiment of the invention, the lyophilization process involves filling of the Cyclophosphamide solution at desired concentration in vials at a temperature 2°C to 10°C, more preferably at 5°C± 3°C. According to an embodiment of the invention, the lyophilization process involves loading of the vials into a lyophilizer chamber having a shelf temperature of 0°C - 25°C followed by evacuation of lyophilizer chamber to a pressure of less than 1200 mTorr to ensure complete tightness of the chamber. Product is then held for 0.5 to 15 hours at this temperature to achieve thermal equilibrium prior to freezing step. According to an embodiment of the invention, the freezing of the product solution was performed at– 30°C to -75°C with an average ramp rate of 5°C per hour to 90°C per hour, to achieve uniform frozen mass of the product. Product is then held for 0.5 to 20 hours at this temperature with chamber pressure of less than 1200 mTorr, preferably at 1000 mTorr to achieve complete solidification of product prior to drying step. In one embodiment of the invention, freezing step contains optional annealing steps. Annealing is performed by warming shelf temperature to -7°C, at a ramp rate of 22°C per hour followed by cooling back to the shelf temperature of -45°C at a ramp rate of 12°C per hour. Product is then held for 0.5 to 15 hours at this temperature with chamber pressure of less than 1200 mTorr, preferably at 1000 mTorr to achieve compete solidification of product prior to drying step. The condenser of the lyophilizer is cooled to below -70°C to trap water vapor came from product during drying step to prevent damage of the vacuum pump. The lyophilizer chamber is maintained to a pressure of less than 1200 mTorr, preferably 200-700 mTorr, more preferably 300-650 mTorr. According to an embodiment of the invention, drying of product was achieved by sublimation of frozen mass at shelf temperature ranging between -10°C to -2°C at a ramp rate, such as an average rate of 1°C to 5°C per hour and by maintaining constant chamber pressure below 650 mTorr. Product is then held for 0.5 to 15 hours at this temperature with chamber pressure of less than 650 mTorr to obtain a dried product with uniform water content between the vials. The time required for sublimation step can be determined by regular trials to achieve uniform water content between the vials. In one embodiment of the invention, drying step optionally involve negative temperature rate drying steps, positive temperature rate drying steps or constant temperature drying steps. Resulted lyophilized Cyclophosphamide product is appeared as white cake or powder which on reconstitution forms a clear, colourless solution. Cyclophosphamide solutions were prepared by dissolving Cyclophosphamide monohydrate in ethanol and water, sterilized by filtration through a 0.2 mm sterilizing filter, then solution was filled into vials (10 to 100 ml) to achieve unit dose ranging from 100 mg/vial to 2000 mg/vial of Cyclophosphamide. Vials were half stoppered and loaded into the lyophilizer. The lyophilized chamber was then evacuated to pressure less than 1200 mTorr and the desired lyophilization cycle was performed. After completion of the lyophilization, obtained lyophilized product were inspected physically for the appearance of lyophilized cake or powder. Reconstitution of product was performed by using sterile water for injection and reconstituted solution was evaluated for absence of particulate matter, clarity, reconstitution time. Lyophilized product was evaluated for water content, glass transition temperature, melting point, residual ethanol content. Water content in lyophilized product was determined by using coulometric Karl Fischer Titration while thermogravimetric analysis (TGA) was used to determine residual moisture content. Differential scanning calorimetry (DSC) was used to determine glass transition temperature and melting point of lyophilized product, Residual ethanol content was determined by Gas chromatography. The present invention is illustrated in detail but not limiting to, the following examples. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention EXAMPLES: Example 1: Investigation of Lyophilization process of Cyclophosphamide: 200 mg, 500mg Presentations: Bulk Cyclophosphamide solution was prepared by dissolving Cyclophosphamide in 8 % v/v ethanol and water. The pH of solution was adjusted to 3.0 to 9.0 using pH modifying agents. Then, the solution was filtered and aseptically filled in vials based on required dose and loaded onto the shelves of a lyophilizer at temperature 0°C, and the chamber was evacuated to pressure less than 1200 mTorr after loading and the lyophilizate was prepared by lyophilization parameters mentioned in Table 1. Table 1: Summary of Target Lyophilization cycle

Following lyophilization cycle, the obtained lyophilizate evaluated for physical appearance, reconstitution time and moisture content by KF titration method and the obtained results were mentioned in Table 2. Physical appearance of the lyophilized product showed a white cake or powder without side shrinkage. Reconstitution of lyophilized product was performed with sterile water for Injection USP, resulted in clear, colourless solutions. Table 2: Analytical results of Lyophilizate

The results given in table 2 showed that the moisture content is similar for the drug substance as well as the lyophilizate which confirms that the crystalline monohydrate form of the API was retained in the lyophilized product. The crystalline Cyclophosphamide monohydrate Form contains 5.7 to 6.8% of water which is in agreement with the results obtained and listed in table 2. Example 2: Investigation of Lyophilization process of Cyclophosphamide: 200 mg, 500mg, 1000mg Presentations Bulk Cyclophosphamide solution was prepared by the preparation process similar to that of process used in the example 1 and the lyophilizate was prepared by lyophilization parameters mentioned in Table 3. Table 3: Summary of Target Lyophilization cycle

Following lyophilization cycle, the obtained lyophilizate evaluated for physical appearance, reconstitution time and moisture content by KF titration method and the obtained results were mentioned in Table 4. Physical appearance of the lyophilized product showed a white cake or powder without side shrinkage. Reconstitution of lyophilized product was performed with sterile water for Injection USP, resulted in clear, colourless solutions. Table 4: Analytical results of Lyophilizate The results given in table 4 showed that the moisture content is similar for the drug substance as well as the lyophilizate confirming that the crystalline monohydrate form of the API was retained in the lyophilized product. The crystalline Cyclophosphamide monohydrate Form contains 5.7 to 6.8% of water which is in agreement with the results obtained and listed in table 4. Example 3: Investigation of Lyophilization process of Cyclophosphamide: 200 mg, 500mg, 1000mg Presentations Bulk Cyclophosphamide solution was prepared by the preparation process similar to that of process used in the example 1 and the lyophilizate was prepared by lyophilization parameters mentioned in Table 5. Table 5: Summary of Target Lyophilization cycle

Following lyophilisation cycle, the obtained lyophilizate evaluated for physical appearance, reconstitution time and moisture content by KF titration method and the obtained results were mentioned in Table 6. Physical appearance of the lyophilized product showed a white cake or powder without side shrinkage. Reconstitution of lyophilized product was performed with sterile water for Injection USP, resulted in clear, colourless solutions. Table 6: Analytical results of Lyophilizate

The results given in table 6 showed that the moisture content is similar for the drug substance as well as the lyophilizate confirming that the crystalline monohydrate form of the API was retained in the lyophilized product. The crystalline Cyclophosphamide monohydrate Form contains 5.7 to 6.8% of water which is in agreement with the results obtained and listed in table 6. Example 4: Investigation of Lyophilization process of Cyclophosphamide: 200 mg, 500mg, 1000mg Presentations Bulk Cyclophosphamide solution was prepared by the preparation process similar to that of process used in the example 1 and the lyophilizate was prepared by lyophilization parameters mentioned in Table 7. Table 7: Summary of Target Lyophilization cycle

Following lyophilisation cycle, the obtained lyophilizate evaluated for physical appearance, reconstitution time and moisture content by KF titration method and the obtained results were mentioned in Table 8. Physical appearance of the lyophilized product showed a white cake or powder without side shrinkage. Reconstitution of lyophilized product was performed with sterile water for Injection USP, resulted in clear, colourless solutions.

The moisture content of lyophilizate is similar to that of the drug substance confirming that the crystalline monohydrate form of the API was retained in the lyophilized product. The annealing step in freezing doesn’t affect the reconstitution time, moisture content and lyophilizate structure which is shown by results obtained by (Example 3 & Example 4). The results of both examples showed that the example with and without annealing step in freezing doesn’t affect the reconstitution time, moisture content and lyophilizate structure. Results of the various lyophilization process showed that the annealing steps at a freezing as well as at drying step is not necessary to have a product with monohydrate structure and the moisture level between 5% (w/w) to 7% (w/w). Table 8: Analytical results of Lyophilizate

Example 5: Investigation of Lyophilization process of Cyclophosphamide: 200 mg, 500mg, 1000 mg Presentations Bulk Cyclophosphamide solution was prepared by the preparation process similar to that of process used in the example 1 and the lyophilizate was prepared by lyophilization parameters mentioned in Table 9. Table 9: Summary of Target Lyophilization cycle

Following lyophilisation cycle, the obtained lyophilizate evaluated for physical appearance, reconstitution time and moisture content by KF titration method and the obtained results were mentioned in Table 10. Physical appearance of the lyophilized product showed a white cake or powder without side shrinkage. Reconstitution of lyophilized product was performed with sterile water for Injection USP, resulted in clear, colourless solutions. The monohydrate form of Cyclophosphamide can be achieved by drying at a same temperature with controlled vacuum of the chamber. Further effect of drying at same temperature but different vacuum level on reconstitution time, moisture content and lyophilizate structure was evaluated. It was observed that drying at a same temperature with controlled vacuum level doesn’t affect the reconstitution time, moisture content and lyophilizate structure. The results given in table 10 showed that the moisture content is similar for the drug substance as well as the lyophilizate confirming that the crystalline monohydrate form of the API was retained in the lyophilized product. The crystalline Cyclophosphamide monohydrate Form contains 5.7 to 6.8% of water which is in agreement with the results obtained and confirmed that the lyophilizate is a stable monohydrate. Table 10: Analytical results of Lyophilizate

Example 6: Investigation of Lyophilization process of Cyclophosphamide: 200 mg, 500mg, 1000mg Presentations Bulk Cyclophosphamide solution was prepared by the preparation process similar to that of process used in the example 1 and the lyophilizate was prepared by lyophilization parameters mentioned in Table 11. Table 11: Summary of Target Lyophilization cycle

Following lyophilisation cycle, the obtained lyophilizate evaluated for physical appearance, reconstitution time and moisture content by KF titration method and the obtained results were mentioned in table 12. Physical appearance of the lyophilized product showed a white cake or powder without side shrinkage. Reconstitution of lyophilized product was performed with sterile water for Injection USP, resulted in clear, colourless solutions. Table 12: Analytical results of Lyophilizate

The crystalline Cyclophosphamide monohydrate Form contains 5.7 to 6.8% of water which is in agreement with the results obtained and confirms that the lyophilizate is a stable monohydrate. Example 7: Investigation of Lyophilization process of Cyclophosphamide: 200 mg, 500mg, 1000mg Presentations Bulk Cyclophosphamide solution was prepared by dissolving in water without adding any solubilizer. The pH of solution was adjusted to 3.0 to 9.0 using pH modifying agents. Then, the solution was filtered and aseptically filled in vials based on required dose and loaded onto the shelves of a lyophilizer at temperature 0°C, and the chamber was evacuated to pressure less than 1200 mTorr after loading. Table 13 gives the freeze drying parameters of exemplary processes for lyophilization and the effect of process parameters on the product appearance and water content of the lyophilizate were evaluated which is given below: Table 13: Summary of Target Lyophilization cycle

Following lyophilisation cycle, the obtained lyophilizate were evaluated for physical appearance, reconstitution time and moisture content by KF titration method and the obtained results were mentioned in Table 14. Physical appearance of the lyophilized product showed a white cake or powder without side shrinkage. Reconstitution of lyophilized product was performed with sterile water for Injection USP, resulted in clear, colourless solutions. The results given in table 14 showed that the moisture content is similar for the drug substance as well as the lyophilizate confirming that the crystalline monohydrate form of the API was retained in the lyophilized product. The crystalline Cyclophosphamide monohydrate Form contains 5.7 to 6.8% of water which is in agreement with the results obtained and listed in table 14. Example 7 is prepared without solubilizer i.e. ethanol in vehicle system and it was observed that absence of ethanol does not affect lyophilizate structure and moisture content. Further in drying stage sublimation is achieved by heating the product to negative temperature by maintaining controlled vacuum in chamber and observed that heating the product to negative temperature retains lyophilizate structure and moisture content. Table 14: Analytical results of Lyophilizate

Example 8: Investigation of Lyophilization process of Cyclophosphamide: 200 mg, 500mg, 1000mg Presentations Bulk Cyclophosphamide solution was prepared by dissolving in water without adding any solubilizer and pH modifying agent. Then, the solution was filtered and aseptically filled in vials based on required dose and loaded onto the shelves of a lyophilizer at temperature 0°C, and the chamber was evacuated to pressure less than 1200 mTorr after loading. Table 15 gives the freeze drying parameters of exemplary processes for lyophilization and the effect of process parameters on the product appearance and water content of the lyophilizate were evaluated which is given below: Table 15: Summary of Target Lyophilization cycle

Following lyophilisation cycle, the obtained lyophilizate were evaluated for physical appearance, reconstitution time and moisture content by KF titration method and the obtained results were mentioned in Table 16. Physical appearance of the lyophilized product showed a white cake or powder without side shrinkage. Reconstitution of lyophilized product was performed with sterile water for Injection USP, resulted in clear, colourless solutions. The results given in table 16 showed that the moisture content is similar for the drug substance as well as the lyophilizate confirming that the crystalline monohydrate form of the API was retained in the lyophilized product. The crystalline Cyclophosphamide monohydrate Form contains 5.7 to 6.8% of water which is in agreement with the results obtained and listed in table 16. Example 8 is prepared without solubilizer i.e. ethanol in vehicle system as well as without pH modifying agent and it was observed that absence of ethanol and pH modifying agent does not affect lyophilizate structure and moisture content. Further in drying stage sublimation is achieved by heating the product to negative temperature by maintaining controlled vacuum in chamber and observed that heating the product to negative temperature retains lyophilizate structure and moisture content.

Table 16: Analytical results of Lyophilizate

Further, the DSC, TGA, FTIR, XRD studies were performed using representative Example 6 200 mg and Cyclophosphamide monohydrate API to confirm the obtained lyophilizate is a monohydrate form and also compared with the Cyclophosphamide monohydrate API and data generated as given below: DSC studies:

DSC was performed using TA-Q2000; sample was accurately weighed into aluminum pan. The pan was crimped and DSC was performed at a heating rate of 0.5°C/min from 25°C to 105°C under atmosphere of nitrogen. The DSC thermograms of Cyclophosphamide monohydrate API and Example 6 200 mg lyophilizate are similar as shown in Figure 1. TGA studies:

TGA was performed using TA-Q500; sample was accurately weighed into aluminum crucibles and heated at a programmed rate of 10 °C/min from 20°C to 120°C under atmosphere of nitrogen. The TGA thermograms of Cyclophosphamide monohydrate API and Example 6 200 mg lyophilizate are similar as shown in Figure 2. XRD Studies:

X-ray diffraction (XRD) patterns were recorded at room temperature using Schimadzu XRD-7000 using Cu Ka radiation (1.54 Å), at 40 kV and 40 mA passing through nickel filter with divergence slit (0.5°), anti-scattering slit (0.5°) and receiving slit (1 mm). Figure 3 shows the diffractograms of Cyclophosphamide monohydrate API and Example lyophilizate. The diffractograms of both Cyclophosphamide monohydrate API and lyophilizate are similar confirming that lyophilizate is a monohydrate form. Diffractograms of Cyclophosphamide monohydrate API and Example 6 (200 mg lyophilizate) is given in Figure 3. IR Studies:

The FTIR transmission spectra of Cyclophosphamide monohydrate and lyophilized products were recorded on a Perkin-Elmer FT-IR spectrophotometer. 1 to 2 mg of sample being examined was triturated with 300mg to 400mg of dried Potassium bromide. The sample was then scanned using a FT-IR spectrophotometer. Figure 4 shows the IR spectra of Cyclophosphamide monohydrate API and Example 6 200 mg lyophilizate. The IR spectra of both Cyclophosphamide monohydrate API and lyophilizate are similar confirming that lyophilizate is a monohydrate form.

Finished product was stored at accelerated (25°C± 2°C /60% ±5% RH) and long term (5°C± 3°C) stability conditions as per ICH guideline. The initial sample and samples stored at different stability conditions were analyzed for assay, related substance (%), water content, Completeness and clarity of reconstituted solution, pH of reconstituted solution, particulate matter of reconstituted solution. The results of the representative examples 8 are given in Table 17, Table 18 and Table 19. Accelerated condition shows that product is stable up to 3 months. Melting of product observed on 6 months. The recommended storage condition Cyclophosphamide API is 2– 8 °C. This is because cyclophosphamide having low melting point in range of 41-45°C and it loose or gain water of crystallization on storage at room temperature and converted to glassy material. This results in reduced potency and increased level of degradants. Significant changes occurs between 3 and 6 months testing at the accelerated storage condition, thus proposed shelf life of finished product will be determined based on long term (5°C± 3°C) stability data. The stability condition is inline with API storage i.e. 2– 8 °C. Table 17a: Stability results of Cyclophosphamide for Injection, 200mg/Vial (Example 8) at 25°C± 2°C /60% ±5% RH.

RC- Reconstitution; WC- water content; NA- Not applicable Table 17b: Stability results of Cyclophosphamide for Injection, 200mg/Vial (Example 8) at 5°C± 3°C.

RC- Reconstitution; WC- water content; NA- Not applicable Table 18a: Stability results of Cyclophosphamide for Injection, 500mg/Vial (Example 8) at 25°C± 2°C /60% ±5% RH

RC- Reconstitution; WC: Water content; NA- Not applicable

Table 18b: Stability results of Cyclophosphamide for Injection, 500mg/Vial (Example 8) at 5°C± 3°C

Table 19a: Stability results of Cyclophosphamide for Injection, 1000mg/Vial (Example 8) at 25°C± 2°C /60% ±5% RH

RC- Reconstitution; WC: Water content; NA- Not applicable

Table 19b: Stability results of Cyclophosphamide for Injection, 1000mg/Vial (Example 8) at 5°C± 3°C

R ^{C- Reconstitution; WC: Water content; NA- Not applicable}