WO2014068585A1

WO2014068585A1 - Novel lyophilized compositions of cyclophosphamide

Info

Publication number: WO2014068585A1
Application number: PCT/IN2013/000469
Authority: WO
Inventors: Kocherlakota Chandrashekhar; Banda Nagaraju
Original assignee: Leiutis Pharmaceuticals Pvt. Ltd.
Priority date: 2012-10-29
Filing date: 2013-07-29
Publication date: 2014-05-08

Abstract

The process for producing lyophilized compositions of cyclophosphamide monohydrate, wherein the process does not need rehydration step. The lyophilization is carried out in presence of solvent or mixture of solvent. The compositions of the present invention have greater stability and uniformity.

Description

Novel lyopliilizcd compositions of Cyclophosphamide

Background of the invention

Cyclophosphamide is a synthetic antineoplastic drug chemically related to the nitrogen mustards and has the following structure:

The chemical name of cyclophosphamide is 2-[bis(2-chloroethyI)amino]tetrahydro-2H- 1 ,3,2-oxazaphosphorine 2-oxide monohydrate. The compound along with related novel cyclic phosphoric acid ester amides was disclosed and claimed in U.S patent 3,018,302.

Cyclophosphamide comprises of monohydrate and anhydrous forms. The monohydrate is a stable form but under dry conditions (relative humidities of about 20% or less) the monohydrate begins to lose this water of hydration which can cause problems in manufacture. Hence maintaining the manufacturing and storage temperatures for this product is extremely important.

U.S patent 4,659,699 to Daniel et al. discloses the process for freeze drying of Cyclophosphamide. The two stage process described in the patent involves freeze drying of an aqueous solution of Cyclophosphamide to yield a hydrate of Cyclophosphamide. In the first stage, Cyclophosphamide is freeze dried with an excipient until the moisture content is less than 2% by weight. In the second stage, the freeze dried material is rehydrated until the moisture content of the product is in the critical range i.e 2-7% by weight. The process described in this patent requires the use of high quantity of excipients for maintaining the stability of the product. U.S patent 4,537,883 to Alexander et al. discloses various lyophilizates of Cyclophosphamide. These lyophilizates are prepared by lyophilizing a solution of Cyclophosphamide and one or more excipients and rehydrating the product such that it contains about 4% moisture. The lyophilizates described in this patent used various excipients like mannitol, sodium bicarbonate, lactose, polyvinyl pyrrolidone (PVP), arginine, and tartaric acid.

U.S. patent 4,537,883 to Nageswara R. Palepu et al. discloses a stable rapidly dissolving lyophilized and hydrated composition of Cyclophosphamide with sodium bicarbonate. The disadvantages associated with the product described in this patent are the large size of the vials required for lyophilization and time taken to solubilise the product.

Cyclophosphamide is available as monohydrate in parenteral dosage formulation consisting of sterile packaged dry powder blend mixtures of drug and sodium chloride. The premixes were dissolved in water prior to administration. During the processing and storage of dry powder premix formulation, a glassiness and or stickiness could be acquired by the premix composition giving unattractive material with inferior solubility characteristics and decreased potency.

Hence there is a need to develop formulations of Cyclophosphamide overcoming the disadvantages of products and processes known in the art.

Summary of the invention

It is an object of the invention to develop lyophilized formulations of Cyclophosphamide using a process that does riot need rehydration step. Another aspect of the present invention is to describe process for producing a stable lyophilized cyclophosphamide monohydrate formulation by using a suitable solvent or mixture of solvents in suitable proportions and optionally contains other excipients.

Another aspect of the present invention is to develop formulations of Cyclophosphamide monohydrate with uniform hydrate integrity between the vials.

Another aspect of the present invention is to provide Cyclophosphamide formulations with improved stability and lesser time for reconstitution.

It is yet another object of the invention to provide a stability indicating analytical method to monitor drug product quality.

BRIEF DESCRIPTION OF THE DRAWINGS:

FIG. 1 shows the DSC thermogram of prelyophilizate comprising of Cyclophosphamide dissolved in mixture of Acetone-TBA-Water (35.17: 47.61 : 16.67%v/v).

FIG 2 depicts Diffractogram of Cyclophosphamide drug and lyophilized product.

FIG 3 depicts a DSC thermogram obtained for Cyclophosphamide drug and lyophilized product.

FIG 4 depicts an IR spectra obtained for Cyclophosphamide drug and lyophilized product. DETAILED DESCRIPTION OF THE INVENTION

The objective of the present invention is to prepare a stable lyophilized parenteral pharmaceutical compositions of cyclophosphamide monohydrate.

An aspect of the present invention relates to the process for producing a stable lyophilized cyclophosphamide monohydrate formulation by using a suitable solvent or mixture of solvents in suitable proportions and optionally contains other excipients. Another aspect of the present invention is to describe lyophilization process for producing pharmaceutical compositions of cyclophosphamide monohydrate. A lyophilization process of cyclophosphamide injection comprises of following steps: (a) Dissolving or dispersing cyclophosphamide in organic solvents or mixtures thereof (b) Filling the solution or dispersion in vials (c) Freezing the solution or dispersion (d) drying.

Another aspect of the present invention is to develop formulations of Cyclophosphamide monohydrate with uniform hydrate integrity between the vials and improved stability and lesser time for reconstitution.

Another aspect of the present invention is to describe stability indicating analytical method to monitor drug product quality.

The invention involves forming stable monohydrate "in situ" during lyophilization without the need for a rehydration step as taught in the prior art. This aspect of the invention is achieved by (i) a proper selection of solvent system and/ or (ii) optimal process conditions during the lyophilization process.

The present invention discloses a process for manufacturing a lyophilized preparation of cyclophosphamide intended for use in parenteral administration. The pharmaceutical formulation of the invention comprises Cyclophosphamide monohydrate and at least one organic solvent or mixture of solvents to dissolve the drug. The solvent is later removed during the freeze drying process. The formulation additionally comprises water to facilitate the formation of the stable monohydrate form during lyophilization.

Freeze drying process involves removal of solvent from a frozen mass under reduced atmospheric pressure. In the context of this invention the term freeze drying, drying and Lyophilization shall be used interchangeably. Lyophilization helps stabilize pharmaceutical formulations by reducing the solvent component or components to levels that no longer support chemical reactions or biological growth. Since drying during lyophilization takes place at a low temperature, chemical decomposition is also reduced. Annealing shall be defined as process of transient increase in product temperature from initial set point to higher or lower set point, and then bringing the product temperature back to original set point. Annealing can be done on product during different steps of freeze drying process.

Frozen mass shall be defined as a product kept at temperature where in the physical state of the product changes from liquid to semi solid mass or solid mass. In the context of the present invention a frozen mass shall also mean a product kept at a temperature lower than zero degrees centigrade.

Prelyophilizate shall mean a composition comprising cyclophosphamide dissolved or suspended in solvents and meant to be subjected to freeze drying. The prelyophilizate may comprise suitable pharmaceutically acceptable excipient selected from list of excipients as described in the present invention^" The excipients or drug shall be in dissolved or suspended form in the solvent mixture.

As used herein, the term "freeze-dried formulation" or "Lyophilizate" shall be defined as a dried product obtained as a result of freeze drying or lyophilization or drying process using a freeze dryer.

Freeze drying apparatus comprises of a chamber, condenser, and vacuum system with programmable features to control temperature and vacuum during drying process. The equipment optionally may have solvent trap.

The injectable formulations of the present invention comprises pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier or excipient can be selected from solvent, bulking agent, complexing agents, preservatives, anti-oxidants, stabilizers, tonicity modifiers or any other suitable adjuvant thereof. As used herein the term "solvent" refers to the liquid component of a formulation that is capable of dissolving or suspending one or more solutes. The term "solvent" can refer to a single solvent or a mixture of solvents. The solvent, as mentioned, can be any liquid in which the material dissolves or could be suspended; the solvent can be a single substance or a mixture of co-solvents. Depending on the formulation or the freeze-drying process, it may be desirable to include one or more organic solvents in the liquid formulation. Suitable solvents include the following, but are not limited to Acetone, Acetonitrile, Tertiary butanol, Methanol, Ethanol, Isopropyl alcohol, Ethylacetate, Toulene, Propyl acetate, Nitromethane, 1 ,4-Dioxane, Heptane, Methylcyclohexane, Formic acid, isopropyl acetate, 1 ,2-Dimethoxyethane, 1 , 1 ,2-Trichloroethene, Methylethyl ketone, Cyclohexane, Ethyl formate, Hexane, Tetrahydrofuran, Chloroform, Methyl acetate, 1 ,2-Dichloroethene, tert-Butylmethyl ether, Dichloromethane, Pentane, Ethyl ether, Sulfalone, Ethyleneglycol, Formamide, Tetralin, N-Methylpyrrolidone, Dimethyl sulfoxide, N,N-Dimethylacetamide, 1-Pentanol, 3-Methyl-l -butanol, Anisole, Ν,Ν-Dimethylformamide, 2-Ethoxyethanol, 1- Butanol, 2-Methoxyethanol, Cumen^ Buiyl acetate, 2-Methyl-l-propanol, Chlorobenzene, Acetic acid, 2-Butanol, Isobutyl acetate, 1-Propanol, Pyridine, Methylbutyl ketone, Methyl isobutyl ketone, Xylene, Dimethyl carbonate, Hexafluoroacetone, Chlorobutanol, Dimethyl sulfone, carbon tetrachloride, tetrahydropyran, dioxane, trioxane and other cyclic mono-, di- and tri-ethers, propylene glycol (PG), polyethylene glycol, glycerine. The solvent system may additionally comprise water. Mixtures of solvents selected are used in a suitable proportion and suitable quantity to achieve desirable effect.

The purpose of the bulking agent is to provide bulk to the formulation and enhance cake formation. Bulking agents include saccharides, preferably monosaccharides or oligosaccharides, sugar alcohols, and mixtures thereof. Suitable bulking agents include the following, but are not limited to mannitol, sodium chloride, glucose, sucrose, lactose, trehalose, dextrose, maltose, sorbitol, dextran, raffinose, PVP, histidine, amino acids such as glycine, arginine, aspartic acid and the like. Tonicity modifier can also be optionally added to the formulation. Suitable tonicity modifiers include the following, but are not limited to mannitol, dextrose, sucrose, glycine, glycerol, sodium chloride and the like.

Stabilizing agents are typically added to a formulation to improve stability of the formulation. Suitable examples of stabilizing agents include cryoprotectants, lyoprotectants, crystallization inhibitors or any other suitable stabilizer thereof. Suitable stabilizers include the following, but are not limited to Saccharides, including monosaccharides such as glucose, disaccharides such as sucrose (glucose+fructose), lactose (glucose+galactose), maltose (glucose+glucose), and trehalose (alpha-D- glucopyranosyl alpha-D-glucopyranoside), and polysaccharides such as dextran (polysaccharide containing glucose monomers, Crystallization inhibitors such as PVP (polyvinylpyrrolidone), HPC (hydroxypropyl cellulose), or HPMC (hydroxypropylmethylcellulose) and ,the like can be used. Surfactants also act as suitable stabilizers such as polyoxyethylene Sforbitan monolaurate (Tween.TM. 20, Tween.TM. 80), pluronic F-68, Triton. TM. X-100, and sodium dodecyl sulfate (SDS), polysorbate or any other suitable surfactant can be selected.

Buffers are typically included in pharmaceutical formulations to maintain the pH of the formulation at a physiologically acceptable pH. The desirable pH for a formulation may also be affected by the active agent. Examples of suitable buffers include buffers derived from an acid such as phosphate, aconitic, citric, gluaric, malic, succinic and carbonic acid, alkali or alkaline earth salt of one of these acids, Tris buffer, histidine buffers, meglumine or any suitable buffer thereof. pH adjusting agents such as, but are not limited to sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, ammonium carbonate, hydrochloric acid, citric acid, lactic acid, phosphoric acid, sodium phosphate, sulfuric acid, and the like can also be used.

Other excipients may also be added to the lyophilized preparations of the present inventions. Such excipients may include antioxidants, antimicrobials, cryostabilizers, or any other suitable pharmaceutically acceptable adjuvants thereof. The lyophilized product or the dried product shall be reconstituted and further diluted using suitable diluents. Suitable diluents may include the following but are not limited to water, sodium chloride, dextrose, sugars, polyols, electrolytes or any other parenterally acceptable diluents and mixtures thereof.

Selection of solvent system and ratios

The solubility of Cyclophosphamide monohydrate in various solvents was checked. The quantity of solvent consumed for dissolving lOOmg of Cyclophosphamide monohydrate is given below:

Water 2.45ml

Acetonitrile 0.5ml

Acetone 0.4 ml

Ethanol 0.5ml

Tertiary butyl alcohol 0.5ml

Dimethyacetamide 2.0ml

Lyophilization. of Cyclophosphamide with different solvent systems

The investigators found surprising results that Cyclophosphamide monohydrate was obtained without rehydration step when Cyclophosphamide was freeze dried in presence of solvents.

Binary solvent mixtures were evaluated for lyophilization of Cyclophosphamide for injection. Solvent systems used for the study are given below in Table- 1 :

Table 1 : Binary solvent Mixtures used for Lyophilization

NA: Not Applicable

The above mentioned solvent mixture ratios were lyophilized without rehydration step and the results of lyophilizate are given below

Table 2: Results of Binary solvent Mixtures used for Lyophilization

Observations & Conclusion:

From the analytical data it was observed that the water content was found to be within the acceptable range for monohydrate (5.7% w/w to 7.5%w/w) whereas the solvent levels are elevated.

Freeze drying of Cyclophosphamide using mixture of solvents: Solvent mixtures of Acetone-Acetonitrile-Water and Acetone-Tertiary Butyl Alcohol- Water were evaluated to establish the suitability in lyophilization. The results are tabulated in table 3.

Table 3: Results after lyophilization with Acetone-Acetonitrile- Water and Acetone-TBA- Water:

*Not applicable

From the analytical results it is evident that Cyclophosphamide when freeze dried using mixture of Acetone: Acetonitrile: Water solvent mixture resulted in high residual solvent content and water content whereas the solvent system containing Acetone: TBA: Water showed low residual solvent content and significantly high water content.

The proportions of constituents of the solvent mixture was optimized by varying the % of water in the solvent system varying from about 7.8% v/v to 16.6% v/v to evaluate the effect of water on the drying Acetone, TBA and Water. The results are tabulated in table 4:

Table 4: Results of solvent mixture proportions on the lyophilizate:

The data in the above table shows that the level of residual solvent content in the lyophilizate was reduced by varying the proportions of water in the solvent mixture.

The impact of constituents of solvent composition used to dissolve cyclophosphamide was assessed by varying Acetone, TBA|;and Water in the pre-lyophilizate composition. The results are in the table 5.

Table 5 : Effect of solvent composition used to dissolve Cyclophosphamide on lyophilizate:

4 2 gms 37.5 50.0 12.5 9.4 71.0 6.57 1234 557

47.6

5 2 gms 35.17 16.66 9.9 71.0 6,53 1018 661

1

46.5

6 2 gms 34.88 18.60 10.1 78.7 6.37 792 538

1

45.4

7 2 gms 34.09 20.45 10.3 78.7 6.36 1575 428

5

44.4

8 2 gms 33.33 22.22 10.5 78.7 6.32 1730 387

4

43.4

9 2 gms 32.60 23.91 10.7 78.7 6.46 2010 437

7

33.3

10 2 gms 25.00 41.66 13.0 75.2 6.57 1 122 706

3

From above it is evident that residual solvent content, and target water content in the lyophilizate are within acceptable range.

Optimisation of process conditions

Cyclophosphamide is stable in its crystalline monohydrate form. The change of description of Cyclophosphamide monohydrate at various temperatures was studied by exposing the drug to different temperatures under constant vacuum and details are given in Tables 6 & 7:

Table 6: Description of Cyclophosphamide monohydrate at different temperatures at lOOOmtorr process vacuum:

Table 7: Description of Cyclophosphamide monohydrate at different temperatures and process vacuum:

The results indicate that physical stability or description of Cyclophosphamide is susceptible to temperature and vacuum. At higher temperatures and vacuum, Cyclophosphamide monohydrate loses water and melts. When it starts losing its water molecule, the physical description changes from powder to sticky mass or liquid, hence selection of the process conditions (like temperature, vacuum etc) is critical for lyophilization process so as to obtain monohydrate form. The selection of process conditions that result in insitu formation of monohydrate form of Cyclophosphamide are also dependent on composition of pre-lyophilizate.

The formulation of the present invention preferably has a solvent or mixture of solvents. The inventors have surprisingly found by use of mixture of solvents with or without water, or mixture of solvent with water yielded best results.

Evaluation of process conditions for Drying The drug solution was transferred into sample pan and sealed with lid. DSC Q2000 was purged with nitrogen. Sample was cooled from 25°C to -50°C and heated from -50°C to 25°C at the rate of 1.5°C/min.

From the DSC thermogram of pre-lyophilizate as shown in FIG 1 it was observed that melt of frozen mass started at -32.80°C and complete melt was observed at -20.53°C. So, to enhance the crystallization, for controlled removal of water and to produce a uniform hydrate insitu during the process, the temperature of product during the drying process was kept on hold for -18°C for removal of water and TBA. Duration of hold at -18°C and 600 mtorr is important for insitu formation of Cyclophosphamide monohydrate and effective removal of excess TBA. This was evaluated by varying the duration of hold at -18°C at 600mtorr pressure during drying process as given in table 8:

Table 8: Analysis results of lyophilizate by varying the duration of hold at -18°C, 600 mtorr: ^

*Note: The total volume in all of above compositions was 9.9mL

The results showed that residual solvent content reduced in the lyophilizate with in hold time while retaining the water content of insitu monohydrate obtained.

Evaluation of Lyophilizate:

The monohydrate form present in the lyophilizate was evaluated in comparison with drug substance used in the process by way of following tests: Moisture Content and Water activity:

The moisture content and water activity of the drug substance as well as the lyophilizate was checked to ensure the retention of the crystalline monohydrate form of the drug substance in the Lyophilizate. Water activity instruments measure the amount of free (sometimes referred to as unbound or active) water present in the sample and KF titration measures the total water in a sample. The values are tabulated in tables 9 and 10.

Table 9: Water content results of Drug substance and Lyophilizate

Table 10: Water activity results for Drug substance and Lyophilizate

Results for moisture content as well as the water activity were 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. Crystalline monohydrate form contains 5.7 to 6.8% of water which is in agreement with the results obtained in table 6 to confirm that the lyophilizate is a stable monohydrate.

X-Ray diffraction studies: XRD was performed using Schimadzu XRD-7000 at following conditions:

X-ray Tube: Cu (1.54060 A)

Voltage: 45.0 kV

Current: 40.0 mA

Scan Range: 2.5000 <-> 49.9980 deg

Step Size: 0.0170 deg

Count Time: 0.51 sec

Slit DS: 1 .00 deg

SS: 1.00 deg

RS: 0.15 mm

The diffractograms of drug substance and lyophilizate as shown in FIG 2; are comparable confirming that lyophilizate is a monohydrate form.

DSC studies

DSC was performed using TA-Q20J; °½ mg of sample was accurately weighed into aluminium pan. The pan was crimped and DSC was performed at a heating rate of 2°C/min from 25°C to 80°C under atmosphere of nitrogen.

The DSC thermograms of drug substance and lyophilizate are similar as shown in FIG 3. IR Studies:

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 (Nicolet IS, Therm'oscientific).

The IR spectra of drug substance and lyophilizate are similar as shown in FIG 4. Analytical evaluations

Lyophilized product was analyzed by HPLC for % purity and Assay. The result shows that the sample contains 0.27% impurities and was found to be 99.73% pure. By Assay HPLC was found to be 101% which is similar to the bulk solution assay prior to the lyophilization. These results show that during the lyophilization process sample was not degraded and crystalline monohydrate form is retained in final product. Cyclophosphamide monohydrate is official in USP and Ph.Eur. Both the monographs specify degradation analytical methods by Thin layer Chromatography. The TLC methods are time consuming and require usage of expensive USP reference impurity standards for comparison of Spots on TLC. These methods are not suitable for estimation of other degradation products or any source of contamination during manufacturing and storage of drug product.

Thus there is a need to develop stability indicating HPLC methods for monitoring quality of drug substance and drug product throughout the shelf life. The investigators developed novel HPLC analytical methods where in the polarity of mobile phase was gradually changed in order to separate the major degradation products on single Chromatographic run.

phosphate

HPLC method for estimation of Impurity-A:

Mobile Phase- A: 0.5ml of Triethylamine is diluted with in 1000ml of HPLC grade (Ultrapure) water and pH adjusted to 7.5 with dilute Orthophosphoric acid. Mobile Phase-B: Mobile phase-A and Acetonitrile are mixed in the ratio of (30:70) % v/v and degassed in a sonicator for about 10 min.

Preparation of Diluent: Mobile Phase-A is used as diluent and maintained at temperature about 2-8°C in the refrigerator.

Typical chromatographic conditions:

Column Kinetex C-18 (100mm x 4.6 mm), 2.6μηι , Wave length 195nm

Flow rate 0.5 mL/min

Column oven Temperature 25±2°C

Sample cooling rack 5±1°C

Injection volume ΙΟΟμΙ,

Runtime

Gradient program:

Table 12: chromatographic conditions for detection of Impurity A

Preparation of standard solution:

Preparation of Impurity-A solution: 0.2% Level

4 mg of Impurity-A was weighed into a 100 ml volumetric flask and 50 ml of diluent added and sonicated for dissolution. The volume was made up with the diluent.

Preparation of Sample solution: (20mg/ml) 100 mg of Cyclophosphamide was weighed into a 5 ml volumetric flask and 3 ml of diluent was added and sonicated. Volume was made up with the diluent.

Procedure:

Injection- Blank (as diluent), diluted standard for two times and test preparation in single into the liquid chromatographic system.

System Suitability Parameters:

• The tailing factor for Impurity-A in standard solution is not more than 2.0

• The USP theoretical plates for Impurity-A is not less than 2000

• The relative standard deviation for two replicate injections is not more than

10.0%

Detection of Impurity B and D

Mobile Phase-A: 1ml of Orthophosp¾oric acid is mixed in 1000ml of water (0.1%) and ø

degassed in sonicator for about lOmin.

Mobile Phase-B: Water and Acetonitrile were mixed in the ratio of 20:80% v/v and degassed in a sonicator for about 1 Omin.

Preparation of Diluent: HPLC grade (Ultrapure) water maintained at temperature about 2-

8°C.

Typical chromatographic conditions:

Column Inertsil-ODS 3, 25cm x 4.6 mm, 5μπι (LI)

Wave length 195nm

Flow rate 0.5 mL/min

Column oven Temperature 25±2°C

Sample cooling rack 5±1°C

Injection volume ΙΟΟμί

Runtime 75 minutes

Sample concentration 20 mg/mL

Gradient program: Table 13 : chromatographic conditions for detection of Impurity B and D

Preparation of standard solution: (0.2% level)

40.0 mg of Cyclophosphamide working standard was transferred into a 100 ml volumetric flask. 50 ml of diluent was added sonicated and volume was made up with diluent. 1ml of this solution was diluted to 10ml withvdiluent.

Preparation of Sample:

100 mg sample was transferred into a 5 ml volumetric flask, 3 ml was added and sonicated and made to volume with diluent.

Procedure:

Injection- the Blank (as diluent), diluted standard for two replicate injections and a single test preparation into the liquid Chromatographic system.

System Suitability Parameters:

1. The tailing factor for Cyclophosphamide peak in standard solution is not more than 2.0

2. The USP theoretical plates for Cyclophosphamide peak is not less than 4000

3. The relative standard deviation for two replicate injections is not more than 10.0%

Calculation:

Blank peaks are disregarded and all impurity peaks and Cyclophosphamide peaks are integrated. Peak below 0.05% is disregarded. Table 14: Cyclophosphamide Impurities and their retention times

• RRT - Relative retention time

# RRF- Relative retention factor

Detection of Impurity C

Impurity-C cannot be detected in HPLC UV detector because the impurity-C does not contain the necessary chromophoric, fluophoric or redox groups. However, this problem was overcome by inducing derivatization reaction. In post-column mode, the reaction is performed automatically by adding a derivatization reagent after separation, and before detection, by means of a second HPLC pump.

Fluorescamine is used as dervitizing reagent for Impurity-C. The derivitized impurity-C was well detected at 380-nm.

Mobile Phase -A: 1.712 lg of di-sodium hydrogen phosphate (0.01M) was transferred in 1000ml of HPLC grade (Ultrapure) water and mixed well, pH 10.5 adjusted with diluted Sodium hydroxide.

Mobile Phase -B: Acetonitrile and HPLC grade (Ultrapure) water were mixed in the ratio of (90: 10) v/v and sonicated for 10 mintes for degassing.

Preparation of Diluent: HPLC grade (Ultrapure) water maintained at temperature about 2- 8°C is used as diluent.

Typical chromatographic conditions:

Column Purospher Star RP18, (250mm x 4.6 mm) 5μηι (LI) Wave length 380nm .

Flow rate 1.0 mL/min

Column oven Temperature 25±2°C Sample cooling rack 5±1°C

Injection volume ΙΟΟμΙ,

Runtime 35 minutes

Sample concentration 20 mg/mL Gradient program:

Table 15: Chromatographic conditions for detection of Impurity C

External Pump conditions:

Connector : 'Τ' Joint

Flow rate <: l .Oml/min

Mobile Phase for external pump: Accurately weighed 80mg of Fluroscamine derivitizating reagent was transferred to a 200ml volumetric flask and 100ml of Acetonitrile was added and the volume was made up volume with Acetonitrile and mixed well.

Preparation of Impurity-C: (~10ppm)-0.06% Level

About 5 mg of Cyclophosphamide Impurity-C was accurately weighed and transferred into a 10 ml volumetric flask and about 5 ml of water was added. The contents were sonicated and the volume was made up with water and further mixed well. Subsequently, 1ml of this solution was diluted to 50ml with water and mixed well.

Procedure for post column derivatization technique: A 'T' joint was connected to the column outlet of external pump and the other outlet of the column was connected to the HPLC detector. The mobile phase from HPLC pump and the mobile phase from external pump were mixed at the 'T' Joint connector and then pumped to the detector.

The following examples further describe certain specific aspects and embodiments of the present invention and demonstrate the practice and advantages thereof. It is to be understood that the examples are given by way of illustration only and are not intended to limit the scope of the invention in any manner.

Examples

Example-1

The solvent mixture was prepared by taking acetone and TBA in a mixing vessel and the solution was cooled to 2-8°C. Cyclophosphamide was added to 80% of solvent mixture and stirred till completely dissolved followed by addition of water at 2-8°C. Volume was made up to 100% with solvent mixture and the bulk solution was filled into vials for lyophilisation. The lyophilization cycle parameters are mentioned in table 16.

Table- 16: Freeze drying cycle process parameters:

-

Freezing -45 60 R 12.7

-

Freezing -45 180 H 15.7

Primary Drying -45 60 R 50 16.7

Primary Drying -45 420 H 50 23.7

Primary Drying -30 180 R 600 26.7

Primary Drying -45 60 R 600 27.7

Primary Drying -18 $ 120 R 600 29.7

Primary Drying -42 60 R 600 30.7

Primary Drying -18 120 R 600 32.7

Primary Drying -42 60 R 600 33.7

Primary Drying -18 120 R 600 35.7

Primary Drying -42 60 R 600 36.7

Primary Drying -18 120 R 600 38.7

Primary Drying -42 60 R 600 39.7

Primary Drying -18 120 R 600 41.7

Primary Drying -42 60 R 600 42.7

Primary Drying -18 120 R 600 44.7

Primary Drying -42 60 R 600 45.7

Primary Drying -18 120 R 600 47.7

Primary Drying -42 60 R 600 48.7

Primary Drying -18 120 R 600 50.7

Primary Drying -42 60 R 600 51.7 28 Primary Drying -18 120 R 600 53.7

29 Primary Drying -18 680 H 600 65.0

30 Primary Drying -42 60 R 800 66.0

31 Primary Drying -42 300 H 800 71.0

The product was evaluated for water content, Solvent content and reconstitution time. Results are tabulated in table 17.

Table 17: Analytical results of lyophilizate:

As is evident from the above results, the formation of monohydrate and the reconstitution time are consistent even at different strengths.

The procedures described herein are adapted to prepare other compositions described below:

Example-2 S.No Ingredients Quantity mg Vial

1. Cyclophosphamide 2000

2. Acetonitrile: Water (30:70) Q.s to 16.0 mL

Acetonitrile and water were mixed and cooled to less than 10^°C (Temperature range: -10 to 10^*C). Drug was added while temperature was maintained in the range mentioned. The solution was filtered and filled in vials to target fill volume and freeze dried.

Example-3

Acetonitrile and water were mixed and cooled to less than 10^"C (Temperature range: -10 to 10^°C). Drug was added while temperature was maintained in the range mentioned. The solution was filtered and filled in vials to target fill volume and freeze dried.

Example-4

Acetone and water were mixed and cooled to less than 10^°C (Temperature range: -10 to 10^°C). Drug was added while temperature was maintained in the range mentioned. The solution was filtered and filled in vials to target fill volume and freeze dried.

Example-5

Acetone, Tertiary butanol and water were mixed and cooled to less tha 10^°C (Temperature range: - 10 to 10^°C). Drug was added while temperature was maintained in the range mentioned. The solution was filtered and filled in vials^' to target fill volume and freeze dried.

Example-6

Acetonitrile, Tertiary butanol and water were mixed and cooled to less than 10^°C (Temperature range: -10 to 10^°C). Drug was added while temperature was maintained in the range mentioned. The solution was filtered and filled in vials to target fill volume and freeze dried. i^;

Example-7

Acetonitrile, Tertiary butanol and water were mixed and cooled to less than 10^°C (Temperature range: -10 to 10^°C). Drug was added while temperature was maintained in the range mentioned. The solution was filtered and filled in vials to target fill volume and freeze dried.

Example-8

ExampIe-9

Cyclophosphamide was dissolved in cool acetone (Temperature range: -10 to 10^°C). Tertiary butanol was added with continuous mixing. The solution was filtered and filled in vials to target fill volume and freeze dried.

Example-10:

Cyclophosphamide was dissolved in cool acetone (Temperature range: -10 to I0^°C). Tertiary butanol and water were added with continuous mixing. The solution was filtered and filled in vials to target fill volume and freeze dried. Example-11 :

Cyclophosphamide was dissolved in cool acetone (Temperature range: -10 to 10^°C). Tertiary butanol and water were added with continuous mixing. The solution was filtered and filled in vials to target fill volume and freeze dried.

Example-12:

Examnle-13;

Cyclophosphamide was dissolved in cool acetone (Temperature range: - 10 to 10^°C). Tertiary butanol was added with continuous mixing. The solution was filtered and filled in vials to target fill volume and freeze dried.

Example-14

Cyclophosphamide - was dissolved in cool acetone (Temperature range: -10 to 10^"C). Tertiary butanol and water were added with continuous mixing. The solution was filtered and filled in vials to target fill volume arid freeze dried.

Examnle-15

Lyophihzation experiments were performed using solvents that had lower vapour pressure than water for example: Dimethyl acetamide and Dimethylsulfoxide. It was found that, the use of these solvents did not result in stable lyophilizate. if

Composition:

Cyclophosphamide was dissolved in cool Dimethylacetamide (Temperature range: -10 to 10^°C) and water was added to the solution and mixed. The solution was filtered and filled in vials to target fill volume and freeze dried. Example-16

The solvent mixture was prepared by taking acetonitrile and water in a mixing vessel and the solution was cooled to 2-8°C. Cyclophosphamide was added to 80% of solvent mixture and stirred well, until the drug is completely dissolved at 2-8°C. Volume was made up to 100% with solvent mixture and the bulk solution was filled into vials for lyophilization. The lyophilization cycle parameters are mentioned in below table 18:

Table 18: Lyophilization Cycle

6 -50 150 R -

Freezing 15.166

7 -50 200 H -

Freezing 18.50

8 Primary Drying -50 60 R 50 19.50

9 Primary Drying -50 180 H 50 22.50

10 Primary Drying -30 120 R 600 24.50

1 1 Primary Drying -50 60 R 600 25.50

12 Primary Drying -30 120 R 600 27.50

13 Primary Drying -50 60 R 600 28.50

14 Primary Drying -30 120 R 600 30.50

15 Primary Drying -50 60 R 600 31.50

16 Primary Drying -30 120 R 600 33.50

17 Primary Drying 180 H 600 36.50

18 Primary Drying -fb 960 H 600 52.50

19 Primary Drying -30 120 H 350 54.50

Results: Water content (%) was 5.8% and Solvent Content (ppm) was 2500.

Example 17

Initially mannitol was dissolved in required quantity of water in a mixing vessel and cooled to 10°C- 15°C. In another mixing vessel Cyclophosphamide was dissolved in 80% of required quantity of Tertiary butanol. Mannitol solution was added to the drug solution and stirred at 10°C-15°C to get a uniform solution. Volume was made up to 100% with Tertiary butanol and the bulk solution was filled into vials for lyophihsation. The sample was then freeze dried.

Example 18

Initially mannitol was dissolved in required quantity of water in a mixing vessel and was cooled to 10°C-15°C. In another mixing vessel Cyclophosphamide was dissolved in 80% of required quantity of Acetone. Mannitol solution was added to the drug solution and stirred at 10°C-15°C to get a unif|fcni^'"'solution. Volume was made up to 100% with Acetone and the bulk solution was filled into vials for lyophihsation. The sample was then freeze dried.

Example 19

Initially mannitol was dissolved in required quantity of water in a mixing vessel and was cooled to 10°C-15°C. In another mixing vessel Cyclophosphamide was dissolved in 80% of required quantity of Acetonitrile. Mannitol solution was added to the drug solution and stirred at 10°C-15°C to get a uniform solution. Volume was made up to 100% with Acetonitrile and the bulk solution was filled into vials for lyophihsation. The sample was then freeze dried. Example 20

Initially sodium chloride was dissolved in required quantity of water in a mixing vessel. In another mixing vessel solvent mixture was prepared by taking acetone and acetonitrile and the solution was cooled to 2-8°C. Cyclophosphamide was added to 80% of solvent mixture and stirred well, until the drug is completely dissolved at 2-8°C. Sodium chloride solution was added to solvent mixture and stirrbcHo get a uniform solution. Volume was made up to 100% with solvent mixture and the bulk solution was filled into vials for lyophilisation. The sample was then freeze dried.

Example 21

Initially sodium chloride was dissolved in required quantity of water in a mixing vessel. In another mixing vessel 80% of required quantity of Acetone was taken cooled to 2-8°C. Cyclophosphamide was added to above solvent and stirred well, until the drug is completely dissolved at 2-8°C. Sodium chloride solution was added to drug solution and stirred to get uniform solution. Volume was made up to 100% with Acetone and the bulk solution was filled into vials for lyophilisation. The sample was then freeze dried.

Claims

We Claim:

1. A composition comprising lyophilized cyclophosphamide monohydrate where in lyophilization process is carried out without a rehydration step.

2. A composition comprising lyophilized cyclophosphamide monohydrate where in lyophi ization is carried out in presence of solvent or mixtures of solvents.

3. The composition according to claim 2, wherein the solvent is selected from group comprising Acetone, Acetonitrile, Tertiary butanol, Methanol, Ethanol, Isopropyl alcohol, Ethylacetate, Toluene, Propyl acetate, Nitromethane, 1,4-Dioxane, Heptane, Methylcyclohexane, Formic acid, isopropyl acetate, 1,2- Dimethoxyethane, 1 , 1 ,2-Trichloroethene, Methylethyl ketone, Cyclohexane, Ethyl formate, Hexane, Tetrahydrofuran, Chloroform, Methyl acetate, 1,2- Dichloroethene, tert-Butylmethyl ether, Dichloromethane, Pentane, Ethyl ether, Sulfalone, Ethyleneglycol, Formamide, Tetralin, N-Methylpyrrolidone, Dimethyl sulfoxide, N,N-Dimethylacef¾mide, 1-Pentanol, 3 -Methyl- 1 -butanol, Anisole, N,N- Dimethylformamide, 2-Ethoxyethanol, 1 -Butanol, 2-Methoxyethanol, Cumene, Butyl acetate, 2-Methyl-l-propanol, Chlorobenzene, Acetic acid, 2-Butanol, Isobutyl acetate, 1 -Propanol, Pyridine, Methylbutyl ketone, Methyl isobutyl ketone, Xylene, Dimethyl carbonate, Hexafluoroacetone, Chlorobutanol, Dimethylsulfone, carbon tetrachloride and tetrahydropyran.

4. The composition according to claim 2, comprising of a mixture of two or more solvents selected from Acetone, Acetonitrile, Tertiary butanol, Methanol, Ethanol, Isopropyl alcohol, Ethylacetate, Toluene, Propyl acetate, Nitromethane, 1 ,4- Dioxane, Heptane, Methylcyclohexane, Formic acid, isopropyl acetate, 1,2- Dimethoxyethane, 1 , 1 ,2-Trichloroethene, Methylethyl ketone, Cyclohexane, Ethyl formate, Hexane, Tetrahydrofuran, Chloroform, Methyl acetate, 1,2- Dichloroethene, tert-Butylmethyl ether, Dichloromethane, Pentane, Ethyl ether, Sulfalone, Ethyleneglycol, Formamide, Tetralin, N-Methylpyrrolidone, Dimethyl sulfoxide, Ν,Ν-Dimethylacetamide, 1-Pentanol, 3 -Methyl- 1 -butanol, Anisole, N,N- Dimethylformamide, 2-Ethoxyethanol, 1 -Butanol, 2-Methoxyethanol, Cumene, Butyl acetate, 2-Methyl-l-propanol, Chlorobenzene, Acetic acid, 2-Butanol, Isobutyl acetate, 1-Propanol, Pyridine, Methylbutyl ketone, Methyl isobutyl ketone, Xylene, Dimethyl carbonate, Hexafluoroacetone, Chlorobutanol, Dimethylsulfone, Carbon tetrachloride and tetrahydropyran.

5. The composition according to claim 2, comprising of mixture of two or more solvents where in atleast one solvent shall have vapor pressure higher than or equal to water selected from Toluene, Propyl acetate, Nitromethane, 1 ,4-Dioxane, 2- Propanol , Heptane, Methylcyclohexane, Ethanol , Formic acid, Isopropyl acetate , 1 ,2-Dimethoxyethane, 1, 1,2-Trichloroethene, Methylethyl ketone, Ethyl acetate, Acetonitrile, Cyclohexane, Ethyl formate, Methanol, Hexane, Tetrahydrofuran, Chloroform, Methyl acetate, 1 ,2-Dichloroethene, Acetone, tert-Butylmethyl ether, Dichloromethane, Pentane, Ethyl ether and atleast one solvent shall have vapor pressure lower than or equal to water and selected from Tertiary butanol, Sulfalone, Ethylene glycol, Formamide, Tetralin, N-Methylpyrrolidone, Dimethyl sulfoxide, Ν,Ν-Dimethylacetamide, lrPentanol, 3-Methyl-l -butanol, Anisole, N,N- Dimethylformamide, 2-EthcfKyethanol, 1 -Butanol, 2-Methoxy ethanol, Cumene, Butyl acetate, 2-Methyl-l-propanol, Chlorobenzene, Acetic acid, 2-Butanol, Isobutyl acetate, 1 -Propanol, Pyridine, Methylbutyl ketone, Methyl isobutyl ketone, Dimethyl carbonate, Hexafluoroacetone, carbon tetrachloride and tetrahydropyran.

6. The composition according to claim 2, comprising of mixture of two or more solvents, wherein atleast one solvent is tertiary butanol, and other solvent or mixture of solvents shall be selected from Acetone, Acetonitrile, Methanol, Ethanol, Isopropyl alcohol, Ethylacetate, Toluene, Propyl acetate, Nitromethane, 1 ,4-Dioxane, Heptane, Methylcyclohexane, Formic acid, isopropyl acetate, 1,2- Dimethoxyethane, 1 ,1,2-Trichloroethene, Methylethyl ketone, Cyclohexane, Ethyl formate, Hexane, Tetrahydrofuran, Chloroform, Methyl acetate, 1 ,2- Dichloroethene, tert-Butylmethyl ether, Dichloromethane, Pentane, Ethyl ether, Sulfalone, Ethyleneglycol, Formamide, Tetralin, N-Methylpyrrolidone, Dimethyl sulfoxide, N,N-Dimethylacetamide, 1-Pentanol, 3-Methyl-l -butanol, Anisole, N,N- Dimethylformamide, 2-Ethoxyethanol, 1 -Butanol, 2-Methoxyethanol, Cumene, Butyl acetate, 2-Methyl-l-propanol, Chlorobenzene, Acetic - acid, 2-Butanol, Isobutyl acetate, 1 -Propanol, Pyridine, Methylbutyl ketone, Methyl isobutyl ketone, Xylene, Dimethyl carbonate, Hexafluoroacetone, Chlorobutanol, Dimethylsulfone, carbon tetrachloride and tetrahydropyran.

7. The composition according to claim 2, where in solvent shall be selected from Acetone, Acetonitrile, Methanol, Ethanol, Isopropyl alcohol, Toluene, Propyl acetate, Nitromethane, Heptane, Methylcyclohexane, isopropyl acetate, 1,2- Dimethoxyethane, 1, 1 ,2-Trichloroethane, Methylethyl ketone, Ethyl acetate, Ethyl formate, Hexane, Tetrahydrofuran, Chloroform, Methyl acetate, 1 ,2- Dichloroethene, tert-Butylmethyl ether, Dichloromethane, Pentane, Ethyl ether, Tetralin, Ν,Ν-Dimethylacetamide, 1 -Pentanol, 3-Methyl-l -butanol, Anisole, N,N- Dimethylformamide, 2-Ethoxyethanol, 1-Butanol, 2-Methoxy ethanol, Cumene, Butyl acetate, 2-Methyl- l-propanol, Chlorobenzene, 2-Butanol, Isobutyl acetate, 1 - Propanol, Pyridine, Ethyleneglycol, Formamide, N-Methylpyrrolidone, Dimethyl sulfoxide, Sulfalone, acetic acid, 1,4-Dioxane, Formic acid, Methylbutyl ketone, Cyclohexane, Methyl isobutyl, ketone, Hexafluoroacetone, tetrahydropyran and shall have freezing point below -5°C

8. The composition according to claim 2, where in the solvent mixture additionally comprises water.

9. The composition according to claim 2, wherein lyophilization is carried out using the solvent combinations selected from Acetone and Acetonitrile, Acetone and Tertiary butanol, Acetonitrile and Tertiary butanol, Isopropyl alcohol and Tertiary butanol, Methanol and Tertiary butanol, Ethanol and Tertiary butanol, Methanol and Isopropyl alcohol, Ethanol and Isopropyl alcohol, Acetonitrile and Isopropyl alcohol.

10. The compositions according to claim 9, wherein solvent mixture additionally comprises water.

1 1. The composition according to claim 2, comprising of solvent or mixture of solvents atleast 25% volume by volume.

12. The compositions according to claim 7, where in solvent mixture additionally comprises water ranging between 0.5% to 60% volume by volume.

13. A process of lyophilizing cyclophosphamide for injection without involving rehydration step.

14. A [yophilisation process for manufacturing Cyclophosphamide injection wherein the process is carried out using organic solvents, a mixture of organic solvents or a mixture of organic solvents and water.

15. A lyophilization process of cyclophosphamide injection comprising of following steps:' (a) Dissolving or dispersing cyclophosphamide in organic solvents or mixtures thereof (b) Filling the solution or dispersion in vials (c) Freezing the solution or dispersion (d) drying.

16. The process of lyophilization of cyclophosphamide according to claim 15, wherein freezing is performed at temperatures below -12°C.

17. The process of freezing according to claim 15, further comprises of one or more annealing steps.

8. The process of lyophilization of cyclophosphamide according to claim 15, wherein drying is performed at temperatures between -50°C and -5°C.

19. The process of lyophilization of cyclophosphamide according to claim 15, where in vacuum used for drying steps*shall be selected between 10 mtorr and 1500mbar.

20. The vacuum range according to claim 19, shall be selected between lOmtorr to 800mtorr.

21. The process of lyophilization of cyclophosphamide that shall comprise of atleast one annealing step with temperature ranging between -10°C and -90°C during freezing, and drying steps.

22. A lyophilized product of cyclophosphamide prepared according to claim 15, comprising of water content in finished product ranging between 5.5% to 8.5%w/w and residual solvent content within acceptable ICH limits for the respective solvent.

23. A lyophilized product of cyclophosphamide prepared according to claim 2 having reconstitution time of less than 60 seconds

24. A lyophilized product of cyclophosphamide prepared according to claim 15, having reconstitution time of less than 60 seconds

25. A lyophilized product of cyclophosphamide prepared according to claim 15, comprising of one or more pharmaceutically acceptable carriers or excipients

26. A lyophilized product of cyclophosphamide prepared according to claim 2, comprising of one or more pharmaceutically acceptable excipients

27. The process of lyophilization of cyclophosphamide according to claim 15, comprising of atleast one of the last three drying steps at a temperature less than or equal +5°C.

28. The process of lyophilization of cyclophosphamide according to claim 27,

comprising of performing secondary drying at a temperature below -15°C for a duration of atleast 2 hours.

29. A lyophilized composition of Cyclophosphamide obtained by the process of claim 1.

30; A reverse phased HPLC method for quantitative analysis of impurities in preparations containing Cyclophosphamide.

31. A reversed phase HPLC method comprising of use of post column derivatization technique for quantitative analysis of impurities in preparations containing Cyclophosphamide.

32. A reversed phase HPLC method described in Claim 30 used for quantitative analysis consisting of: &

(a) Mobile Phase-A comprising of triethylamine dissolved in water at a pH of 7.5

(b) Mobile Phase-B comprising of atleast 40% of Acetonitrile mixed with Mobile phase-A

(c) The temperature of diluted sample maintained between 2-25°C

(d) The chromatographic conditions comprising of C-18 column having a particle size of 2.6μπι or lesser, and flow rate of atleast 0.5mL/min, and run time of atleast 30 minutes, at a temperature of 25±2°C.

33. A chromatographic separation of impurity-A using chromatographic conditions according to claim 32, and with a gradient program comprising of combining mobile phase-A and mobile phase-b in a programmed manner over a run time of atleast 15 minutes

34. A reversed phase HPLC method according to Claim 30, used for quantitative analysis consisting of:

a) Mobile Phase-A comprising of orthophosphoric acid dissolved in water

b) Mobile Phase-B comprising of atleast 10% of Acetonitrile mixed with Mobile phase-A c) The temperature of diluted sample maintained between 2-25°C

d) The chromatographic conditions comprising of C-18 column having a particle size of 5μιτι or lesser, and flow rate of atleast 0.5mL/min, and run time of atleast 30 minutes, at a temperature of 25±2°C

35. A chromatographic separation of impurity-B and impurity-D using chromatographic conditions according to claim 34, and with a gradient program comprising of combining mobile phase-A and mobile phase-b in a programmed manner over a run time of atleast 15 minutes.

36. A reversed phase HPLC method according to Claim 31, used for quantitative analysis using post column derivatization technique for determination of impurity- C consisting of:

(a) Using a derivatizing agent Fluorescamine

(b) Mobile Phase-A comprising ofdisodium hydrogen phosphate buffer at pH 10.5

(c) Mobile Phase-B comprisingJbf atleast 50% of Acetonitrile mixed with Mobile phase-A

(d) The temperature of diluted sample maintained between 2-25°C

(e) The chromatographic conditions comprising of C-18 column having a particle size of 5μιτι or lesser, and flow rate of atleast 0.5mL/min, and run time of atleast 15 minutes, at a temperature of 25±2°C.

37. A chromatographic separation of impurity-C using chromatographic conditions according to claim 35, with a gradient program comprising of combining mobile phase-A and mobile phase-b in a programmed manner over a run time of atleast 15 minutes.

38. A lyophilized product of cyclophosphamide prepared according to claim 1, having a shelf life of atleast 1 year when stored below 25°C.

39. A lyophilized product of cyclophosphamide prepared according to claim 2, having a shelf life of atleast 1 year when stored below 25°C.

40. A lyophilized product of cyclophosphamide prepared according to claim 1,

comprising of suitable pharmaceutically acceptable carriers or excipients