FORM 2
THE PATENTS ACT 1970
(39 of 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rulel3)
1. TITLE OF THE INVENTION:
"STABLE LYOPHILIZED CYCLOPHOSPHAMIDE INJECTION"
2. APPLICANT:
(a) NAME: NEON LABORATORIES LTD.
(b) NATIONALITY: Indian Company incorporated under the
Companies Act, 1956
(c) ADDRESS: 140, Damji Shamji Industrial Complex, Mahakali Caves
Road, Andheri (East), Mumbai - 400093, Maharashtra, India.
3.PREAMBLE TO THE DESCRIPTION:
The following specification describes the nature of this invention and the manner
in which it is to be performed:

TECHNICAL FILED:
This invention relates to new stable lyophilized preparations of Cyclophosphamide for injection with excellent storage stability, less reconstitution time and to a process for their preparation. The invention further relate to Cyclophosphamide for injection obtained by reconstitution of the lyophilized products described herein.
BACKGROUND AND PRIOR ART:
Cyclophosphamide is nitrogen mustard containing alkylating agent and is used to treat various types of cancers and few autoimmune disorders. In the past, a pharmaceutical composition has been marketed, containing the cyclophosphamide-monohydrate in the form of a coarse powder, mixed with common salt for the purpose of making it flowable through hopper. Currently available formulation of Cyclophosphamide for injection is sterile powder filling which includes sodium chloride as an aid to increase flow property. Such powder filling may not be readily reconstituted in water and hence external heating of the glass vials may be required with long reconstitution time.
Further, it is a known fact that Powder filling is far more difficult to prepare in terms of achieving the highly desirable pharmaceuticals characteristics of homogeneity compared to cryodesicated from a homogenous solution is, by its very nature of optimum uniformity. Therefore, the above compositions have been replaced by the freeze-dried mixture of cyclophosphamide-monohydrate and mannitol. The technique known as lyophilization is often employed for freeze-dried injectable pharmaceuticals which exhibit poor stability in aqueous solution. This process involves freeze drying, whereby ice is sublimed from frozen solutions leaving only the solid, dried components of the original liquid.
US4537883 discloses a hydrated lyophilizate composition with improved stability, superior solubility characteristics and enhanced appearance comprising about 20 parts by weight of cyclophosphamide, taken as the anhydride, about 1/4-2 parts by weight of water and from about 10 to 85 parts by weight of mannitol.

US5413995 discloses a process for producing a lyophilized cyclophosphamide pharmaceutical solid composition which comprises (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 said solution of desired cyclophosphamide content in said 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.
In another process variant, US'995 discloses a process for producing lyophilized cyclophosphamide which comprises (a) dissolving about 2 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 said solution at a chamber pressure of about 500 millitorr and an initial shelf temperature of about 10 DEG and an initial lyophilization condenser temperature of about -60 DEG for about 10-12 hours; (d) continuing the lyophilization of said solution at a chamber pressure of about 500 millitorr but with a shelf temperature of about -10 DEG and a condenser temperature of about -30 DEG for a total lyophilization time of 36-48 hours at which point the amount of water remaining is approximately equimolar with the cyclophosphamide on an anhydrous basis. It is known that cyclophosphamide is stable only when it is in monohydrate form. Absorption of further water or desorption of associated water molecule may leads to degradation of cyclophosphamide. Optimization of lyophilization cycle is very difficult with such products particularly with Cyclophosphamide. The time required to form reconstituted solution for 0.5 gm dosage form (which contains about 0.225 gm sodium chloride) range from about 2 minutes to 2 hours or more, depending upon the particular physical/chemical characteristics of Cyclophosphamide and sodium chloride in particular batch of powder measured into the vial.

In view of the above art, it becomes apparent that selection of a suitable excipient or excipients for a pharmaceutical product containing cyclophosphamide is not sufficient to obtain a good lyophillized cake, however, obtaining stable lyophillized cake with good lyo-protection using industrially feasible process is also important.The processes disclosed in US'995 involves Lyophilization either Lyophilizationfollowed by Humidification or Direct Lyophilization process with long Lyophilization cycles as described above.
Also, desired drug assay is required for the development of such a composition to check the storage stability and the ease of reconstitution of the product as and when required within the duration of its shelf-life. The compositions discussed according to the state of the art also have the drawback that the cyclophosphamide is always delivered in a mixture with another substance.
In view of the foregoing, the current invention provides a lyophilized Cyclophosphamide injection in a unit dosage form with appropriate lyo-protection using the Direct Lyophilization process with short lyo cycle with reproducible product characteristics to overcome the cumbersome lyophillization processes employed in prior art. The lyophillized product of the instant invention demonstrates improved stability, solubility characteristics and enhanced appearance compared with currently available dry powder pre-mix composition of Cyclophosphamide as discussed herein below.
DETAILED DESCRIPTION:
The therapeutically-active component of this invention, cyclophosphamide, is a well-known and widely used anticancer agent. Cyclophosphamide chemically is 2-[bis-(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine-2-oxide monohydrate, shown in Formula I


The instant invention provides a lyophilized Cyclophosphamide injection in a unit dosage form with appropriate lyo-protection using the Direct Lyophilization process with short lyo cycle to overcome the cumbersome lyophillization processes employed in prior art. The lyophillized product of the instant invention demonstrates improved stability, solubility characteristics and enhanced appearance compared with currently available dry powder pre-mix composition of Cyclophosphamide The Iyophilized cake is better in appearance; storage stable and short reconstitution times provide an advantage in that the therapeutic agent has not decomposed from exposure in a solution for an extended period of time prior to administration.
Accordingly, in a preferred embodiment, the invention provides a lyophillized product comprising Cyclophosphamide and mannitol in the ratio of 4:3 in water as vehicle. The lyophillezed product of the present invention is prepared by a process which comprises:
a) dissolving the mannitol in a part of water for injection followed by dissolving cyclophosphamide;
b) checking the pH of the solution and the required volume is made up with water for injection;
c) asecptically filtering the solution with 0.2μ filter followed by filling in the flint vial, partially stoppered with gray bromo-butyl slotted rubber stoppers and
d) Lyophilizing the solution by optimized the lyo recipe without subjecting to the pressure rise test to get the desired moisture content.
According to another embodiment, the lyo cake thus obtained is subjected to stability and solubility studies as discussed herein below.
The present invention is further illustrated in detail with reference to the following examples. It is desired that the examples be considered in all respect as illustrative and are not intended to limit the scope of the claimed invention.

Examples:
Table 1

FORMULA: Batch Size: 100 vials
Sr. No. Ingredients Quantity / vial OA Batch Formula Use
1. Cyclophosphamide 100.0 mg — 10.0 gmXF Active
2. Mannitol 75.0 mg 7.5 gm Lyoprotecta nt
3. Water for Injection Upto 3.0 ml - Upto 300 ml Vehicle
Procedure tor manufacture:
Following steps are involved in preparation of formulation:
1. Dissolving the Lyoprotectantin a part of water for injection,
2. Adding and dissolving cyclophosphamide in the solution of step 1.
3. Checking the pH of the solutionand the required volume is made up with water for injection;
4. Filtering the solution aseptically with 0.2μ. filter and filled in flint vial, partially stoppered with gray bromo-butyl slotted rubber stoppers and Lyophilised and
5. Optimizing the lyo recipe without subjecting to the pressure rise test to get the desired moisture content.
The process of lyophilization step comprises rapidly freezing said solution of desired cyclophosphamide content and then employing a chamber pressure of about 0.1000 mbar with condenser temperature of about -55°C for initial 2 to3 hours. Then, raising the shelf temperature to about -15°C with 0.8000 mbar chamber pressure for about 13 to 15 hours and continuing the lyophilization of said solution at 0.0°C with maximum vacuum for further 2 to 4 hours followed by cycle end without pressure rise test.
The lyophilized product prepared according to the invention is evaluated for its water content, storage stability for upto 6 months as well as the assay upon reconstitution.The detailed study the same is provided below in tables 3 to 8.The Assay of the batches prepared according to the invention at a temperature of 30°C ranges from 92.5-107.5%, with a water content: NMT 5.0 % w/v.

Result of all the batches subjected to accelerated stability test and the results are summarized in table 2 below.
Table-2

Sr. No. B.No. Stability duration 30° C-75%RH

Assay (By HPLC) Water Content
1. 11081378 12M 96.88% 4.79%
2. 12071856 6M 100.47% 4.55%
3. 12071866 6M 99.76% 4.34%
4. 12071867 6M 101.05% 4.27%
5. 12071870 6M 99.72% 4.57%
As a preferred form of packaging freeze dried cyclophosphamide is in glass vial, it will immediately be evident that a cake or plug tightly held in a glass container, near its bottom, has greater visual appeal than a loose powder, particularly if the powder has a proclivity to dust.

Table 3

Product Name: CYCLOPHOSPHAMIDE FOR INJECTION Pharmacopoeia: NP
Generic Name: CYCLOPHOSPHAMIDE FOR INJECTION (LYOPHILIZED) Strength: 100 mg/ Vial Generic Name:
CYCLOPHOSPHAMIDE FOR INJECTION (LYOPHILIZED)
MFG Date: 07/08/2011 Setup Date: 16/08/2011 MFG Date: 07/08/2011
Primary packing: 20mm ; 10ml flint USP Type I Tubular Vials
Storage condition: Store below 30°c, protected from light
Objective: First Stability batch

Sr. No. Parameters STD Limit Withdrawal Period

Initial
16/08/201 1 1M 2M 3M 6M 12M

Below 25°C 30° C-75%RH Below
25°C 30° C-75%RH Below
25°C 30° C-75%RH Below
25°C 30° C-75%RH Below 25°C 30° C-75%RH
1. Description * * * * * * * * * * * *
2. Acidity (pH)
(2% w/v solution) Between 3.0 to 9.0 4.65 4.589 4.593 4.329 4.194 4.223 4.173 4.117 4.103 4.21 4.18
3. Particulate matter Visual Inspection ** ** ** ** ** ** +* *+ ** ** ** **
4. Water 4.0 to 6.0
% w/v 4.8% 5.50% 8.39% Not Done Not Done 5.31% 4.47% Not Done Not Done 4.68% 4.79%
5. Related substances (By TLC) To Comply Complies Complies Complies Complies Complies Complies Complies Complies Complies Complies Complies
6. Assay (By HPLC)
(O.A.2%) N.L.T.
92.5% and
N.M.T.
107.5% 99.79% 100.28% 99.03% 100.81% 100.72% 100.86% 100.45% 98.93% 96.27% 99.70% 96.88%
* A White coloured Lyophilised mass. ** Reconstitute solution free from particulate matter. Remarks:

Product is stable till 12 M Below 25°C / 30°C-75%RH (when the water content of lyophilized mass is with in specified range of 4.0 to 6.0 %w/v) there is no significant change.
Table 4

Product Name: CYCLOPHOSPHAMIDE FOR INJECTION Pharmacopoeia: NP
Generic Name: CYCLOPHOSPHAMIDE FOR INJECTION Strength: 100 MG/VIAL Batch No.: 12071856
MFG Date: 24/07/2012 Setup Date: 30/07/2012 EXP Date:
Primary packing: 20 mm; 10 ml Flint USP Type I Tubular vial.
Storage condition: Store Below 25°C, Protected from light.
Objective: Second Stability batch Date: 15/01/2013

Sr.
No. Parameters STD Limit Withdrawal Period

Initial
(27/07/2012) 1M 2M 3M 6M

Below
25°C 30° C-75%RH Below
25°C 30° C-75%RH Below
25°C 30° C-75%RH Below
25°C 30° C-75%RH
1. Description * * * * * * * * + *
2. pH (2.0% solution) Between 3.0 to9.0 4. 14 3.962 3.920 4.007 3.773 4.022 4.080 4.004 3.908
3. Particulate matter Visual Inspection ** ** ** ** ** ** ** ** **
4. % Transmittance of reconstituted (solution- after 1 min of reconstitution)
A) At 430 nm
B) At 550 nm NLT 90.0% NLT 98.0% 98.75% 99.88% 100.023% 99.981% 98.345% 99.026% 99.347% 99.993% 99.775% 100.125% Not Done Not Done 98.206% 99.008% 98.211%
99.050%
5. Related Substance (By TLC) To Comply Complies Complies Complies Complies Complies Complies Compile
s Complies Complies
6. Water 4,0 to 6.0 % w/v 4. 35% Not Done Not Done 4.31% 4.29% 4.918% 5. 326% 4.49% 4.55%
7. Assay (By HPLC)
OA = Nil NLT 92.5% and NMT 107.5% 102.70% 101.47% 100.65% 98.84% 98.30% 102.23% 101.128 % 101.70% 100.47%

* A White coloured Lyophilised mass. ** Reconstitute solution free from particulate matter.
Remarks:
Product is stable till 6 M Below 25°C / 30°C-75%RH (when the water content of lyophilized mass is with in specified range of 4.0 to 6.0 %w/v)
there is no significant change.
Table 5

Product Name: CYCLOPHOSPHAMIDE FOR INJECTION Pharmacopoeia: NP
Generic Name: CYCLOPHOSPHAMIDE FOR INJECTION Strength: 100 MG/VIAL Batch No.: 12071866
MFG Date: 24/07/2012 Setup Date: 30/07/2012 EXP Date:
Primary packing: 20 mm; 10 ml Flint USP Type I Tubular vial.
Storage condition: Store Below 25°C, Protected from light. -
Objective: Third Stability batch Date: 15/01/2013

Sr.
No. Parameters STD Limit Withdrawal Period

Initial
(27/07/2012) 1M 2M 3M 6M

Below
25°C 30° C-75%RH Below 25°C 30° C-75%RH Below 25°C 30° C-75%RH Below 25°C 30° C-75%RH
1 Description * * * * * * * * * #
2 pH (2.0 %solution) Between 3.0 to
9.0 4.20 3.958 3. 922 3.713 3.852 3.924 3.713 3.819 3.86
3 Particulate matter Visual Inspection ** ** ** ** ** ** ** ** ** **
4 % Transmittance of reconstituted (solution- after 1 min of reconstitution)
A) At 430 nm
B) At 550 nm NLT 95.0% NLT 98.0% Not Done 99.279% 99.655% 98.725% 99.358% 98.591% 99.491% 99.630%
100.173% Not Done Not Done 97.510% 98.325% 96.272% 98.156%
5 Related Substance To Comply Complies Complies Complies Complies Complies Complies Complies Complies Complies

6 Water (By KF Method ) 4.0 - 6.0% Not Done Not Done Not Done 4.07% 4.11% 4.9762% 4. 9180% 4.38% 4.34%
7. Assay (By HPLC)
OA = Nil NLT 90.0% and NMT 110% 99.38% 102. 88% 102. 55% 101.30% 96.95% 99.24% 101.94% 100. 93% 99.76%
* A White coloured Lyophilised mass. ** Reconstitute solution free from particulate matter.
Remarks:
Product is stable till 6 M Below 25°C / 30°C-75%RH (when the water content of lyophilized mass is with in specified range of 4.0 to 6.0 %w/v)
there is no significant change.
Table 6

Product Name: CYCLOPHOSPHAMIDE FOR INJECTION Pharmacopoeia: NP
Generic Name: CYCLOPHOSPHAMIDE FOR INJECTION Strength: 100 MG/VIAL Batch No.: 12071867
MFG Date: 26/07/2012 Setup Date: 30/07/2012 EXP Date:
Primary packing: 20 mm; 10 ml Flint USP Type I Tubular vial.
Storage condition: Store Below 25°C, Protected from light.
Objective: Fourth Stability batch Date: 15/01/2013

Sr.
No. Parameters STD Limit Withdrawal Period

Initial
(09/08/2012) IM 2M 3M 6M

Below
25°C 30° O 75%RH Below
25°C 30° C-75%RH Below
25°C 30° C-75%RH Below 25°C 30° C-75%RH
1 Description * * * * * * * * * *
2 pH (2.0 % solution) Between 3.0 to 9.0 4.20 3.963 3.945 4.073 3.977 4.142 4.163 3.884 3.915
3 Particulate matter Visual Inspection ** ** ** ** ** ** ** ** ** **

4
% Transmittance of reconstituted (solution- after 1 min ofreconstitution)
A) At 430 nm
B) At 550 nm NLT 95.0% NLT 98-0% 100.87% 100.76% 99.703% 99.911% 99.716% 100.0% 99.130% 99.711% 100.01% 100.34% Not Done Not Done 99.440% 99.697% 99.302% 99.728%
5 Related Substance To Comply Complies Complies Complies Complies Complies Complies Complies Complies Complies
6 Water (By KF
Method) 4.0 to 6.0 % w/v Not Done Not Done Not Done 4. 36% 4. 39% 4.0864% 4.8204% 4.28% 4.27%
7 Assay (By H PLC) OA = Nil NLT 90.0% and NMTU0% 100.27% 102.76% 98.83% 98.59% 98.05% 101.78% 101.09% 101.02% 101.05%
* A White coloured Lyophilised mass. ** Reconstitute solution free from particulate matter.
Remarks:
Product is stable till 6 M Below 25°C / 30°C-75%RH (when the water content of lyophilized mass is with in specified range of 4.0 to 6.0 %w/v)
there is no significant change.
Table 7

Product Name: CYCLOPHOSPHAMIDE FOR INJECTION Pharmacopoeia: NP
Generic Name: CYCLOPHOSPHAMIDE FOR INJECTION Strength: 100 MG/VIAL Batch No.: 12071870
MFC Date: 28/07/2012 Setup Date: 30/07/2012 EXP Date:
Primary packing: 20 mm; 10 ml Flint USP Type I Tubular vial.
Storage condition: Store Below 25°C, Protected from light.
Objective: Fifth Stability batch Date: 15/01/2013

Sr. No. Parameters STD Limit Withdrawal Period

Initial
(09/08/201
2) 1 VI 2M 3M 6M

Below
25°C 30° C-75%RH Below
25°C 30° C-75%RH Below
25°C 30° C-75%RH Below
25°C 30° C-75%RH
1 Description * * * * * * * * * *

2 pH (2.0 % solution) Between 3.0 to9.0 4.22 4.084 4.066 4.083 3.946 4.224 4.099 3-994 3.893
3 Particulate matter Visual Inspection ** ** ** ** ** ** ** ** ** **
4 % Transmittance of reconstituted (solution- after 1 min of reconstitution)
A) At 430 nm
B) At 550 nm NLT 95.0% NLT98.0% 97.18% 98.11% 98.119%
99.016% 99.056%
99.542% 99.441% 99.947% 99.376% 99.912% Not Done Not Done 98.373%
99.140% 97.457% 98.642%
5 Related Substance To Comply Complies Complies Complies Complies Complies Complies Complies Complies Complies
6 Water (By KF Method) 4.0to6.0%w/v Not Done Not Done Not Done 4.40% 4.49% 4.1213%) 4.4244% 4.42% 4.57%
7 Assay (By HPLC) OA = Nil NLT 90.0% and NMT 110% 102.08% 102.90% 98.51% 97.56% 98.07% 101.41% 100.88%) 101.09% 99.72%
* A White coloured Lyophilised mass. ** Reconstitute solution free from particulate matter. -
Remarks:
Product is stable till 6 M Below 25°C / 30°C-75%RH (when the water content of lyophilized mass is with in specified range of 4.0 to 6.0 %w/v)
there is no significant change.

We claim,
1. Stable Lyophillized injectable product with opted appearance, to get super
solubility characteristics comprising cyclophosphamide and mannitol in the ratio
of 4:3 in water as vehicle prepared by direct lyophilization process with short lyo
cycle comprising;
a) dissolving the mannitol in a part of water for injection followed by dissolving cyclophosphamide;
b) checking the pH of the solution and the required volume is made up with water for injection;
c) asecptically filtering the solution with 0.2μ filter followed by filling in the flint vial, partially stoppered with gray bromo-butyl slotted rubber stoppers; and
d) Lyophilizing the solution by optimizing the lyo recipe without subjecting to the pressure rise test to get the optimum moisture content.

2. The stable lyophillized injectable product according to claim 1, wherein the moisture content of the product is up to 4 to 6%.
3. The process of claim 1 wherein lyophilization step comprises
i) Rapidly freezing said solution of desired cyclophosphamide content and then employing a chamber pressure of about 0.1000 mbar with condenser temperature of about -55°C for initial 2 to3 hours. Then raising the shelf temperature to about -15°C with 0.8000 mbar chamber pressure for about 13 to 15 hours.
ii) Continuing the lyophilization of said solution at 0.0°C with maximum vacuum for further 2 to 4 hours followed by cycle end without pressure rise test.