FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2006
COMPLETE SPECIFICATION (See Section 10 and Rule 13)
STABLE LYOPHILIZED CRYSTALLINE PHARMACEUTICAL COMPOSITIONS OF
BENDAMUSTINE HYDROCHLORIDE
PANACEA BIOTEC LIMITED,
an Indian Company incorporated under the Companies Act 1956 B-l Ext. A-27. Mohan Co-operative Industrial Estate, Mathura Road,
New Delhi -110 044,
The following specification particularly describes the invention

FIELD OF THE INVENTION
The present invention pertains to the field of pharmaceutical compositions for the treatment of various disease states, especially neoplastic diseases and autoimmune diseases. Particularly, it relates to pharmaceutical formulation comprising nitrogen mustards, particularly the nitrogen mustard bendamustine, e.g., bendamustine hydrochloride,
BACKGROUND:
The following discussion of the prior art is intended to present the invention in an appropriate technical context and allow its significance to be properly appreciated. Unless clearly indicated to the contrary, however, reference to any prior art in this specification should be construed as an admission that such art is widely known or forms part of common general knowledge in the field.
Bendamustine is a drug selected from the group of alkylating agents, which is used in the treatment of neoplastic diseases. Bendamustine is an atypical structure that includes a benzimidazole ring, an active nitrogen mustard fragment and a residue of butanoic acid. Bendamustine is chemically known as (4-{5-[Bis(2-chloroethyl)amino]-l-methyl-2-benzimidazolyl}butyric acid hydrochloride and is represented by the following Formula I.

Bendamustine was initially synthesized in 1963 by Ozegowski and Krebs in East Germany (the former German Democratic Republic) and was available from 1971 to 1992 in that location under the name Cytostasan®. Since that time, it has been marketed in Germany under the trade name Ribomustin®. East German investigators found that it was useful for treating chronic

lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and lung cancer.
In March 2008, Cephalon received approval from the United States Food and Drug Administration to market bendamustine in the US, where it is sold under the trade name TREANDA, for treatment of chronic lymphocytic leukemia. In October 2008, the FDA granted further approval to market TREANDA for the treatment of indolent B-cell non-Hodgkin's lymphoma that has progressed during or within six months of treatment with rituximab or a rituximab-containing regimen.
Bendamustine is a white, water soluble microcrystalline powder with amphoteric properties. It acts as an alkylating agent causing intra-strand and inter-strand cross-links between DNA bases. After intravenous infusion it is extensively metabolized in the liver by cytochrome p450. More than 95% of the drug is bound to protein - primarily albumin. Only free bendamustine is active. Elimination is biphasic with a half-life of 6-10 minutes and a terminal half-life of approximately 30 minutes. It is eliminated primarily through the kidneys.
Because of their high reactivity in aqueous solutions, nitrogen mustards are difficult to formulate as pharmaceuticals and are often supplied for administration in a lyophilized form that requires reconstitution, usually in water, by skilled hospital personnel prior to administration. Once in aqueous solution, nitrogen mustards are subj ect to degradation by hydrolysis, thus, the reconstituted product should be administered to a patient as soon as possible after its reconstitution. The current lyophilized formulation of bendamustine (Ribomustin®) contains bendamustine hydrochloride and mannitol in a sterile lyophilized form as a white powder for intravenous use following reconstitution. The finished lyophilisate is unstable when exposed to light. Therefore, the product is stored in brown or amber-colored glass bottles. The current lyophilized formulation of bendamustine contains degradation products that may occur during manufacturing of the drug substance and/or during the lyophilization process to make the finished drug product.
Currently bendamustine is formulated as a lyophilized powder for injection with 100 mg of drug

per 50 mL vial or 25 mg of drug per 20 mL vial. The vials are opened and reconstituted as close to the time of patient administration as possible. The product is reconstituted with 40 mL (for the 100 rng presentation) or 10 mL (for the 25 mg presentation) of Sterile Water for Injection. The reconstituted product is further diluted into 500 mL, q.s., 0.9% Sodium Chloride for Injection. The route of administration is by intravenous infusion over 30 to 60 minutes. Following reconstitution with 40 mL Sterile Water for Injection, vials of bendamustine are stable for a period of 7 hours under room temperature storage or for 6 days upon storage at 2-8°C. The 500 mL admixture solution must be administered to the patient within 7 hours of vial reconstitution (assuming room temperature storage of the admixture).
German (GDR) Patent No. 34727 discloses a method of preparing co-[5-bis-(p-chloroethyl)-amino-benzimidazolyl-(2)]-alkane carboxylic acids substituted in the 1-position from 4-nitro-2-amino-N-methylaniline and the recrystallization of the product from water.
German (GDR) Patent No. 80967 discloses an injectable preparation of bendamustine hydrochloride monohydrate, ascorbic acid, and water. GDR 80967 describes that lyophilization of compounds such as bendamustine is only possible if the compound is of sufficient stability that it can withstand the processing conditions. The preparation described in GDR 80967 is not lyophilized.
German (GDR) Patent No. 159877 discloses a method for preparing bendamustine free base by reaction of the bis-hydroxyl precursor with thionyl chloride followed by recrystallization from water.
German (GDR) Patent No. 159289 discloses an injectable solution of bendamustine that avoids lyophilization and describes an anhydrous solution of bendamustine hydrochloride in 1.2-propylene glycol or ethanol. Further GDR 159289 mentions that a solution of bendamustine in ethanol is stable for up to 8 weeks.

US patent application number 2006/159713 Al, and PCX international patent application number WO2010/036702 disclose formulations of bendamustine hydrochloride. These patent applications also include impurities of bendamustine.
International application publication number WO 2009/120386 A2 (hereinafter WO '386) describes solid forms of bendamustine hydrochloride designated as bendamustine hydrochloride Form 1, bendamustine hydrochloride Form 2, bendamustine hydrochloride Form 3. bendamustine hydrochloride Form 4, amorphous bendamustine hydrochloride or a mixture thereof, processes for their preparation along with lyophilized compositions comprising these forms. Further, in the disclosure it is also mentioned that monohydrate of bendamustine hydrochloride has been prepared previously as per W. Ozegowski and D. Krebs. The earlier monohydrate has a reported melting point of 152-156°C which is similar to that of the observed melting point of bendamustine hydrochloride Form 2.
Further, WO '386 also mentions the various form conversions that take place with respect to each of the disclosed forms; for example, Form 1 gets converted to a hydrate of bendamustine hydrochloride (Form 2) during 2 months of storage at 25° C./94% RH; Form 2 on heating between ambient and 100° C. converts to Form 4; Form 3 converted to Form 2 during 1 week storage at 40° C/75% RH; Form 4 converted to Form 2 during 24 hours under ambient conditions; and the amorphous bendamustine hydrochloride undergoes partial crystallization during 1 week at 40° C/75% RH, and converts possibly to a mixture of Forms 2 and 3.
Tn view of the existence of various known crystalline forms of bendamustine hydrochloride & its monohydrate, there appears to be a need for a stable form of lyophilized composition of bendamustine hydrochloride that retains its stability even after long s torage which may be amenable to scale up and provides improved yields & quality.
SUMMARY OF THE INVENTION
The present invention pertains to the field of lyophilized pharmaceutical compositions for the treatment of various disease states, especially neoplastic diseases and autoimmune diseases.

Particularly, it relates to pharmaceutical formulations comprising nitrogen mustards, particularly the nitrogen mustard bendamustine, e.g., bendamustine hydrochloride.
In one aspect, the present invention provides a pharmaceutical composition of bendamustine hydrochloride wherein the said pharmaceutical composition is a lyophilized solid which has improved stability on storage at ambient temperature and which is capable of re-dissolving in an aqueous diluent prior to administration.
In another aspect of the invention, there is provided a lyophilized pharmaceutical composition of bendamustine hydrochloride with a novel XRPD pattern which has adequate stability for storage at ambient temperature and which is capable of re-dissolving in an aqueous diluent prior to administration.
In another aspect the present invention provides a storage stable lyophilized pharmaceutical composition comprising Bendamustine Hydrochloride.
Tn further preferred embodiments the novel crystalline pharmaceutical compositions according to the present invention may further comprise other pharmaceutically acceptable excipients like mannitol.
In a most preferred embodiment the present invention relates to novel crystalline pharmaceutical compositions comprising bendamustine, wherein the composition is free of tertiary butyl alcohol and/or acetone and/or acetonitrile and mixtures thereof.
In a specific aspect, the present invention provides a lyophilized crystalline composition "form PB-1", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.7647, 10.6988, 15.0462, 20.0877, 20.4728. 21.1464, 26.3467 and 36.1533 ±0.2 °2 theta.
In a further aspect the present invention provides a lyophilized crystalline composition "form PB-1", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern is

further characterized by peaks at 7.5181, 9,7647, 10.1710, 10.6988, 11.4612, 12.4871, 13.6848, 15.0462, 17.3397 , 18.7380, 19.4907, 20.0877, 20.4728, 21.1464, 21.2859, 22.1307, 22.9664, 24.7214, 25.3075, 25.4567, 26.3467, 27.9233, 28.8312, 30.1837, 32.1186, 33.1792, 33.9529, 34.9601, 36.1533, 36.5464 and 37.9271 ±0.2 °2 theta.
In another specific aspect, the present invention provides a lyophilized crystalline composition "form PB-2", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.7936, 15.0244, 20.5218, 21.2014, 24.3684 and 36.1304 ±0.2 ° 2 theta.
In a further aspect the present invention provides a lyophilized crystalline composition "form PB-2:', comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern is further characterized by peaks at 7.4564, 9.7936, 10.6951, 11.3897, 12.4210, 13.6685, 15.0244, 18.7484, 19.5547, 20.5218,21.2014,22.2135, 22.9472, 24.3684, 24.7637, 25.3746, 26.0932, 26.5448, 28.0185, 28.9361, 30.2580, 32.2187, 35.0589, 36.1304 and 37.9319 ±0.2 ° 2 theta.
In another specific aspect, the present invention provides a lyophilized crystalline composition "form PB-3", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.7089, 20.0198, 21.1176, 22.8902, 26.4924 and 36.0927 ±0.2 ° 2 theta.
In a further aspect the present invention provides a lyophilized crystalline composition "form PB-3", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern is farther characterized by peaks at 3.7263, 7.4408, 9.7089, 10.1256, 10.6197, 11.3928, 12.4441, 13.6255, ]4.9765, 18.6632, 18.9712, 19.4604, 20.0198, 20.3990, 21.1176, 21.2755, 22.0906, 22.8902, 23.2712, 24.6496, 25.2143, 25.3627, 26.0222, 26.4924, 27.0214, 27.8812, 28.7434, 29.0752, 30.1482. 30.9657, 31.8949, 32.1634, 33.9228, 34.8819, 36.0927, 36.4936, 37.8653 and 39.3098 ±0.2° 2 theta.
In another specific aspect, the present invention provides a lyophilized crystalline composition "form PB-4", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.7429, 12.4563, 21.0756, 30.1736 and 36.1208 ±0.2 °2 theta.

In a further aspect the present invention provides a lyophilized crystalline composition "form PB-4", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern is further characterized by peaks at 3.7696, 7.4779, 9.7429, 10.6497, 11.4406, 12.4563, 13.6602, 14.9697, 18.7273, 19.4637, 20.4480, 21.0756, 21.3187, 22.1212, 22.8995, 23.3095, 24.6733, 25.2882, 26.0002, 26.5331, 27.8960, 29.0880, 30.1736, 30.6201, 31.9384, 34.9259, 36.1208, 37.8518 and 39.4477 ±0.2 ° 2 theta.
In another specific aspect, the present invention provides a lyophilized crystalline composition "form PB-5", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.9168, 10.8427, 20.2554, 20.6012, 20.726 and 22.4254 ±0.2 D 2 theta.
In a further aspect the present invention provides a lyophilized crystalline composition "form PB-5", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern is further characterized by peaks at 7.4852, 9.9168, 10.8427, 12.6206, 13.8330, 15.2400, 18.9818, 19.7967, 20.2554, 20.6012, 20.7260, 21.3922, 22.4254, 23.1708, 24.9521, 25.5490, 26.6616, 28.2199, 29.0342, 30.5861, 32.2334, 35.0543, 36.3079 and 38.1188 ±0.2 ° 2 theta.
In another specific aspect, the present invention provides a lyophilized crystalline composition "form PB-6", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.9058, 10.8374, 20.6491, 23.2002 and 36.3908 ±0.2 ° 2 theta.
In a further aspect the present invention provides a lyophilized crystalline composition "form PB-6". comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern is further characterized by peaks at 9.9058, 10.8374, 12.5979, 13.7317, 15.1319, 18.8887, 19.6641, 20.6491, 21.3587, 22.3682, 23.2002, 24.9025, 25.6135, 26.2143, 26.6549, 28.1846, 29.0925, 30.3881, 32,2330, 35.2102 and 36.3908 ±0.2 ° 2 theta.
In another specific aspect, the present invention provides a lyophilized crystalline composition "form PB-7", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.6738, 14.8888, 20.368, 21.0845, 22.8545, 26.275 and 36.0567 ±0.2 ° 2 theta.

In a further aspect the present invention provides a lyophilized crystalline composition "form PB-7", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern is further characterized by peaks at 3.7128, 7.4092, 9.6738, 10.6056, 11.3708, 12.3786, 13.5814, 14.8888, 18.6203, 19.4268, 20.3680, 21.0845, 22.0467, 22.8545, 24.5953, 25.2204, 26.2750, 27.8504, 29.0602, 30.1448, 31.8419, 34.9144 and 36.0567 ±0.2 ° 2 theta.
In another specific aspect, the present invention provides a lyophilized crystalline composition "form PB-8". comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.714, 18.7136, 19.4655, 24.6835 and 36.1437 ±0.2 ° 2 theta.
In a further aspect the present invention provides a lyophilized crystalline composition "form PB-8", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern is further characterized by peaks at 3.7576, 7.4896, 9.7140, 10.1364, 10.6398, 11.4091, 12.4185, 13.6507, 14.9906, 18.7136, 19.4655, 20.0424, 20.2777, 20.4088, 21.0715, 21.2698, 22.1097, 22.9035, 24.4662, 24.6835, 25.2394, 26.3319, 27.9104, 28.7586, 30.1755, 31.9490, 34.9381 and 36.1437 ±0.2° 2 theta.
In another specific aspect, the present invention provides a lyophilized crystalline composition "form PB-9", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.7297, 21.0649, 21.8695, 24.6868 and 36.0778 ±0.2 ° 2 theta.
In a further aspect the present invention provides a lyophilized crystalline composition "form PB-9", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern is further characterized by peaks at 3.7299, 7.4366, 9.7297, 10.6516, 11.3405, 12.4378, 13.6438, 14.9679, 18.6758, 19.4586, 20.3996, 21.0649, 21.8695, 22.1294, 22.9093, 24.4248, 24.6868, 25.2642, 26.4553, 27.8965, 28.8912, 30.1309, 31.9303, 34.9261 and 36.0778 ±0.2 ° 2 theta.
In a preferred aspect, the present invention provides a lyophilized crystalline composition "form PB-1", "form PB-2", "form PB-3", "form PB-4", "form PB-5", "form PB-6", "form PB-7", "form PB-8" and "form PB-9", comprising bendamustine hydrochloride wherein

the lyophilized crystalline composition exhibits increased storage stability and/or a lower impurity content, and wherein the lyophilized crystalline composition may further comprise mannitol.
In still another aspect the present invention provides a process for lyophilizing bendamustine hydrochloride wherein the mannitol is present in an amount of at least 150 to 200 parts by weight on the basis of 100 parts by weight of bendamustine hydrochloride.
In a specific aspect the present invention provides a process for the preparation of novel lyophilized crystalline compositions "form PB-1", "form PB-2'\ "form PB-3", "form PB-4", "form PB-5", "form PB-6", "form PB-7", "form PB-8" and "form PB-9",comprising bendamustine hydrochloride, wherein the process comprises the steps of:
a) solubilizing bendamustine Hydrochloride;
b) adding water along with cryoprotectant; and
c) lyophilizing.
In a further preferred aspect the present invention provides a process for producing lyophilized
crystalline composition "form PB-1", "form PB-2'\ "form PB-3", "form PB~4", "form PB-5",
"form PB-6". "form PB-7'\ "form PB-8:" and "form PB-9". comprising bendamustine
hydrochloride;
wherein the process comprises the steps of:
a) dissolving bendamustine hydrochloride in a solvent;
b) adding water for injection;
c) dissolving an excipient;
d) filtering; and
e) freeze drying.
In another aspect, the present invention provides lyophilized crystalline composition comprising bendamustine with lower impurity content.

The present invention also provides coexistence of an amorphous phase and a crystalline phase which has the consequence of stabilizing the freeze-dried pharmaceutical active ingredient.
These and other features, aspects, and advantages of the present subject matter will become better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an X-ray powder diffractogram (XRPD) of crystalline lyophilized composition "form
PB-1" of the present invention comprising bendamustine hydrochloride.
FIG. 2 is an X-ray powder diffractogram (XRPD) of crystalline lyophilized composition "form
PB-2" of the present invention comprising bendamustine hydrochloride.
FIG. 3 is an X-ray powder diffractogram (XRPD) of crystalline lyophilized composition "form
PB-3" of the present invention comprising bendamustine hydrochloride.
FIG. 4 is an X-ray powder diffractogram (XRPD) of crystalline lyophilized composition "form
PB-4'' of the present invention comprising bendamustine hydrochloride.
FIG. 5 is an X-ray powder diffractogram (XRPD) of crystalline lyophilized composition "form
PB-5" of the present invention comprising bendamustine hydrochloride.
FIG. 6 is an X-ray powder diffractogram (XRPD) of crystalline lyophilized composition "form
PB-6" of the present invention comprising bendamustine hydrochloride.
FIG. 7 is an X-ray powder diffractogram (XRPD) of crystalline lyophilized composition "form
PB-7" of the present invention comprising bendamustine hydrochloride.
FIG. 8 is an X-ray powder diffractogram (XRPD) of crystalline lyophilized composition "form
PB-8'" of the present invention comprising bendamustine hydrochloride.
FIG. 9 is an X-ray powder diffractogram (XRPD) of crystalline lyophilized composition "form
PB-9" of the present invention comprising bendamustine hydrochloride.
FIG. 10 is a DSC thermogram of bendamustine Hydrochloride used in the preparation of
crystalline lyophilized compositions of the present invention.

DETAILED DESCRIPTION OF THE INVENTION
It is known that lyophilization may have a considerable effect on the degradation of the pharmaceutical active ingredients in a formulation, as well as a strong impact on their stability in freeze-dried form. The various variables which affect these parameters are mainly the pH, the quantity of salts present, the type and quantity of excipients in the formulation, the type of cryoprotection chosen, as well as the temperatures, pressure and time chosen for the freezing, sublimation and drying operations. These different variables influence the physical state of the freeze-dried product obtained, namely: vitreous amorphous, soft amorphous, crystalline or a combination of these states.
Lyophilization involves the addition of water to a compound, followed by freezing of the resultant suspension or solution, and sublimation of the water from the compound. In preferred embodiments, at least one organic solvent is added to the suspension/solution. In other preferred embodiments, the suspension/solution further comprises a lyophilization excipient.
In a typical lyophilization procedure, water, a pharmaceutically acceptable lyophilizing excipient, an organic solvent, and a compound are combined to form a solution, which is then sterilized, preferably using sterile filtration methodology. This solution is then lyophilized using standard lyophilization equipment and techniques.
An aspect of the present invention provides a pharmaceutical composition comprising bendamustine hydrochloride wherein the said pharmaceutical composition is a lyophilized solid which has adequate stability for storage at ambient temperature and which is capable of re-dissolving in an aqueous diluent prior to administration.
In another aspect the present invention provides a storage stable lyophilized pharmaceutical compositions comprising Bendamustine Hydrochloride.

The storage stable lyophilized pharmaceutical compositions of the present invention comprising Bendamustine Hydrochloride demonstrates superior stability particularly upon storage under varying conditions of temperature and humidity.
In another aspect of the invention, there is provided a lyophilized pharmaceutical composition of bendamustine hydrochloride with a novel XRPD pattern which has adequate stability for storage at ambient temperature and which is capable of re-dissolving in an aqueous diluent prior to administration.
In a most preferred embodiment the present invention relates to novel crystalline pharmaceutical compositions comprising bendamustine. wherein the composition is free of tertiary butyl alcohol and/or acetone and/or acetonitrile and mixtures thereof.
In an embodiment, the present invention provides a lyophilized crystalline composition "form PB-1", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.7647, 10.6988, 15.0462, 20.0877, 20.4728, 21.1464, 26.3467 and 36.1533 ±0.2 °2 theta.
In a further embodiment, the present invention provides a lyophilized crystalline composition "form PB-1", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern of the lyophilized crystalline composition "form PB-1" exhibits peaks at 7.5181, 9.7647, 10.1710, 10.6988, 11.4612, 12.4871, 13.6848, 15.0462, 17.3397, 18.7380, 19.4907, 20.0877, 20.4728. 21.1464, 21.2859, 22.1307, 22.9664, 24.7214, 25.3075, 25.4567, 26.3467, 27.9233, 28.8312, 30.1837, 32.1186, 33.1792, 33.9529, 34.9601, 36.1533, 36.5464 and 37.9271 ±0.2 °2 theta; wherein the lyophilized crystalline preparation exhibits increased storage stability and/or a lower impurity content, wherein the lyophilized crystalline composition may further comprise mannitol.
In an embodiment, a typical X-ray Powder Diffractogram, XRPD of the lyophilized composition "form PB-1", prepared in accordance with the lyophilization procedures described herein and

comprising bendamustine hydrochloride and mannitol is shown in FIG. 1. The XPRD data corresponding to this Diffractogram is shown below.

Angle [2-Theta] d value [Angstrom] Relative Intensity [%]
7.5181 11.7495 2.47
9.7647 9.05064 61.38
10.1710 8.68995 2.44
10.6988 8.26242 37.11
11.4612 7.71448 2.90
12.4871 7.08290 2.86
13.6848 6.46556 6.15
15.0462 5.88347 4.32
17.3397 5.11011 4.96
18.7380 4.73179 15.90 .
19.4907 4.55073 11.65
20.0877 4.41682 35.05
20.4728 4.33459 100.00
21.1464 4.19802 50,39
21.2859 4.17081 37.78
22.1307 4.01346 39.72
22.9664 3.86928 39.10
24.7214 3.59842 36.42
25.3075 3.51641 51.20
25.4567 3.49613 30.35
26.3467 3.38002 9.48
27.9233 3.19265 10.32
28.8312 3.09415 2.86
30.1837 2.95851 7.26
32.1186 2.78456 5.38
33.1792 2.69793 1.13

33.9529 2.63820 1.89
34.9601 2.56447 5.73
36.1533 2.48252 30.61
36.5464 2.45972 7.79
37.9271 2.37040 0.82
In another embodiment the present invention provides a lyophihzed crystalline composition "form PB-2", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.7936, 15.0244, 20.5218, 21.2014, 24.3684 and 36.1304 ±0.2 ° 2 theta..
In another specific embodiment the present invention provides a lyophilized crystalline composition "form PB-2", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 7.4564, 9.7936, 10.6951, 11.3897, 12.4210, 13.6685, 15.0244, 18.7484, 19.5547, 20.5218,21.2014,22.2135, 22.9472, 24.3684, 24.7637, 25.3746, 26.0932. 26.5448, 28.0185, 28.9361, 30.2580, 32.2187. 35.0589, 36.1304 and 37.9319 ±0.2° 2 theta.
In other embodiments, the novel crystalline "form PB-2" of lyophilized composition comprising bendamustine hydrochloride is characterized by an XRD pattern substantially in accordance with Fig.2. The XPRD data corresponding to this Diffractogram is shown below.

Angle [2-Theta] d value [Angstrom] Relative Intensity [%]
7.4564 11.84647 0.92
9.7936 9.02397 68.45
10.6951 8.26530 20.60
11.3897 7.76276 1.39
12.4210 7.12044 1.49
13.6685 6.47322 2.22
15.0244 5.89197 2.98
18.7484 4.72918 6.97

19.5547 4.53597 12.08
20.5218 4.32435 100
21.2014 4.18724 53.82
22.2135 3.99870 34.19
22.9472 3.87248 26.93
24.3684 3.64976 14.74
24.7637 3.59238 40.87
25.3746 3.50726 55.53
26.0932 3.41227 5.72
26.5448 3.35524 6.33
28.0185 3.18202 9.85
28.9361 3.08317 0.85
30.2580 2.95141 5.97
32.2187 2.77614 5.06
35.0589 2.55747 5.47
36.1304 2.48404 33.03
37.9319 2.37011 0.86
In another embodiment the present invention provides a lyophilized crystalline composition "form PB-3", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.7089, 20.0198, 21.1176, 22.8902, 26.4924 and 36.0927 ±0.2 ° 2 theta.
In another specific embodiment the present invention provides a lyophilized crystalline composition "form PB-3", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 3.7263, 7.4408, 9.7089, 10.1256, 10.6197, 11.3928, 12.4441, 13.6255, 14.9765, 18.6632, 18.9712, 19.4604, 20.0198, 20.3990, 21.1176, 21.2755, 22.0906, 22.8902, 23.2712, 24.6496, 25.2143, 25.3627, 26.0222, 26.4924, 27.0214, 27.8812, 28.7434, 29.0752, 30.1482, 30.9657, 31.8949, 32.1634, 33.9228, 34.8819, 36.0927, 36.4936, 37.8653 and 39.3098 ±0.2 ° 2 theta.

In other embodiments, the novel crystalline "form PB-3" of lyophilized composition comprising bendamustine hydrochloride is characterized by an XRD pattern substantially in accordance with Fig.3. The XPRD data corresponding to this Diffractogram is shown below.

Angle [2-Theta] d value [Angstrom] Relative Intensity [%]
3.7263 23.68231 1.64
7.4408 11.87122 3.22
9.7089 9.10254 72.78
10.1256 8.72882 4.26
10.6197 8.32381 54.55
11.3928 7.76061 4.75
12.4441 7.10725 3.37
13.6255 6.49358 4.97
14.9765 5.91070 6.56
18.6632 4.75059 11.84
18.9712 4.67415 1.81
19.4604 4.55773 9.36
20.0198 4.43164 33.44
20.3990 4.35010 100
21.1176 4.20368 48.56
21.2755 4.17283 27.67
22.0906 4.02067 38.06
22.8902 3.88199 47.53
23.2712 3.81929 12.82
24.6496 3.60875 35.88
25.2143 3.52919 46.37
25.3627 3.50888 34.58
26.0222 3.42124 7.97
26.4924 3.36175 10.01
27.0214 3.29713 2.35

27.8812 3.19737 10.71
28.7434 3.10340 3.04
29.0752 3.06873 3.66
30.1482 2.96191 11.34
30.9657 2.88555 2.01
31.8949 2.80358 10.10
32.1634 2.78078 4.97
33.9228 2.64048 1.62
34.8819 2.57004 5.25
36.0927 2.48655 29.37
36.4936 2.46015 7.01
37.8653 2.37412 1.19
39.3098 2.29014 ' 0.84
In another embodiment the present invention provides a lyophilized crystalline composition "form PB-4". comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.7429, 12.4563, 21.0756, 30.1736 and 36.1208 ±0.2 ° 2 theta.
In another specific embodiment, the present invention provides a lyophilized crystalline composition "form PB-4", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 3.7696, 7.4779, 9.7429, 10.6497, 11.4406, 12.4563, 13.6602, 14.9697, 18.7273, 19.4637, 20.4480, 21.0756, 21.3187, 22.1212, 22.8995, 23.3095, 24.6733, 25.2882, 26.0002, 26.5331, 27.8960, 29.0880, 30.1736, 30.6201, 31.9384, 34.9259, 36.1208, 37.8518 and 39.4477 ±0.2 ° 2 theta.
In other embodiments, the novel crystalline "form PB-4" of lyophilized composition comprising bendamustine hydrochloride is characterized by an XRD pattern substantially in accordance with Fig.4. The XPRD data corresponding to this Diffractogram is shown below.

Angle [2-Theta]
d value [Angstrom] Relative Intensity [%]
3.7696 23.42038 1.27
7.4779 11.81241 2.76
9.7429 9.07081 65,46
10.6497 8.30042 38.67
11.4406 7.72828 3.37
12.4563 7.10032 3.25
13.6602 6.47715 4.65
14.9697 5.91338 5.79
18.7273 4.73447 12.12
19.4637 4.55697 11.25
20.4480 4.33980 100
21.0756 4.21195 48.94
21.3187 4.16446 25.60
22.1212 4.01517 39.95
22.8995 3.88044 43.83
23.3095 3.81310 10.88
24.6733 3.60534 38.63
25.2882 3.51904 52.53
26.0002 3.42427 7.28
26.5331 3.35670 11.23
27.8960 3.19572 10.98
29.0880 3.06741 4.19
30.1736 2.95948 9.85
30.6201 2.91733 2.17
31.9384 2.79986 10.31
34.9259 2.56690 5.98
36.1208 2.48468 32.67
37.8518 2.37494 1.52
39.4477 2.28246 1.04

In another embodiment the present invention provides a lyophilized crystalline composition "form PB-5". comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.9168, 10.8427, 20.2554, 20.6012, 20.726 and 22.4254 ±0.2 ° 2 theta..
In another specific embodiment the present invention provides a lyophilized crystalline composition "form PB-5", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 7.4852, 9.9168, 10.8427, 12.6206, 13.8330, 15.2400, 18.9818, 19.7967, 20.2554, 20.6012, 20.7260, 21.3922, 22.4254, 23.1708, 24.9521, 25.5490, 26.6616, 28.2199, 29.0342, 30.5861, 32.2334, 35.0543, 36.3079 and 38.1188 ±0.2 ° 2 theta.
In other embodiments, the novel crystalline "form PB-5" of lyophilized composition comprising bendamustine hydrochloride is characterized by an XRD pattern substantially in accordance with Fig.5. The XPRD data corresponding to this Diffractogram is shown below.

Angle [2-Theta] d value [Angstrom] Relative Intensity [%]
7.4852 11.80103 0.53
9.9168 8.91219 21.54
10.8427 8.15313 15.49
12.6206 7.00827 1.70
13.8330 6.39661 2.98
15.2400 5.80910 3.87
18.9818 4.67157 8.24
19.7967 4.48106 12.31
20.2554 4.38062 48.57
20.6012 4.30785 100
20.7260 4.28220 81.14
21.3922 4.15032 32.67
22.4254 3.96138 27.76
23.1708 3.83561 47.92
24.9521 3.56568 40.77

25.5490 3.48371 39.84
26.6616 3.34080 12.34
28.2199 3.15977 10.28
29.0342 3.07297 2.77
30.5861 2.92050 11.94
32.2334 2.77491 9.36
35.0543 2.55779 3.92
36.3079 2.47230 32.51
38.1188 2.35891 1.70
In another embodiment the present invention provides a lyophilized crystalline composition "form PB-6", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.9058, 10.8374, 20.6491, 23.2002 and 36.3908 ±0.2 ° 2 theta.
In another specific embodiment the present invention provides a lyophilized crystalline composition "form PB-6", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.9058, 10.8374, 12.5979, 13.7317, 15.1319, 18.8887, 19.6641, 20.6491, 21.3587, 22.3682, 23.2002, 24.9025, 25.6135, 26.2143, 26.6549, 28.1846, 29.0925, 30.3881, 32.2330, 35.2102 and 36.3908 ±0.2 ° 2 theta.
In other embodiments, the novel crystalline "form PB-6" of lyophilized composition comprising bendamustine hydrochloride is characterized by an XRD pattern substantially in accordance with Fig.6. The XPRD data corresponding to this Diffractogram is shown below.

Angle [2-Theta] d value [Angstrom] Relative Intensity [%]
9.9058 8.92202 30.46
10.8374 8.15706 12.31
12.5979 7.02081 1.55
13.7317 6.44358 2.76
15.1319 5.85034 2.95

18.8887 4.69438 6.48
19.6641 4.51099 9.79
20.6491 4.29798 100
21.3587 4.15675 40.36
22.3682 3.97139 28.89
23.2002 3.83082 29.98
24.9025 3.57266 48.40
25.6135 3.47509 10.97
26.2143 3.39679 8.25
26.6549 3.34163 10.33
28.1846 3.16365 10.87
29.0925 3.06695 2.19
30.3881 2.93907 9.46
32.2330 2.77494 8.76
35.2102 2.54683 7.28
36.3908 2.46686 33.47
In another embodiment, the present invention provides a lyophilized crystalline composition "form PB-7", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.6738, 14.8888, 20.368, 21.0845, 22.8545, 26.275 and 36.0567 ±0.2 ° 2 theta.
In another specific embodiment the present invention provides a lyophilized crystalline composition "form PB-7", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 3.7128, 7.4092, 9.6738, 10.6056, 11.3708, 12.3786, 13.5814, 14.8888, 18.6203, 19.4268, 20.3680, 21.0845, 22.0467, 22.8545, 24.5953, 25.2204, 26.2750, 27.8504, 29.0602, 30.1448, 31.8419, 34.9144 and 36.0567 ±0.2 °2 theta.
In other embodiments, the novel crystalline "form PB-7" of lyophilized composition comprising bendamustine hydrochloride is characterized by an XRD pattern substantially in accordance with Fig.7. The XPRD data corresponding to this Diffractogram is shown below.

Angle [2-Theta] d value [Angstrom] Relative Intensity [%]
3.7128 23.77883 2.43
7.4092 11.92183 3.59
9.6738 9.13547 71.95
10.6056 8.33481 40.64
11.3708 7.77560 4.70
12.3786 7.14470 4.13
13.5814 6.51456 5.94
14.8888 5.94533 7.32
18.6203 4.76144 14.00
19.4268 4.56555 9.23
20.3680 4.35665 100
21.0845 4.21019 52.55
22.0467 4.02857 36.25
22.8545 3.88798 43.13
24.5953 3.61660 34.49
25.2204 3.52835 49.00
26.2750 3.38908 12.85
27.8504 3.20084 9.13
29.0602 3.07028 3.35
30.1448 2.96224 8.61
31.8419 2.80812 10.06
34.9144 2.56772 6.01
36.0567 2.48895 29.94
In another embodiment the present invention provides a lyophilized crystalline composition "form PB-8". comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.714, 18.7136,. 19.4655, 24.6835 and 36.1437 ±0.2 ° 2 theta.

In another specific embodiment the present invention provides a lyophilized crystalline composition "form PB-8". comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 3.7576, 7.4896, 9.7140, 10.1364, 10.6398, 11.4091, 12.4185, 13.6507, 14.9906, 18.7136, 19.4655, 20.0424, 20.2777, 20.4088, 21.0715, 21.2698, 22.1097, 22.9035, 24.4662, 24.6835, 25.2394, 26.3319, 27.9104, 28.7586, 30.1755, 31.9490, 34.9381 and 36.1437 ±0.2° 2 theta.
In other embodiments, the novel crystalline "form PB-8" of lyophilized composition comprising bendamustine hydrochloride is characterized by an XRD pattern substantially in accordance with Fig.8. The XPRD data corresponding to this Diffractogram is shown below.

Angle [2-Theta] d value [Angstrom] Relative Intensity [%]
3.7576 23.49501 3.46
7.4896 11.79407 6.11
9.7140 9.09771 76.32
10.1364 8.71953 3.12
10.6398 8.30814 57.26
11.4091 7.74957 4.47
12.4185 7.12186 4.74
13.6507 6.48163 6.64
14.9906 5.90517 8.69
18.7136 4.73791 20.00
19.4655 4.55655 10.02
20.0424 4.42669 42.93
20.2777 4.37585 83.77
20.4088 4.34802 100
21.0715 4.21276 51.57
21.2698 4.17393 29.23
22.1097 4.01722 32.32
22.9035 3.87976 56.47

24.4662 3.63538 21.32
24.6835 3.60386 33.21
25.2394 3.52573 48.90
26.3319 3.38188 17.83
27.9104 3.19410 9.16
28.7586 3.10179 5.59
30.1755 2.95930 11.41
31.9490 2.79895 9.73
34.9381 2.56603 5.86
36.1437 2.48316 25.69
In another embodiment the present invention provides a lyophilized crystalline composition "form PB-9". comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 9.7297, 21.0649, 21.8695, 24.6868 and 36.0778 ±0.2 ° 2 theta
In another specific embodiment the present invention provides a lyophilized crystalline composition "form PB-9", comprising bendamustine hydrochloride, wherein the powder X-ray diffraction pattern exhibits peaks at 3.7299, 7.4366, 9.7297, 10.6516, 11.3405, 12.4378, 13.6438, 14.9679, 18.6758, 19.4586, 20.3996, 21.0649, 21.8695, 22.1294, 22.9093, 24.4248, 24.6868, 25.2642, 26.4553, 27.8965, 28.8912, 30.1309, 31.9303, 34.9261 and 36.0778 ±0.2 ° 2 theta.
In other embodiments, the novel crystalline "form PB-9" of lyophilized composition comprising bendamustine hydrochloride is characterized by an XRD pattern substantially in accordance with Fig.9. The XPRD data corresponding to this Diffractogram is shown below.

Angle [2-ThetaJ d value [Angstrom] Relative Intensity [%]
3.7299 23.66969 2.49
7.4366 11.87798 3.93
9.7297 9.08315 72.13

10.6516 8.29892 37.64
11.3405 7.79629 2.94
12.4378 7.11085 3.96
13.6438 6.48490 5.76
14.9679 5.91407 7.26
18.6758 4.74741 14.58
19.4586 4.55817 9.71
20.3996 4.34997 100
21.0649 4.21407 58.11
21.8695 4.06080 30.44
22.1294 4.01371 28.38
22.9093 3.87880 46.97
24.4248 3.64145 24.60
24.6868 3.60340 32.03
25.2642 3.52233 48.74
26.4553 3.36639 13.00
27.8965 3.19565 9.17
28.8912 3.08785 3.90
30.1309 2.96357 8.82
31.9303 2.80055 11.65
34.9261 2.56688 6.28
36.0778 2.48754 30.54
In another embodiment, the present invention provides crystalline lyophilized compositions form "PB-2", "PB-3" and "PB-4", wherein the composition contains 40% ethanol and 5 ml fill volume and characterized by powder X-ray diffraction pattern which exhibits peaks at 9.7. 12.4. 15.0, 20.5, 21.1, 22.8 and 36.1±0.2 ° 2 theta.
The novel crystalline forms of lyophilized composition comprising bendamustine hydrochloride will be analyzed for its thermal behavior by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC); for its spectroscopic properties by solid state nuclear magnetic

resonance (NMR) spectrometry, Raman spectroscopy and infrared (IR) spectrometry; and for the moisture content by Karl Fischer analysis.
Another embodiment of the present invention provides a process for producing lyophilized crystalline composition "form PB-1" "form PB-2", "form PB-3"; "form PB-4", "form PB-5:', "form PB-6", "form PB-7", "form PB-8" and "form PB-9'. comprising bendamustine hydrochloride; wherein (he process comprises the steps of:
a) solubilizing bendamustine Hydrochloride;
b) adding water along with cryoprotectant; and
c) lyophilizing.
The details of the above embodiment are as follows:
Step a) involves solubilizing bendamustine hydrochloride. The solubilization is done by dissolving bendamustine hydrochloride in a solvent selected from the group consisting of but not limited to methanol, ethanol. n-propanol, iso-propanol, n-butanol, tert-butanol, or mixtures thereof, preferably ethanol.
Step b) invofves adding water with cryoprotectant. The amount of water used in step (b) is in the range of 6-10 ml with respect to 100 mg of bendamustine hydrochloride.
The cryoprotectants used in step b) are selected from the group consisting of but not limited to glucose, mannitol or trehalose, preferably mannitol.
Step c) involves lyophilization. This step involves a process in which bendamustine, together with the cryoprotectant is dissolved in a solvent and then subjected to a procedure that involves placing into a dryer and establishing a low shelf temperature, e.g., from -30°C to 25°C. applying vacuum to obtain a residue, and subsequently drying under reduced vacuum to remove residual solvent.
Another aspect of the present invention provides a process for producing lyophilized crystalline composition "form PB-1", "form PB-2", "form PB-3,;, "form PB-4", "form PB-5", "form PB-6",

"form PB-7", "form PB-8" and "form PB-9", comprising bendamustine hydrochloride; wherein the process comprises the steps of:
a) dissolving bendamustine hydrochloride in a solvent;
b) adding water for injection;
c) dissolving an excipient:
d) filtering: and
e) freeze drying.
The details of the above aspect are as follows:
Step (a): Step (a) of the above process involves the dissolution of bendamustine hydrochloride in
a solvent.
The solvent used in step (a) include water and organic solvents that form stable solutions with
bendamustine hydrochloride without appreciably degrading the bendamustine, and which are
capable of being evaporated/sublimed through lyophilization. Examples of suitable organic
solvents include, for example, methanol, ethanol, n-propanol, iso-propanol, n-butanol, tert-
butanol, or mixtures thereof. A preferred organic solvent is ethanol.
The amount of solvent used for dissolving bendamustine hydrochloride ranges between 1 to 3 ml
with respect to 100 mg of bendamustine hydrochloride used. Preferably, the amount of solvent
used is 2 ml.
The temperature for carrying out the dissolution of the above step (a) ranges between 2°C to
10°C.
Step (b): The step (b) involves addition of water for injection.
The amount of water used in step (b) is in the range of 6-10 ml with respect to 100 mg of bendamustine hydrochloride. Pre ferably, the amount of water used is 8 ml for 100 mg of bendamustine hydrochloride.
Step (c): Step (c) involves dissolving an excipient.
The excipient used in step (c) may be selected from the group of sugar alcohols more particularly
mannitol. The amount of mannitol used in the above step (c) is in the range of 150 to 200 parts

by weight on the basis of 100 parts by weight of bendamustine hydrochloride. Preferably the amount of mannitol used is 170 mg with respect to 100 mg of bendamustine hydrochloride used. After the excipient is dissolved, additional water may be added to make up the solution to the desired volume.
Step (d): Step (d) involves filtration. The filtration is carried out through a 0.2 micron filter. After filtration, the liquid obtained is filled in vials and partially stoppered for the next step.
Step (e): The step (e) of the above process involves freeze drying.
In an embodiment, the freeze-drying process of step (e) consists of three stages; that is, freezing,
primary drying, and secondary drying.
The solid/cake obtained after step (e) is sealed, labeled and packaged.
In a preferred embodiment, the present invention provides a process for lyophilizing bendamustine hydrochloride wherein the mannitol is present in an amount of at least 170 parts by weight on the basis of 100 parts by weight of bendamustine hydrochloride.
In yet another related embodiment, the novel crystalline form of lyophilized compositions comprising bendamustine hydrochloride are prepared using varying percentages of ethanol.
In an embodiment, the percentage of ethanol may range between 1-50%, preferably 20-45%, more preferably 40%.
In an embodiment, the lyophilized pharmaceutical composition comprising bendamustine hydrochloride of the present invention contains a maximum fill volume of 10 ml.
The novel crystalline forms of lyophilized compositions of bendamustine hydrochloride of the present invention are expected to have a higher purity level, lesser impurity content and greater storage stability.

in an embodiment, the present invention provides a storage stable lyophilized crystalline composition comprising bendamustine with lower impurity content.
The level of product and process related impurities in the lyophilized pharmaceutical compositions of the present invention comprising bendamustine hydrochloride are lower. The major impurities associated with bendamustine hydrochloride are shown below:



In an embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of HP-1 impurity is not more than 0.4% at time zero after reconstitution.
In a further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of HP-1 impurity is not more than 0.25% when stored at 2-8°C for 24 hours.
In a still further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2". "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of HP-1 impurity is not more than 2% when stored in a controlled room temperature (CRT) for 24 hours.
In another embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of the HP-1 ester impurity is not more than 0.02% at time zero after reconstitution.

In a further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of HP-1 ester impurity is not more than 0.05% when stored at 2-8°C for 24 hours.
In a still further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of HP-1 ester impurity is not more than 0.7% when stored in a controlled room temperature (CRT) for 24 hours.
In yet another embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2". "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of the HP-2 impurity is below the limit of detection at time zero after reconstitution.
In a further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of HP-2 impurity is below the limit of detection when stored at 2-8°C for 24 hours.
In a still further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2". "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of HP-2 impurity is below the limit of detection when stored in a controlled room temperature (CRT) for 24 hours.
hi another embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of the methyl ester impurity is not more than 0.08% at time zero after reconstitution.

In a further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of methyl ester impurity is not more than 0.08% when stored at 2-8°C for 24 hours.
In a still further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of methyl ester impurity is not more than 0.07% when stored in a controlled room temperature (CRT) for 24 hours.
In another embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2". "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of the BEN-2 impurity is below the limit of detection at time zero after reconstitution.
In a further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2". "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of BEN-2 impurity is below the limit of detection when stored at 2-8°C for 24 hours.
In a still further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of BEN-2 impurity is below the limit of detection when stored in a controlled room temperature (CRT) for 24 hours.
In a preferred embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2", "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of total impurity is not more than 0.5% at time zero after reconstitution.

In a further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2"; "form PB-3:' and "form PB-4" comprising bendamustine hydrochloride wherein the content of total impurity is not more than 0.5% when stored at 2-8°C for 24 hours.
In a still further embodiment, the present invention provides a lyophilized pharmaceutical composition "form PB-2". "form PB-3" and "form PB-4" comprising bendamustine hydrochloride wherein the content of total impurity is not more than 3.05% when stored in a controlled room temperature (CRT) for 24 hours.
Controlled room temperature (CRT), as used herein means a temperature maintained thermostatically that encompasses the usual and customary working environment of 20° to 25°C. All of the impurity contents herein are expressed as percentages of the drug content.
The below table shows the level of impurities analyzed on two samples A and B (containing 2ml elhanol and 5 ml fill volume) of lyophilized pharmaceutical composition comprising bendamustine hydrochloride and the commercially available composition of bendamustine hydrochloride, TREANDA.

Impurity TRENDA Sample A Sample B
pH 97.98 95.1 100.9
Drug Assay 2.964 2.962 2.951
Drug related substance
HP-1 Impurity 0.42 0.30 0.36
HP-1 Ester 0.02 ND* ND*
HP-2 (mpurity ND* ND* ND*
Methyl Ester ND* ND* 0.07
BEN-2A ND* ND* ND*
Single highest impurity 0.34 0.01 0.02
Total impurity 1.45 0.40 0.49

* ND - not detected
Although there are no specific regulation or guidance documents on bulk product holding times, good manufacturing practice dictates that the holding time should be validated to ensure that in-process and bulk product can be held, without any adverse effect to the quality of the material.
The below table provides the results of the solution hold time study of lyophilized pharmaceutical composition comprising bendamustine hydrochloride; Sample C (2 ml ethanol and 5 ml fill volume)

Solution hold time study of Bendamustine hydrochloride fc r injection
Sampl eC
Condition Initial 2°-8°C CRT†

lhr 3hr 5hr 9hr 24 hr 2hr 4hr 8hr 24 hr
Assay 104 104.3 103.6 103.1 101.9 103.3 105.7 102.3 101.7 101
pH 2.971 2.95 2.948 2.955 9.956 3.005 2.96 2.972 2.96! 2.801
Rel ated su )stance
HP-1 Impurity 0.19 0.16 0.17 0.19 0.19 0.24 0.35 0.52 0.77 1.98
HP-1 Ester 0.02 0.02 0.02 0.02 0.02 0.05 0.08 0.13 0.22 0.63
HP-2 Impurity ND* ND* ND* ND* ND* ND* ND* ND* ND* ND*
Methyl Ester 0.06 0.07 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06
BEN-2A ND* ND* ND* ND* ND* ND* ND* ND* ND* ND*
Single highest impurity 0.01 0.01 0.01 0.01 0.02 0.03 0.03 0.04 0.05 0.15
Total impurity 0.3 0.28 0.29 0.32 0.33 0.42 0,57 0.81 1.19 3.02
*ND-Not detected
t CRT - controlled room temperature
In an embodiment, the present invention provides an improved process for preparing the novel crystalline lyophilizate compositions wherein the lyophilization process generally includes any or all of following steps:

1) dissolving bendamustine hydrochloride and excipient mannitol in a suitable solvent, generally water for injection;
2) sterilizing the bulk solution;
3) filling into individual sterile containers and partially stoppering the containers under aseptic conditions;
4) transporting the partially stoppered containers to the lyophilizer and loading into the chamber under aseptic conditions;
5) freezing the solution by placing the partially stoppered containers on cooled shelves in a freeze-drying chamber or pre-freezing in another chamber;
6) applying a vacuum to the chamber and heating the shelves in order to evaporate the water from the frozen state: and
7) complete stoppering of the vials usually by hydraulic screw rod stoppering mechanisms installed in the lyophilizers.
The number of lyophilization steps/cycles may be varied and such variations are within the scope of the present invention.
A lyophilization excipient as used herein can be any pharmaceutically acceptable excipient that, when used during the lyophilization process, results in a lyophilized product that has improved properties, for example, improved handling properties, solubility properties, and the like.
A lyophilization excipient can be, for example, a bulking agent: suitable bulking agents are known in the art. Examples of suitable lyophilization excipients include, for example, sodium phosphate, potassium phosphate, citric acid, tartaric acid, gelatin, glycine, mannitol, lactose, sucrose, maltose, glycerin, dextrose, dextran, trehalose, hetastarch, or mixtures thereof.
A lyophilization excipient may also comprise a pharmaceutically acceptable antioxidant, such as. for example, ascorbic acid, acetylcysteine, cysteine, sodium hydrogen sulfite, butyl-hydroxylanisole. butyl-hydroxytoluene, or alpha-tocopherol acetate. A preferred lyophilization excipient is mannitol.

Solvents for use in the present invention include water and organic solvents that form stable solutions with bendamustine hydrochloride without appreciably degrading the bendamustine, and which are capable of being evaporated/sublimed through lyophilization. Examples of suitable organic solvents include, for example, methanol, ethanol, n-propanol, iso-propanol. n-butanol, tert-butanol, or mixtures thereof. A preferred organic solvent is ethanol.
In yet another related embodiment, the novel crystalline form of lyophilized compositions comprising bendamustine hydrochloride are prepared using varying percentage of ethanol. In an embodiment, the percentage of ethanol may range between 1-50%, preferably 20-45%. more preferably 40%.
Preferably, the pharmaceutical compositions of bendamustine hydrochloride described herein are reconstituted into an injectable preparation. More preferably, the lyophilized pharmaceutical compositions of bendamustine hydrochloride of the present invention will have a shorter reconstitution time.
In another preferred embodiment the conversion of bendamustine hydrochloride to bendamustine base during or after lyophilization is in the range of 1-10 % or 1-20 % or 1-30% or 1-40% with respect to the weight of bendamustine hydrochloride. In a preferred embodiment the conversion of bendamustine hydrochloride to bendamustine base during or after lyophilization is below 5%.
In an embodiment, the bendamustine Hydrochloride used in the preparation of crystalline lyophilized compositions has an XRD pattern which is consistent.
In another embodiment, the bendamustine Hydrochloride used in the preparation of crystalline lyophilized compositions has a DSC thermogram substantially in accordance with Fig. 10. The DSC is analyzed using the Universal Analysis 2000 software version 4.2E (TA Instruments), ramp 10°C/minute to 200°C.
The bendamustine hydrochloride used in the present invention can be prepared according to any of the known procedures; preferably the bendamustine hydrochloride is prepared according to

the procedure described in International application publication number WO 2011/079193 A2, which involves:
(1) preparation of isopropyl-4-(5-(bis (2-hydroxyethylamino)-l-methyl-lH-benzo[d]imidazol-2-yl) butanoate by reacting 4-(l-methyl-5-amino-lH-benzo[d]imidazol-2-yl) butanoate with 2-chloroethanol in the presence of base.
(2) converting isopropyl-4-(5-(bis (2-hydroxyethylamino)-l-methyl-lH-benzo[d]imidazol-2-yl) butanoate to isopropyl-4-(5-(bis (2-chloroethylamino)-l-methyl-lH-benzo[d]imidazol-2-yl) butanoate; and
(3) hydrolysis of isopropyl-4-(5-(bis(2-chloroethylamino)-l-methyl-lH-benzo[d]imidazo]-2-yl) butanoate using aqueous hydrochloric acid to give crude bendamustine hydrochloride.
In a further embodiment, the novel crystalline pharmaceutical compositions according to the present invention may be used for the treatment, induction, salvage therapy, conditioning prior to stem cell transplantation, maintenance therapy, treatment of residual disease of a medical condition in a human or animal, preferably a human, which medical condition is selected from chronic lymphocytic leukemia (CLL). acute lymphocytic leukaemia (ALL), chronic myelocytic leukaemia (CML), acute myelocytic leukaemia (AML), Hodgkin's disease, non-Hodgkin's lymphoma (NHL), multiple myeloma, breast cancer, ovarian cancer, small cell lung cancer, non-small cell lung cancer, and an autoimmune disease. Preferred autoimmune diseases include rheumatoid arthritis, multiple sclerosis or lupus.
In yet another further embodiment, the present invention also comprises a method of treatment of a medical condition selected from chronic lymphocytic leukemia, acute lymphocytic leukaemia, chronic myelocytic leukaemia acute myelocytic leukaemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, breast cancer, ovarian cancer, small cell lung cancer, non-small cell lung cancer, and an autoimmune disease, in a human or animal body comprising administering to the human or animal body in need thereof an effective amount of the pharmaceutical preparation of this invention. Preferably the medical condition is non-Hodgkin's lymphoma.

In an embodiment, the novel crystalline pharmaceutical compositions according to the present invention may be administered in combination with at least one further active agent, wherein said further active agent is given prior, concurrently, or subsequently to the administration of the pharmaceutical composition. This at least one further active agent is preferably an antibody specific for CD20, such as rituximab or ofaturnumab, an anthracyclin derivative, such as doxorubicin or daunorubicin. a vinca alkaloid, such as vincristine, a platin derivative, such as cisplatin or carboplatin, daporinad (FK866). YM155, thalidomide and analogues thereof, such as thalidomide or lenalidomide. or a proteasome inhibitor, such as bortezomib.
In a preferred embodiment, the pharmaceutical composition of this invention may also be administered in combination with at least one corticosteroid, wherein said corticosteroid is given prior, concurrently, or subsequently to the administration of the pharmaceutical composition. Examples of the corticosteroids are prednisone, prednisolone or dexamethasone.
EXAMPLES:
The invention is further described by reference to the following examples which are given solely for the purpose of illustration only and therefore should not be construed to limit the scope of the invention.
Example-l: Preparation of lyophilized composition of Bendamustine hydrochloride
100 mg of Bendamustine hydrochloride is weighed and dissolved in 2 ml ethanol at 2-10°C. After the complete dissolution of bendamustine hydrochloride in ethanol. sterile water (8 ml) is added followed by addition of 170 mg of mannitol until dissolution. The volume is made up by addition of sterile water and the liquid is filtered through 0.2 micron filter. The filtrate is filled in vials and partially stoppered and conditioned to freeze drying. The freeze dried cake is sealed, labeled and packaged.

We Claim:
1. A novel crystalline pharmaceutical compositions comprising bendamustine hydrochloride,
wherein the composition is free of tertiary butyl alcohol and/or acetone and /or acetonitrile.
2. A novel crystalline lyophilized composition according to claim 1, wherein the composition
is "form PB-1" characterized by powder X-ray diffraction pattern which exhibits peaks at
9.7647.. 10.6988, 15.0462, 20.0877, 20.4728, 21.1464, 26.3467 and 36.1533 ±0.2 °2 theta.
3. A novel crystalline lyophilized composition according to claim 2, which is further characterized by peaks at 7.5181, 9.7647, 10.1710, 10.6988, 11.4612, 12.4871, 13.6848, 15.0462, 17.3397, 18.7380, 19.4907, 20.0877, 20.4728, 21.1464, 21.2859, 22.1307, 22.9664, 24.7214, 25.3075, 25.4567, 26.3467, 27.9233, 28.8312, 30.1837, 32.1186, 33.1792, 33.9529, 34.9601, 36.1533, 36.5464 and 37.9271 ±0.2 °2 theta.
4. A novel crystalline lyophilized composition according to claim 1, wherein the composition is "form PB-2" characterized by powder X-ray diffraction pattern which exhibits peaks at 9.7936, 15.0244, 20.5218, 21.2014, 24.3684 and 36.1304 ±0.2 ° 2 theta.
5. A novel crystalline lyophilized composition according to claim 4, which is further characterized by peaks at 7.4564, 9.7936, 10.6953, 11.3897, 12.4210, 13.6685, 15.0244, 18.7484, 19.5547, 20.5218,21.2014,22.2135, 22.9472, 24.3684, 24.7637, 25.3746, 26.0932, 26.5448, 28.0185, 28.9361, 30.2580, 32.2187, 35.0589, 36.1304 and 37.9319 ±0.2 ° 2 theta.
6. A novel crystalline lyophilized composition according to claim 1, wherein the composition is "form PB-3" characterized by powder X-ray diffraction pattern which exhibits peaks at 9.7089, 20.0198, 21.1176, 22.8902, 26.4924 and 36.0927 ±0.2 ° 2 theta..
7. A novel crystalline lyophilized composition according to claim 6, which is further characterized by peaks at 3.7263, 7.4408, 9.7089, 10.1256, 10.6197, 11.3928, 12.4441, 13.6255, 14.9765, 18.6632, 18.9712, 19.4604, 20.0198, 20.3990, 21.1176, 21.2755,

22.0906, 22.8902, 23.2712, 24.6496, 25.2143, 25.3627, 26.0222, 26.4924, 27.0214, 27.8812, 28.7434, 29.0752, 30.1482, 30.9657, 31.8949, 32.1634, 33.9228, 34.8819, 36.0927, 36.4936, 37.8653 and 39.3098 ±0.2 ° 2 theta.
8. A novel crystalline lyophilized composition according to claim 1, wherein the composition
is "form PB-4" characterized by powder X-ray diffraction pattern which exhibits peaks at
9.7429, 12.4563, 21.0756, 30.1736 and 36.1208 ±0.2 ° 2 theta.
9. A novel crystalline lyophilized composition according to claim 8, which is further
characterized by peaks at 3.7696, 7.4779, 9.7429, 10.6497, 11.4406, 12.4563, 13.6602,
14.9697, 18.7273, 19.4637, 20.4480, 21.0756, 21.3187, 22.1212, 22.8995, 23.3095,
24.6733, 25.2882, 26.0002, 26.5331, 27.8960, 29.0880, 30.1736, 30.6201, 31.9384,
34.9259, 36.1208, 37.8518 and 39.4477 ±0.2 ° 2 theta.
10. A novel crystalline lyophilized composition according to claim 1. wherein the composition is "form PB-5" characterized by powder X-ray diffraction pattern which exhibits peaks at 9.9168, 10.8427, 20.2554, 20.6012, 20.726 and 22.4254 ±0.2 ° 2 theta.
11. A novel crystalline lyophilized composition according to claim 10, which is further characterized by peaks at 7.4852, 9.9168, 10.8427, 12.6206, 13.8330, 15.2400, 18.9818, 19.7967, 20.2554, 20.6012, 20.7260, 21.3922, 22.4254, 23.1708, 24.9521, 25.5490, 26.6616, 28.2199, 29.0342, 30.5861, 32.2334, 35.0543, 36.3079 and 38.1188 ±0.2 ° 2 theta.
12. A novel crystalline lyophilized composition according to claim 1, wherein the composition is "form PB-6" characterized by powder X-ray diffraction pattern which exhibits peaks at 9.9058, 10.8374, 20.6491, 23.2002 and 36.3908 ±0.2 ° 2 theta.
13. A novel crystalline lyophilized composition according to claim 12, which is further characterized by peaks at 9.90^8, 10.8374, 12.5979, 13.7317, 15.1319, 18.8887, 19.6641, 20.6491, 21.3587, 22.3682, 23.2002, 24.9025, 25.6135, 26.2143, 26.6549, 28.1846, 29.0925, 30.3881, 32.2330, 35.2102 and 36.3908 ±0.2 °2 theta.

14. A novel crystalline lyophilized composition according to claim 1, wherein the composition
is "form PB-7" characterized by powder X-ray diffraction pattern which exhibits peaks at
9.6738, 14.8888, 20.368, 21.0845, 22.8545, 26.275 and 36.0567 ±0.2 ° 2 theta.
15. A novel crystalline lyophilized composition according to claim 14, which is further characterized by peaks at 3.7128, 7.4092, 9.6738, 10.6056, 11.3708, 12.3786, 13.5814, 14.8888, 18.6203, 19.4268, 20.3680, 21.0845, 22.0467, 22.8545, 24.5953, 25.2204, 26.2750, 27.8504, 29.0602, 30.1448, 31.8419, 34.9144 and 36.0567 ±0.2 ° 2 theta.
16. A novel crystalline lyophilized composition according to claim 1, wherein the composition is "form PB-8" characterized by powder X-ray diffraction pattern which exhibits peaks at 9.714, 18.7136, 19.4655, 24.6835 and 36.1437 ±0.2 ° 2 theta.
17. A novel crystalline lyophilized composition according to claim 16, which is further characterized by peaks at 3.7576, 7.4896, 9.7140, 10.1364, 10.6398, 11.4091, 12.4185, 13.6507, 14.9906, 18.7136, 19.4655, 20.0424, 20.2777, 20.4088, 21.0715, 21.2698, 22.1097, 22.9035, 24.4662, 24.6835, 25.2394, 26.3319, 27.9104, 28.7586, 30.1755, 31.9490, 34.9381 and 36.1437 ±0.2 ° 2 theta.
18. A novel crystalline lyophilized composition according to claim 1, wherein the composition is "form PB-9" characterized by powder X-ray diffraction pattern which exhibits peaks at 9.7297, 21.0649, 21.8695, 24.6868 and 36.0778 ±0.2 ° 2 theta.
19. A novel crystalline lyophilized composition according to claim 18, which is further characterized by peaks at 3.7299, 7.4366, 9.7297, 10.6516, 11.3405, 12.4378; 13.6438, 14.9679, 18.6758, 19.4586, 20.3996, 21.0649, 21.8695, 22.1294, 22.9093, 24.4248, 24.6868, 25.2642, 26.4553, 27.8965, 28.8912, 30.1309, 31.9303, 34.9261 and 36.0778 ±0.2 ° 2 theta.

20. The novel crystalline lyophilized composition according to any of the preceding claims,
wherein the lyophilized crystalline composition further comprises mannitol.
21. A storage stable crystalline lyophilized compositions according to any of the preceding
claims.
22. A process for the preparation of lyophilized crystalline composition according to any of the
preceding claims, wherein the process comprises the steps of:
a) solubilizing bendamustine hydrochloride;
b) adding water along with cryoprotectant; and
c) lyophilizing.

23. The process according to claim 22, wherein bendamustine hydrochloride is solubilized in a solvent selected from water methanol, ethanol, n-propanol, iso-propanol, n-butanol, tert-butanol, or mixtures thereof.
24. The process according to claim 23, wherein the amount of solvent used for dissolving bendamustine hydrochloride ranges between 1 to 3 ml with respect to 100 mg of bendamustine hydrochloride used.
25. The process according to claim 22. wherein the cryoprotectant used in step b) is selected from the group of sugar alcohols, such as mannitol.
26. The process according to claim 25, wherein the amount of mannitol used is between 150 to 200 parts by weight on the basis of 100 parts by weight of bendamustine hydrochloride.
27. The crystalline lyophilized compositions according to any of the preceding claims comprising Bendamustine Hydrochloride, wherein the compositions are prepared using ethanol.

28. The crystalline lyophilized compositions according to claim 27, wherein the composition is
prepared using 40% ethanol.
29. The crystalline lyophilized composition according to any of the preceding claims wherein
the composition contains a maximum fill volume of 10 ml.
30. The crystalline lyophilized compositions according to claims 4. 6 and 8, wherein the composition contains 40% ethanol and 5 ml fill volume.
31. The crystalline composition according to claim 30 characterized by powder X-ray diffraction pattern which exhibits peaks at 9.7, 12.4, 15.0, 20.5, 21.J, 22.8 and 36.1±0.2 0 2 theta.
32. The crystalline lyophilized composition according to claim 30, wherein the content of the HP1 impurity is:

a) not more than 0.4% at time zero after reconstitution; or
b) not more than 0.25% when stored at 2-8°C for 24 hours; or
c) not more than 2% when stored in a controlled room temperature (CRT) for 24 hours.
33. The crystalline lyophilized composition according to claim 30, wherein the content of HP-1
ester impurity is:
a) not more than 0.02% at time zero after reconstitution; or
b) not more than 0.05% when stored at 2-8°C for 24 hours; or
c) not more than 0.7% when stored in a controlled room temperature (CRT) for 24 hours.
34. The crystalline lyophilized composition according to claim 30, wherein the content of HP-2
impurity is below the limit of detection:
a) at time zero after reconstitution; or
b) when stored at 2-8°C for 24 hours; or
c) when stored in a controlled room temperature (CRT) for 24 hours.

35. The crystalline lyophilized composition according to claim 30, wherein the content of
methyl ester impurity is:
a) not more than 0.08% at time zero after reconstitution; or
b) not more than 0.08% when stored at 2-8°C for 24 hours; or
c) not more than 0.07% when stored in a controlled room temperature (CRT) for 24 hours.
36. The crystalline lyophilized composition according to claim 30, wherein the content of BEN-
2 impurity is below the limit of detection:
a) at time zero after reconstitution; or
b) when stored at 2-8°C for 24 hours; or
c) when stored in a controlled room temperature (CRT) for 24 hours.
37. The crystalline lyophilized composition according to claim 30, wherein the content of total
impurity is:
a) not more than 0.5% at time zero after reconstitution; or
b) not more than 0.5% when stored at 2-8°C for 24 hours.; or
c) not more than 3.05%) when stored in a controlled room temperature (CRT) for 24 hours.