WO2011103150A2

WO2011103150A2 - Lyophilized preparations of bendamustine

Info

Publication number: WO2011103150A2
Application number: PCT/US2011/025031
Authority: WO
Inventors: Michael L. Cappola; Piyush R. Patel
Original assignee: Cephalon, Inc.
Priority date: 2010-02-18
Filing date: 2011-02-16
Publication date: 2011-08-25
Also published as: WO2011103150A3

Abstract

Lyophilized preparations comprising bendamustine, and pharmaceutically acceptable salts thereof, and cyclodextrins are described, as well as methods for their production and use.

Description

LYOPHILIZED PREPARATIONS OF BENDAMUSTINE

TECHNICAL FIELD

Lyophilized preparations of bendamustine and bendamustine salts, as well as methods for their preparation and use, are described.

BACKGROUND

There are many pharmaceuticals that are known to be highly effective for the treatment of debilitating diseases such as cancer. Unfortunately, many of these

pharmaceuticals are not stable to commercial shipping and storage conditions, which may include temperatures above 23 °C (ambient room temperature), relative humidity greater than ambient, and time. In an effort to preserve the integrity of the pharmaceutical, non- routine shipping and storage conditions are sometimes employed. For example, the pharmaceutical is shipped and stored at refrigerated temperatures, under an inert atmosphere, and/or it is provided with instructions to use or discard within just a few days of receipt. Oftentimes, the pharmaceutical must be discarded because it has not retained sufficient integrity during the shipping and storage process. This is undesirable because these sensitive pharmaceuticals are generally developed and manufactured at great expense.

Lyophilization, or "freeze-drying," is a method used in the manufacture of unstable and/or sensitive pharmaceuticals. Many technical challenges, for example, identifying appropriate shelf temperatures, product temperatures, vacuum levels, freezing, primary drying parameters, and secondary drying parameters, must be overcome in the

development of a commercially viable lyophilization process. In addition, the

pharmaceutical is usually sensitive to the lyophilization process, which typically involves the use of water. Moreover, lyophilization usually involves the addition of pharmaceutical excipients, such as bulking agents and the like. The sensitivity of the particular pharmaceutical to excipients is generally unknown and must be exhaustively evaluated.

Another factor in identifying a suitable lyophilization process is the evaluation of the properties of the lyophilized "cake." The cake must be stable to the storage and shipping conditions for a reasonable amount of time. Additionally, if the pharmaceutical is to be used for injection, the lyophilized cake should be readily reconstituted with an appropriate intravenous solution such as Sodium Chloride for Injection, Sterile Water for Injection, Mannitol I.V., and the like, to form a particulate-free injectable solution.

Indeed, lyophilized cakes that do not readily form a clear solution with little to no particulate must be discarded.

As such, lyophilization conditions and methods for producing stable, readily reconstitutable lyophilized alkylating are needed.

SUMMARY

The present invention is directed to lyophilized preparations comprising (a) bendamustine or a pharmaceutically acceptable salt thereof; and a cyclodextrin. Methods of preparing and using the lyophilized preparations of the invention are also described.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

It has now been discovered that the addition of a cyclodextrin to lyophilized preparations of bendamustine, or pharmaceutically acceptable salts thereof, results in preparations that are stable and readily and reproducibly reconstituted to form solutions suitable for injection into humans. Lyophilization processes suitable for use with bendamustine or pharmaceutically acceptable salts thereof have also been discovered.

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 personal prior to administration. Once in aqueous solution, nitrogen mustards are subject to degradation by hydrolysis, thus, the reconstituted product should be administered to a patient as soon as possible after its reconstitution.

Bendamustine, (4-{5-[bis(2-chloroethyl)amino]-l-methyl-2-benzimidazolyl} butyric acid, is an atypical structure with a benzimidazole ring, and the structure of which includes an active nitrogen mustard (see Formula I, which shows bendamustine hydrochloride).

Formula I Bendamustine was initially synthesized in 1963 in the German Democratic

Republic (GDR) 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 tradename Ribomustin®. It has been widely used in Germany to treat chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, and breast cancer.

Due to its degradation in aqueous solutions (like other nitrogen mustards), bendamustine is supplied as a lyophilized product. The current lyophilized formulation of bendamustine that is marketed in the United States (Treanda®) 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 manufactured in brown or amber-colored glass bottles. The current lyophilized formulation of bendamustine may contain 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 in a 20 mL single-use, amber vial or 25 mg of drug in an 8 mL vial. The product is reconstituted as close to the time of patient administration as possible. The 25 mg product is reconstituted by aseptically adding 5 mL of Sterile Water for Injection, USP (SWFI). For the 100 mg product, 20 mL of SWFI is used. The vial is shaken well and complete dissolution should occur within 5 minutes.

Following dissolution in SWFI, the volume required for the appropriate dose is aseptically withdrawn from the vial, and immediately transferred to a 500 ml infusion bag of 0.9% Sodium Chloride Injection USP (normal saline), or of 2.5% Dextrose/0.45% Sodium Chloride Injection, USP. Transfer to the infusion bag must be done within 30 minutes of reconstitution.

The route of administration is by intravenous infusion over 30 to 60 minutes. Once diluted with either 0.9% Sodium Chloride Injection, USP, or 2.5%

Dextrose/0.45% Sodium Chloride Injection, USP, the final admixture is stable for 24 hours when refrigerated (2-8°C or 36-47°F) or for 3 hours when stored at room

temperature. Administration must be completed within this period.

Thus, a need exists for lyophilized formulations of bendamustine that are easier to reconstitute and which have a better impurity profile than the current lyophilate

(lyophilized powder) formulations of bendamustine.

It should be pointed out that the two-step reconstitution procedure described above is necessary because bendamustine hydrochloride, for example, is relatively insoluble at the concentration required in normal saline. Thus, it is one aspect of the present invention to provide a bendamustine preparation having a greatly reduced reconstitution time in water. It is another aspect to provide a bendamustine preparation that is soluble in, and can be reconstituted, for example, in normal saline, thus eliminating an initial dissolution step using SWFI in the reconstitution process.

The present invention is also applicable to the pharmaceutically acceptable salts of bendamustine. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

A preferred salt of bendamustine is bendamustine hydrochloride.

The pharmaceutically acceptable salts of the present invention can be prepared from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of the compound with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack

Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is hereby incorporated by reference.

Embodiments of the present invention are directed to lyophilized preparations of bendamustine or a pharmaceutically acceptable salt thereof, in combination with a cyclodextrin. "Cyclodextrin" refers to a family of cyclic oligosaccharides containing five or more a-D-glucopyranoside units. The cyclodextrins of the present invention can include the naturally occurring cyclodextrins and their derivatives. The natural cyclodextrins include a-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. Derivatives are typically prepared by modifying the hydroxyl groups located on the exterior, or hydrophilic side, of the cyclodextrin. The types and degree of modification, as well as their preparation, are well known in the art. See, for example, Szejtli, J., Cyclodextrins and Their Inclusion Complexes, Akademiai Kiado: Budapest, 1982; U.S. Pat. No.

5,024,998; U.S. Pat. No. 5,874,418 and U.S. Pat. No. 5,660,845, and references contained therein, all of which are incorporated herein in their entireties.

Any of the natural cyclodextrins can be derivatized. Cyclodextrin derivatives include alkylated cyclodextrins, preferably methyl-, dimethyl-, trimethyl- and ethyl-β- cyclodextrins; hydroxyalkylated cyclodextrins, including hydroxyethyl-, hydroxypropyl-, and dihydroxypropyl-P-cyclodextrin; ethylcarboxymethyl cyclodextrins; sulfate, sulfonate and sulfoalkyl cyclodextrins, preferably β-cyclodextrin sulfate, β-cyclodextrin sulfonate, and sulfobutyl ether β-cyclodextrin; as well as polymeric cyclodextrins. Other cyclodextrin derivatives can be made by substitution of the hydroxy groups with saccharides, such as glucosyl- and maltosyl^-cyclodextrin.

Preferred cyclodextrins include the naturally occurring cyclodextrins, methyl-β- cyclodextrin, dimethyl^-cyclodextrin, trimethyl^-cyclodextrin, 2-hydroxymethyl^- cyclodextrin, hydroxyethyl^-cyclodextrin, hydroxypropyl^-cyclodextrin, β-cyclodextrin sulfate, β-cyclodextrin sulfonate, or sulfobutyl ether β-cyclodextrin. Most of these are commercially available from such suppliers as Aldrich Chemical Company, Milwaukee Wisconsin and Wacker Chemicals, New Canaan, Connecticut. Preferred cyclodextrins are hydroxypropyl- -cyclodextrin, hydroxypropyl-y-cyclodextrin, sulfobutyl ether β- cyclodextrin, or a mixture thereof. In the most preferred embodiments of the present invention, the cyclodextrin is sulfobutyl ether β-cyclodextrin. Preferably, the cyclodextrin is present in an amount effective to stabilize bendamustine or a pharmaceutically acceptable salt thereof during and/or after, lyophilization. The cyclodextrin will typically be present in an amount up to about 99% w/w of the lyophilized preparation. Preferably, the cyclodextrin will be present in an amount from about 20% to about 90% w/w of the lyophilized preparation. In preferred embodiments, the cyclodextrin is present in an amount from about 40% to about 70% w/w of the lyophilized preparation. Even more preferred are those embodiments wherein the cyclodextrin is present in an amount of from about 45% to about 65% w/w of the lyophilized preparation. Most preferred embodiments comprise 40%>, 45%, or 55% w/w of cyclodextrin.

The lyophilized preparations of the present invention also include one or more pharmaceutically acceptable excipients. For example, the lyophilized preparations of the invention may contain one or more bulking agents.

Bulking agents that have "generally regarded as safe" (GRAS) status from the United States Food and Drug Administration (FDA) are well known in the art of pharmaceutical lyophilization and tend to strengthen the structure of the resulting lyophilized cake. Bulking agents include saccharides, preferably monosaccharides or oligosaccharides, sugar alcohols, and mixtures thereof. More specifically, bulking agents used in the present invention include sucrose, dextrose, maltose, lactose, sorbitol, glycine, mannitol and dextran. A most preferred bulking agent is mannitol.

The bulking agent is typically present in an amount up to about 99% w/w of the lyophilized preparation. Preferably, the bulking agent comprises about 20% to about 90% w/w of the lyophilized preparation. In preferred embodiments, the bulking agent comprises about 30%> to about 60%> w/w of the lyophilized preparation. In other embodiments, the bulking agent comprises about 35% or about 45% w/w of the lyophilized preparation.

Other excipients may also be added to the lyophilized preparations of the present inventions. Such excipients may include antioxidants, antimicrobials, cryostabilizers, and the like.

In certain embodiments, the pre-lyophilization solution is prepared with a pharmaceutically acceptable organic solvent. The addition of the alcohol may assist in the solubilizing of the compound of Formula I or Formula II when preparing the pre- lyophilization solution. In certain embodiments, the pre-lyophilization solution will comprise up to about 90% v/v of organic solvent, such as up to about 75% v/v, up to about 60% v/v, or up to about 40%> v/v. Preferably, the pre-lyophilization solution comprises about 5% to about 75% v/v of organic solvent, such as about 10% to about 70% v/v. In preferred embodiments, the pre-lyophilization solution comprises about 20%> to about 60%> v/v of organic solvent, such as about 30%> to about 50%> v/v. Preferably, the pre- lyophilization solution comprises about 40% v/v of organic solvent.

Suitable organic solvents include solvents that are miscible with water and removable by lyophilization. Examples include alcohols, preferably Ci_6alcohols such as ethanol, propanol, t-butanol, and propylene glycol, and polar aprotic solvents such as dimethylsulfoxide. A preferred organic solvent is tert-butanol. Typically, about 3% w/w, or less, of the organic solvent will be present in lyophilized preparations of the present invention. Preferably, the organic solvent will be present in an amount of from about 1% w/w, or less, of the lyophilized preparation.

Preferred lyophilized preparations of the present invention include those comprising bendamustine, or a pharmaceutically acceptable salt thereof, and a

cyclodextrin, preferably hydroxypropyl- -cyclodextrin, hydroxypropyl-y-cyclodextrin, sulfobutyl ether β-cyclodextrin, or a mixture thereof. Particularly preferred lyophilized preparations of the present invention include those comprising bendamustine, or a pharmaceutically acceptable salt thereof, a cyclodextrin, preferably hydroxypropyl-β- cyclodextrin, hydroxypropyl-y-cyclodextrin, sulfobutyl ether β-cyclodextrin, or a mixture thereof, and a bulking agent, preferably mannitol. Any of the lyophilized preparations of the invention may further comprise, an organic solvent, such as a Ci_6alcohol, preferably tert-butanol, preferably in an amount less than about 3% w/w of the lyophilized

preparation.

A particular advantage of the lyophilized preparations of the present invention is that they are readily reconstituted to form clear, near-colorless to colorless solutions that are free of discoloration (i.e., are colorless or near colorless) and/or particulate matter, suitable for injection into humans in about 180 seconds or less. Preferably, the lyophilized preparations of the present invention are readily reconstituted to form clear, colorless solutions that are free of particulate matter, suitable for injection into humans in about 180 seconds or less. In certain preferred embodiments, the lyophilized preparations are reconstituted in about 120 seconds or less to form clear, colorless solutions that are free of particulate matter, suitable for injection into humans. Preferably, the lyophilized preparations are reconstituted in about 90 seconds or less to form clear, colorless solutions that are free of particulate matter, suitable for injection into humans. More preferably, the lyophilized preparations are reconstituted in about 60 seconds or less to form clear, colorless solutions that are free of particulate matter, suitable for injection into humans. More preferably, the lyophilized preparations are reconstituted in about 30 seconds or less to form clear, colorless solutions that are free of particulate matter, suitable for injection into humans. More preferably, the lyophilized preparations are reconstituted in about 10 seconds or less to form clear, colorless solutions that are free of particulate matter, suitable for injection into humans.

Lyophilized preparations of the invention may be prepared using any of the techniques known to those skilled in the art.

In those embodiments of the invention comprising bendamustine or a

pharmaceutically acceptable salt thereof, a cyclodextrin, and a bulking agent, one preferred method includes preparing a first solution containing the cyclodextrin, the bulking agent, and water. Preferred first solutions include hydroxypropyl- -cyclodextrin, hydroxypropyl-y-cyclodextrin, sulfobutyl ether β-cyclodextrin, or a mixture thereof, as the cyclodextrin and mannitol as the bulking agent. A compound solution containing bendamustine or a pharmaceutically acceptable salt thereof, and an organic solvent, preferably tert-butanol, is prepared. The first solution and the compound solution are combined to provide a pre-lyophilization solution. In some embodiments, the pre- lyophilization solution is sterilized using techniques known in the art, for example, sterile filtration. The pre-lyophilization solution is then lyophilized using lyophilization techniques known in the art.

It is envisioned that in some embodiments of the present invention, the described lyophilized preparations can be administered, either serially or simultaneously, with another antineoplastic agent. There are large numbers of antineoplastic agents available in commercial use, in clinical evaluation and in pre-clinical development, which could be selected for treatment of neoplasia by combination drug chemotherapy. Such

antineoplastic agents fall into several major categories, namely, antibiotic-type agents, covalent DNA-binding drugs, antimetabolite agents, hormonal agents, including glucocorticoids such as prednisone and dexamethasone, immunological agents, interferon- type agents, differentiating agents such as the retinoids, pro-apoptotic agents, and a category of miscellaneous agents, including compounds such as antisense, small interfering R A, and the like. Alternatively, other anti-neoplastic agents, such as metallomatrix proteases (MMP) inhibitors, SOD mimics or alpha_vbeta3 inhibitors may be used.

The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

EXAMPLES Reconstitution times, and stability data were collected for the following, lyophilized preparations. Preparation and lyophilization may be carried out as follows:

1.50g of bendamustine hydrochloride is weighed into a glass vessel, then 23.4g of t-butanol is weighed into the vessel, and the mixture stirred until the material dissolves. 2.55 g of mannitol and the appropriate amount of the cyclodextrin are weighed into a separate glass vessel. In the case of Preparation A, below, 4.90 g of hydroxypropyl-β- cyclodextrin is used. In the case of Preparation B, below, 4.90g of sulfobutyl ether β- cyclodextrin is used. In the case of Preparation C, below, 4.90g of hydroxypropyl-γ- cyclodextrin is used, and in the case of Preparation D, below, 2.45g of hydroxypropyl-γ- cyclodextrin is used. 67.65g of sterile water for injection (SWFI) is then weighed into the vessel containing the cyclodextrin. The mixture is stirred until all solids dissolve. The t- butanol solution is added slowly to the aqueous solution with stirring. This resulting bulk solution is filled into 20 mL glass vials in 6.67 mL aliquots. A lyophilization stopper is placed in lyophilization position on each vial, and loaded in a lyophilizer at 5°C. The solution is held at 5°C for 2 hours, ramped to -40°C at 30°C/hour, held at -40°C for 8 hours, ramped to -13°C at 30°C/hour, held at -13°C for 3 hours, then ramped to -40°C at 30°C/hour, then held at -40°C for 8 hours, all at atmospheric pressure. The vials are then placed under vacuum at 80 μιη Hg. The temperature is ramped up to 25°C at 30°C/hour, and held at 80 μιη Hg for 18 hours. Finally, the temperature is ramped up to 30°C, and held at 80 μιη Hg for 20 hours. After the lyophilization cycle is complete, the vials are mechanically stoppered under a slight vacuum of 8.6 psia and removed from the chamber. An aluminum crimp cap is then applied to each vial. In the following examples, the hydroxypropyl-P-cyclodextrin (HPpCD) used was Kleptose HPB, made by Roquette; the hydroxypropyl-y-cyclodextrin (HPyCD) used was Cavasol W8, made by ISP; and the sulfobutyl ether β-cyclodextrin (SBEpCD) used was Captisol, made by Cydex.

The bulk solutions described above were prepared such that the 6.67 ml aliquots that went into each vial as described above contained 100 mg of bendamustine hydrochloride. After lyophilization the composition of the dry lyophilate for Preparations A, B, C and D are as follows. The "Control" contained only bendamustine hydrochloride and mannitol.

Preparation A

Bendamustine hydrochloride 100.0 mg

Mannitol 170.0 mg

HPPCD 326.8 mg

Preparation B

Bendamustine hydrochloride 100.0 mg

Mannitol 170.0 mg

SBEPCD 326.8 mg

Preparation C

Bendamustine hydrochloride 100.0 mg

Mannitol 170.0 mg

HPyCD 326.8 mg

Preparation D

Bendamustine hydrochloride 100.0 mg

Mannitol 170.0 mg

HPyCD 433.4 mg

Control

Bendamustine hydrochloride 100.0 mg

Mannitol 170.0 mg Reconstitution Times

In each case, 20 ml SWFI, or normal saline is added, and the mixture shaken vigorously. Table I shows the reconstitution times for the preparations described above. The time given is that to form a clear solution.

Table I

SWFI Normal Saline

Control: 3 to 5 minutes Insoluble

Preparation A 15 to 25 seconds

Preparation B 30 to 35 seconds 40 to 55 seconds

Preparation C 10 seconds 45 seconds

Preparation D 2.5 minutes 5 minutes

Stability Data

Assay of the control material, and of the cyclodextrin preparations, in SWFI and in normal saline at various times, were determined by HPLC, under the following conditions:

Column: Zorbax Bonus-RP, 3.5 μιη, 150 x 4.6 mm (length x ID)

Column Temp.: 30°C

Tray Temp.: 5°C

Detector: UV, 254 nm

Inject: 5 |iL

Flow Rate: 1.5 mL/min.

Mobile phases: A. 0.1% trifluoroacetic acid in water

B. 0.1% trifluoroacetic acid in acetonitrile

Gradient: Time (min.) %A %B

0.0 93 7

3.3 93 7

8.7 73 27

10.7 73 27

16.0 43 57

16.1 10 90

18.0 10 90

18.1 93 7

20.0 93 7 In each case, 20 ml of SWFI, or normal saline, as indicated, was added to each of the preparations, and assays performed at the times, and under the conditions indicated. Concentration and purity of bendamustine, and in some cases amounts and percentages of a number of impurities were determined.

In the tables that follow, "BMl" is bendamustine. Listed impurities/degradation products are as follows:

BMl Dimer Table II shows an average of the assay of three batches of Control preparations at times from 0 (initial) to 5 hours following reconstitution in SWFI. Average BMl (bendamustine) assays, in mg/vial; average % purity of BMl : average % of the HPl, HP2 and BMl Dimer; and the concentration of bendamustine at the given time divided by initial concentration of bendamustine (C/CO) are shown. The data of Table II were collected at 25°C, at 60% relative humidity, in the presence of light.

Table II

Table III shows an average of the assay of three batches of Preparation A, at times from 0 to 24 hours following reconstitution in SWFI. Time in hours, percent purity of bendamustine, and the concentration of bendamustine at the given time divided by the initial concentration of bendamustine (C/CO) are shown. The data of Table III were collected at 25°C in room light.

Table III

Time %Purity BMl C/CO

0 99.37 1.00

1 hr 98.25 0.992 5 hr 93.95 0.942 24 hr 78.53 0.777

Table IV shows assays for a batch Preparation B, at times from 0 to 25 hours following reconstitution in SWFI, at room temperature, in room light. Abbreviations are as given above. Table IV

Sample % HP2 HP1 BM1 c/c,

Purity Dimer (%purity)

Initial 99.4 nd 0.3 0.1 100.0

1 hour 98.5 nd 1.2 0.1 99.0

1.5 hour 98.3 nd 1.3 0.1 98.9

2 hour 98.3 nd 1.4 0.1 98.9

5 hour 95.0 0.1 4.4 0.3 95.5

6 hour 94.6 0.1 4.7 0.3 95.2

6.5 hour 94.1 0.2 5.2 0.3 94.7

25 hour 82.4 2.0 14.7 0.5 82.9

nd= not detected

Table V shows an average of the assay of two batches of Preparation B, at times from 0 to 24 hours, following reconstitution in normal saline. Time in hours, percent purity of bendamustine, percents of certain impurities, and the concentration of bendamustine at the given time divided by the initial concentration of bendamustine (C/C0) are shown. The data of Table V were collected at 25°C in room light.

Table V

Sample % HP2 HP1 BM1 c/c,

Purity Dimer (%purity)

Initial 99.5 nd 0.2 0.1 100.0

1 hour 99.0 nd 0.7 0.1 99.5

5 hour 97.5 <0.05 2.2 0.1 98.0

24 hour 91.4 0.4 7.7 0.3 91.9

Table VI shows an average of the assay of two batches of Preparation C, at times from 0 to 24 hours, following reconstitution in SWFI, with abbreviations as given above. The data of Table VI were collected at room temperature, in room light. Table VI

Time % Purity BM1 C/CO

0 99.45 1.00

2 hours 96.74 0.976

5 hours 93.05 0.932

24 hours 75.28 0.741

Table VII shows an average of the assay of two batches of Preparation C, at times from 0 to 24 hours, following reconstitution in normal saline, with abbreviations as given above. The data of Table VII were collected at room temperature, in room light.

Table VII

Time % Purity BM1 C/C0

0 99.56 1.00

2 hours 98.56 0.990

5 hours 97.18 0.979

24 hours 89.36 0.892

Table VIII shows an average of the assay of two batches of Preparation D, at times from 0 to 24 hours, following reconstitution in SWFI, with abbreviations as given above. The data of Table VIII were collected at room temperature, in room light. Table VIII

Time % Purity BM1 C/CO

0 99.40 1.00

2 hours 96.11 0.977

5 hours 91.92 0.924

24 hours 71.26 0.702

Table IX shows an average of the assay of two batches of Preparation D, at times from 0 to 24 hours, following reconstitution in normal saline, with abbreviations as given above. The data of Table IX were collected at room temperature, in room light.

Table IX

Time % Purity BM1 C/C0

0 99.44 1.00

2 hours 98.14 0.987 5 hours 96.42 0.969

24 hours 87.15 0.872

Thus, it can be seen that the present invention provides preparations that are more easily, and more quickly reconstituted than previous formulations. Further, solutions of the preparations of the present invention, following reconstitution, show greater stability with regard to bendamustine.

Accordingly, a first aspect of the present invention provides a lyophilized preparation comprising bendamustine, or a pharmaceutically acceptable salt thereof, and a cyclodextrin. A second aspect provides a lyophilized preparation according to the first aspect, wherein the cyclodextrin is methyl-P-cyclodextrin, dimethyl-P-cyclodextrin, trimethyl-β- cyclodextrin, 2-hydroxymethyl-P-cyclodextrin, hydroxyethyl-P-cyclodextrin,

hydroxypropyl-P-cyclodextrin, hydoxypropyl-y-cyclodextrin, β-cyclodextrin sulfate, β- cyclodextrin sulfonate, sulfobutyl ether β-cyclodextrin, or a mixture thereof.

A third aspect provides a lyophilized preparation according to the second aspect wherein the cyclodextrin is hydroxypropyl-P-cyclodextrin, hydroxypropyl-y-cyclodextrin, or sulfobutyl ether β-cyclodextrin.

A fourth aspect provides a lyophilized preparation according any of the first, second or third aspects, further comprising a bulking agent.

A fifth aspect provides a lyophilized preparation according to the fourth aspect, wherein the bulking agent is a monosaccharide, an oligosaccharide, a sugar alcohol, an amino acid, or a mixture thereof.

A sixth aspect provides a lyophilized preparation according to the fifth aspect, wherein the bulking agent is mannitol.

A seventh aspect of the present invention provides a lyophilized preparation according to the third aspect, further comprising a bulking agent, wherein the bulking agent is mannitol.

An eighth aspect provides a lyophilized preparation according to any of the first, second or third aspects, wherein the lyophilized preparation reconstitutes in about 180 seconds or less to provide a clear solution free of particulate matter.

A ninth aspect provides a lyophilized preparation according to any of the first, second or third aspects, wherein the lyophilized preparation reconstitutes in about 90 seconds or less to provide a clear solution free of particulate matter.

A tenth aspect provides a lyophilized preparation according to any of the first, second or third aspects, wherein the reconstituted solution is colorless. An eleventh aspect of the present invention provides a lyophilized preparation according to any of the fourth, fifth, sixth or seventh aspects, wherein the lyophilized preparation reconstitutes in about 180 seconds or less to provide a clear solution free of particulate matter.

A twelfth aspect provides a lyophilized preparation according to any of the fourth, fifth, sixth or seventh aspects, wherein the lyophilized preparation reconstitutes in about 90 seconds or less to provide a clear solution free of particulate matter.

A thirteenth aspect provides a lyophilized preparation according to any of the fourth, fifth, sixth or seventh aspects, wherein the reconstituted solution is colorless.

A fourteenth aspect provides a method for manufacturing a lyophilized preparation according to any of the first, second or thirds aspects, comprising:

providing a first solution comprising a cyclodextrin and water; providing a second solution comprising bendamustine, or a

pharmaceutically acceptable salt thereof, and t-butanol;

combining the first and the second solution to form a pre-lyophilization solution; and

lyophilizing the pre-lyophilization solution.

A fifteenth aspect provides a method for manufacturing a lyophilized preparation according to any of the fourth, fifth, sixth and seventh aspects, comprising:

providing a first solution comprising a cyclodextrin, a bulking agent, and water;

providing a second solution comprising bendamustine, or a

pharmaceutically acceptable salt thereof, and t-butanol;

lyophilizing the pre-lyophilization solution.

A sixteenth aspect provides a method according to the fourteenth aspect comprising: providing a first solution comprising a cyclodextrin, and water;

providing a second solution comprising bendamustine hydrochloride, and t- butanol;

lyophilizing the pre-lyophilization solution.

A seventeenth aspect provides a method according to the fifteenth aspect comprising:

lyophilizing the pre-lyophilization solution.

An eighteenth aspect provides a method according to the seventeenth aspect, wherein the bulking agent is mannitol.

A nineteenth aspect provides a method according to the sixteenth aspect, wherein the cyclodextrin is hydroxypropyl-P-cyclodextrin, hydoxypropyl-y-cyclodextrin, or sulfobutyl ether β-cyclodextrin.

A twentieth aspect provides a method according to the seventeenth aspect wherein the cyclodextrin is hydroxypropyl-P-cyclodextrin, hydoxypropyl-y-cyclodextrin, or sulfobutyl ether β-cyclodextrin.

A twenty-first aspect provides a method according to the twentieth aspect, wherein the bulking agent is mannitol.

A twenty-second aspect of the present invention provides a method of treating chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma or breast cancer comprising administering to a patient in need thereof a therapeutically effective amount of a reconstitution solution prepared from the lyophilized preparation according to any of the first, second or third aspects.

A twenty-third aspect provides a method of treating chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma or breast cancer comprising administering to a patient in need thereof a therapeutically effective amount of a reconstitution solution prepared from the lyophilized preparation according to any of the fourth, fifth, sixth or seventh aspects.

A twenty-fourth aspect provides a method according to the twenty-second aspect, further comprising administering to said patient, either serially or simultaneously, another antineoplastic agent.

A twenty-fifth aspect provides a method according to the twenty-third aspect, further comprising administering to said patient, either serially or simultaneously, another antineoplastic agent.

Claims

What is Claimed is:

1. A lyophilized preparation comprising bendamustine, or a pharmaceutically acceptable salt thereof, and a cyclodextrin.

2. The lyophilized preparation of claim 1, wherein the cyclodextrin is methyl-β- cyclodextrin, dimethyl-P-cyclodextrin, trimethyl-P-cyclodextrin, 2-hydroxymethyl-P- cyclodextrin, hydroxyethyl-P-cyclodextrin, hydroxypropyl-P-cyclodextrin,

hydoxypropyl-y-cyclodextrin, β-cyclodextrin sulfate, β-cyclodextrin sulfonate, sulfobutyl ether β-cyclodextrin, or a mixture thereof.

3. The lyophilized preparation of claim 2 wherein the cyclodextrin is hydroxypropyl- β-cyclodextrin, hydroxypropyl-y-cyclodextrin, or sulfobutyl ether β-cyclodextrin.

4. A lyophilized preparation according to claim 1, further comprising a bulking agent.

5. The lyophilized preparation of claim 4, wherein the bulking agent is a

monosaccharide, an oligosaccharide, a sugar alcohol, an amino acid, or a mixture thereof.

6. The lyophilized preparation of claim 5, wherein the bulking agent is mannitol.

7. The lyophilized preparation of claim 3, further comprising a bulking agent, wherein the bulking agent is mannitol.

8. The lyophilized preparation of claim 1, wherein the lyophilized preparation reconstitutes in about 180 seconds or less to provide a clear solution free of particulate matter.

9. The lyophilized preparation of claim 1, wherein the lyophilized preparation reconstitutes in about 90 seconds or less to provide a clear solution free of particulate matter.

10. The lyophilized preparation of claim 1, wherein the reconstituted solution is colorless.

11. The lyophilized preparation of claim 4, wherein the lyophilized preparation reconstitutes in about 180 seconds or less to provide a clear solution free of particulate matter.

12. The lyophilized preparation of claim 4, wherein the lyophilized preparation reconstitutes in about 90 seconds or less to provide a clear solution free of particulate matter.

13. The lyophilized preparation of claim 4, wherein the reconstituted solution is colorless.

14. A method for manufacturing a lyophilized preparation according to claim 1 comprising:

pharmaceutically acceptable salt thereof, and t-butanol;

lyophilizing the pre-lyophilization solution.

15. A method for manufacturing a lyophilized preparation according to claim 4 comprising:

providing a second solution comprising bendamustine, or a

pharmaceutically acceptable salt thereof, and t-butanol;

lyophilizing the pre-lyophilization solution.

16. A method according to claim 14 comprising:

providing a first solution comprising a cyclodextrin, and water; providing a second solution comprising bendamustine hydrochloride, and t- butanol;

lyophilizing the pre-lyophilization solution.

17. A method according to claim 15 comprising:

lyophilizing the pre-lyophilization solution.

18. A method according to claim 17, wherein the bulking agent is mannitol.

19. A method according to claim 16, wherein the cyclodextrin is hydroxypropyl-β- cyclodextrin, hydoxypropyl-y-cyclodextrin, or sulfobutyl ether β-cyclodextrin.

20. A method according to claim 17 wherein the cyclodextrin is hydroxypropyl-β- cyclodextrin, hydoxypropyl-y-cyclodextrin, or sulfobutyl ether β-cyclodextrin.

21. A method according to claim 20, wherein the bulking agent is mannitol.

22. A method of treating chronic lymphocytic leukemia, Hodgkin's disease, non- Hodgkin's lymphoma, multiple myeloma or breast cancer comprising administering to a patient in need thereof a therapeutically effective amount of a reconstitution solution prepared from the lyophilized preparation according to claim 1.

23. A method of treating chronic lymphocytic leukemia, Hodgkin's disease, non-

Hodgkin's lymphoma, multiple myeloma or breast cancer comprising administering to a patient in need thereof a therapeutically effective amount of a reconstitution solution prepared from the lyophilized preparation according to claim 4.

24. A method according to claim 22, further comprising administering to said patient, either serially or simultaneously, another antineoplastic agent.

25. A method according to claim 23, further comprising administering to said patient, either serially or simultaneously, another antineoplastic agent.