Patent ID: 12246007

DETAILED DESCRIPTION

Bendamustine

The concentrated liquid pharmaceutical compositions of the invention described herein comprise bendamustine or a pharmaceutically acceptable bendamustine salt. Thus, concentrated liquid pharmaceutical compositions of the present invention can comprise bendamustine free base or any pharmaceutically acceptable bendamustine salt, for example bendamustine hydrochloride. Amorphous or hydrated forms of bendamustine or bendamustine pharmaceutically acceptable salt, for example anhydrous bendamustine hydrochloride or bendamustine hydrochloride monohydrate, may be used to make the compositions of the present invention. Other examples of pharmaceutically acceptable salts, besides the hydrochloride salt, that may be used in combination with bendamustine include, but are not limited to hydrobromide, citrate, formate, acetate, and tartrate.

The bendamustine or bendamustine salt is present in the concentrated liquid pharmaceutical compositions of the present invention in a pharmaceutically-effective amount. The treatment of a cancer in a patient, such as a human, may be realized over a dosing regimen requiring multiple effective doses of a composition of the present invention containing bendamustine or a bendamustine salt, separated at intervals, over a period of time. A composition of the present invention may for example contain an effective dose of bendamustine or bendamustine salt of from about 1 mg to about 500 mg, from about 10 mg to about 200 mg, from about 20 mg to about 150 mg, from about 20 mg to about 100 mg, or from about 25 mg to about 100 mg. Preferably a composition of the present invention contains an effective amount of bendamustine or bendamustine salt of about 25 mg, about 50 mg, about 100 mg, or about 200 mg. Preferably the bendamustine or bendamustine salt is bendamustine hydrochloride.

In different embodiments, the concentration of the bendamustine or bendamustine salt in a concentrated liquid pharmaceutical composition of the present invention is from about 1 mg/ml to about 100 mg/ml, from about 10 mg/ml to about 100 mg/ml, from about 20 mg/ml to about 60 mg/ml, from about 20 mg/ml to about 50 mg/ml, from about 25 mg/ml to about 60 mg/ml, and from about 25 mg/ml to about 50 mg/ml. Preferably the concentration of the bendamustine or bendamustine salt in a concentrated liquid pharmaceutical composition of the present invention is about 25 mg/ml, about 30 mg/ml, or about 50 mg/ml. A concentrated liquid pharmaceutical composition of the present invention may contain about 5 mg/ml, about 10 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, or about 60 mg/ml bendamustine or bendamustine salt.

The term “v/v” means “volume per volume” and is used herein to express the concentration of a substance in a solution on a volume per volume basis. By way of example, a solution containing 50% v/v non-aqueous solvents means that there are about 50 ml of combined organic compound solvents in every 100 ml of said solution. As another example, if a solution containing 13.1% v/v ethanol, then every 100 ml of said solution contains about 13.1 ml of ethanol. As further examples, one liter of a pharmaceutical composition of the present invention containing 2.5% v/v water contains 25 ml water, and one liter of a pharmaceutical composition of the present invention containing 3% water contains 30 ml water.

Non-Aqueous Solvent System

The concentrated liquid pharmaceutical compositions of the present invention comprise a non-aqueous solvent system (a “non-aqueous solvent”) to assist in the dissolution of the bendamustine or pharmaceutically acceptable bendamustine salt. A non-aqueous solvent system is a single organic compound solvent or combination of organic compound solvents. The non-aqueous solvent system may comprise one or more organic compounds selected from organic polymeric compound solvents and organic small molecule solvents. Organic compound solvents (solvents that are organic compounds) are well known in the art, for example numerous organic alcohols, including organic alcohols of low molecular weight (organic small molecule alcohols) and organic compounds of higher molecular weight containing hydroxy moieties (organic polymeric compounds with hydroxy moieties). In one embodiment, the non-aqueous solvent system comprises an organic polymeric compound solvent, examples including polysorbates (such as polysorbate 80 or polysorbate 20), polyethylene glycols (such as polyethylene glycol 400 or polyethylene glycol 300), and polyalkoxylated castor oils for example a polyethoxylated castor oil (such as CREMOPHOR). In another embodiment, the non-aqueous solvent system comprises an organic small molecule solvent, such as N,N-dimethylacetamide (DMA), dimethyl sulfoxide, 1-methyl-2-pyrrolidinone, or an organic small molecule alcohol (for example ethanol, isopropyl alcohol, benzyl alcohol, or propylene glycol). An organic small molecule solvent generally has a molecular weight of less than about 200 atomic mass units, whereas a polymeric compound generally has an average molecular weight of at least about 300 atomic mass units.

The non-aqueous solvent system in the concentrated liquid pharmaceutical composition of the present invention can comprise one, two, three, four, five, six, seven, eight, nine, ten or more different organic compound solvents.

In different embodiments, the total amount of the organic compound solvents in a concentrated liquid pharmaceutical composition of the present invention is any amount within from about 70% to about 98% v/v of the composition. In other embodiments, the total amount of the organic compound solvents in a composition of the present invention is from about 75% to about 98% v/v, from about 80% to about 98% v/v, from about 85% to about 98% v/v, from about 90% to about 98% v/v, or from about 95% to about 98% v/v. In other embodiments, the total amount of the organic compound solvents in a composition of the present invention is from about 70% to about 96.6% v/v, from about 75% to about 97.6% v/v, from about 80% to about 97.6% v/v, from about 85% to about 97.6% v/v, from about 90% to about 97.6% v/v, or from about 95% to about 97.6% v/v. In other embodiments, the total amount of the organic compound solvents in a composition of the present invention is from about 70% to about 97% v/v, from about 75% to about 97% v/v, from about 80% to about 97% v/v, from about 85% to about 97% v/v, from about 90% to about 97% v/v, or from about 95% to about 97% v/v. In other embodiments, the total amount of the organic compound solvents in a composition of the present invention is from about 70% to about 96.5% v/v, from about 75% to about 96.5% v/v, from about 80% to about 96.5% v/v, from about 85% to about 96.5% v/v, from about 90% to about 96.5% v/v, or from about 95% to about 96.5% v/v. In other embodiments, the total amount of the organic compound solvents in a composition of the present invention is from about 70% to about 96% v/v, from about 75% to about 96% v/v, from about 80% to about 96% v/v, from about 85% to about 96% v/v, from about 90% to about 96% v/v, or from about 95% to about 96% v/v. In other embodiments, the total amount of the organic compound solvents in a concentrated liquid pharmaceutical composition of the present invention is from about 70% to about 95% v/v, from about 75% to about 95% v/v, from about 80% to about 95% v/v, from about 85% to about 95% v/v, or from about 90% to about 95% v/v. In other embodiments, the total amount of the organic compound solvents in a concentrated liquid pharmaceutical composition of the present invention is from about 70% to about 94% v/v, from about 80% to about 94% v/v, from about 85% to about 94% v/v, or from about 90% to about 94% v/v.

Preferably, a concentrated liquid pharmaceutical composition of the present invention comprises one organic polymeric compound solvent and one organic small molecule solvent, and the amount of the organic polymeric compound solvent in the composition is greater (on a v/v basis) than the amount of the organic small molecule solvent. Without being bound by theory, the inventors understand that the organic polymeric compounds are generally less reactive with the bendamustine in the composition than are any single organic small molecule solvent in the composition, and have thus found it possible to include a greater amount of an organic polymeric compound solvent in the composition than an organic small molecule solvent without compromising the long-term stability of the bendamustine or bendamustine salt in the composition. On the other hand, the inclusion of an organic small molecule solvent aids in reducing the viscosity of the composition, thereby rendering a composition that is more suitable for injection.

In one embodiment, the non-aqueous solvent system in a concentrated liquid pharmaceutical composition of the invention comprises one or more organic polymeric compound solvents in a total amount of from about 68% to about 96% v/v in the composition and one or more organic small molecule solvents in a total amount of from about 2% to about 10% v/v in the composition, with a total amount of organic compound solvents in the composition being a maximum of about 98% v/v of the composition. In one embodiment, the non-aqueous solvent system in a concentrated liquid pharmaceutical composition of the present invention contains a polyethylene glycol and an organic small molecule solvent. In another embodiment, the non-aqueous solvent system in a concentrated liquid pharmaceutical composition of the present invention contains a polyethylene glycol and ethanol. In one embodiment, the non-aqueous solvent system in a concentrated liquid pharmaceutical composition of the present invention contains polyethylene glycol 400 and ethanol. In another embodiment, the non-aqueous solvent system in a concentrated liquid pharmaceutical composition of the present invention contains polyethylene glycol 300 and ethanol. In another embodiment, the non-aqueous solvent system in a concentrated liquid pharmaceutical composition of the present invention contains a mixture of polyethylene glycol 300 and polyethylene glycol 400 and ethanol.

Additional Components

The concentrated liquid pharmaceutical compositions of the present invention additionally comprise at least about 2% v/v water. In different embodiments, a concentrated liquid pharmaceutical composition of the present invention comprises at least about 2.1%, at least about 2.2%, at least about 2.3%, at least about 2.4%, at least about 2.5%, at least about 2.8%, at least about 3% v/v water, at least about 3.5% v/v water, at least about 4% v/v water, at least about 5% v/v water, or at least about 6% v/v water. Preferably, a concentrated liquid pharmaceutical composition of the present invention comprises from at least about 2% v/v water to about 30% v/v water, to about 25% v/v water, to about 20% v/v water, to about 15% v/v water, to about 10% v/v water, or to about 5% v/v water (i.e. from about 2% v/v water to about 5% v/v water). In different embodiments, a concentrated liquid pharmaceutical composition of the present invention comprises from about 2% to about 30%, about 2.1% to about 30%, about 2.2% to about 30%, about 2.3% to about 30%, about 2.4% to about 30%, about 2.5% to about 30%, about 2.8% to about 30%, about 3% to about 30%, about 3.5% to about 30%, about 4% to about 30%, about 5% to about 30% v/v water, or about 6% to about 30% v/v water.

In other embodiments, a concentrated liquid pharmaceutical composition of the present invention comprises from about 2% to about 25%, about 2.1% to about 25%, about 2.2% to about 25%, about 2.3% to about 25%, about 2.4% to about 25%, about 2.5% to about 25%, about 2.8% to about 25%, about 3% to about 25%, about 3.5% to about 25%, about 4% to about 25%, about 5% to about 25%, or about 6% to about 25% v/v water.

In other embodiments, a concentrated liquid pharmaceutical composition of the present invention comprises from about 2% to about 20%, about 2.1% to about 20%, about 2.2% to about 20%, about 2.3% to about 20%, about 2.4% to about 20%, about 2.5% to about 20%, about 2.8% to about 20%, about 3% to about 20%, about 3.5% to about 20%, about 4% to about 20%, about 5% to about 20%, or about 6% to about 20% v/v water.

In other embodiments, a concentrated liquid pharmaceutical composition of the present invention comprises from about 2% to about 15%, about 2.1% to about 15%, about 2.2% to about 15%, about 2.3% to about 15%, about 2.4% to about 15%, about 2.5% to about 15%, about 2.8% to about 15%, about 3% to about 15%, about 3.5% to about 15%, about 4% to about 15%, about 5% to about 15%, or about 6% to about 15% v/v water.

In other embodiments, a concentrated liquid pharmaceutical composition of the present invention comprises from about 2% to about 10%, about 2.1% to about 10%, about 2.2% to about 10%, about 2.3% to about 10%, about 2.4% to about 10%, about 2.5% to about 10%, about 2.8% to about 10%, about 3% to about 10%, about 3.5% to about 10%, about 4% to about 10%, about 5% to about 10%, or about 6% to about 10% v/v water.

In other embodiments, a concentrated liquid pharmaceutical composition of the present invention comprises from about 2% to about 5%, about 2.1% to about 5%, about 2.2% to about 5%, about 2.3% to about 5%, about 2.4% to about 5%, about 2.5% to about 5%, about 2.8% to about 5%, about 3% to about 5%, about 3.5% to about 5%, or about 4% to about 5% v/v water.

In other embodiments, a concentrated liquid pharmaceutical composition of the present invention comprises from about 2% to about 4%, about 2.1% to about 4%, about 2.2% to about 4%, about 2.3% to about 4%, about 2.4% to about 4%, about 2.5% to about 4%, about 2.8% to about 4%, about 2.8% to about 3.5%, about 3% to about 4%, about 2% to about 3.5%, about 2.4% to about 3.5%, about 2.8% to about 3.5%, or about 3% to about 3.5% v/v water.

A concentrated liquid pharmaceutical composition of the present invention may optionally contain additional ingredients known in the art to be used in connection with injectable pharmaceutical compositions.

For example, a concentrated liquid pharmaceutical composition of the present invention may optionally contain one or more antioxidants. Antioxidants known in the art may be used in the compositions of the invention, for example sodium bisulfite, sodium sulfite, sodium metabisulfite, ascorbic acid, sodium EDTA, monothioglycerol, L-cysteine, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, glutathione, gentisic acid, lipoic acid, ascorbityl palmitate, propyl gallate, nordihydroguaiaretic acid, or a combination thereof. In one embodiment of the invention, the antioxidant is a chain terminator, i.e. a chemical substance capable of donating a hydrogen radical, thereby possessing the ability to terminate free radical chain reactions occurring in other compounds due to oxidative stress. In one embodiment of the invention, the antioxidant is a sulfhydryl compound, for example an antioxidant comprising an —SH moiety such as monothioglycerol, L-cysteine, thioglycolic acid or thiosorbitol. Preferably, an antioxidant used in a composition of the present invention is a phenol antioxidant, i.e. an antioxidant comprising one or more phenolic hydroxy groups for example butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate or nordihydroguaiaretic acid.

When calculated on a v/v basis, the amounts of bendamustine or bendamustine salt and optional additional ingredients (e.g. pH adjustor or antioxidant) in a concentrated liquid pharmaceutical composition of the present invention are negligible on a v/v basis. Thus, the organic solvents and the water comprise the detectable volume of a composition of the present invention when measured by standard volumetric glassware methods. About 100% of the volume of a concentrated liquid pharmaceutical composition of the present invention is the organic solvents and the water. A small, negligible amount of volume is taken up by the 1) bendamustine or bendamustine salt, and 2) pH adjuster and antioxidant (to the extent either is included in the composition of the invention).

pH

In one embodiment, the pH of a concentrated liquid pharmaceutical composition of the present invention is between about 3.30 to about 3.70, when the pH of such composition is tested in a water solution at about 12.4% v/v. In another embodiment, the pH of a concentrated liquid pharmaceutical composition of the present invention is between about 3.40 to about 3.60, when the pH of such composition is tested in a water solution at about 12.4% v/v. In different embodiments, the pH of a concentrated liquid pharmaceutical composition of the present invention is from about 3.30, about 3.37, or about 3.45 to about 3.70, when the pH of such composition is tested in a water solution at about 12.4% v/v. In different embodiments, the pH of a concentrated liquid pharmaceutical composition of the present invention is from about 3.30, about 3.37, or about 3.45 to about 3.66, when the pH of such composition is tested in a water solution at about 12.4% v/v. In different embodiments, the pH of a concentrated liquid pharmaceutical composition of the present invention is from about 3.30, about 3.37, or about 3.45 to about 3.57, when the pH of such composition is tested in a water solution at about 12.4% v/v.

Methods for adjusting pH of a pharmaceutical composition are well known in the art, and any such method can be used to adjust the pH of a composition of the present invention to achieve a preferred pH as described herein. If necessary, for example, to achieve a preferred pH as described herein, a strong base may be added to a composition of the present invention. Examples of strong bases that can be used in pharmaceutical compositions of the present invention to adjust pH are well known in the art and include, but are not limited to, NaOH and KOH.

For example, the strong base may be increasingly titrated into a formulation until a desired pH is obtained. After adding a small amount of strong base, an aliquot of the concentrated liquid pharmaceutical composition may be taken and combined with water to about 12.4% v/v composition/water. The pH of the resulting solution may be tested with a calibrated pH meter. Thus, the pH of the concentrated liquid pharmaceutical composition may be determined. If necessary, the process is repeated until the desired pH for the concentrated liquid pharmaceutical composition is obtained.

Stable Presentations and Dosage Forms

Compositions of the present invention can be provided in unit presentations. Each unit presentation can contain a single dose or multiple-doses of a composition of the present invention. For example a unit containing a composition of the present invention may contain one, two, three, four, five, six, seven, eight, nine, ten or more doses. The units may be provided in any suitable type of sealed container known to those in the art. For example the units may be packaged and provided in vials, syringes, sealed bottles, or sealed bags made of pharmaceutically acceptable material, such as glass or pharmaceutically acceptable plastic.

In different embodiments, the present invention provides a sealed container containing a concentrated liquid pharmaceutical composition of the present invention containing 25 mg bendamustine HCl, 50 mg bendamustine HCl, 100 mg bendamustine HCl, 150 mg bendamustine HCl, 200 mg bendamustine HCl, 250 mg bendamustine HCl, 300 mg bendamustine HCl, 500 mg bendamustine HCl, or 1000 mg bendamustine HCl.

The sealed units containing the concentrated liquid pharmaceutical compositions of the present invention may be suitable for long term storage, prior to administration. The sealed units containing the concentrated liquid pharmaceutical compositions of the present invention may be suitable for long term storage under standard refrigeration temperatures. A standard refrigeration temperature is from 2° C. to 8° C. For example, sealed units containing the concentrated liquid pharmaceutical compositions of the present invention may be suitable for storage under standard refrigeration conditions for up to about one week, up to about three weeks, up to about 1 month, up to about six weeks, up to about 3 months, up to about 6 months, up to about 12 months, up to about 18 months, up to about 24 months, or up to about 36 months. “Suitable for storage” means that a concentrated liquid pharmaceutical composition of the present invention is stable overtime, it remains liquid, does not significantly degrade and does not significantly lose its potency, over time, under standard refrigeration conditions.

Scheme 1 illustrates different bendamustine degradation reactions that may occur in various environments. Upon reaction with water over time, bendamustine hydrolysis products BM HP1 and BM HP2 may occur, with BM HP1 predominating over BM HP2. As depicted in Scheme 1, increasing pH, i.e. a relatively more basic environment, favors bendamustine hydrolysis degradation. Bendamustine reaction with alcohols at relatively higher pH's (the “Base” direction in Scheme 1) is believed to result in bendamustine alkyl ether degradants. For example, reaction at a higher pH with ethanol and polyethylene glycol can result in, respectively, bendamustine ethyl ether (“BM Ethyl Ether”) and bendamustine polyethylene glycol ethers (“BM PEG Ether”). As pH decreases (the “Acid” direction in Scheme 1), dimerisation may occur, resulting in bendamustine trichloro dimer over time (“BM Trichloro Dimer”). Additionally, at lower pH (“Acid” direction in Scheme 1), reaction with alcohols forms bendamustine alkyl ester degradants, for example bendamustine ethyl ester (“BM EE”) from bendamustine reaction with ethanol and bendamustine polyethylene glycol esters (“BM PEG Esters”) from bendamustine reaction with polyethylene glycol. An additional bendamustine degradant is bendamustine de-chloroethyl (“BM DOE”), which the present inventors hypothesize (without being bound by such theory) is formed by a radical mechanism. Such bendamustine degradants are alternatively referred to herein as “bendamustine related substances”, “related substances” or “impurities”.

The sealed containers and concentrated liquid pharmaceutical compositions therein may be sterilized by methods known in the art, for example by aseptic filtration, terminal sterilization (autoclaving or irradiation), or any combination thereof, thus obtaining sealed containers of sterile concentrated liquid pharmaceutical compositions of the present invention.

Methods of Treatment

The present invention provides a method of treating a cancer in a mammal, comprising administering an effective amount of a concentrated liquid pharmaceutical composition as described herein to the mammal. In different embodiments of the method of treating cancer in a mammal of the present invention, the cancer is a leukemia or a lymphoma. In other aspects of the invented method for treating cancer in a mammal, the cancer is chronic lymphocytic leukemia or non-Hodgkin lymphoma.

The therapeutic methods of the invention comprise diluting an effective amount of a concentrated liquid pharmaceutical composition of the present invention with a pharmaceutically acceptable diluent, and administering the resulting diluted pharmaceutical composition containing the effective amount of the concentrated liquid pharmaceutical composition to a mammal in need thereof by injection. The effective amount of a concentrated liquid pharmaceutical composition of the present invention for treating cancer, for example chronic lymphocytic leukemia (CLL) or non-Hodgkin Lymphoma (NHL), can be determined by a person of ordinary skill in the art, for example by reference to approved methods for treating CLL or NHL using already approved drugs containing bendamustine or a bendamustine salt, such as the drugs BENDEKA™ and TREANDA®. The effective amount can be adjusted by a person of ordinary skill in the art based on the condition and size (body surface area in, for example, square meters) of the patient being treated.

The concentrated liquid pharmaceutical compositions of the present invention are designed for administration, after dilution, to a mammal by injection. The diluted compositions may be administered to a patient by intravenous (IV) infusion. For administration by IV infusion, a pharmaceutically effective volume of concentrated liquid pharmaceutical composition of the invention may be aseptically withdrawn from the container containing the composition and transferred to, or may be injected aseptically into (from the container (e.g. syringe) containing the composition), an infusion bag containing a suitable pharmaceutically acceptable diluent, for example 0.9% sodium chloride, 2.5% dextrose/0.45% sodium chloride, or 5% dextrose. The concentration of bendamustine or bendamustine salt in a diluted formulation just prior to administration to the patient is preferably within a range of about 0.1 mg/mL to about 10 mg/mL. In different embodiments of the present invention, the concentration of bendamustine or bendamustine salt in a diluted formulation just prior to administration to a patient is from about 1 mg/mL or about 1.5 mg/mL to about 7 mg/mL or about 8 mg/mL, for example from about 1.85 mg/mL to about 5.6 mg/mL. In other embodiments of the present invention, the concentration of bendamustine salt in a diluted formulation just prior to administration to a patient is within a range of about 0.1 mg/mL to about 1.85 mg/mL, for example from about 0.2 mg/mL about 0.6 mg/mL or 0.7 mg/mL. As described herein, a concentrated liquid pharmaceutical composition of the present invention may be diluted in a diluent suitable for injection (for example a pharmaceutically acceptable diluent contained in an infusion bag) just prior to administration to a mammal in need thereof to achieve such bendamustine (or bendamustine salt) concentration of from about 0.1 mg/mL to about 10 mg/mL.

Methods of Preparation

The present invention also provides methods of preparing concentrated liquid pharmaceutical compositions as described herein. In one embodiment, the invention provides a method for preparing a concentrated liquid pharmaceutical composition, which method comprises combining: a) bendamustine or a pharmaceutically acceptable salt thereof, b) one or more organic compounds selected from organic polymeric compound solvents and organic small molecule solvents, c) at least about 2% water v/v (water/pharmaceutical composition), and d) optionally, one or more antioxidants. Preferably, ingredient (c) is from about 2% to a maximum of about 10% water v/v (water/pharmaceutical composition). In a further embodiment of this method, after combining the ingredients (a)-(c), and optionally (d): i) the pH of an aqueous solution containing about 12.4% volume of said concentrated liquid pharmaceutical composition is measured; ii) if the pH of (i) is not from about 3.4 to about 3.6, a pH adjuster is added to the concentrated liquid pharmaceutical composition; and iii) steps (i) and (ii) are repeated, if necessary, until the pH of an aqueous solution containing about 12.4% of the concentrated liquid pharmaceutical composition v/v is from about 3.4 to about 3.6.

In another embodiment, the invention provides a method for preparing a concentrated liquid pharmaceutical composition, which method comprises 1) combining a) bendamustine or a pharmaceutically acceptable salt thereof, b) optionally, one or more antioxidants, c) water, d) one or more organic small molecule solvents, and e) one or more organic polymeric compound solvents; 2) agitating the combination resulting from step (1) of the method until the bendamustine or pharmaceutically acceptable salt thereof and the antioxidant or antioxidants, if present, are dissolved; and 3) adding a further amount of the organic polymeric compound solvent or organic polymeric compound solvents sufficient to obtain a concentrated liquid pharmaceutical composition comprising at least about 2% water v/v (water/pharmaceutical composition). Preferably, the water and organic solvents in steps (1) and (2) of the method are combined in amounts sufficient to obtain a concentrated liquid pharmaceutical composition comprising from about 2% to a maximum of about 10% water v/v (water/pharmaceutical composition). In a further aspect of this method, the method further comprises, after step (3), (i) measuring the pH of an aqueous solution containing about 12.4% volume of the concentrated liquid pharmaceutical composition v/v; (ii) if the pH of (i) is not from about 3.4 to about 3.6, adding a pH adjuster to the concentrated liquid pharmaceutical composition; and (iii) repeating steps (i) and (ii), if necessary, until the pH of an aqueous solution containing about 12.4% of the concentrated liquid pharmaceutical composition v/v is from about 3.4 to about 3.6.

The steps of the methods of preparation described herein are carried out under pharmaceutically acceptable conditions for sterility and manufacturing.

The bendamustine or pharmaceutically acceptable bendamustine salt used in the aforementioned methods of preparation of the invention may be a pharmaceutically acceptable amount of bendamustine or pharmaceutically acceptable bendamustine salt. However, concentrated liquid pharmaceutical compositions of the present invention can be prepared in batch quantities, if desired, using the aforementioned methods of the invention.

Preferably, the amount of the organic polymeric compound solvents in the methods of preparation described herein is greater than the amount of the organic small molecule solvents used, when the amounts of such solvents are calculated on a v/v basis relative to the resulting concentrated liquid pharmaceutical composition. The organic small molecule solvents and the organic polymeric compound solvents that can be used in the methods of preparation of the invention are as described herein. Preferably, the organic polymeric compound solvent is a polyethylene glycol, such as PEG 300 or PEG 400. Preferably, the organic compound small molecule solvent is a small molecule alcohol, for example ethanol, propylene glycol, benzyl alcohol or isopropyl alcohol).

Any pH adjuster known in the art can be used in the methods of preparation described herein. The pH adjuster may, for example, be a strong base such as NaOH or KOH. The bendamustine, bendamustine salt, optional antioxidant or antioxidants and optional pH adjuster may be dissolved into the concentrated liquid pharmaceutical compositions in the methods described herein by agitation during the methods, and such techniques (e.g. mixing) are known to those of ordinary skill in the art.

As described hereinabove, the concentrated liquid bendamustine compositions so prepared can be packaged in sterile, sealed containers, such as vials, syringes, bottles or bags, thereby providing pharmaceutically acceptable unit presentations of concentrated liquid pharmaceutical compositions of the present invention suitable for storage and transport.

EXAMPLES

The following examples are provided to illustrate certain aspects of the present invention. These Examples should not be considered as limiting the scope of the invention, as more fully described herein and set forth in the appended claims.

Example 1: Preparation of 25 m/mL Bendamustine HCl Liquid Injection Formulations

One mL units of 25 mg/mL bendamustine HCl formulations according to the present invention were made consisting of the materials outlined in the following Table 1:

TABLE 1Bulk Formulation of Bendamustine Formulation B TypeMaterialQuantityBendamustine HCl25 mg/mLButylated hydroxyanisole1 mg/mLEthanolSee Table 2Water for Injection (WFI)See Table 2PEG 400Quantity sufficient to 1 mL(“PEG 400” is polyethylene glycol 400)

To prepare the above described one mL units, a bulk solution was manufactured as follows, under yellow light as far as practical: 6.25 grams of bendamustine hydrochloride was added to a 250 mL volumetric flask. 250 mg of butylated hydroxyanisole (BHA) was added. Approximately 200 mL of PEG 400 was added to the 250 mL flask. A predetermined amount of absolute ethanol was added to the flask (see the following Table 2). The predetermined volume of Water for Injection (WFI) was then added to the flask (see the following Table 2). The bendamustine HCl and the BHA were then dissolved by swirling or shaking the flask. The solution was then filled to volume with PEG 400 so as to obtain a 25 mg/mL bendamustine HCl solution. The flask was repeatedly inverted until a homogenous solution was achieved. The pH was adjusted using 5N NaOH to a target of 3.5 (range of 3.4 to 3.6). The bulk solution was filtered through a 0.2-micron pore size polyvinylidene fluoride (PVDF) membrane filter.

TABLE 225 mg/mL Bendamustine Formulations “B”Amount per 250 mLEthanol (%FormulationFormulationWFI (% v/v)v/v)WFI (mL)Ethanol (mL)B10.05.0012.5B25.05.012.512.5B35.00.012.50B45.010.012.525B55.05.012.512.5B610.05.02512.5B75.05.012.512.5B85.05.012.512.5B98.58.521.2521.25B108.51.521.253.75B111.58.53.7521.25B121.51.53.753.75B135.05.012.512.5

Example 2

PH Assessment of Bendamustine Formulations

A study was conducted to assess the effect of different pH levels on the formulation stability of bendamustine HCl formulations. Bendamustine bulk liquid formulation was prepared using bendamustine HCl, absolute alcohol (ethanol), Water for Injection (WFI), and PEG 400 (polyethylene glycol 400). The bulk formulation contained 25 mg/mL bendamustine HCl, 1 mg/mL butylated hydroxyanisole, 3.0% absolute alcohol and 3% v/v WFI (water for injection), made up to volume with PEG 400. The bulk was then pH-adjusted with 5N NaOH to obtain said formulation having the different pH values set forth in Table 3.

To determine the pH of a 25 mg/mL bendamustine HCl formulation prepared as set forth in this Example, a 0.55±0.05 gram aliquot of a pH-adjusted formulation was prepared. 3.5 mL water was added, and the combination was mixed gently until visually homogenous, thereby obtaining a 12.3% v/v solution of the chosen bendamustine HCl liquid formulation in water. The pH of the solution in the flask was then measured using a calibrated pH meter.

Having thus obtained 25 mg/mL bendamustine HCl formulations with different identified pH, said formulations were then filled into 2 mL vials, at approximately 1.5 mL per vial.

The bendamustine stability was measured over time for the formulations having the various pH. Bendamustine related substances were detected in the formulations over time by HPLC (high performance liquid chromatography).

The HPLC test method used was as follows:

Method RequirementDescriptionColumnL1(Octyldecylsilane, 250 × 4.6 mm, 5 μmColumn temperature25° C.Sample temperature5° C.Detector wavelength260 nmFlow rate1.1 mL/minuteInjection volume15 μLMobile phase A0.06M ammonium formate in WFI (pH 3.2):Acetonitrile (90:10)Mobile Phase B0.06M ammonium formate in WFI (pH 3.2):Acetonitrile (30:70)TimeA (%)B (%)Gradient01000310007937128020334060360100381000551000Run time55 minutesDiluentMethanolSample solution0.4 mg/mLconcentration

Amounts of related substances in a sample are expressed as a percentage-namely area under the curve (AUC) of the peak located for the related substance in a test sample output relative to the AUC of the bendamustine peak in the bendamustine standard. In the following Tables and description, “ND” means “not detected”. “RRT” means “relative retention time”. “T” equals “number of months”, such that “TO” is the initial starting time, and “T6”, for example, is six months later.

The following Table 3 lists the test results from the bendamustine HCl pH stability studies at various points over time for the BM DCE impurity at 25° C.:

TABLE 3Effect of pH on BM DCE levelSample at 25° C.T0T1T2T4T6pH 3.23ND0.09%0.13%0.20%0.30%pH 3.30ND0.05%0.07%0.11%0.15%pH 3.37ND0.06%0.09%0.14%0.20%pH 3.45ND0.07%0.11%0.18%0.25%pH 3.57ND0.09%0.15%0.25%0.36%pH 3.66ND0.11%0.19%0.32%0.49%pH 3.78ND0.13%0.25%0.42%0.61%

Table 3 illustrates that with time at pH levels higher than 3.30 at 25° C., the BM DCE impurity levels increase.

The following Table 4 lists the test results from the bendamustine HCl pH studies from TO to T6 for the BM HP1 impurity at 25° C.:

TABLE 4Effect of pH on BM HP1 levelSample at25° C.T0T1T2T4T6pH 3.23ND0.02%0.02%0.03%0.04%pH 3.30ND0.03%0.03%0.06%0.08%pH 3.37ND0.04%0.05%0.10%0.14%pH 3.450.03%0.05%0.07%0.13%0.19%pH 3.570.02%0.06%0.10%0.18%0.27%pH 3.660.03%0.08%0.13%0.24%0.36%pH 3.780.02%0.09%0.17%0.31%0.47%

Table 4 illustrates that with time at higher pH levels at 25° C., the BM HP1 impurity levels increase.

TABLE 5Effect of pH on BM PEG Ethers levelSample at25° C.T0T1T2T4T6pH 3.23NDNDND0.02%0.05%pH 3.30NDND0.02%0.07%0.08%pH 3.370.01%ND0.05%0.11%0.11%pH 3.450.02%0.02%0.08%0.13%0.14%pH 3.570.02%0.03%0.12%0.21%0.23%pH 3.660.03%0.07%0.14%0.26%0.38%pH 3.780.04%0.08%0.19%0.35%0.48%

Table 5 illustrates that with time at higher pH levels at 25° C., the BM PEG Ether group of impurities increases.

TABLE 6Effect of pH on BM PEG Esters levelSample at25° C.T0T1T2T4T6pH 3.23ND1.64%3.36%6.54%10.92%pH 3.30ND0.17%0.41%0.82%1.17%pH 3.37ND0.11%0.25%0.51%0.71%pH 3.45ND0.09%0.22%0.40%0.61%pH 3.57ND0.09%0.20%0.35%0.62%pH 3.66ND0.07%0.19%0.30%0.65%pH 3.78ND0.04%0.18%0.30%0.96%

Table 6 illustrates that with time a lower pH levels less than 3.30 at 25° C., the BM PEG Ester group of impurities increases significantly than compared to higher pH levels.

TABLE 7Effect of pH on BM Trichloro Dimer levelSample at 25° C.T0T1T2T4T6pH 3.23ND0.06%0.21%0.38%0.35%pH 3.30ND0.02%0.03%0.06%0.08%pH 3.37ND0.02%0.04%0.05%*pH 3.45ND0.03%0.05%0.12%*pH 3.57ND0.04%0.07%0.16%*pH 3.66ND0.06%0.11%0.23%*pH 3.78ND0.09%0.18%0.35%**Co-elution with BM PEG Esters therefore could not accurately determine their levels.

Table 7 illustrates that with time the BM Trichloro dimer impurity is lower as the pH increases, however the actual amount of dimer (at higher pH levels) is difficult to determine as it is eluting with BM PEG Esters.

TABLE 8Effect of pH on BM EE levelSample at25° C.T0T1T2T4T6pH 3.230.02%0.83%1.79%3.28%5.76%pH 3.30ND0.10%0.21%0.40%0.61%pH 3.37ND0.06%0.13%0.24%0.41%pH 3.45ND0.05%0.10%0.18%0.28%pH 3.57ND0.04%0.09%0.16%0.24%pH 3.66ND0.04%0.08%0.14%0.22%pH 3.78ND0.04%0.07%0.13%0.20%

Table 8 illustrates that that with time the BM EE impurity is lower as the pH increases.

TABLE 9Effect of pH on Total Impurity levelSample at25° C.T0T1T2T4T6pH 3.230.022.64%5.71%10.46%18.05%pH 3.30ND0.36%0.78%1.53%2.19%pH 3.370.01%0.29%0.61%1.17%1.70%pH 3.450.04%0.31%0.65%1.17%1.57%pH 3.570.05%0.35%0.73%1.34%1.80%pH 3.660.06%0.43%0.86%1.53%2.25%pH 3.780.06%0.49%1.08%1.91%2.75%

Table 9 is comparing the total impurity percentage at all pH levels. The optimum pH range was 3.37-3.57.

TABLE 10AEffect of pH on Impurity Levels at 5° C. (the impurity quantitiesare %, based on the standard bendamustine HCl)Sampleat 5° C.T0T4T6with in-T0T4T6T0T4T6BMBMBMdicatedBMBMBMBMBMBMPEGPEGPEGpHDCEDCEDCEHP1HP1HP1EthersEthersEthers3.23ND0.020.20ND0.01NDNDNDND3.30ND0.020.02ND0.02NDND0.010.013.37ND0.020.030.010.01ND0.010.010.023.45ND0.030.030.020.01ND0.020.010.033.57ND0.030.040.020.020.020.020.020.043.66ND0.040.060.030.020.020.030.030.053.78ND0.050.080.040.020.030.040.030.07

TABLE 10BEffect of pH on Impurity Levels at 5° C. (the impurity quantitiesare %, based on the standard bendamustine HCl)Sampleat 5° C.T0T4T6withBMBMBMT0T4T6T0T4T6indicatedPEGPEGPEGTri-ClTri-ClTri-ClBMBMBMpHEstersEstersEstersDimerDimerDimerEEEEEE3.23ND0.420.60ND0.030.040.020.240.373.30ND0.060.06ND0.01NDND0.030.043.37ND0.040.04ND0.01NDND0.020.033.45ND0.040.06ND0.01NDND0.020.023.57ND0.050.07ND0.02NDND0.020.023.66ND0.030.07ND0.02NDND0.010.023.78ND0.030.08ND0.03NDND0.030.02

Note, in Table 10B, “Tri-CI dimer” is BM Trichloro Dimer.

As shown in Tables 10A and 10B, at 5° C. from T0 to T6 months, a similar fashion was observed to the accelerated 25° C. data: The BM EE and BM PEG Esters impurity levels are high when the pH of the formulation is lower than 3.3.

The experiments in this Example 2 demonstrated that pH affected the impurity profile of a bendamustine HCl formulation containing a small molecule organic solvent, an organic polymeric compound solvent (with multiple hydroxy moieties), and water. Where the pH of the formulation was lower than 3.30, high levels of BM EE and BM PEG Ester impurities were observed over time. Where the pH of the formulation was higher than 3.66, high levels of BM PEG Ether impurities were observed overtime.

Example 3: Long Term Stability of Liquid Bendamustine Formulations at Refrigeration Temperatures

A study was performed to assess the difference in stability profile (as indicated by assay and impurity levels) of a bendamustine formulation according to the present invention and BENDEKA™ (comprising the ingredients listed in Table 11 below) when stored long term at both 2° C. and 8° C.

TABLE 11Product/Formulations DetailsDrug ProductBendamustine Formulation ABENDEKA ™FormulationAPI25 mg/mLAPI25 mg/mLBHA1 mg/mLMTG5 mg/mLAA23.7 mg/ml (3% v/V)PG0.1 mLWFI30 mg/ml (3% v/V)PEG400Qs.PEG400Qs.NaOH**AR pH 3.2-NaOH**AR pH 3.4-3.6*3.3*Note on Table 11:“API” is bendamustine HCl;“BHA” is butylated hydroxyanisole;“WFI” is Water for Injection;“AA” is ethanol;“AR” is “as required”;“MTG” is monothioglycerol;“PG” is propylene glycol;“Qs” is “quantity sufficient”; and“PEG” is polyethylene glycol”.*Formulation in a 12.4% aqueous solution (v/v).**NaOH is used to adjust pH.

All samples were stored at 2-8° C. prior to the study start and after study completion. The samples were tested for pH, assay and related substances. After four months storage at 2° C. and 8° C., the pH of and related substances in the samples were tested. After ten months storage at 2° C. and 8° C., the physical state of the samples was observed and sample photos were taken. After 12 months storage at 2° C. and 8° C., samples were tested for description, pH, assay and related substances. The presence and the amount of related substances were determined by HPLC using the following parameters:

Method RequirementDescriptionColumnPhenomenex CuroSil-PFP, 150 × 4.6 mm, 5 μm(pentaflurophenyl USP L43 packing)Column temperature30° C.Sample temperature5° C.Detector wavelength260 nmFlow rate1.0 mL/minuteInjection volume10 μLMobile phase A0.06M ammonium formate in WFI (pH 3.2)Mobile Phase BAcetonitrileTimeA (%)B (%)Gradient09010157327406634455446474852509010609010Run time60 minutesDiluentMethanolSample solution2.0 mg/mLconcentration

TABLE 12Stability dataBM TRICLMajorTotalTimeStorageBM DCEBM HP1DIMERBM EEUnknownImpuritiesSamplePointTemperatureDescriptionpHAssay(Δ T0)(Δ T0)(Δ T0)(Δ T0)(Δ T0)(Δ T0)BendamustineT0N/ANot tested3.4599.4%<0.05%<0.05%NDNDND<0.05%HCl injecttionT42° C.Not tested3.48Not tested<0.05%<0.05%NDND<0.05%<0.05%PB1008° C.Not tested3.47Not tested<0.05%<0.05%ND<0.05%<0.05%<0.05%T122° C.Complies3.5398.9%<0.05%<0.05%<0.05%<0.05%<0.05%<0.05%4-6° C.*Complies3.5298.7%<0.05%<0.05%<0.05%0.05%<0.05%0.05%8° C.Complies3.5298.4%<0.05%<0.05%<0.05%0.06%<0.05%0.06%Bendeka ™T0N/ANot tested3.1797.1%0.39%0.05%NCND0.07%0.74%ZBR049T42° C.Not tested3.19Not tested0.39%0.06%ND<0.05%0.22%1.2%8° C.Not tested3.19Not tested0.36%0.06%ND<0.05%0.30%1.6%T122° C.Complies3.2394.3%0.32%0.05%<0.05%ND0.92%3.7%4-6° C.*Complies3.2491.1%0.31%0.05%<0.05%ND1.67%6.7%8° C.Complies3.2382.7%0.36%0.05%<0.05%ND3.5%14.5%

Description for Table 12: “Complies” was defined as a clear, colourless to yellow-coloured solution, practically free from visible particulates. ND=Not Detected. Reporting threshold is >0.05%. “Δ T0” refers to the change in the amount of the specified related substance from T0 to either four months later or twelve months later, as indicated, when stored at the indicated temperature or temperature range.

*Stored at 2-8° C., but chamber temperature was typically 4-6° C.

The results of this study show that both BENDEKA™ and Bendamustine Formulation A were clear and colorless-to-yellow after 12 months storage at 2° C. and 8° C. The stability of Bendamustine Formulation A after storage at either 2° C. or 8° C. for 12 months was comparable; additionally, there was minimal degradation with Bendamustine Formulation A over the 12 month study period at any temperature. BENDEKA™ degraded significantly over the 12 month study period and there was a vast difference in both assay and impurity levels between 2° C. and 8° C. temperatures. The most significant change was observed with the unknown impurities in BENDEKA™. These impurities are in the “esters” group, specifically propylene glycol ester and PEG esters, which form when bendamustine HCl reacts with the propylene glycol and the PEG 400 in BENDEKA™. Based on the studies described herein (as demonstrated Example 2 above), low pH promotes formation of esters, and this may be responsible for the high levels of the propylene glycol and PEG esters observed in BENDEKA™ when stored over time in this study.

Additionally, the product vials on storage at 2° C. and 8° C. were observed and photos were taken. The results demonstrated that Bendamustine Formulation A was a liquid at both 2° C. and 8° C., but BENDEKA™ was a solid at 2° C. and a liquid at 8° C.

The study in this Example 3 demonstrated that BENDEKA™ is a frozen solid at 2° C. and a liquid at 8° C. An additional issue is the vast difference in impurity levels for BENDEKA™ when stored at 2° C. and 8° C., with total impurities of 3.7% and 14.5% respectively. The BENDEKA™ sample which was stored in the stability chamber at 2-8° C. (typical temperature was 4-6° C.) had a total impurity levels of 6.7%, which was between that of the 2° C. and 8° C. BENDEKA™ samples. In contrast, Bendamustine Formulation A was a liquid at both 2° C. and 8° C. There was no significant difference in impurity levels in Bendamustine formulation A when stored at either 2° C. or 8° C., with total impurities of <0.05% and 0.06% respectively. Bendamustine Formulation A stored in the stability chamber at 2-8° C. (typical temperature was 4-6° C.) had total impurity levels of 0.05%.

PEG 400, a suitable solvent for bendamustine HCl, has a freezing point of approximately 4° C. The proposed long term storage temperature of a bendamustine drug product is refrigeration temperature (2-8° C.). Thus, if the bendamustine drug product formulation contains PEG 400, there is a possibility that freezing may occur during storage that may result in significant differences in impurity levels over the temperature range depending on whether the product is a liquid or a frozen solid. Additionally, having a frozen product is not end-user friendly, as it has to be warmed prior to administration. The study in this Example 3 shows that a bendamustine formulation of the present invention, Bendamustine Formulation A, avoids the problem of freezing when stored under standard refrigeration conditions.

Example 4: Organic Solvents

An initial study was performed to assess bendamustine HCl solubility and brief stability in various organic solvents. Amounts of approximately 415.4 mg bendamustine HCl API (active pharmaceutical ingredient) were weighed into 4 mL amber glass vials. 2 mL of each indicated test solvent was added to two of each of the vials. One of each set of two vials containing the same solvent was placed on a sample roller at 2-8° C., and the other vial in each set was placed on a sample roller at laboratory temperature overnight. The following day, the solution from each vial was filtered through a Pall, 0.2 μm Fluorodyne II (Hydrophilic modified PVDF) syringe filter into a fresh 4 mL amber glass vial.

The solubility of bendamustine HCl was given by the TO assay analysis, and the results are summarized in Table 13. The temperature in the laboratory was measured to be 23° C., thus solubility was determined at 2-8° C. and at 23° C. Mostly, bendamustine HCl solubility was found to be higher at 23° C. than at 2-8° C.

Benzyl alcohol, dimethyl sulfoxide, 1-methyl-2-pyrrolidinone and the dimethyl acetamide showed high bendamustine HCl solubility (>180 mg/mL). They all completely dissolved the API, at both temperatures tested, indicating bendamustine HCl solubility is approximately 180 mg/mL or above in these solvents.

Propylene glycol also had high bendamustine HCl solubility, giving approximately 155 mg/mL at 2-8° C. and 170 mg/mL at 23° C. Negligible differences were observed between three propylene glycol suppliers (Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc.; The Dow Chemical Company; and Croda International Plc).

The remaining solvents had bendamustine HCl solubility limits below the concentration of 90 mg/mL in TREANDA® Injection (i.e. the TREANDA® (bendamustine hydrochloride), injection, for intravenous use, solution product). Ethanol was approximately 50-60 mg/mL, PEG 300 approximately 60 mg/mL and PEG 400 approximately 30 mg/mL. Thus, ethanol, PEG 300 and PEG 400 would require co-solvent(s) to reach 90 mg/mL. Isopropyl alcohol was even lower with only approximately 5 mg/mL of bendamustine HCl dissolving.

TABLE 13Solubility of Bendamustine HCl in Organic Solvents2-8° C.23° C.SolubilityCompleteSolubilityCompleteSolvent(mg/mL)dissolution?(mg/mL)dissolution?Absolute Ethanol52.1N61.1NBenzyl alcohol176.9Y174.0YDimethyl sulfoxide181.5Y181.3YIsopropyl alcohol4.5N6.7N1-methyl-2-181.3Y180.3YpyrrolidinoneDimethyl acetamide177.6Y179.3Y(high purity)-FinarDimethyl acetamide180.5Y174.2Y(EP)-FinarDimethyl acetamide182.2Y179.7Y(EP)-PanreacPEG 30060.2N64.5NPEG 40032.8N34.4NPropylene glycol158.7N172.3Y(USP)-MerckPropylene glycol153.7N165.8Y(USP)-DowPropylene glycol153.8N170.3Y(USP)-Croda“USP” is “United States Pharmacopeia”, meaning the substance has been determined to be pure according to the standards published by the United States Pharmacopeia.“EP” is European Pharmacopeia“PEG” is polyethylene glycol

Example 5: Bendamustine HCl Injection Formulation “C”

Bendamustine HCl monohydrate was used to prepare a liquid bendamustine formulation suitable for injection, wherein each mL of the formulation contains the following ingredients in polyethylene glycol 400: 25 mg bendamustine HCl, 1 mg butylated hydroxyanisole, 23.7 mg dehydrated ethanol, 30 mg water, and sodium hydroxide as needed to adjust the pH to from about 3.4 to about 3.6. To adjust and calculate the pH, the composition is tested in a water solution at about 12.4% v/v, and sodium hydroxide was added, if needed, until a pH of from about 3.4 to about 3.6 was achieved. The liquid bendamustine HCl formulation was filled into amber glass vials to make the following single-dose or multiple-dose presentations of ready-to-dilute bendamustine HCl solution: 1 mL presentation (containing 25 mg bendamustine HCl), 4 mL presentation (containing 100 mg bendamustine HCl), and 8 mL presentation (containing 200 mg bendamustine HCl). These formulations maintained a pH target range of from about 3.3 to about 3.7 over the stability program.

Example 6: Bendamustine Formulations with Increasing Water Content

A study was performed to assess the effect of water content (10-30% v/v) on the stability of a Bendamustine HCl formulation. Formulations containing identical amounts of Bendamustine HCl, Butylated Hydroxy Anisole and Absolute alcohol but with increasing amounts of water were prepared as shown in the table below.

FORMULATION(F)12345Bendamustine25 mg/25 mg/25 mg/25 mg/25 mg/HClmLmLmLmLmLButylated1 mg/1 mg/1 mg/1 mg/1 mg/Hydroxy AnisolemLmLmLmLmLAbsolute Alcohol3% v/V3% v/V3% v/V3% v/V3% v/VWater10% v/V15% v/V20% v/V25% v/V30% v/VPEG400Qs. to 1Qs. to 1Qs. to 1Qs. to 1Qs. to 1mLmLmLmLmLpHApprox.Approx.Approx.Approx.Approx.3.53.53.53.53.5

These formulations were placed on stability for 7 months at 2-8° C. and 25° C. Related substances testing was performed at the initial time point (0 M), after 1 month (1 M) and after 7 months (7M) storage. The results are shown in the Tables 14 and 15 below.

TABLE 14BENDAMUSTINEBENDAMUSTINEBENDAMUSTINEHYDROLYSISTRI-CHLOROBENDAMUSTINEMAJOR2-8° C.DECHLOROETHYLPRODUCT 1DIMERETHYL ESTERUNKNOWNTOTALSTORAGEIMPURITYIMPURITYIMPURITYIMPURITYIMPURITYIMPURITIESTest Point0 M1 M7 M0 M1 M7 M0 M1 M7 M0 M1 M7 M0 M1 M7 M0 M1 M7 MF1 -10% waterNDND<QLNDND0.07NDND<QLNDNDNDNDNDNDNDND0.1F2 -15% waterNDND<QLND<QL0.21NDND0.09NDNDNDNDNDNDNDND0.3F3 -20% waterNDND<QLND0.090.55ND<QL0.26NDNDNDNDND0.05NDND0.9F4 -25% waterNDND<QLND0.181.08ND0.100.57NDNDNDNDND0.08ND0.281.8F5 -30% waterNDND<QLND0.341.94ND0.211.11NDND<QLNDND0.1ND0.553.5M = months;ND = Not detected,QL = Quantitation Limit

TABLE 15BENDAMUSTINEBENDAMUSTINEBENDAMUSTINEHYDROLYSISTRI-CHLOROBENDAMUSTINEMAJOR25° C.DECHLOROETHYLPRODUCT 1DIMERETHYL ESTERUNKNOWNTOTALSTORAGEIMPURITYIMPURITYIMPURITYIMPURITYIMPURITYIMPURITIESTest Point0 M1 M7 M0M1 M7 M0 M1 M7 MOM1 M7 M0 M1 M7 M0 M1 M7 M10% waterNDND0.16ND0.171.02NC0.050.29ND<QL0.22ND<QL0.17ND0.23.315% waterND0.070.31ND0.522.73ND0.150.66ND<QL0.24ND<QL0.23ND0.76.320% waterNDND0.21ND1.305.15ND0.411.09ND<QL0.32ND0.090.29ND1.910.125% waterNDND0.18ND2.597.14ND0.831.26ND<QL0.84ND0.130.45NC4.014.730% waterNDND0.47ND4.329.16ND1.341.12ND<QL2.11ND0.151.03ND6.722.8M = months;ND = Not detected,QL = Quantitation Limit