Pharmaceutical compositions suitable for intravenous administration

Abstract

This invention is directed to compositions comprising bisantrene, particularly formulations of bisantrene dihydrochloride, wherein the formulations are suitable for intravenous administration into peripheral veins as well as methods for their use and methods for preparation of such compositions. The compositions according to the present invention eliminate the need to administer bisantrene by central venous catheter administration. The compositions can be used to treat malignancies and other conditions, and may be used for cardioprotection in patients undergoing chemotherapy with anthracycline-based anti-neoplastic agents. The inclusion of α-hydroxy acids in compositions according to the present invention improves the stability of such compositions and prevents precipitation of the bisantrene.

Description

PHARMACEUTICAL COMPOSITIONS SUITABLE FOR INTRAVENOUS ADMINISTRATION FIELD OF THE INVENTION

[0001] This invention is directed to formulations of bisantrene and pharmaceutical compositions wherein the formulations are suitable for intravenous administration, including peripheral veins, as well as methods for manufacturing such formulations. BACKGROUND OF THE INVENTION

[0002] Bisantrene, generally employed as the dihydrochloride salt, is an antineoplastic agent with several cytotoxic and immunologic mechanisms of action. The chemical name for bisantrene dihydrochloride is 9,10-anthracenedicarboxaldehyde-bis [(4, 5-dihydro-1H-imidazole-2-yl) hydrazine] dihydrochloride. Bisantrene was found to kill tumor cells in clonogenic assays and inhibits both DNA and RNA synthesis. Bisantrene can also induce double-stranded DNA breaks resulting from inhibition of the topoisomerase II. Bisantrene is inactive when given orally, but effective when given intravenously (i.v.), intraperitoneally (i.p.), or subcutaneously (s.c.) in a number of cancer models, including colon 26, Lewis lung, Ridgway osteosarcoma, B16, Lieberman plasma cell, P388 and L1210 cancer cells. Anticancer activity in clonogenic assays was seen in breast, small cell lung, large cell lung, squamous cell lung, ovarian, pancreatic, renal, adrenal, head and neck, sarcoma, gastric, lymphoma and melanoma tumor cells, but not in colorectal cancer. A lack of cross-resistance with Adriamycin (doxorubicin) was noted in metastatic breast cancer (Yap, H-Y et al (1983), Cancer Research 43: 1402-1404).

[0003] With broad activity demonstrated historically across several cancer indications, as stated above, clinical (re)development of bisantrene in the modern oncology setting could be used to target multiple cancer types. However, phlebitis was observed during intravenous (i.v.) administration in early clinical trials resulting in the need to deliver bisantrene dihydrochloride through a central venous catheter to avoid the occurrence of phlebitis. Clinical administration of bisantrene requires the use ofslow, central venous catheter infusions due to the drug’s blood solubility properties. In modern oncology clinics, the use of central venous catheter infusions is often restricted, potentially limiting applications for bisantrene. Thus, to expand the clinical utility of bisantrene, there is a need for the development of new bisantrene formulations (and methods for their manufacturing) that improve the blood solubility of bisantrene and enable the use of commonly practiced peripheral vein infusions for administration. SUMMARY OF THE INVENTION

[0004] The pharmaceutical compositions of the present invention improve the blood solubility of bisantrene, thereby reducing the potential for phlebitis and enabling safer administration of bisantrene by intravenous infusions, including into peripheral veins, and expanding the clinical utility of bisantrene. These compositions can be used to treat a range of malignancies, as well as other diseases and conditions, and can be used in conjunction with other drugs and treatments. These compositions and methods can also be used to provide cardioprotection in patients treated with anthracyclines and other cardiovascular damaging drugs. Elimination of exposure of patients receiving infusions of bisantrene dihydrochloride to precipitates or particulates by use of methods according to the present invention to prepare pharmaceutical compositions including bisantrene dihydrochloride reduces or eliminates phlebitis at the site of infusion, and reduces the risk of other side effects, such as venous irritation, hyperpigmentation, drug extravasation, and anaphylactoid reactions. Elimination of exposure of patients receiving infusions of bisantrene dihydrochloride to precipitates or particulates when the pharmaceutical compositions are prepared by use of methods according to the present invention allows the use of standard peripheral vein i.v. infusions, avoiding the need for central line infusion. Additionally, elimination of exposure of patients receiving infusions of bisantrene dihydrochloride to precipitates or particulates increases the safety of administration of bisantrene dihydrochloride as a therapeutic agent while not reducing its effectiveness.

[0005] One aspect of the present invention is therefore a pharmaceutical composition in one or more dosage units comprising: (1) a therapeutically effective quantity of bisantrene;(2) a cyclodextrin; and (3) an ^-hydroxy acid.

[0006] In another aspect, the pharmaceutical composition in one or more dosage units comprises: (1) a therapeutically effective quantity of bisantrene; and (2) a cyclodextrin.

[0007] The bisantrene can be in salt form or in free base form. When the bisantrene is in salt form, the bisantrene salt is typically bisantrene dihydrochloride. However, other salts of bisantrene can alternatively be used.

[0008] Typically, the cyclodextrin is ^-cyclodextrin, ^-cyclodextrin, ^-cyclodextrin, a sulfobutyl ether ^-cyclodextrin (SBECD), 2-hydroxypropyl-^-cyclodextrin (HPBCD), 2- hydroxypropyl-^-cyclodextrin (HPGCD), or a randomly methylated ^-cyclodextrin (RMBCD). Typically, when the cyclodextrin is an SBECD, the SBECD comprises from 1 to 7 sulfobutyl ether moieties.

[0009] Typically, the ^-hydroxy acid, when present, is glycolic acid, lactic acid, mandelic acid, tartaric acid, malic acid, or citric acid, including all stereoisomers of these ^-hydroxy acids. Lactic acid has two stereoisomers, L-lactic acid and D-lactic acid. Mandelic acid also has two stereoisomers, (R)-mandelic acid and (S)-mandelic acid. Tartaric acid has three stereoisomers, (2R,3R)-tartaric acid (the naturally occurring form, also known as L-tartaric acid), its enantiomer, (2S,3S)-tartaric acid (also known asD-tartaric acid), and the meso form, (2R,3S)-tartaric acid or (2S,3R)-tartaric acid. Malic acid has two stereoisomers,L-malic acid andD-malic acid. When the ^-hydroxy acid is tartaric acid, preferably, the stereoisomer of tartaric acid is L-tartaric acid.

[0010] Typically, the molar ratio of the bisantrene to the cyclodextrin is from about 1:1 to about 1:6. Typically, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:5.

[0011] Typically, the composition in one or more dosage units is a solution, a suspension, a lyophilized powder or cake, or a spray-dried powder, wherein the lyophilized powder or cake or the spray-dried powder is suitable for reconstitution with vehicle (e.g., water for injection) prior to use. Typically, the pH of the solution, the pH of the suspension, the pH of the lyophilized powder or cake on reconstitution with water, orthe pH of the spray-dried powder on reconstitution with water, is from about 2.0 to about 6.0. Typically, the composition is packaged in vials. In one embodiment, the vials are plastic vials. In another embodiment, the vials are silanized or unsilanized glass vials. The silanized or unsilanized glass vials can be clear or amber glass. When the vials are silanized, typically, silanization is performed by coating the interior of the vials with an organofunctional alkoxysilane that is (3-aminopropyl)-triethoxysilane, (3-aminopropyl)- diethoxymethylsilane, (3-aminopropyl)-dimethyl-ethoxysilane, (3-aminopropyl)- trimethoxysilane, (3-glycidoxypropyl)-dimethyl-ethoxysilane, (3-mercaptopropyl)- trimethoxysilane, (3-mercaptopropyl)-methyl dimethoxysilane, or a derivative thereof. When the vials are plastic vials, typically, the plastic vials are constructed of a plastic that is cyclic olefin polymer (COP) plastic, cyclic olefin copolymer (COC) plastic, high- density polyethylene plastic, or high-density non-nucleated polypropylene plastic.

[0012] In an embodiment, the composition can further comprise a suitable quantity of an additional pharmaceutically acceptable carrier. Typically, the additional pharmaceutically acceptable carrier is in one or more of the following categories: acidifying agents; alkalizing agents; antimicrobial preservatives; antioxidants; buffering agents or pH-adjusting agents; chelating agents; complexing agents; emulsifying and / or solubilizing agents; polymers; solvents; or vehicles (sterile).

[0013] The present invention also provides methods for preparing bisantrene formulations, particularly formulations of bisantrene dihydrochloride, that improve blood solubility and enable the use of more commonly practiced peripheral vein infusions for the administration of bisantrene.

[0014] Thus, in another aspect, the present invention provides a method for preparing a pharmaceutical composition of bisantrene, wherein the pharmaceutical composition comprises: (i) bisantrene; (ii) a cyclodextrin; and (iii) an ^-hydroxy acid, wherein the method comprises the steps of: (a) preparing a stock solution of a cyclodextrin and an ^-hydroxy acid in water; (b) combining a stock solution of bisantrene with the stock solution of the cyclodextrin and the ^-hydroxy acid from step (a) to produce a combined stock solution of the bisantrene, the cyclodextrin, and the ^- hydroxy acid; and (c) adjusting the pH of the combined stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid from step (b).

[0015] The stock solution of a cyclodextrin and an ^-hydroxy acid in water may be prepared by: a) preparing a stock solution of a cyclodextrin in water and dissolving the ^-hydroxy acid into the stock solution of the cyclodextrin; b) preparing a stock solution of an ^-hydroxy acid in water and dissolving the cyclodextrin into the stock solution of the ^-hydroxy acid; or c) combining a cyclodextrin in solid form with an ^- hydroxy acid in solid form and then dissolving the resulting solid mixture in water.

[0016] The method of preparation may further comprise a step of sterile filtration of the pH-adjusted combined stock solution, and in certain embodiments that step may comprise filtration through one or more filters, at least one of which has a filtration cutoff of about 0.2 ^m.

[0017] The method of preparation may comprise placing the pharmaceutical composition in vials, and in particular embodiments the vials are silanized or unsilanized amber glass.

[0018] The method of preparation may comprise a step of lyophilizing the combined stock solution, and in particular embodiments, the step of lyophilizing the combined stock solution is performed in vials.

[0019] In particular embodiments of the methods of preparation according to the present invention, the cyclodextrin is selected from the group consisting of ^- cyclodextrin, an SBECD, and HPBCD, and in even more particular embodiments the cyclodextrin is an SBECD. In particular embodiments, the molar ratio of the bisantrene to the cyclodextrin is from about 1:1 to about 1:6, preferably from about 1:2.5 to about 1:4.5.

[0020] In particular embodiments of the methods of preparation according to the present invention, the ^-hydroxy acid is selected from lactic acid, tartaric acid, and malic acid, and in even more particular embodiments, the ^-hydroxy acid is tartaric acid, preferablyL-tartaric acid. In particular embodiments the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:5, preferably from about 1:0.75 to about 1:1.25.

[0021] In particular embodiments of the methods of preparation according to the present invention, the pH of the combined stock solution including the bisantrene, the cyclodextrin, and the ^-hydroxy acid is adjusted to between about 3.0 and about 6.0. Infurther particular embodiments where the combined stock solution is lyophilized, the pH of the lyophilized powder or cake on reconstitution with water is from about 2.5 to about 6.0, more preferably from 4.0 to 6.0, and even more preferably from 5.0 to 6.0.

[0022] In particular embodiments of the present invention, there is provided a method for producing a pharmaceutical composition comprising bisantrene dihydrochloride, L-tartaric acid, and SBECD, wherein the method comprises the steps of: (a) preparing a solution of theL-tartaric acid and the SBECD in water for injection; (b) adding the bisantrene dihydrochloride to the solution of (a); (c) adjusting the pH of the solution from step (b) with aqueous sodium hydroxide to a pH of about 5.5; and (d) filtering the solution of step (c) through a 0.22-^m filter, optionally further comprising the step of filling the filtered solution into vials, and optionally further comprising the step of freezing and lyophilizing the solution in the vials.

[0023] In particular embodiments of the present invention, there is provided a method for producing a pharmaceutical composition comprising bisantrene dihydrochloride, L-tartaric acid, and SBECD wherein the ratio of SBECD to bisantrene is 3:1, wherein the method comprises the steps of: (a) preparing a solution of the L-tartaric acid and the SBECD in water for injection; (b) adjusting the pH of the solution of (a) with an aqueous solution of sodium hydroxide to about 5.3 to about 5.7; (c) adding the bisantrene dihydrochloride to the solution of (b); (d) adjusting the pH of the solution including the bisantrene dihydrochloride from step (c) with aqueous sodium hydroxide to a pH of about 5.3; (e) adding additional water for injection to adjust the concentrations of the bisantrene dihydrochloride, the L-tartaric acid, and the SBECD; and (f) filtering the solution of step (e) through a 0.22-^m filter, optionally further comprising the step of filling the filtered solution into vials, and optionally further comprising the step of freezing and lyophilizing the solution in the vials.

[0024] Pharmaceutical compositions of bisantrene comprising bisantrene, a cyclodextrin and an ^-hydroxy acid, prepared by a method according to the invention are also provided.

[0025] In particular embodiments of the pharmaceutical compositions and methods of preparation according to the present invention, greater than 60%, such as greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%, orgreater than 99% of the bisantrene in the pharmaceutical composition, or the pharmaceutical composition resulting from the method of preparation, is (E,E)- bisantrene or a pharmaceutically acceptable salt thereof, where (E,E) refers to the stereochemistry about the two hydrazone C=N double bonds.

[0026] In particular embodiments of the pharmaceutical compositions and methods of preparation according to the present invention, the form of bisantrene used for producing the pharmaceutical composition is the dihydrochloride salt in crystalline form having the following properties: (a) a weight loss of up to 10% up to 150^C in thermogravimetric analysis (TGA); (b) a sharp endothermic peak at between 95^C and 115^ C in differential scanning calorimetry (DSC); and (c) predominant peaks at 2^ of about 9.3^ and 14.0^ in X-ray powder diffraction (XRPD).

[0027] In another aspect, the present invention provides a method for treating a disease or condition treatable by administration of bisantrene comprising administering a pharmaceutical composition according to the invention, wherein the pharmaceutical composition is administered by infusion to the patient to treat the disease or condition treatable by administration of bisantrene. Typically, the disease or condition treatable by administration of bisantrene is cancer. However, the disease or condition treatable by administration of bisantrene can alternatively be a non-malignant disease or condition. The non-malignant disease or condition can be, but is not limited to, immunodeficiency, obesity, diabetes, metabolic syndrome, pancreatitis, and sequelae of cystic fibrosis affecting the pancreas. In related embodiments, the present invention also provides: use of: (i) a therapeutically effective quantity of bisantrene; (ii) a cyclodextrin; and optionally (iii) an ^-hydroxy acid for the manufacture of a medicament for infusion to a patient for treating a disease or condition treatable by administration of bisantrene; and a pharmaceutical composition comprising: (i) a therapeutically effective quantity of bisantrene; (ii) a cyclodextrin; and optionally (iii) an ^-hydroxy acid for use in treatment of a disease or condition treatable by administration of bisantrene.

[0028] In certain embodiments, the method comprises the steps of: (a) reconstituting the contents of one or more vials comprising the pharmaceutical composition using sterile water for injection; (b) diluting the reconstituted contents of a portion of or all of the one or more vials from step (a) into an intravenous infusionvehicle; and (c) administering the reconstituted and diluted pharmaceutical composition from step (b) into a patient with cancer or other disease treatable by administration of bisantrene, wherein the administration is performed by infusion into a vein. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings, where:

[0030] Figure 1 provides images of aqueous mixtures at pH 5.5 – 7.0 containing 25 mg / mL Bis.Free (i.e. bisantrene free base equivalents) and either 20% w / v SBECD or 20% w / v HPBCD (cyclodextrins) after stirring for 24 h at room temperature.

[0031] Figure 2 provides light microscopy images showing the extent of precipitation in solutions containing 7.5 mg / mL bisantrene, 6% w / v SBECD and the indicated excipient (0.5, 1% w / v) after dilution (1:1) into 5% dextrose in water (5DW) followed by dilution (1:1) into 0.1 M Sorenson’s buffer (pH 7.4) at the time points indicated. Bisantrene Only Control (containing no SBECD or excipient) and Bisantrene + SBECD Control (containing no additional excipient) were correspondingly diluted in 5DW and Sorensen’s buffer.

[0032] Figure 3 provides images (top) and descriptions (bottom) of bisantrene solutions containing 0.5% w / v tartrate (buffered disodium tartrate / L-(+)-tartaric acid) and the indicated molar ratios of SBECD:bisantrene. Solutions were initially produced in water (to mimic reconstitution of solid drug product), diluted to 0.5 mg / mL bisantrene in 5DW (to mimic a clinical solution for infusion) and left to stand for 18 h.

[0033] Figure 4 provides images of flowthrough collected from an in vitro venous infusion model immediately, 1 h and 3 h after injection of bisantrene formulations (dilution in 5DW) into flowing Sorensen’s buffer pH 7.4 or (after dilution in Hartmann’s Solution) fetal calf serum.

[0034] Figure 5 shows the process for calculation of the number of substitutions of R=CH2CH2CH2CH2SO3Na for SBECD.

[0035] Figure 6 shows the process for calculation of the molecular weight of substitutions of R=CH2CH2CH2CH2SO3Na for SBECD.

[0036] Figure 7 shows the process for calculation of the molecular weight of SBECD including the substitutions.

[0037] Figure 8 is an example calculation of the quantity of SBECD per vial in an embodiment wherein the ratio of SBECD to bisantrene is 3:1.

[0038] Figure 9 is an example calculation of the quantity of SBECD per vial in an embodiment wherein the ratio of SBECD to bisantrene is 4.5:1.

[0039] Figure 10 shows the process for calculation of the number of substitutions of R=CH2CH2CH2CH2SO3Na for SBECD (lot 47090322) for Examples 9-12.

[0040] Figure 11 shows the process for calculation of the molecular weight of substitutions of R=CH2CH2CH2CH2SO3Na for SBECD (lot 47090322) for Examples 9- 12.

[0041] Figure 12 shows the process for the calculation of the molecular weight of SBECD including the substitutions (lot 47090322) for Examples 9-12.

[0042] Figure 13 shows the process for calculation of the bisantrene potency for lot CDS-0067 / STG-02 / 00422P.

[0043] Figure 14 is an example calculation of the quantity of SBECD per vial in an embodiment wherein the ratio of SBECD to bisantrene is 3:1 for Examples 9-12.

[0044] Figure 15 is an example calculation of the quantity of SBECD per vial in an embodiment wherein the ratio of SBECD to bisantrene is 4.5:1 for Examples 9-12.

[0045] Figure 16 provides representative images from rabbit ear vein infusion studies showing in-vein precipitation of bisantrene when administered as bisantrene dihydrochloride diluted in 5DW (left) versus no precipitation when infused as formulated bisantrene in 5DW (right).

[0046] Figure 17 shows the1H-NMR spectrum for an exemplary sample of (E,E)- bisantrene (400 MHz, DMSO-d6).

[0047] Figure 18 shows a differential scanning calorimetry analysis of an exemplary sample of (E,E)-bisantrene.

[0048] Figure 19 shows a thermogravimetric analysis of an exemplary sample of (E,E)-bisantrene.

[0049] Figure 20 shows an X-ray powder diffractogram for an exemplary sample of (E,E)-bisantrene.DETAILED DESCRIPTION OF THE INVENTION

[0050] Definitions

[0051] Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of embodiments described herein or other embodiments within the scope of the invention, some preferred methods, compositions, materials, and devices are described herein. However, in this context, it is to be understood that this invention is not limited to the particular components, compositions, methodologies, or protocols described herein, as these aspects of the invention may vary in accordance with routine experimentation and optimization as is generally known in the art. It is also to be understood that the terminology used in the description and the claims is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments as described herein as understood by one of skill in the art.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of any conflict of meanings, the present specification and claims, including definitions therein, shall control. Accordingly, in the context of the embodiments described herein, the following definitions apply. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include references to the plural unless the context clearly dictates otherwise. Thus, for example, a reference to “a cyclodextrin” is a reference to one or more cyclodextrins or equivalents thereof known to those skilled in the art. Similarly, a reference to “a vial” refers to one or more vials. As used herein, the terms “comprise,” “include,” and linguistic variations thereof denote the presence of recited features, elements, method steps, or other components of the invention without the exclusion of the presence of additional or recited features, elements, method steps, or other components. Conversely, the terms “consisting of” and linguistic variations thereof, when used in the claims of the present application, denote the presence of recited features, elements, method steps, or other components of the invention and exclude any unrecited recited features, elements, method steps, or other components of the invention except for ordinarily-associatedimpurities. The phrase “consisting essentially of” and linguistic variations thereof, when used in the claims of the present application, denote the presence of recited features, elements, method steps, or other components of the invention and any additional features, elements, method steps, or other components of the invention that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language in the claims of the present application; such embodiments also encompass embodiments described in terms of “consisting essentially of” or “consisting of” language, which may be alternatively claimed or described using such language, unless the context clearly excludes “consisting essentially of” or “consisting of” language. The terms “comprising,” “consisting essentially of,” and “consisting of” are applicable to both composition claims and method claims.

[0053] As used herein, the terms “administration,” “administering,” or other equivalent terminology, refer to the act of giving a drug, prodrug, pharmaceutical composition, or other agent intended to provide therapeutic treatment to a subject or in vivo, in vitro, or ex vivo to cells, tissues, or organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs or other portions of the respiratory tract (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (such as, but not limited to, intravenously, subcutaneously, intraperitoneally, or by other injection routes as known in the art). Suitable routes of administration will depend on the particular formulation being administered, the quantity of bisantrene being administered, and the particular excipients in the formulation. Suitable routes of administration for therapeutic agents and pharmaceutical compositions described herein are as described below. Typically, bisantrene is administered intravenously, but other routes of administration are contemplated and can be employed where appropriate.

[0054] All chemical names used herein, including names of substituents, should be interpreted in light of the chemical nomenclature conventions of IUPAC and / or a modified format in which functional groups within a substituent are read in the order in which they branch from the scaffold or main structure. As used herein, however, theterm “bisantrene” includes: (i) bisantrene dihydrochloride; (ii) another salt of bisantrene with a counterion other than chloride; and (iii) the unionized free-base form of bisantrene, unless otherwise limited.

[0055] As used herein, the term “formulated for treating” refers to a composition produced by a method according to the present invention that is suitable for affecting a subject, tissue, organ, or cell to obtain a desired pharmacological and / or physiological effect, and can include inhibiting a condition, i.e., slowing or arresting its development, or relieving or ameliorating the effects of the condition, such as by causing reversal or regression of the effects of the condition. Such conditions can include, but are not necessarily limited to, abnormal or uncontrolled cellular proliferation such as the abnormal or uncontrolled cellular proliferation that is characteristic of malignancy. However, compositions produced by a method according to the present invention can be formulated for treating conditions other than malignancy as described herein according to clinical uses for bisantrene, particularly for the clinical uses of bisantrene dihydrochloride.

[0056] As used herein, the term “formulated for preventing” refers to a composition produced by a method according to the present invention that is suitable for preventing a condition from occurring in a subject, tissue, organ, or cell that may be at risk of having the condition, but does not necessarily mean that the condition will not eventually develop, or that a subject, tissue, organ, or cell will not eventually develop a condition. Preventing includes delaying the onset of a condition in a subject, tissue, organ, or cell. Such conditions can include, but are not necessarily limited to, abnormal or uncontrolled cellular proliferation such as the abnormal or uncontrolled cellular proliferation that is characteristic of malignancy. However, compositions produced by methods according to the present invention can be formulated for preventing other

[0057] As used herein, the term “subject” broadly refers to any animal, including, but not limited to, humans and non-human mammals. The reference to non-human mammals includes, but is not limited to, socially or economically important animals or animals used for research including cattle, sheep, goats, horses, donkeys, pigs, llamas, alpacas, dogs, cats, rabbits, guinea pigs, rats, hamsters, gerbils, or mice. The reference to non-human mammals can also include captive wild animals such as foxes or deerand can also include non-human primates. Typically, the mammal is a human or a non- human primate. More typically, the mammal is a human. Unless specified, methods and compositions according to the present invention are not limited to treatment of humans. In general, when treatment of humans is intended, the term “patient” can be used in place of “subject.”

[0058] As used herein, the terms “effective amount,” “therapeutically effective amount,” or other equivalent terminology refer to the amount of a compound or compounds or to the amount of a composition including such a compound or such compounds sufficient to effect beneficial or desired results. The beneficial or desired results are typically a reduction in severity, symptoms, or duration of a disease or condition being treated and can generally be characterized as an amount of a therapeutic agent or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The use of such terminology cannot, unless specifically indicated, be interpreted as implying a complete cure for any disease or condition as recited herein. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or administration route unless a particular formulation or administration route is specified. The effect induced by the administration of a therapeutically effective amount can be detected by, for example, chemical markers, antigen levels, or changes in pathological indicators such as tumor burden in the case of malignancies or other markers for other diseases or conditions. Therapeutic effects also can include subjective improvements in well-being, reduction of fatigue, or increased energy noted by the subjects or their caregivers. Therapeutic effects can also include reduction in cardiotoxicity. In the case of malignancies, therapeutic effects can include improvements in clinical parameters such as the Karnofsky performance score or other clinical parameters known in the art. The precise therapeutically effective amount for a subject will depend upon the subject’s size, weight, and health, the nature and extent of the condition affecting the subject, the administration of other therapeutic agents administered to treat the particular disease or condition being treated or other diseases or conditions affecting the subject, as well as variables such as liver and kidney function that affect the pharmacokinetics ofadministered therapeutics. Thus, it is not useful to specify an exact effective amount in advance for pharmaceutical compositions produced by methods according to the present invention. However, the therapeutically effective amount for a given situation can be determined by routine experimentation and is within the judgment of the clinician.

[0059] As used herein, the term “pharmaceutical composition” refers to the combination of one or more therapeutically active agents with at least one carrier or excipient, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. Typically, in the context of the present invention, the therapeutically active agent is bisantrene, or a pharmaceutically acceptable salt of bisantrene. A particularly suitable pharmaceutically acceptable salt of bisantrene is bisantrene dihydrochloride, but other pharmaceutically acceptable salts of bisantrene exist. As used herein with respect to bisantrene or a pharmaceutically acceptable salt of bisantrene, the term “therapeutically active agent” includes both agents for treatment of a disease or condition, wherein the disease or condition includes both malignancy and other disease or conditions treatable by administration of bisantrene or a pharmaceutically acceptable salt of bisantrene, and agents that are cardioprotective or reduce cardiotoxicity.

[0060] As used herein, the term “stock solution” refers to a solution containing one or more components of the eventual pharmaceutical composition produced by a method according to the present invention. The stock solution can be subject to various operations such as pH adjustment in the course of the method. Depending on the details of a method according to the present invention as described herein, one or more stock solutions can be prepared. The one or more stock solutions are used in the preparation of the pharmaceutical composition according to one or more alternatives as described in the present invention.

[0061] As used herein, the terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions, or components included in compositions, that do not substantially produce adverse reactions, such as, but not limited to, toxic, allergic, or unwanted immunological reactions, whenadministered to a subject and also that do not substantially interact in a deleterious manner with any of the other components on the composition in which it is included.

[0062] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, water, emulsions, such as oil / water or water / oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants such as potato starch or sodium starch glycolate), and the like. The carriers also can include stabilizers and preservatives. Suitable pharmaceutically acceptable carriers are described below. As used herein, the term “carrier” can include any and all solvents, dispersion media, vehicles, coatings, diluents, bulking agents, carrier solutions, suspensions, colloids, and forming and binding agents, any or all of which may include other pharmaceutical excipients as generally known in the art, including lubricants, antibacterial and antifungal agents, isotonic or absorption-delaying agents, buffers, antioxidants, other stabilizers including physical stabilizers such as thickeners or viscosity enhancers, coloring agents, flavoring or sweetening agents, and the like. The use of such media and agents with therapeutically active agents is well known in the art. Except insofar as any conventional medium or agent is incompatible with any therapeutically active agent or with any other medium or agent whose use is contemplated, its use in pharmaceutical compositions as described herein is contemplated.

[0063] As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound that is used in a method of the present invention or is a component of a pharmaceutical composition of the present invention, which, upon administration to a subject, is capable of providing a therapeutically active compound as described in the present application, including bisantrene or another therapeutically active compound as described below, or an active metabolite or residue thereof. As is known to those of skill in the art, salts of the therapeutically active compounds described herein may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and other acids known in the art as suitable for formation of pharmaceutically acceptable salts. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Examples of bases include, but are not limited to, alkali metals (such as sodium or potassium) hydroxides, alkaline earth metals (such as calcium or magnesium), hydroxides, ammonia, and compounds of formula NW4+, wherein W is C1- C4 alkyl, and the like. Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, NH4+, and NW4+, wherein W is a C1-C4 alkyl group), and the like. For therapeutic use, salts of the compounds herein are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0064] The following description of analogs and derivatives applies with respect only to potentially active analogs and derivatives of cyclodextrins or ^-hydroxy acids as included in pharmaceutical compositions prepared by methods as described herein. As used herein, “analog” refers to a chemical compound that is structurally similar to a parent compound, but differs slightly in composition (e.g., one atom or functional group is different, added, or removed). The analogue may or may not have different chemical or physical properties than the original compound and may or may not have improved biological and / or chemical activity or physical properties. For example, the analogue may be more hydrophilic or hydrophobic or it may have altered reactivity as comparedto the parent compound. The analogue may mimic the chemical and / or biological activity of the parent compound (i.e., it may have similar or identical activity), or, in some cases, may have increased or decreased activity. The analogue may be a naturally or non-naturally occurring variant of the original compound. Other types of analogues include isomers (enantiomers, diastereomers, and the like) and other types of chiral variants of a compound, as well as structural isomers. As used herein, “derivative” refers to a chemically or biologically modified version of a chemical compound that is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound. A “derivative” differs from an “analog” in that a parent compound may be the starting material to generate a “derivative,” whereas the parent compound may not necessarily be used as the starting material to generate an “analog.” A derivative may or may not have different chemical or physical properties than the parent compound. For example, the derivative may be more hydrophilic or hydrophobic or it may have altered reactivity as compared to the parent compound. Derivatization (i.e., modification) may involve substitution of one or more moieties within the molecule (e.g., a change in functional group). The analogs or derivatives can include functional groups as described below but are not limited to inclusion of such functional groups. Definitions for such functional groups are known in the art. Such functional groups include, but are not limited to, alkyl, carbocyclyl, cycloalkyl, heteroalkyl, heteroalkylenyl, haloalkyl, aryl, heterocyclyl, non-aromatic heterocyclyl, heteroaryl, hydroxyaryl, ester, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, amino, amido, acyl, arylalkyl, heteroarylalkyl, alkanoyl, alkoxy, sulfo, sulfamoyl, sulfobutyl, carboxyl, carbamoyl, monoaminoalkyl, dialkylaminoalkyl, alkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, alkyloxycarbonyl, aryloxycarbonyl, aryloxyalkylcarbonyl, thiocarbonyl, and alkylidene functional groups. In particular, the term “sulfobutyl” refers to a (–(CH2)4–SO3−) substituent, wherein the hydrogens of the CH2 moieties can be optionally substituted; this is relevant with respect to the cyclodextrins described below which can be substituted with sulfobutyl moieties.

[0065] As used herein, the term “solvate” means a compound formed by solvation (the combination of solvent molecules with molecules or ions of the solute), or an aggregate that consists of a solute ion or molecule, i.e., a compound used in amethod according to the present invention or a product of such a method, with one or more solvent molecules. The term “solvate” typically means a physical association of a compound involving varying degrees of ionic and / or covalent bonding, including hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example when one or more solvent atoms are incorporated into the crystal lattice of the crystalline solid. The term “solvate” encompasses both solution-phase and isolatable solvates. Suitable solvates in which the solvent is other than water include, but are not limited to, ethanolates or methanolates. When water is the solvent, the corresponding solvate is a “hydrate.” Examples of hydrates include, but are not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, hexahydrate, and other hydrated forms. It should be understood by one of ordinary skill in the art that the pharmaceutically acceptable salts and / or prodrugs of compounds described herein for use in methods for preparation or compositions according to the present invention may also exist in a solvate form. When the solvate is a hydrate, the hydrate is typically formed via hydration which is either part of the preparation of the original compound or through natural absorption of moisture by the original anhydrous compound. Additionally, compounds may exist as clathrates or other complexes, which are therapeutic agent-host inclusion complexes wherein the therapeutic agent and the host are present in stoichiometric or non-stoichiometric amounts.

[0066] Certain compounds described herein for use in methods and compositions produced by methods according to the present invention possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids or certain other compounds wherein the use of such terminology for stereoisomers is conventional, and individual isomers are encompassed within the scope of the present invention unless specific isomers are excluded. The present disclosure is meant to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L) -isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, andunless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. The term “tautomer,” as used herein, refers to one of two or more structural isomers that exist in equilibrium and that are readily converted from one form to another. Examples of tautomerism include, but are not limited to, keto-enol tautomerism, enamine-imine tautomerism, and lactam-lactim tautomerism. Unless one of the tautomeric alternatives is expressly excluded, all such tautomeric forms are intended to be within the scope of the invention. For example, with respect to stereoisomerism, for certain ^-hydroxy acids that are ingredients in methods for preparing formulations of bisantrene according to the present invention, tartaric acid exists in the following forms: D-tartaric acid, L-tartaric acid, meso-tartaric acid, and a racemic mixture of D-tartaric acid and L-tartaric acid. Lactic acid exists in the enantiomersL-lactic acid andD-lactic acid as well as in the racemic formDL-lactic acid. Mandelic acid exists in the enantiomers (+)-mandelic acid and (-)-mandelic acid as well as in the racemic form. Malic acid exists in the enantiomers L-malic acid and D-malic acid as well as in the racemic form.

[0067] Furthermore, in methods according to the present invention, small molecules used in such methods, such as cyclodextrins or ^-hydroxy acids, can be substituted by analogs or derivatives thereof in which the analogs or derivatives include substitutions that are considered to be bioisosteric. Such analogs or derivatives of cyclodextrins or ^-hydroxy acids are within the scope of the present invention, provided that such analogs or derivatives do not significantly alter the pharmacological or physical properties of the cyclodextrins or ^-hydroxy acids or interfere with the pharmacological activity of the bisantrene, particularly bisantrene dihydrochloride, included in pharmaceutical compositions whose preparation is described in the reference. Bioisosterism is a well-known tool for predicting the biological activity of compounds, based on the premise that compounds with similar size, shape, and electron density can have similar biological activity. To form a bioisostere of a given molecule, one can replace one or more atoms or groups in the original molecule with known bioisosteric replacements for that atom or group. Known bioisosteric replacements include, but are not necessarily limited to, the interchangeability of –F, -- OH, --NH2, --Cl, and –CH3, the interchangeability of –Br and -i-C3H7; theinterchangeability of –I and -t-C4H9; the interchangeability of –O--, --S--, --NH2--, --CH2-- , and –Se--; the interchangeability of –N=, --CH=, and –P= in cyclic or noncyclic moieties; the interchangeability of phenyl and pyridyl groups; the interchangeability of – C=C- and –S— (for example, benzene and thiophene); the interchangeability of an aromatic nitrogen (R1-N(R3)-R2) for an unsaturated carbon ((R1-C(=R3)-R2); and the interchangeability of –CO--, --SO--, and –SO2--. Other alternatives for bioisosteric replacements are known in the art.

[0068] The major dose-limiting toxic effect of bisantrene is leukopenia (Von Hoff et al.1981b; Alberts et al.1982, supra; Spiegel et al.1982, supra; Yap et al 1982, supra)). On a schedule of dosing once every 3 to 4 weeks, the nadir for myelosuppression was 9 days with recovery by 19 days (Von Hoff et al.1981b). Thrombocytopenia was mild, although bisantrene can reportedly inhibit platelet aggregation (M.E. Rybak et al., “The Effects of Bisantrene on Human Platelets,” Invest. New Drugs 4: 119-125 (1986)). Anemia and cumulative myelosuppressive toxic effects were not encountered with bisantrene.

[0069] In addition to myelosuppression, bisantrene produced severe phlebitis along peripheral veins used for drug infusion (Von Hoff et al.1981b; Alberts et al.1982). This resulted from drug precipitation in veins, which has been documented in experimental models (G. Powis & J.S. Kovach 1983). The drug is a potent vesicant and produces severe local tissue necrosis if inadvertently extravasated (Von Hoff et al 1981b). Severe arm swelling, hyperpigmented veins, and punctate perivenous orange discolorations have been occasionally observed following bisantrene infusions given via peripheral veins. The arm swelling appeared to be the result of a localized capillary leak syndrome in the arm used for infusion. In an experimental mouse skin model, extravasation necrosis was blocked with a local injection of sodium bicarbonate (R.T. Dorr et al., “Bisantrene Solubility and Skin Toxicity Studies: Effect of Sodium Bicarbonate as a Local Ulceration Antidote,” Invest. New Drugs 2: 351-357 (1984)). The occurrence of phlebitis is one of the major reasons for the preparation of the pharmaceutical compositions described herein, as the pharmaceutical compositions of the present invention enable bisantrene to be administered via peripheral veins with a low risk of phlebitis.

[0070] Various formulations for administering bisantrene or derivatives or analogs thereof are known in the art. United States Patent No.4,784,845 to Desai et al. discloses a composition for delivery of a hydrophobic drug (i.e., bisantrene or a derivative or analog thereof) comprising: (i) the hydrophobic drug; (ii) an oleaginous vehicle or oil phase that is substantially free of butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT); (iii) a co-surfactant or emulsifier; (iv) a co-surfactant or auxiliary emulsifier; and (v) benzyl alcohol as a co-solvent. United States Patent No. 4,816,247 by Desai et al. discloses a composition for delivery by intravenous, intramuscular, or intraarticular routes of hydrophobic drugs (such as bisantrene or a derivative or analog thereof) comprising: (i) the hydrophobic drug; (ii) a pharmaceutically acceptable oleaginous vehicle or oil selected from the group consisting of: (a) naturally occurring vegetable oils and (b) semisynthetic mono-, di-, and triglycerides, wherein the oleaginous vehicle or oil is free of BHT or BHA; (iii) a surfactant or emulsifier; (iv) a co-surfactant or emulsifier; (v) an ion-pair former selected from C6-C20 saturated or unsaturated aliphatic acids when the hydrophobic drug is basic and a pharmaceutically acceptable aromatic amine when the hydrophobic drug is acidic; and (vi) water. United States Patent No.5,000,886 to Lawter et al. and United States Patent No.5,143,661 to Lawter et al. disclose compositions for delivery of pharmaceutical agents such as bisantrene or a derivative or analog thereof comprising a microcapsule, wherein the microcapsule includes a hardening agent that is a volatile silicone fluid. United States Patent No.5,070,082 to Murdock et al., United States Patent No.5,077,282 to Murdock et al., and United States Patent No.5,077,283 to Murdock et al. disclose prodrug forms of poorly soluble hydrophobic drugs, including bisantrene and derivatives and analogs, that are functionalized with phosphoramidic acid. United States Patent No.5,116,827 to Murdock et al. and United States Patent No. 5,212,291 to Murdock et al. disclose prodrug forms of poorly soluble hydrophobic drugs, including bisantrene and derivatives and analogs, that are quinolinecarboxylic acid derivatives. United States Patent No.5,378,456 to Tsou discloses compositions containing an anthracene antitumor agent, such as bisantrene or a derivative or analog thereof, in which the bisantrene or derivative or analog thereof is conjugated to or admixed with a divinyl ether-maleic acid (MVE) copolymer. United States Patent No.5,609,867 to Tsou discloses polymeric 1,4-bis derivatives of bisantrene and copolymers of bisantrene and another monomer, such as a dianhydride.

[0071] In certain formulations previously known in the art, bisantrene dihydrochloride for injection was produced by combining solid bisantrene dihydrochloride with sterile water for injection at a concentration of about 40 mg / mL. The resulting mixture was filtered through a 5-^m first filter, then through a 1.2 µm second filter, and finally through an 0.2-^m third filter in order to produce a 40 mg / mL bisantrene solution. Other filtration regimens could also be used for such alternative formulations previously known in the art. Removal of particulates at this stage does not diminish the bisantrene dihydrochloride content in solution, possibly because the insoluble material is a poorly soluble form of bisantrene or bisantrene dihydrochloride that is removed in HPLC prefilters when the bisantrene content is analyzed by HPLC, and therefore never assayed either before or after filtration. The resulting particle-free bisantrene dihydrochloride solution (6.25 mL) is filled into 10-mL vials and lyophilized. Finished vials are sealed under nitrogen and partial vacuum for storage. Storage of the vials is typically at 18^C to 25^C.

[0072] In formulations previously known in the art, bisantrene dihydrochloride lyophilized powder, when reconstituted, contains particulates. Although not wishing to be bound by this hypothesis, it is likely that the particulates are microcrystalline forms with low dissolution rates. The source of these particles may be the freezing step of the lyophilization process. During the freezing step, low temperature induced crystallization may be occurring and possibly in concert with nucleation sites on the surfaces of the manufacturing equipment and / or vials. The occurrence of particulates is substantially reduced in formulations produced by methods according to the present invention.

[0073] When bisantrene dihydrochloride formulations previously known in the art are reconstituted and injected into an i.v. bag, analytical results have demonstrated the following. (1) The assay of fully diluted bisantrene dihydrochloride in the i.v. bag was approximately 5% lower than the assay of the reconstituted bisantrene dihydrochloride in the finished vial when samples were collected in glass vials; however, when samples were collected in plastic vials, there was no reduction in bisantrene dihydrochloride concentration. (2) The assay of fully diluted bisantrene dihydrochloride in the i.v. bagwhen passed through an 0.2-^m filter was approximately 5% lower than the i.v. bag assay when sample was collected in glass vials for analysis. (3) The assay of fully diluted bisantrene dihydrochloride in the i.v. bag when passed through an 0.2-^m filter approximately matched the i.v. bag assay when sample was collected in plastic vials (rather than glass vials). (4) These results suggest that bisantrene or bisantrene dihydrochloride adheres to glass. However, these results were obtained with formulations previously known in the art and the occurrence of such reductions in concentrations as determined by assays and the likelihood of adherence of the bisantrene dihydrochloride to glass is greatly reduced in formulations according to the present invention, and produced by methods according to the present invention.

[0074] For formulations previously known in the art, reconstituted bisantrene dihydrochloride formulations can be cleared of particulates by initial filtration of reconstituted bisantrene dihydrochloride through an 0.2-^m syringe filter while injecting the formulation into an i.v. infusion vehicle for administration to a patient. Additional safety regarding particulates is achieved using an i.v. infusion set equipped with an in- line 0.2-^m filter. Without pre-filtration, reconstituted and diluted bisantrene dihydrochloride formulations have the tendency to block 0.2-^m i.v. infusion filters. However, additional filtration steps were found to be required for formulations previously known in the art, which could only be administered by central line infusion. Minimizing or eliminating phlebitis at peripheral vein i.v. infusion sites would make bisantrene safer, more acceptable to patients, and reduce treatment costs. Similarly, eliminating the need for central line infusion would make bisantrene safer, more acceptable to patients, and reduce treatment costs.

[0075] The present application is therefore directed to new bisantrene formulations that improve the blood solubility of bisantrene, particularly bisantrene dihydrochloride, and that also would enable the administration of bisantrene through more commonly practiced peripheral vein infusions with a reduced risk of phlebitis or other significant side effects caused by peripheral vein infusion, as compared to the risk associated with administration of bisantrene formulations previously known in the art. The present invention is also directed to methods for the preparation and packaging of such formulations, as well as methods of treatment comprising the administration ofsuch formulations, uses of such formulations for the manufacture of medicaments for treating conditions indicating administration of bisantrene, and such formulations for use in the treatment of conditions indicating administration of bisantrene.

[0076] Pharmaceutical Compositions Comprising Bisantrene

[0077] One aspect of the present invention is a pharmaceutical composition in one or more dosage units comprising: (1) bisantrene; (2) a cyclodextrin; and (3) optionally, an ^-hydroxy acid.

[0078] The bisantrene can be in salt form or in free base form. When the bisantrene is in salt form, the bisantrene salt is typically bisantrene dihydrochloride. However, other salts of bisantrene can alternatively be used, including, but not limited to, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propionates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, ^-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates, as well as salts with other negatively- charged counterions. When a salt other than the dihydrochloride salt of bisantrene is used, the quantity of the salt used in a pharmaceutical composition must be adjusted to account for the formula weight of the counterion.

[0079] Typically, the cyclodextrin is ^-cyclodextrin, ^-cyclodextrin, ^-cyclodextrin, a sulfobutyl ether ^-cyclodextrin (SBECD), 2-hydroxypropyl-^-cyclodextrin (HPBCD), 2- hydroxypropyl-^-cyclodextrin (HPGCD), or a randomly methylated ^-cyclodextrin (RMBCD). Alternatively, derivatives and analogs of these cyclodextrins can be used. Preferably, the cyclodextrin is ^-cyclodextrin, an SBECD, or HPBCD. More preferably, the cyclodextrin is an SBECD. Typically, when the cyclodextrin is an SBECD, typically,the SBECD comprises from 1 to 7 sulfobutyl ether moieties. Preferably, the SBECD comprises from 1 to 2 sulfobutyl ether moieties, from 3 to 4 sulfobutyl ether moieties, or from 5 to 7 sulfobutyl ether moieties. Typically, when the cyclodextrin is an SBECD the sulfonic acid groups of the SBECD sulfobutyl ether moieties are in acid form, salt form, or a mixture of acid form and salt form. Typically, when the sulfonic acid groups of the SBECD sulfobutyl either moieties are in either salt form or a mixture of acid form and salt form, the counterions of the sulfobutyl ether moieties that are in salt form are Li+, Na+, K+, ammonium, or another pharmaceutically acceptable cation. Preferably, when the sulfonic acid groups of the SBECD sulfobutyl either moieties are in either salt form or a mixture of acid form and salt form, the counterions of the sulfobutyl ether moieties that are in salt form are Na+. Alternatively, other cyclodextrins compatible with administration of bisantrene can be used, including derivatives and analogs of the cyclodextrins recited above.

[0080] Typically, the ^-hydroxy acid is glycolic acid, lactic acid, mandelic acid, tartaric acid, malic acid, citric acid, or any stereoisomers thereof. Lactic acid has two stereoisomers, L-lactic acid and D-lactic acid. Mandelic acid also has two stereoisomers, (R)-mandelic acid and (S)-mandelic acid. Tartaric acid has three stereoisomers, (2R,3R)-tartaric acid (L-tartaric acid (the naturally occurring form), its enantiomer, (2S,3S)-tartaric acid (D-tartaric acid), and the meso form, (2R,3S)-tartaric acid or (2S,3R)-tartaric acid. Tartaric acid may also occur in a racemic form that includes (2R,3R)-tartaric acid and (2S,3S)-tartaric acid. Malic acid has two stereoisomers, L- malic acid andD-malic acid. Preferably, the ^-hydroxy acid is lactic acid, tartaric acid, or malic acid. More preferably, the ^-hydroxy acid is tartaric acid. When the ^-hydroxy acid is tartaric acid, typically, the tartaric acid is D-tartaric acid, L-tartaric acid, meso-tartaric acid, or a racemic mixture of D-tartaric acid and L-tartaric acid. When the ^-hydroxy acid is tartaric acid, preferably the tartaric acid isL-tartaric acid. However, other pharmaceutically compatible ^-hydroxy acids known in the art can alternatively be used, including derivatives and analogs of the ^-hydroxy acids recited above. In most embodiments, the use of the ^-hydroxy acid is preferable.

[0081] Typically, the molar ratio of the bisantrene to the cyclodextrin is from about 1:1 to about 1:6. Preferably, the molar ratio of the bisantrene to the cyclodextrin isfrom about 1:2 to about 1:5. Still more preferably, the molar ratio of the bisantrene to the cyclodextrin is from about 1:2.5 to about 1:4.5. Still even more preferably, the molar ratio of the bisantrene to the cyclodextrin is about 1:2.5, about 1:3, about 1:3.5, or about 1.4.

[0082] Typically, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:5. Preferably, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:4. More preferably, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:3. Still more preferably, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:2. Yet more preferably, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:1.5. Even more preferably, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.75 to about 1:1.25.

[0083] In another embodiment, the pharmaceutical composition comprises bisantrene and a cyclodextrin as described above. When the pharmaceutical composition comprises bisantrene and a cyclodextrin, suitable cyclodextrins are as described above.

[0084] Typically, the composition is a solution, a suspension, a lyophilized powder or cake, or a spray-dried powder, wherein the lyophilized powder or cake or the spray-dried powder is suitable for reconstitution with water prior to use. Typically, the pH of the solution, the pH of the suspension, the pH of the lyophilized powder or cake on reconstitution with water, or the pH of the spray-dried powder on reconstitution with water is from about 2.0 to about 6.0. Preferably, the pH of the solution, the pH of the suspension, the pH of the lyophilized powder or cake on reconstitution with water, or the pH of the spray-dried powder on reconstitution with water is from about 2.5 to about 6.0. More preferably, the pH of the solution, the pH of the suspension, the pH of the lyophilized powder or cake on reconstitution with water, or the pH of the spray-dried powder on reconstitution with water is from about 3.0 to about 6.0. Still more preferably, the pH of the solution, the pH of the suspension, the pH of the lyophilized powder or cake on reconstitution with water, or the pH of the spray-dried powder on reconstitution with water is from about 3.5 to about 6.0, such as from about 4.0 to about 6.0, such as from about 4.5 to about 6.0, from about 5.0 to about 6.0, from about 5.0 to about 5.5,from about 5.25 to about 5.75, from about 5.5 to about 6.0, from about 4.0 to about 5.5, from about 4.0 to about 5.0. from about 4.5 to about 5.5, from about 4.5 to about 5.0, about 4.0, about 4.5, about 5.0, about 5.5, or about 6.0.

[0085] In certain embodiments of pharmaceutical compositions according to the invention, the hydrazone linkages of bisantrene are both in the trans (E) orientation, as illustrated by compound 1 in the scheme below. In other embodiments of pharmaceutical compositions according to the present invention, one or both of the hydrazone linkages in bisantrene are in the cis (Z) orientation, as illustrated by compounds 2 and 3.

[0086] As described in co-pending and co-owned PCT application titled “ISOMERIC FORMS OF BISANTRENE” filed on 12 September 2025, herein incorporated in its entirety by cross-reference, the all-trans (E,E) version of bisantrene, illustrated by compound 1 ((E,E)-bisantrene), is photosensitive and will partially convert to compound 2 and, to a much lesser extent, compound 3 when exposed to ambient light, especially blue or purple light. This effect is observed primarily in the solution phase. This conversion (also described as isomerization or photoisomerism) is to be prevented or at least minimized. The cis,trans (E,Z) version of bisantrene illustrated by compound 2 ((E,Z)-bisantrene) is also photosensitive and will partially convert to compound 1 and, to a lesser extent, compound 3 ((Z,Z)-bisantrene) when exposed to ambient light, especially blue or purple light. This effect is also observed primarily in the solution phase. Therefore, care must be taken in the preparation, storage, and use of the compositions, especially solutions of the present invention to prevent exposure toambient light if conversion between isomers is to be avoided. Strategies for protection from light include, but are not limited to, wrapping vessels containing bisantrene solutions in an opaque covering (e.g. aluminum foil) or at least a covering opaque to blue and purple light, preparing the bisantrene formulation or components thereof under red or yellow light, use of amber glass vials, and covering prepared dosage units in amber plastic sheaths during infusion. Therefore, protection from ambient light, especially blue or purple light, is an element of some embodiments of the present invention.

[0087] In particular embodiments of all and any pharmaceutical compositions according to the present invention, greater than 60% of the bisantrene in the pharmaceutical composition is present as compound 1, such as greater than about 70%, or greater than 80%, or greater than 90%, or greater than 95%, or greater than 96%, or greater than 97%, or greater than 98%, or greater than 99%. In all embodiments of pharmaceutical compositions according to the present invention where the bisantrene is not 100% compound 1, the balance is made of primarily compound 2 and, to a lesser extent, of compound 3.

[0088] In particular embodiments, the form of bisantrene used for producing the pharmaceutical composition is the dihydrochloride salt in crystalline form, preferably as compound 1, having the following properties: (a) a weight loss of up to 10% up to 150^C in thermogravimetric analysis (TGA); (b) a sharp endothermic peak at between 95^C and 115^C in differential scanning calorimetry (DSC); and (c) predominant peaks at 2^ of about 9.3^ and 14.0^ in X-ray powder diffraction (XRPD). As described in co-pending and co-owned PCT application titled “PROCESS FOR MANUFACTURING HIGH PURITY (E,E)-BISANTRENE DIHYDROCHLORIDE WITH MINIMIZATION OF ISOMER FORMATION FIELD OF THE INVENTION” filed on 12 September 2025, herein incorporated in its entirety by cross-reference, this particular crystalline form has been found to be particularly stable and quicker to dissolve, facilitating processing and handling.

[0089] Typically, the composition is packaged in vials. In one embodiment, the vials are plastic vials. In another embodiment, the vials are silanized or unsilanized glass vials. The silanized or unsilanized glass vials can be clear or amber glass. Whenthe vials are silanized, typically, silanization is performed by coating the interior of the vials with an organofunctional alkoxysilane that can be (3-aminopropyl)-triethoxysilane, (3-aminopropyl)-diethoxymethylsilane, (3-aminopropyl)-dimethyl-ethoxysilane, (3- aminopropyl)-trimethoxysilane, (3-glycidoxypropyl)-dimethyl-ethoxysilane, (3- mercaptopropyl)-trimethoxysilane, (3-mercaptopropyl)-methyl dimethoxysilane, or derivatives thereof. When the vials are plastic vials, typically, the plastic vials are constructed of a plastic that can be cyclic olefin polymer (COP) plastic, cyclic olefin copolymer (COC) plastic, high-density polyethylene plastic, and high-density non- nucleated polypropylene plastic. Other plastics known in the art that are compatible with the bisantrene and with any carriers included in the pharmaceutical composition can alternatively be used.

[0090] Pharmaceutically acceptable excipients may be added to facilitate manufacture, enhance stability, control release, enhance product characteristics, enhance bioavailability, drug absorption or solubility, optimize other pharmacokinetic considerations, optimize the pharmaceutical formulation for a route of administration, enhance patient acceptability, or for another reason related to manufacture, storage, or use of a pharmaceutical composition.

[0091] Typically, the pharmaceutically acceptable excipient(s) is / are one or more of: acidifying agents; alkalizing agents; antimicrobial preservatives; antioxidants; buffering agents or pH-adjusting agents; chelating agents; complexing agents. Excipients used in pharmaceutical compositions according to the present invention are compatible with the pharmaceutically active agent or agents included in the pharmaceutical composition, are compatible with other excipients included in the pharmaceutical composition, and are not injurious to and are tolerated by any patients to whom the pharmaceutical composition is administered. The particular excipient or excipients in any pharmaceutical composition according to the present invention can be varied according to such factors as the intended route of administration of the pharmaceutical composition, the quantity of bisantrene, ^-hydroxy acid if present, and cyclodextrin in a unit dose of the composition, and other factors generally understood in the art. Excipients for a pharmaceutical composition according to the present invention are selected such that they do not interfere with the activity of the bisantrene that isincluded in the pharmaceutical composition. Excipients for a pharmaceutical composition according to the present invention are also selected so that they do not interfere with the activity of other excipients or cause phase separation in the composition. The quantities of any excipient included in a composition according to the present invention can be determined by one of ordinary skill in the art in order to ensure suitable physical properties of the composition and also in order to ensure suitable pharmacokinetics for the bisantrene included in the composition. As is generally known in the art of pharmaceutical formulation, a particular excipient can fulfill one or more of these functions in a particular pharmaceutical composition, depending on the concentration of the excipient, the other excipients in the composition, the physical form of the composition, the concentration of active agent in the composition, the intended route of administration of the composition, and other factors. The recitation of a particular excipient in a category below is not intended to exclude the possible use of the excipient in another category or categories.

[0092] Typically, the acidifying agent is acetic acid, citric acid, fumaric acid, hydrochloric acid, diluted hydrochloric acid, malic acid, nitric acid, phosphoric acid, diluted phosphoric acid, sulfuric acid, or tartaric acid. Other acidifying agents known in the art can be used.

[0093] Typically, the alkalizing agent is strong ammonia solution, ammonium carbonate, diethanolamine, diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, sodium hydroxide, or trolamine. Other alkalizing agents known in the art can be used.

[0094] Typically, the buffering agent or pH-adjusting agent is acetic acid, ammonium carbonate, ammonium phosphate, hydrochloric acid, sulphuric acid, boric acid, citric acid, lactic acid, phosphoric acid, potassium citrate, potassium metaphosphate, potassium phosphate monobasic, sodium acetate, sodium citrate, sodium lactate solution, dibasic sodium phosphate, monobasic sodium phosphate, sodium bicarbonate, sodium hydroxide. The selection of a suitable buffering agent or pH-adjusting agent depends on the desired pH and on the presence and quantity of acidic or basic components in the composition. Other buffering agents or pH-adjusting agents known in the art can be used.

[0095] Typically, sterile water is used as a vehicle for compositions according to the invention, such as for reconstitution of lyophilizates or spray-dried compositions of the present invention for injection.

[0096] Methods for Preparing Pharmaceutical Compositions Comprising Bisantrene

[0097] Another aspect of the present invention is a method for preparing a pharmaceutical composition of bisantrene wherein the pharmaceutical composition comprises: (i) bisantrene; and (ii) a cyclodextrin, wherein the method comprises the steps of: (1) preparing a stock solution of a cyclodextrin in water; (2) combining a stock solution of bisantrene with the stock solution of the cyclodextrin from step (1) to produce a combined stock solution of the bisantrene and the cyclodextrin; (3) adjusting the pH of the combined stock solution of the bisantrene and the cyclodextrin from step (2); and (4) optionally filling the pH-adjusted stock solution of the bisantrene and the cyclodextrin from step (3) into vials.

[0098] A more specific aspect of the present invention is a method for preparing a pharmaceutical composition of bisantrene, wherein the pharmaceutical composition comprises: (i) bisantrene; (ii) a cyclodextrin; and (iii) an ^-hydroxy acid, wherein the method comprises the steps of: (a) preparing a stock solution of a cyclodextrin and an ^-hydroxy acid in water; (b) combining a stock solution of bisantrene with the stock solution of the cyclodextrin and the ^-hydroxy acid from step (a) to produce a combined stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid; and (c) adjusting the pH of the combined stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid from step (b).

[0099] The stock solution of a cyclodextrin and an ^-hydroxy acid in water may be prepared by: a) dissolving cyclodextrin in solid form with an ^-hydroxy acid in solid form in water; b) combining a cyclodextrin in solid form with an ^-hydroxy acid in solid form and then dissolving the resulting solid mixture in water; c) preparing a stocksolution of an ^-hydroxy acid in water and dissolving the cyclodextrin into the stock solution of the ^-hydroxy acid; or d) preparing a stock solution of a cyclodextrin in water and dissolving the ^-hydroxy acid into the stock solution of the cyclodextrin.

[0100] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the pH-adjusted combined solution of bisantrene, cyclodextrin and α-hydroxy acid, especially prior to packaging into vials (if this is to be done) may be sterile filtered. The step of sterile filtration may comprise filtration through 1 to 3 filters. In one embodiment, the step of sterile filtration is filtration through 1 filter; typically, in this embodiment, the filter has a filtration cutoff of about 0.2 ^m. In another embodiment, the step of sterile filtration is filtration through 2 filters; typically, in this embodiment, at least one of the two filters has a filtration cutoff of about 0.2 ^m. In yet another embodiment, the step of sterile filtration is filtration through 3 filters; typically, in this embodiment, one of the three filters has a filtration cutoff of about 0.45 ^m and at least two of the three filters each have a filtration cutoff of about 0.2 ^m. In this arrangement of the three filters, typically, the three filters are ordered such that the pH-adjusted stock solution of the bisantrene and the cyclodextrin first passes through a filter with a filtration cutoff of about 0.45 ^m and then passes through two filters each with a filtration cutoff of about 0.2 ^m. The three filters can be provided as three separate filter units. In another embodiment, at least two of the three filters are contained in sequence in a single filter unit. When at least two of the three filters are contained in sequence in a single filter unit, typically, the filter having a filtration cutoff of about 0.45 ^m and the first of two filters each having a filtration cutoff of about 0.2 ^m are contained in sequence in a single filter unit. In still another alternative, when three filters are used as described above, the three filters can be contained in sequence in a single filter unit. In another alternative, when two 0.2 ^m filters are used, they can be contained in a single filter unit.

[0101] Unlike with previously known bisantrene formulations, the requirements for additional filtration steps for removal of particulates are eliminated for formulations produced by methods according to the present invention. When compositions prepared by methods according to the present invention are administered, no additional filtrationsteps would be required and patient safety is ensured without such additional filtration steps.

[0102] An additional advantage of formulations produced by methods according to the present invention, is that pre-filtration of a stock bisantrene dihydrochloride solution according to the invention (formulated with cyclodextrin and ^-hydroxy acid) eliminates particulates prior to the lyophilization process.

[0103] The method of preparation may comprise placing the pharmaceutical composition in vials. In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the vials used are plastic vials. In other embodiments, the vials used are silanized or unsilanized glass vials. The silanized or unsilanized glass vials are typically clear or amber glass; however, glass with other colors or tints can be used. Preferably, when glass vials are used, amber vials are used in order to ensure photostability upon exposure to ambient light and to prevent the occurrence of photoisomerization, particularly after reconstitution with water for injection in a pharmacy. When the vials are silanized, typically, silanization is performed by coating the interior of the vials with an organofunctional alkoxysilane selected from the group consisting of (3-aminopropyl)- triethoxysilane, (3-aminopropyl)-diethoxymethylsilane, (3-aminopropyl)-dimethyl- ethoxysilane, (3-aminopropyl)-trimethoxysilane, (3-glycidoxypropyl)-dimethyl- ethoxysilane, (3-mercaptopropyl)-trimethoxysilane, (3-mercaptopropyl)-methyl dimethoxysilane, and derivatives thereof; however, other organofunctional alkoxysilanes are known in the art and can also be used. When the vials are plastic vials, typically, the plastic vials are constructed of a plastic selected from the group consisting of cyclic olefin polymer (COP) plastic, cyclic olefin copolymer (COC) plastic, high-density polyethylene plastic, and high-density non-nucleated polypropylene plastic; however, other types of plastic that are compatible with the ingredients of the composition and do not cause nucleation, aggregation, or precipitation can be used.

[0104] The method of preparation may comprise a step of lyophilizing the combined stock solution.

[0105] The resulting pharmaceutical composition of bisantrene may be a solution or a lyophilized powder or cake, wherein the lyophilized powder or cake is suitable for reconstitution with water prior to use.

[0106] The step of filling the pH-adjusted combined stock solution into vials may further comprise a step of lyophilizing the stock solution that is filled into the vials. When the stock solution is not filled into vials, the step of lyophilizing the stock solution can be a bulk lyophilization. Alternatively, when the stock solution is not filled into vials, the stock solution can be spray-dried. In instances of bulk lyophilization or spray-drying, the resulting composition is filled into vials or other packaging after lyophilization or spray- drying is complete.

[0107] Alternatives for preparation of the bisantrene dihydrochloride lyophilized powder include: (i) preparation in plastic vials; (ii) preparation in glass vials; (iii) preparation at about 25 mg / mL; or (iv) preparation at about 40 mg / mL. Alternatively, as described below, preparation using methods according to the present invention as described herein can be done at any concentration from about 10 mg / mL to about 100 mg / mL, including, but not limited to, 10 mg / mL, such as of at least 20 mg / mL, or of at least 30 mg / mL, or of at least 40 mg / mL. or of at least 50 mg / mL. or of at least 60 mg / mL. or of at least 70 mg / mL. or of at least 80 mg / mL. or of at least 90 mg / mL. or of at least 100 mg / mL, or any value between these values.

[0108] Aqueous stock solutions of cyclodextrin, ^-hydroxy acid and / or bisantrene may be prepared at a temperature of from about 20^C to about 25^C. Other temperatures outside this range can alternatively be used. Typically, stock solutions of cyclodextrin, ^-hydroxy acid and / or bisantrene are prepared in sterile water for injection.

[0109] Stock solutions of cyclodextrin may be prepared at a concentration of from about 0.5 g / mL to about 2 g / mL. Preferably, the stock solution of the cyclodextrin is prepared at a concentration of from about 0.8 g / mL to about 1.2 g / mL.

[0110] Stock solutions of ^-hydroxy acid in water may be prepared at a concentration of from about 0.01 g / mL to about 0.1 g / mL. Preferably, stock solutions of ^-hydroxy acid are prepared at a concentration of from about 0.02 g / mL to about 0.05 g / mL.

[0111] Stock solutions of bisantrene may be prepared at a concentration of from about 10 mg / mL to about 100 mg / mL, calculated as bisantrene free-base, such as about 10 mg / mL, such as about 20mg / mL, or of about 30 mg / mL, or about 40 mg / mL. or about 50mg / mL. or about 60mg / mL. or about 70mg / mL. or about 80mg / mL. or about 90mg / mL. or about 100mg / mL, or any value between these values. Preferably, the stock solution of the bisantrene is prepared at a bisantrene free-base concentration of from about 25 mg / mL to about 45 mg / mL. In preferred embodiments, the stock solution of the bisantrene can be prepared at a bisantrene free-base concentration of from about 25 mg / mL to about 35 mg / mL or from about 35 mg / mL to about 45 mg / mL. In more preferred embodiments, the stock solution of the bisantrene can be prepared at a bisantrene free-base concentration of about 30 mg / mL or of about 75 mg / mL.

[0112] Bisantrene may be in salt form or in free base form. When the bisantrene is in salt form, the bisantrene salt is typically bisantrene dihydrochloride. However, other salts of bisantrene can alternatively be used, including, but not limited to, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propionates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, ^-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates, as well as salts with other negatively- charged counterions. When a salt of bisantrene is used, such as bisantrene dihydrochloride, the quantity of the salt used in a method according to the present invention must be adjusted to account for the formula weight of the counterion.

[0113] In certain embodiments of any and all aspects of the invention described herein, the hydrazone linkages of bisantrene are both in the trans (E) orientation, as described earlier. In particular embodiments of all and any methods of preparing pharmaceutical compositions according to the present invention, greater than 60% ofthe bisantrene in the pharmaceutical composition is present as compound 1, such as greater than about 70%, or greater than about 80%, or greater than about 90%, or greater than about 95%, or greater than about 96%, or greater than about 97%, or greater than about 98%, or greater than about 99% of compound 1. In all embodiments of pharmaceutical compositions according to the present invention where the bisantrene is not 100% compound 1, the balance is made of primarily compound 2 and, to a lesser extent, of compound 3.

[0114] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the form of bisantrene used for producing the pharmaceutical composition is the dihydrochloride salt in crystalline form, preferably as compound 1, having the following properties: (a) a weight loss of up to 10% up to 150^C in thermogravimetric analysis (TGA); (b) a sharp endothermic peak at between 95^C and 115^C in differential scanning calorimetry (DSC); and (c) predominant peaks at 2^ of about 9.3^ and 14.0^ in X-ray powder diffraction (XRPD).

[0115] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the cyclodextrin may be selected from the group consisting of ^-cyclodextrin, ^-cyclodextrin, ^-cyclodextrin, a sulfobutyl ether ^-cyclodextrin (SBECD), 2-hydroxypropyl-^-cyclodextrin (HPBCD), 2-hydroxypropyl-^- cyclodextrin (HPGCD), a randomly methylated ^-cyclodextrin (RMBCD), and analogs and derivatives thereof. Preferably, the cyclodextrin is selected from the group consisting of ^-cyclodextrin, an SBECD, and HPBCD. More preferably, the cyclodextrin is an SBECD. Typically, when the cyclodextrin is an SBECD, typically, the SBECD comprises from 1 to 7 sulfobutyl ether moieties. Preferably, the SBECD comprises from 1 to 2 sulfobutyl ether moieties, from 3 to 4 sulfobutyl ether moieties, or from 5 to 7 sulfobutyl ether moieties. Typically, when the cyclodextrin is an SBECD the sulfonic acid groups of the SBECD sulfobutyl ether moieties are in acid form, salt form, or a mixture of acid form and salt form. Typically, when the sulfonic acid groups of the SBECD sulfobutyl either moieties are in either salt form or a mixture of acid form and salt form, the counterions of the sulfobutyl ether moieties that are in salt form are selected from the group consisting of Li+, Na+, K+, ammonium, and another pharmaceutically acceptable cations. Preferably, when the sulfonic acid groups of the SBECD sulfobutyleither moieties are in either salt form or a mixture of acid form and salt form, the counterions of the sulfobutyl ether moieties that are in salt form are Na+.

[0116] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the ^-hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, mandelic acid, tartaric acid, malic acid, citric acid, and analogs and derivatives thereof. Preferably, the ^-hydroxy acid is selected from the group consisting of lactic acid, tartaric acid, and malic acid. More preferably, the ^-hydroxy acid is tartaric acid. When the ^-hydroxy acid is tartaric acid, typically, the tartaric acid is selected from the group consisting ofD-tartaric acid,L- tartaric acid, meso-tartaric acid, and a racemic mixture of D-tartaric acid and L-tartaric acid. Preferably, the tartaric acid is L-tartaric acid. However, other pharmaceutically compatible ^-hydroxy acids known in the art can alternatively be used. Lactic acid exists in the enantiomersL-lactic acid andD-lactic acid as well as in the racemic formDL-lactic acid. Mandelic acid exists in the enantiomers (+)-mandelic acid and (-)- mandelic acid as well as in the racemic form. Malic acid exists in the enantiomers L- malic acid and D-malic acid as well as in the racemic form.

[0117] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the molar ratio of the bisantrene to the cyclodextrin may be from about 1:1 to about 1:6. Preferably, the molar ratio of the bisantrene to the cyclodextrin is from about 1:2 to about 1:5. Still more preferably, the molar ratio of the bisantrene to the cyclodextrin is from about 1:2.5 to about 1:4.5. Still even more preferably, the molar ratio of the bisantrene to the cyclodextrin is about 1:2.5, about 1:3, about 1:3.5, or about 1.4.

[0118] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the molar ratio of the bisantrene to the ^-hydroxy acid may be from about 1:0.5 to about 1:5. Preferably, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:4. More preferably, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:3. Still more preferably, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:2. Yet more preferably, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:1.5. Even more preferably, the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.75 to about 1:1.25.

[0119] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the weight-to-weight ratio of the cyclodextrin to the ^-hydroxy acid may be from about 30:1 to about 70:1. Preferably, the weight-to-weight ratio of the cyclodextrin to the ^-hydroxy acid is from about 40:1 to about 60:1. In preferred embodiments, the weight-to-weight ratio of the cyclodextrin to the ^-hydroxy acid can be 40:1, 50:1, or 60:1.

[0120] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, where pH adjustment is required, pH adjustment may be performed using an aqueous solution of sodium hydroxide. Typically, in the aqueous solution of sodium hydroxide, the sodium hydroxide concentration in the aqueous solution is between about 0.01 M and about 4 M. Preferably, the sodium hydroxide concentration in the aqueous solution is about 0.01 M, about 0.05 M, about 1 M, about 2 M, about 3 M, or about 4 M. However, in other embodiments, a base other than sodium hydroxide can be used, such as potassium hydroxide, ammonium hydroxide, or an alkylamine, as long as the base to be used is compatible with the ingredients of the composition and does not cause degradation of any of the components of the composition. Any alternative to sodium hydroxide as the base used for pH adjustment needs to be suitable for use in a formulation intended for administration to a human patient.

[0121] Typically, the pH of the stock solution including the bisantrene, the cyclodextrin, and the ^-hydroxy acid may be adjusted to between about 2.5 and about 6.5. Preferably, the pH of the stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid is adjusted to between about 3.0 and about 6.0. More preferably, the pH of the stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid is adjusted to between about 3.5 and about 6.0. Still more preferably, the pH of the stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid is adjusted to between about 4.0 and about 6.0. Yet more preferably, the pH of the stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid is adjusted to between about 4.5 and about 6.0. Still more preferably, the pH of the stock solution of the bisantrene,the cyclodextrin, and the ^-hydroxy acid is adjusted to between about 5.0 and about 6.0. In particularly preferred embodiments, the pH of the stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid is adjusted to between about 5.0 and about 5.5, to between about 5.5 and about 6.0, or to between about 5.25 and about5.75.

[0122] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the pH of the solution, the pH of the lyophilized powder or cake on reconstitution with water, or the pH of the spray-dried powder on reconstitution with water, is from about 2.5 to about 6.0. Preferably, the pH of the solution, the pH of the lyophilized powder or cake on reconstitution with water, or the pH of the spray-dried powder on reconstitution with water is from about 3.0 to about 6.0, such as from about 3.5 to about 6.0, more preferably from about 4.0 to about 6.0, still more preferably from about 4.5 to about 6.0, yet more preferably from about 5.0 to about 6.0, such as from about 5.0 to about 5.5; from about 5.25 to about 5.75 or from about 5.5 to about 6.0.

[0123] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the weight-to-weight ratio of the cyclodextrin to the ^-hydroxy acid is from about 30:1 to about 70:1. Preferably, the weight-to-weight ratio of the cyclodextrin to the ^-hydroxy acid is from about 40:1 to about 60:1. In preferred embodiments, the weight-to-weight ratio of the cyclodextrin to the ^-hydroxy acid can be about 40:1, about 50:1, or about 60:1.

[0124] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the step of filling the pH-adjusted stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid into vials further comprises the step of stoppering vials containing the lyophilized stock solution under reduced pressure. Suitable rubber stoppers for use in the step of stoppering vials containing the lyophilized stock solution under reduced pressure include, but are not necessarily limited to, natural rubber stoppers, butyl rubber stoppers, bromobutyl rubber stoppers, chlorobutyl rubber stoppers, fluoroelastomer stoppers, ethylene propylene monomer stoppers, and rubber stoppers coated with polyfluorotetraethylene.

[0125] In particular embodiments of any methods of preparing pharmaceutical compositions according to the present invention, the resulting pharmaceutical composition is formulated for treating a condition treatable by administration of bisantrene. The condition treatable by administration of bisantrene can be, but is not limited to, a malignancy. When the condition treatable by administration of bisantrene is a malignancy, the malignancy can be, but is not limited to, a malignancy selected from the group consisting of: breast cancer; acute myelocytic leukemia; acute lymphocytic leukemia of childhood; myelodysplastic syndrome; chronic myelocytic leukemia; chronic lymphocytic leukemia; Hodgkin’s lymphoma; non-Hodgkin’s lymphoma; mycosis fungoides; prostate cancer; lung small-cell carcinoma; lung non-small-cell carcinoma; glioblastoma; neuroblastoma; a malignancy characterized by overexpressed topoisomerase II; a malignancy characterized by an overexpressed and / or mutated receptor tyrosine kinase that can be EGFR or another receptor tyrosine kinase that can be, but is not limited to, HER2 / ErbB2, MET, KIT, a FGFR-family receptor tyrosine kinase, IR, FLT3, PDGFRA, ALK, RET, or a TRK-family receptor tyrosine kinase; ovarian cancer; renal cancer; melanoma; gastric cancer; adrenal cancer; head and neck cancer; hepatocellular cancer; hypernephroma; bladder cancer; myeloma; and localized polyp stage colon cancer. The composition can be formulated for administration of another malignancy. Alternatively, the resulting composition can be formulated for treatment of a non-malignant disease or condition. When the condition treatable by administration is a non-malignant disease or condition, the non-malignant disease or condition can be, but is not limited to, immunodeficiency, obesity, Type 2 diabetes, or metabolic syndrome. In still another embodiment, the resulting composition is formulated for reducing cardiotoxicity or is formulated for cardioprotective activity.

[0126] According to a specific aspect of the present invention, there is provided a method for producing a pharmaceutical composition comprising bisantrene dihydrochloride,L-tartaric acid, and SBECD wherein the molar ratio of SBECD to bisantrene is 3:1, wherein the method comprises the steps of: (1) preparing a solution of theL-tartaric acid and the SBECD in water for injection; (2) adding the bisantrene dihydrochloride to the solution of (1);(3) adjusting the pH of the solution from step (2) with aqueous sodium hydroxide to a pH of about 5.5; and (4) filtering through a 0.22-^m filter.

[0127] In particular embodiments of this aspect, the method may further comprise a filtration step and / or a step of filling the solution into vials, and / or a lyophilization step, or any combination thereof, as described further above.

[0128] In particular embodiments, the method may further comprise a step of stirring the pH-adjusted solution of step (3) for about an additional 10 minutes at room temperature.

[0129] According to another specific aspect of the present invention, there is provided a method for producing a pharmaceutical composition comprising bisantrene dihydrochloride,L-tartaric acid, and SBECD wherein the molar ratio of SBECD to bisantrene is 4.5:1, wherein the method comprises the steps of: (1) preparing a solution of the L-tartaric acid and the SBECD in water for injection; (2) adding the bisantrene dihydrochloride to the solution of (1); (3) adjusting the pH of the solution from step (2) with aqueous sodium hydroxide to a pH of about 5.5; (4) adding additional water for injection to adjust the concentrations of the bisantrene dihydrochloride, theL-tartaric acid, and the SBECD; and (5) filtering through a 0.22-^m filter.

[0130] In particular embodiments of this aspect, the method may further comprise a filtration step and / or a step of filling the solution into vials, and / or a lyophilization step, or any combination thereof, as described further above.

[0131] In particular embodiments of this aspect, the method can further comprise a step of stirring the solution of step (1) for about 10 minutes at room temperature, sonicating for about 10 minutes at room temperature, and stirring the solution again for about 10 minutes at room temperature.

[0132] In particular embodiments of this aspect, the method can also further comprise a step of stirring the pH-adjusted solution of step (4) for about 10 minutes at room temperature.

[0133] Yet another specific aspect of the present invention is a method for producing a pharmaceutical composition comprising bisantrene dihydrochloride,L- tartaric acid, and SBECD wherein the molar ratio of SBECD to bisantrene is 3:1, wherein the method comprises the steps of: (1) preparing a solution of the L-tartaric acid and the SBECD in water for injection; (2) adjusting the pH of the solution of (1) with an aqueous solution of sodium hydroxide to about 5.3 to about 5.7; (3) adding the bisantrene dihydrochloride to the solution of (2); (4) adjusting the pH of the solution including the bisantrene dihydrochloride from step (3) with aqueous sodium hydroxide to a pH of about 5.3 to about 5.7; (5) adding additional water for injection to adjust the concentrations of the bisantrene dihydrochloride, theL-tartaric acid, and the SBECD; and (6) filtering through a 0.22-^m filter.

[0134] In particular embodiments of this aspect, the method may further comprise a filtration step and / or a step of filling the solution into vials, and / or a lyophilization step, or any combination thereof, as described further above.

[0135] In particular embodiments of this aspect, the pH at steps (b) and (d) is adjusted to about 5.3. In alternative particular aspects, the pH at steps (b) and (d) is adjusted to about 5.5. In alternative particular aspects, the pH at steps (b) and (d) is adjusted to about 5.7.

[0136] In particular embodiments of this aspect, the aqueous sodium hydroxide used to adjust the pH in step (2) is about 2 M. In particular embodiments of this aspect, the aqueous sodium hydroxide used to adjust the pH in step (4) is about 0.5 M.

[0137] In this method, the method can further comprise a step of stirring the solution of step (1) for about 10 minutes at room temperature, sonicating for about 10 minutes at room temperature, and stirring again for about 10 minutes.

[0138] In this method, the method can also further comprise a step of stirring the solution of step (5) for about 10 minutes at room temperature.

[0139] Therapeutic Uses of Bisantrene and Pharmaceutical Compositions Including Bisantrene

[0140] Another aspect of the present invention is a method for treating a disease or condition treatable by administration of bisantrene comprising administering a pharmaceutical composition comprising: (i) bisantrene; (ii) a cyclodextrin; and (iii) optionally, an ^-hydroxy acid to a patient with a disease or condition treatable by administration of bisantrene, wherein the pharmaceutical composition is administered by infusion to the patient to treat the disease or condition treatable by administration of bisantrene. As stated above, in most applications, the use of the ^-hydroxy acid is preferable.

[0141] As detailed further below, bisantrene, particularly in the form of bisantrene dihydrochloride, has potential clinical utility for the treatment of a number of malignancies. The malignancies that are potentially treatable by administration of bisantrene, particularly in the form of bisantrene dihydrochloride, include, but are not limited to: breast cancer; acute myelocytic leukemia; acute lymphocytic leukemia of childhood; myelodysplastic syndrome; chronic myelocytic leukemia; chronic lymphocytic leukemia; Hodgkin’s lymphoma; non-Hodgkin’s lymphoma; mycosis fungoides; prostate cancer; lung small-cell carcinoma; lung non-small-cell carcinoma; glioblastoma; neuroblastoma; a malignancy characterized by overexpressed topoisomerase II; a malignancy characterized by overexpressed and / or mutated tyrosine kinases, wherein the overexpressed and / or mutated tyrosine kinases include, but are not limited to, EGFR or another receptor tyrosine kinase that can be, but is not limited to, HER2 / ErbB2, MET, KIT, a FGFR-family receptor tyrosine kinase, IR, FLT3, PDGFRA, ALK, RET, or a TRK-family receptor tyrosine kinase; ovarian cancer; renal cancer; melanoma; gastric cancer, adrenal cancer; head and neck cancer; hepatocellular cancer; hypernephroma; bladder cancer; myeloma; and localized polyp stage colon cancer. Other malignancies can also be treated by administration of bisantrene.

[0142] In addition, bisantrene, particularly in the form of bisantrene dihydrochloride, can be administered to treat a number of non-malignant diseases and conditions. In particular, these diseases and conditions can include immunodeficiency, as bisantrene acts as an immunopotentiator. Furthermore, these diseases andconditions include obesity, as bisantrene acts as an inhibitor of the FTO demethylase. Individuals with one or two copies of the rs9939609 single nucleotide polymorphism (SNP) allele in FTO demethylase had a higher rate of obesity than individuals with no copies of this allele. Therefore, inhibition of FTO demethylase by administration of bisantrene can be used to treat or prevent obesity, particularly in individuals with one or two copies of the rs9939609 SNP allele. As the occurrence of both Type 2 diabetes and metabolic syndrome is strongly correlated with obesity, this suggests that bisantrene could also be useful for the prevention or treatment of Type 2 diabetes and metabolic syndrome.

[0143] In certain methods, the method may comprise the steps of: (1) reconstituting one or more vials comprising the pharmaceutical composition using sterile water for injection or a pharmaceutically acceptable diluent; (2) diluting the reconstituted contents of a portion of, or all of, the one or more vials from step (2) into an intravenous infusion vehicle; and (3) administering the reconstituted and diluted pharmaceutical composition from step (2) into a patient, wherein administration is performed by infusion, to treat the disease or condition treatable by administration of bisantrene.

[0144] Typically, in this method, the bisantrene is bisantrene dihydrochloride. However, other salts of bisantrene can alternatively be used as described above.

[0145] In certain embodiments, the hydrazone linkages of bisantrene are both in the trans (E) orientation, as described earlier. In particular embodiments of all and any methods of preparing pharmaceutical compositions according to the present invention, greater than 60% of the bisantrene in the pharmaceutical composition is present as compound 1, such as greater than about 70%, or greater than about 80%, or greater than about 90%,or greater than about 95%, or greater than about 96%, or greater than about 97%, or greater than about 98%, or greater than about 99% of compound 1. In all embodiments of pharmaceutical compositions according to the present invention where the bisantrene is not 100% compound 1, the balance is made of primarily compound 2 and, to a lesser extent, of compound 3.

[0146] In particular embodiments of any methods of treatment according to the present invention, the bisantrene is provided as the dihydrochloride salt in crystallineform, preferably as compound 1, having the following properties: (a) a weight loss of up to 10% up to 150^C in thermogravimetric analysis (TGA); (b) a sharp endothermic peak at between 95^C and 115^C in differential scanning calorimetry (DSC); and (c) predominant peaks at 2^ of about 9.3^ and 14.0^ in X-ray powder diffraction (XRPD).

[0147] Although the method described above recites infusion into a peripheral vein, other alternative routes of infusion can be employed, such as, but not limited to, infusion into a central vein. However, infusion into a peripheral vein is preferred.

[0148] Typically, the cyclodextrin is ^-cyclodextrin, ^-cyclodextrin, ^-cyclodextrin, a sulfobutyl ether ^-cyclodextrin (SBECD), 2-hydroxypropyl-^-cyclodextrin (HPBCD), 2- hydroxypropyl-^-cyclodextrin (HPGCD), or a randomly methylated ^-cyclodextrin (RMBCD). Preferably, the cyclodextrin is ^-cyclodextrin, an SBECD, or HPBCD. More preferably, the cyclodextrin is an SBECD, even more preferably comprising from 1 to 7 sulfobutyl ether moieties as described above. Other cyclodextrins known in the art can be employed.

[0149] Typically, the ^-hydroxy acid is glycolic acid, lactic acid, mandelic acid, tartaric acid, malic acid, citric acid, or all stereoisomers thereof. Lactic acid has two stereoisomers,L-lactic acid andD-lactic acid. Mandelic acid also has two stereoisomers, (R)-mandelic acid and (S)-mandelic acid. Tartaric acid has three stereoisomers, (2R,3R)-tartaric acid (L-tartaric acid (the naturally occurring form), its enantiomer, (2S,3S)-tartaric acid (D-tartaric acid), and the meso form, (2R,3S)-tartaric acid or (2S,3R)-tartaric acid. Tartaric acid may also occur in a racemic form that includes (2R,3R)-tartaric acid and (2S,3S)-tartaric acid. Malic acid has two stereoisomers, L- malic acid and D-malic acid. Preferably, the ^-hydroxy acid is lactic acid, tartaric acid, or malic acid. More preferably, the ^-hydroxy acid is tartaric acid. When the ^-hydroxy acid is tartaric acid, typically, the tartaric acid is D-tartaric acid, L-tartaric acid, meso-tartaric acid, or a racemic mixture ofD-tartaric acid andL-tartaric acid; preferably, the tartaric acid is L-tartaric acid. However, other pharmaceutically compatible ^-hydroxy acids known in the art can alternatively be used.

[0150] Typically, the pharmaceutical composition used in this method is a solution, a suspension, a lyophilized powder or cake, or a spray-dried powder, whereinthe lyophilized powder or cake or the spray-dried powder is suitable for reconstitution with water prior to use.

[0151] When the method includes reconstituting the contents of one or more vials comprising the pharmaceutical composition, in one embodiment, each vial is a plastic vial. Typically, the plastic vial is constructed of a plastic that is cyclic olefin polymer (COP) plastic, cyclic olefin copolymer (COC) plastic, high-density polyethylene plastic, or high-density non-nucleated polypropylene plastic. In another embodiment, each of the vials is a silanized or unsilanized glass vial comprising clear glass or amber glass. When the vial is silanized, typically, silanization is performed by coating the interior of the vials with an organofunctional alkoxysilane that is (3-aminopropyl)- triethoxysilane, (3-aminopropyl)-diethoxymethylsilane, (3-aminopropyl)-dimethyl- ethoxysilane, (3-aminopropyl)-trimethoxysilane, (3-glycidoxypropyl)-dimethyl- ethoxysilane, (3-mercaptopropyl)-trimethoxysilane, (3-mercaptopropyl)-methyl dimethoxysilane, or derivatives thereof. Typically, the vial is stoppered with a stopper that is a natural rubber stopper, a butyl rubber stopper, a bromobutyl rubber stopper, a chlorobutyl rubber stopper, a fluoroelastomer stopper, an ethylene propylene monomer stopper, or a rubber stopper coated with polyfluorotetraethylene.

[0152] Typically, in this method for treating a disease or condition treatable by the administration of a therapeutically effective quantity of bisantrene to a patient, the intravenous infusion vehicle is typically selected from, but not limited to, 5% dextrose in water, lactated Ringer’s solution, and Sorenson’s phosphate buffer. Typically, the volume of the intravenous infusion vehicle is between 50 mL and 1 L. As stated above, typically, the disease or condition treatable by the administration of a therapeutically effective quantity of bisantrene is cancer. However, as stated above, the disease or condition treatable by administration of bisantrene can alternatively be a non-malignant disease or condition as described herein.

[0153] Typically, in this method for treating a disease or condition treatable by the administration of a therapeutically effective quantity of bisantrene, the duration of the infusion to the patient is from about 0.5 hours to about 2.5 hours.

[0154] Typically, in this method for treating a disease or condition treatable by the administration of a therapeutically effective quantity of bisantrene, the dosage receivedby the patient is from about 5 mg / m2to about 500 mg / m2body surface area, such as from about 10 mg / m2 / day to about 450 mg / m2 / day, such as from about 20 mg / m2 / day to about 400 mg / m2 / day, from about 40 mg / m2 / day to about 350 mg / m2 / day, from about 50 mg / m2 / day to about 300 mg / m2 / day, from about 100 mg / m2 / day to about 250 mg / m2 / day, from about 100 mg / m2 / day to about 200 mg / m2 / day, from about 5 mg / m2 / day to about 400 mg / m2 / day, from about 5 mg / m2 / day to about 300 mg / m2 / day, from about 5 mg / m2 / day to about 200 mg / m2 / day, from about 5 mg / m2 / day to about 100 mg / m2 / day, from about 5 mg / m2 / day to about 80 mg / m2 / day, from about 5 mg / m2 / day to about 50 mg / m2 / day, about 10 mg / m2 / day, about 20 mg / m2 / day, about 30 mg / m2 / day, about 40 mg / m2 / day, about 50 mg / m2 / day, about 80 mg / m2 / day, about 100 mg / m2 / day, about 150 mg / m2 / day, about 200 mg / m2 / day, or about 300 mg / m2 / day. In some embodiments, bisantrene is administered daily or weekly, once every two weeks, once every three weeks, once every four weeks, over a period of, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days or 63 days. In certain embodiments, bisantrene is administered once or multiple times over a period of 28 days, optionally once or multiple times daily or weekly, once every two weeks, once every three weeks, once every four weeks, at a dosage of, for example, about 5 to about 500 mg / m2 / 28 days. For example, in certain embodiments, bisantrene may be administered to a patient once weekly over a period of four weeks, with a dosing regimen of from about 5 mg / m2 / week to about 100 mg / m2 / week, from about 5 mg / m2 / week to about 50 mg / m2 / week, from about 10 mg / m2 / week to about 40 mg / m2 / week, from about 5 mg / m2 / week to about 30 mg / m2 / week, about 10 mg / m2 / week, about 25 mg / m2 / week, about 50 mg / m2 / week, about 75 mg / m2 / week, or about 100 mg / m2 / week. The administration of pharmaceutically acceptable salts of bisantrene may be performed at similar dosage rates, adjusted for molar equivalence.

[0155] Typically, the disease or condition treatable by administration of bisantrene is cancer. However, the disease or condition treatable by administration of bisantrene can alternatively be a non-malignant disease or condition as described herein. The non-malignant disease or condition can be, but is not limited to,immunodeficiency, obesity, diabetes, metabolic syndrome, pancreatitis, and sequelae of cystic fibrosis affecting the pancreas.

[0156] Methods according to the present invention can be used either simultaneously with either surgery or radiation, or can be used subsequent to the use of surgery or radiation as adjuvant therapy when the disease or condition being treated is cancer. The specific methods of surgery or radiation to be employed, and whether surgery or radiation should be employed simultaneously with administration of bisantrene or prior to administration of bisantrene, where bisantrene is used as adjuvant therapy, can be determined by one of skill in the art, depending on factors such as the particular malignancy affecting the patient, the accessibility of the malignancy to surgical intervention, the susceptibility of the malignancy to radiation, the stage of the malignancy, the particular cells or tissues comprising the malignancy, other chemotherapy that has previously been administered or is concurrently being administered to the patient, the age and weight of the patient, and pharmacokinetic parameters including liver and kidney function.

[0157] The invention is illustrated by the following Examples. These Examples are included for illustrative purposes only, and are not intended to limit the invention. EXAMPLES Example 1 Development of Novel Bisantrene Formulations

[0158] Bisantrene is soluble in aqueous solutions at acidic pH as the dicationic salt but precipitates as the uncharged free base (pKa16.8; T.P. Wunz et al., “DNA Binding by Antitumor Anthracene Derivatives,” J. Med. Chem.33: 1549-1553 (1990)) in blood due to the neutral pH (7.4). In principle, a formulation of bisantrene that could avoid precipitation in blood when administered into a peripheral vein requires excipients that enhance the solubility of bisantrene at neutral pH, or alternatively, prevent precipitation upon contact with blood long enough to allow sufficient dilution. Discovery of such a formulation was approached by: (1) screening solubility-enhancing cyclodextrins, (2) screening of additives, and (3) optimization of SBECD content.1. Screening of Solubility-Enhancing Cyclodextrins

[0159] Cyclodextrins and their derivatives are known to enhance the aqueous solubility of certain compounds, including small molecule active pharmaceutical ingredients (APIs; U.S. Patent No.5,134,127). The two ^-cyclodextrin derivatives sulfobutylether β-cyclodextrin (SBECD) and 2-hydroxypropyl β-cyclodextrin (HPBCD) were assessed for their ability to limit bisantrene precipitation under pH neutral conditions since these are represented in the United States of America’s Food and Drug Administration (FDA) register of acceptable inactive excipients for use in pharmaceutical compositions administered to humans via the intravenous route.

[0160] Aqueous solutions containing either 25 mg / mL or 2.5 mg / mL bisantrene (free base equivalents, Bis.Free) and 20% w / v of either SBECD or HPBCD were prepared and subsequently diluted into 5% w / v dextrose in water (5DW; 8 mL bisantrene / cyclodextrin solution added to 492 mL 5DW, a vehicle typically used for intravenous infusions). No precipitation was observed in any solutions at this step (Table 1). Table 1: Volume of bisantrene / cyclodextrin (SBECD or HPBCD) / 5DW solution (50 mL maximum) that could be added to 50 mL phosphate buffered saline (pH 7.4) or 50 mL human plasma before observing precipitation of bisantrene.Bis.Free + 20% w / v SBECD 2.5 mg / mL Clear Additional 10 mins required No precipitation Bis.Free + after all 50 mL added after all 50 mL 20% w / v addedBis.Free + 20% w / v HPBCD 2.5 mg / mL Clear 28 mL No precipitation Bis.Free + after all 50 mL 20% w / v added HPBCD

[0161] The bisantrene / cyclodextrin / 5DW solutions were then added dropwise (up to 50 mL maximum) to 50 mL of either phosphate buffered saline (PBS, pH 7.4) at room temperature or human blood plasma at 37°C and observed visually for the first visible signs of precipitation. The volume of solution able to be added before observing precipitation was found to be higher for bisantrene solutions containing SBECD relative to those containing HPBCD (Table 1).

[0162] Bisantrene was also found to be qualitatively more soluble in aqueous solutions containing SBECD at pHs approaching neutral, compared to HPBCD- containing solutions. Clear solutions were obtained when 25 mg / mL bisantrene was stirred with 20% w / v SBECD for 24 h at room temperature under near-neutral conditions (pH 6.0), whereas voluminous undissolved solids were present under the equivalent conditions with HPBCD (Figure 1). Dynamic light scattering measurements showed that human plasma treated with mixtures containing bisantrene and SBECD had fewer and smaller particles present and were less polydisperse than cyclodextrin-free bisantrene solutions. In contrast, HPBCD showed similar particle content and polydispersity to bisantrene solutions. Collectively, the data supported selection of SBECD over HPBCD as a superior cyclodextrin-based excipient for enhancing the aqueous solubility of bisantrene at physiological pH. 2. Screening of Additives

[0163] A selection of non-ionic surfactants and ^-hydroxy acids were assessed as additives for preventing or prolonging the time to formation of bisantrene precipitates in standing (unstirred) solutions at room temperature. Aqueous samples were prepared containing bisantrene dihydrochloride (7.5 mg / mL free base equivalents), 6% w / v SBECD and additives. Each additive was assessed in the concentration range 0.1-1.0% w / v at 0.1% w / v increments. Once prepared, the bisantrene / SBECD / additive solutions were diluted 1:1 in 5DW (to mimic dilution into infusion vehicle in the clinic) and then further diluted (1:1) into 0.1 M Sorenson’s buffer (pH 7.4; pH neutral aqueous surrogate for human blood). Diluted solutions were allowed to stand at room temperature and observed over time (up to 19 hours) for evidence of precipitation using light microscopy. Figure 2 (images) and Table 2 (qualitative scoring) illustrate the relative amounts of precipitation observed in representative solutions.Table 2: Qualitative scoring of the extent of precipitation in solutions containing 7.5 mg / mL bisantrene, 6% w / v SBECD and the indicated excipient (0.1 – 1% w / v) after dilution (1:1) into 5% dextrose in water (5DW) followed by dilution (1:1) into 0.1 M Sorenson’s buffer (pH 7.4) at the time points indicated. Voluminous precipitation (+++); numerous precipitated solids (++); small / few observable solid particles (+); no observable solids (Clear Solution). Appearance AfterDilutionsSample Components Prior to Dilution 0.1 M Sorensen’s Buffer (pH 7.4)0 hours 1 hour 19 hours 7.5 mg / mL bisantrene +++ +++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD + + ++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.1% w / v Polysorbate-20 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.2% w / v Polysorbate-20 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.3% w / v Polysorbate-20 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.4% w / v Polysorbate-20 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.5% w / v Polysorbate-20 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.6% w / v Polysorbate-20 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.7% w / v Polysorbate-20 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.8% w / v Polysorbate-20 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.9% w / v Polysorbate-20 Clear Solution + +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 1.0% w / v Polysorbate-20 Clear Solution + +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.1% w / v Polysorbate-80 + + +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.2% w / v Polysorbate-80 + + +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.3% w / v Polysorbate-80 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.4% w / v Polysorbate-80 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.5% w / v Polysorbate-80 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.6% w / v Polysorbate-80 + ++ +++Appearance AfterDilutions Sample Components Prior to 19 0.1 M Sorensen’s Buffer (pH 7.4)0 hours 1 hour hours 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.7% w / v Polysorbate-80 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.8% w / v Polysorbate-80 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.9% w / v Polysorbate-80 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 1.0% w / v Polysorbate-80 + ++ +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.1% w / v L-ascorbic acid Clear Clear Solution Solution ++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.2% w / v L-ascorbic acid Clear Solution + +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.3% w / v L-ascorbic acid Clear Solution + + 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.4% w / v L-ascorbic acid Clear Solution + +++ 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.5% w / v L-ascorbic acid Clear Solution + + 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.6% w / v L-ascorbic acid Clear Solution + + 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.7% w / v L-ascorbic acid Clear Solution + + 7.5 mg / mL bisantrene, 6% w / v SBECD, 0.8% w / v L-ascorbic acid Clear Clear Solution Solution ++

[0164] These results combined with the results shown in Figure 2 clearly demonstrate that ternary formulations containing bisantrene, SBECD and ^-hydroxy acids are superior for enhancing bisantrene solubility, solution stability and preventing precipitation relative to binary formulations containing bisantrene and SBECD only and no ^-hydroxy acid.

[0165] Solutions containing bisantrene on its own showed obvious, voluminous precipitation immediately upon dilution into Sorenson’s buffer. Reduced precipitation was observed for samples of bisantrene and 6% SBECD upon initial dilution into Sorenson’s buffer, but large crystalline precipitates formed after 1 hour that increased insize and number after 19 hours. SBECD-containing bisantrene solutions in the presence of alpha-hydroxy acids showed increased stabilization, where little (DL-malic) to no (L-(+)-tartaric acid) precipitation was evident from first dilution into Sorenson’s buffer or even after 19 hours of standing. These results emphasize the superiority of formulations containing ^-hydroxy acids as additives in terms of stability and prevention of precipitation over formulations without ^-hydroxy acids.

[0166] None of the other additives tested, including polysorbate-20, polysorbate- 80, Kolliphor HS-15, Kolliphor ELP, Kolliphor RH40, pluronic F-68, pluronic F-127, citric acid, L-ascorbic acid, propylene glycol, PEG-400, carbitol and DMSO, showed equivalent or better stabilization of bisantrene solutions containing ^-hydroxy acids. 3. Optimization of SBECD Content

[0167] The optimal ratio of SBECD:bisantrene for solubilization in the presence of L-(+)-tartaric acid was evaluated in water and 5DW. Solutions containing bisantrene (30 mg / g free base equivalents) and 0.5% w / w tartrate (buffer of disodium L-(+)-tartrate and L-(+)-tartaric acid) were prepared with SBECD present in the molar ratios (SBECD:bisantrene) 0.74:1, 1:1, 1.235:1, 1.47:1, 2.2:1 and 3:1. Dilution of the solutions with water to 12.5 mg / mL bisantrene (free base equivalents; to mimic reconstitution of lyophilized solid drug product) produced clear, homogenous solutions only when the SBECD:bisantrene ratio was 1.47:1 or greater (Figure 3). Subsequent dilution of the 12.5 mg / mL aqueous solutions in 5DW to 0.5 mg / mL bisantrene (representative of a clinically applicable concentration) produced clear solutions. No difference in solution clarity was observed for solutions containing 3:1 SBECD:bisantrene with either buffered disodium L-(+)-tartrate / L-(+)-tartaric acid or L-(+)-tartaric acid, indicating that the solutions are stable across the pH range 3.4 (L-(+)-tartaric acid) to 4.8 (disodium L-(+)- tartrate / L-(+)-tartaric acid).

[0168] Applying the results of the above studies, new test formulations were evaluated for solution stability and the ability to infuse such formulations without precipitation of bisantrene (in vitro and in vivo).Example 2 In vitro Venous Infusion Model

[0169] An in vitro model was used to simulate infusion of bisantrene formulations into veins and assess the extent of precipitation. Methods were adapted from J.L.H Johnson et al., “Prediction of Precipitation-Induced Phlebitis: A Statistical Validation of an in Vitro Model,” J. Pharm. Sci.1574-1581 (2003). Formulations evaluated contained 12.5 mg / mL bisantrene (free base equivalents), 3:1 molar ratio of SBECD:bisantrene (if SBECD present), 0.5% w / v L-(+)-tartaric acid and NaOH (for pH adjustment). The formulations were diluted to 0.5 mg / mL bisantrene (free base equivalents) in either 5DW (final pH 4.9, 5.8) or Hartmann’s solution (final pH 5.5, 5.8).

[0170] The infusion model consisted of Tygon tubing (3.2 mm inner diameter) connected to a GoodPump HandyPump 02 dual channel peristaltic pump. A solution containing 0.1 M Sorensen’s buffer (pH 7.4) or Fetal Calf Serum (FCS; to mimic human blood) was flowed through the system at a rate of 5 mL per minute to mimic blood flow in human veins. A 1 mL tuberculin syringe fitted with a 19G hypodermic needle was used to inject 1 mL of test solutions through the wall of the tubing into the flowing solutions at an angle of 45° approximately 10 cm from the open end. The solutions were manually injected at a steady rate over 30 seconds and the flow-through was collected from the end of the tube (total of 60 seconds following the start of injection) into 20 mL clear glass vials. Approximately 6-6.5 mL of flow-through solution was collected in each test. Flow-through solutions were mixed by capping the collecting vial and inverting three times within 2 minutes of ceasing pump flow. Collected samples were left to stand at room temperature and observed periodically for visual evidence of precipitation. The experiments were recorded on video and photographs of flow through solutions were taken soon after injection and after standing for 1 to 3 hours (Figure 4). Qualitative observations of precipitation were scored (Table 3).Table 3: Summary of visual observations from in vitro venous infusion model: no precipitation (–); precipitation (+); significant precipitation (++).N / A Not available

[0171] No solution showed precipitation during injection into either Sorensen’s buffer or FCS, or immediately after collection of the flow through. After 1 hour, solutions containing bisantrene alone (no excipients) diluted in 5DW infusion vehicle showed strong precipitation following injection into Sorensen’s buffer (Figure 4) and some precipitation when injected into FCS (not shown). Solutions containing bisantrene alone (no excipients) diluted in Hartmann’s infusion vehicle showed negligible precipitation when injected into Sorensen’s buffer (not shown) and some precipitation following injection into FCS (Figure 4). For bisantrene formulations according to the invention diluted in Hartmann’s solution and injected into FCS, a small amount of precipitation was observed 1 hour after injection. No precipitation was observed after 1 hour for formulations diluted in 5DW and injected into either Sorensen’s buffer or FCS. The in vitro venous infusion model confirmed that pH-adjusted formulations containing bisantrene and SBECD and formulations containing bisantrene and SBECD and L-(+)- tartaric acid diluted into 5DW infusion vehicle showed no precipitation during injection and remained stable after standing for up to 3 hours in Sorensen’s buffer (not shown) or FCS (Figure 4).Example 3 Potency and Molar Ratio Calculations for Examples 5-7

[0172] In the Examples, with respect to reference to addition of materials where the quantity of the materials added is expressed in grams, the reference is to the total weight of the materials added, including the solvent, typically water. For example, a reference to the addition of 19.19 grams of a 1 M solution of sodium hydroxide in Example 5 refers to the addition of that quantity of the solution expressed in terms of its weight in grams, including the water used to make up the solution.

[0173] The potency and molar ratios of SBECD (Lot #47K070322) to bisantrene dihydrochloride used in Examples 5-7 were calculated according to Figures 5-9.

[0174] The potency of bisantrene dihydrochloride was calculated according to Table 4. Table 4Example 4 Preparation of Bisantrene Dihydrochloride Solution (40 mg / g Bisantrene Free Base Equivalents)

[0175] To a beaker was added bisantrene dihydrochloride (31.5 grams, calculated from Example 3). Water for injection (568.5 grams) was added and the resulting mixture was stirred for approximately 15 hours. The resulting mixture was filtered through a 0.22-^m filter and then assayed to verify the concentration of the bisantrene dihydrochloride. Using two different lots of bisantrene dihydrochloride, two bulk solutions were prepared with assays as follows: Bulk Solution #1 was assayed at 82.15% label claim and Bulk Solution #2 was assayed at 104.0% label claim.Example 5 Preparation of Prototype Formulation #1

[0176] To a beaker was added water for injection (104.85 grams). With stirring, L- tartaric acid (3 grams) and SBECD (120.96 grams) were added. The resulting mixture was stirred for 10 minutes at room temperature, sonicated for 10 minutes at room temperature, and then stirred for an additional 10 minutes at room temperature to form a visually transparent solution. Bisantrene dihydrochloride (bulk solution #1, 187.5 grams + 40.7 grams) was added to the resulting mixture. An aqueous sodium hydroxide solution (1 M, 19.19 grams) was added to adjust the pH of the mixture to 3.74. After stirring for an additional 10 minutes, the resulting solution was filtered through an 0.22- ^m filter. Following filtration, the resulting solution was filled into vials (15 grams per vial), frozen and lyophilized, resulting in an orange to red cake containing 250 mg of bisantrene free base equivalents. Example 6 Preparation of Prototype Formulation #2

[0177] To a beaker was added water for injection (104.85 grams). With stirring,L- malic acid (3 grams) and SBECD (120.96 grams) were added. The resulting mixture was stirred for 10 minutes at room temperature, sonicated for 10 minutes at room temperature, and then stirred for an additional 10 minutes at room temperature to form a visually transparent solution. Bisantrene dihydrochloride (bulk solution #1, 187.5 grams + 40.7 grams) was added to the resulting mixture. An aqueous sodium hydroxide solution (2 M, 2.59 grams) was added to adjust the pH of the mixture to 3.74. After stirring for an additional 10 minutes, the resulting solution was filtered through a 0.22- ^m filter. Following filtration, the resulting solution was filled into vials (15 grams per vial), frozen and lyophilized, resulting in an orange to red cake containing 250 mg of bisantrene free base equivalents. Example 7 Preparation of Prototype Formulation #3

[0178] To a beaker was added water for injection (177.0 grams). With stirring,L- tartaric acid (3 grams) and SBECD (181.43 grams) were added. The resulting mixturewas stirred for 10 minutes at room temperature, sonicated for 10 minutes at room temperature, and then stirred for an additional 10 minutes at room temperature to form a visually transparent solution. Bisantrene dihydrochloride (bulk solution #2, 187.5 grams) was added to the resulting mixture. An aqueous sodium hydroxide solution (1 M, 18.07 grams) was added to adjust the pH of the mixture to 3.75. Additional water for injection (33.0 grams) was added to achieve target weight. After stirring for an additional 10 minutes, the resulting solution was filtered through a 0.22-^m filter. Following filtration, the resulting solution was filled into vials (20 grams per vial), frozen and lyophilized, resulting in an orange to red cake containing 250 mg of bisantrene free base equivalents. Example 8 Compositions and Analytical Results for Prototype Formulations #1-3

[0179] Typical pre-lyophilized compositions for the three formulations described in Examples 5-7 are summarized in Table 5 below. Table 5

[0180] Analytical results for the three formulations described in Examples 5-7 are summarized in Table 6 below.Table 6Example 9 Potency and Molar Ratio Calculations for Examples 11-14

[0181] The potency and molar ratios of SBECD to bisantrene dihydrochloride used in Examples 11-14 were calculated according to Figures 10-15. Because the same lot of bisantrene dihydrochloride was used for all examples, the potency of the bisantrene dihydrochloride was calculated according to Example 3. Example 10 Preparation of Bisantrene Dihydrochloride Solution (40 mg / g)

[0182] To a beaker was added bisantrene dihydrochloride (47.2 grams, calculated from Example 9). Water for injection (852.8 grams) was added and the resulting mixture was stirred for approximately 15 hours. The resulting mixture was filtered through a 0.22-^m filter and assayed to verify concentration. The resulting bulk solution was assayed at 97.82% label claim. Example 11 Preparation of Prototype Formulation #4

[0183] To a beaker was added water for injection (100.5 grams). With stirring,L- tartaric acid (3 grams) and SBECD (120.1 grams) were added. The resulting mixture was stirred for 10 minutes at room temperature, sonicated for 10 minutes at room temperature, and then stirred for an additional 10 minutes at room temperature to form a visually transparent solution. An aqueous solution of sodium hydroxide (2 M, 15.9 mL) was added to adjust the pH of the resulting solution to 5.3. Bisantrene dihydrochloride (the bulk solution from Example 10, 187.8 grams) was added to the resulting mixture. An aqueous sodium hydroxide solution (0.5 M, 10 mL) was added to adjust the pH of the mixture to 5.3. Additional water for injection (33.8 grams) was added to achieve target weight. After stirring for an additional 10 minutes, the resulting solution was filtered through a 0.22-^m filter. Following filtration, the resulting solution was filled into vials (6.25 grams per vial), frozen and lyophilized, resulting in an orange to red cake containing 100 mg of bisantrene free base equivalents.Example 12 Preparation of Prototype Formulation #5

[0184] To a beaker was added water for injection (107.7 grams). With stirring, L- tartaric acid (3 grams) and SBECD (120.1 grams) were added. The resulting mixture was stirred for 10 minutes at room temperature, sonicated for 10 minutes at room temperature, and then stirred for an additional 10 minutes at room temperature to form a visually transparent solution. An aqueous solution of sodium hydroxide (2 M, 21.1 mL) was added to adjust the pH of the solution to 5.5. Bisantrene dihydrochloride (the bulk solution from Example 10, 188.0 grams) was added to the resulting mixture. Additional water for injection (19.1 grams) was added to achieve target weight. After stirring for an additional 10 minutes, the resulting solution was filtered through a 0.22-^m filter. Following filtration, the resulting solution was filled into vials (6.25 grams per vial), frozen and lyophilized, resulting in an orange to red cake containing 100 mg of bisantrene free base equivalents. Example 13 Preparation of Prototype Formulation #6

[0185] To a beaker was added water for injection (100.2 grams). With stirring, L- tartaric acid (3 grams) and SBECD (120.1 grams) were added. The resulting mixture was stirred for 10 minutes at room temperature, sonicated for 10 minutes at room temperature, and then stirred for an additional 10 minutes at room temperature to form a visually transparent solution. An aqueous solution of sodium hydroxide (2 M, 16.8 mL) was added to adjust the pH of the resulting solution to 5.7. Bisantrene dihydrochloride (the bulk solution from Example 10, 188.2 grams) was added to the resulting mixture. An aqueous solution of sodium hydroxide (0.5 M, 9.5 mL) was added to adjust the pH of the resulting solution to 5.7. Additional water for injection (32.3 grams) was added to achieve target weight. After stirring for an additional 10 minutes, the resulting solution was filtered through a 0.22-^m filter. Following filtration, the resulting solution was filled into vials (6.25 grams per vial), frozen and lyophilized, resulting in an orange to red cake containing 100 mg of bisantrene free base equivalents.Example 14 Preparation of Prototype Formulation #7

[0186] To a beaker was added water for injection (159.9 grams). With stirring, L- tartaric acid (3 grams) and SBECD (180.1 grams) were added. The resulting mixture was stirred for 10 minutes at room temperature, sonicated for 10 minutes at room temperature, and then stirred for an additional 10 minutes at room temperature to form a visually transparent solution. An aqueous solution of sodium hydroxide (2 M, 24.3 mL) was added to adjust the pH of the resulting solution to 5.5. Bisantrene dihydrochloride (the bulk solution from Example 10, 188.0 grams) was added to the resulting mixture. Additional water for injection (44.6 grams) was added to achieve target weight. After stirring for an additional 10 minutes, the resulting solution was filtered through a 0.22- ^m filter. Following filtration, the resulting solution was filled into vials (8 grams per vial), frozen and lyophilized, resulting in an orange to red cake containing 100 mg of bisantrene free base equivalents. Example 15 Compositions and Analytical Results for Formulations #4 to #7

[0187] Typical per-vial pre-lyophilized compositions for the four prototype formulations #4-#7 of Examples 11-14 are shown in Table 7 below. Table 7

[0188] Analytical results for the four formulations described in Examples 13-16 are summarized in Table 8 below. Table 8Example 16 In vivo Characterization of Infusion Site Tolerability – Rabbit Ear Infusion Model

[0189] Infusion site tolerability of a prototype bisantrene formulation (Formulation #5; bisantrene 100 mg free base equivalents, SBECD 1601 mg, L-(+)-Tartaric Acid 40 mg, pH 5.5) was evaluated using the rabbit ear vein infusion model. New Zealand White rabbits were assigned to six study groups, with three animals per group. Four of the groups were infused with Formulation #5 diluted in 5DW and two groups received infusion vehicle (5DW) containing Formulation #5 excipients without bisantrene. Final doses and infusion conditions are summarized in Table 9. Table 9: Study groups evaluated in rabbit ear vein infusion model using Formulation #5.1 5DW Vehicle #1 0 Excipients as per 10 1.0 mg / kg dose (0.5 mg / mL) 2 5DW Vehicle #2 0 Excipients as per 20 0.5 mg / kg dose (2.0 mg / mL) 3 Formulation #5 10 0.5 1.0 Condition 1 4 Formulation #5 10 1.0 0.5 Condition 2 5 Formulation #5 10 2.0 0.25 Condition 3 6 Formulation #5 20 2.0 0.5 Condition 4

[0190] Anesthetized rabbits were administered the test solutions intravenously via the marginal ear vein. Two of the Group 6 animals died soon after infusion, indicating that the dose of 20 mg / kg was not tolerated under the infusion conditions. For the remaining groups, two of the three animals were sacrificed at 1-hour post-dose, while the third animal was sacrificed at 48 hours. At sacrifice, both ears were harvested. For the animals at the 1-hour post dose time point, an approximately 1 cm wide strip of skin overlaying the peripheral ear vein of both infused and contralateral ears waspeeled back to expose approximately 4 cm of vein, just above the point of infusion on the infused ear to the base of the ear (and equivalent region of non-infused ear). The marginal ear veins of infused ears were opened and observed for deposition of drug- related material by visual inspection, with and without light microscopy. Photographs were taken and observations reported.

[0191] For the third rabbit in each group (i.e., 48 h post-dose sacrifice), sections of the marginal ear vein of the infused ear within 1 cm, 3 cm, and 5 cm downstream of the injection site were collected and photographs of both the infused and contralateral ear were taken. The sections were then placed in 10% neutral buffered formalin (NBF). Equivalent sections from the contralateral ear vein of each rabbit were processed in the same way for histology to act as intra-rabbit controls.

[0192] There was no evidence of bisantrene precipitation or other macroscopic changes at the 1 h timepoint, apart from some yellow areas of tissue in two animals due to spillage of the test solution. At 48 h post dosing, there was no visual evidence of precipitation of bisantrene at the injection site or at any of the other sites of evaluation. Some mild to moderate bruising was observed around the injection site, but this was attributed to the infusion procedure, as it also appeared in the control animals.

[0193] Microscopic changes in the ear marginal veins were observed ≥ 1cm downstream from the infusion site in Group 3 (10 mg / kg, 0.5 mg / mL, 1 h infusion), Group 4 (10 mg / kg, 1.0 mg / mL, 0.5 h infusion) and Group 6 (20 mg / kg, 2.0 mg / mL, 0.5 h infusion) animals. These changes consisted of mild fibrinous inflammation and thrombosis at 1 cm from the infusion site (Groups 3 and 6) and minimal (Group 4) to mild (Group 6) fibrinous inflammation at 3 cm.

[0194] In summary, this study confirmed that precipitation of bisantrene does not occur during infusion of Formulation #5 into the marginal rabbit ear vein of rabbits and that there was no association between phlebitis and Formulation #5 administration. Representative images comparing rabbit ear veins infused with Formulation #5 to infusions with bisantrene dihydrochloride (diluted in 5DW; separate study) are provided in Figure 16.Example 17 In vivo Characterization of Infusion Site Tolerability – Dog Infusion Model

[0195] A repeat dose GLP toxicology study was conducted in beagle dogs to assess the effects of Formulation #5 when given once weekly for three doses (Days 1, 8 and 15), followed by a four-week recovery period. Local infusion site tolerability was assessed macroscopically and histologically as part of the study. Thirty-two dogs were divided into four groups of 3 males and 3 females each. An additional 2 males and 2 females were included with each of the control and high-dose groups to monitor for recovery. Animals in three of the groups received weekly 0.5 hour IV infusions (4.17 mL / kg) of 2, 6 and 12 mg / kg (Formulation #5, 0.48, 1.44, 2.88 mg / mL in D5W, respectively), for a total of three doses. Control animals in the fourth group received 3^ weekly 0.5 hour IV infusions (4.17 mL / kg) of 5DW.

[0196] Macroscopic assessment of infusion sites showed dark or purple discoloration, which correlated microscopically to hemorrhage in some animals. This microscopic correlation was attributed to the intravenous infusion procedure as it was also observed in the control infusions. All microscopic infusion site findings were considered indirect and attributed to the procedure and not the test article. Example 18 Preparation of (E,E)-Bisantrene 1

[0197] Anthracene-9,10-dicarbaldehyde (100 grams) and 2-hydrazino-2- imidazoline dihydrochloride (165.22 grams, 2.2 equivalents) were combined with methanol (2 L) and stirred at room temperature for 15 minutes. The reaction was heated to 65ºC and stirred at that temperature for 5 hours. The reaction was cooled to 45ºC and concentrated under vacuum at below 45ºC until approximately 150 mL of solvent remained. At this stage, the content of (E,Z)-bisantrene 2 was typically between 10-15% in the reaction mixture. In order to convert the bisantrene isomeric mixture to (E,E)- bisantrene 1, isopropyl alcohol (200 mL) was added and the reaction was again concentrated under vacuum at below 45ºC until approximately 150 mL of solvent remained. Isopropyl alcohol (200 mL) was again added followed by concentration under vacuum at below 45ºC until approximately 150 mL of solvent remained. The residuewas then diluted with isopropyl alcohol (2 L) and heated to 80ºC. After stirring at 80ºC for 5 hours, the (E,Z)-bisantrene 2 isomer content was typically below 0.5% in the reaction mixture. With the isomerized bisantrene content under control, the reaction was cooled to 30ºC and stirred at that temperature for 2 hours. The resulting mixture was filtered and the isolated solids washed with isopropyl alcohol (400 mL). The washed solids were dried under vacuum at 30ºC for 4 hours, after which drying was continued at 45ºC for an additional 4 hours. The resulting solids were de-lumped and then dried at 45ºC for a final 8 hours. After drying, the solids were combined with acetone (1.6 L) and water (800 mL). The resulting mixture was heated to 55ºC and stirred at that temperature for 40 minutes. The resulting hazy mixture was filtered through Celite (40 grams) at 55ºC. The Celite bed was washed with 25% aqueous acetone (300 mL, 75 mL of water combined with 225 mL of acetone). At this stage, control over the crystal form was achieved by initial heating of the combined filtrates to 55ºC to maintain a solution. The heated solution was twice-filtered through a 0.45 micron filter. Acetone (6.4 L, pre-cooled to 5ºC) was added to the filtrate over 40 minutes. After acetone addition, the reaction temperature was recorded at approximately 20ºC. The reaction was further cooled to 0ºC and stirred at that temperature for 3 hours. The resulting solids were filtered and washed with acetone (400 mL, pre-cooled to 5ºC) under a nitrogen atmosphere. At this point, the (E,Z)-bisantrene 2 isomer content in the reaction mixture was less than 1.0%. The collected solids were dried under vacuum at 30ºC. After 4 hours of drying, the material was de-lumped and dried under vacuum at 30ºC for an additional 16 hours giving (E,E)-bisantrene dihydrochloride 1 in typical yields of greater than 90%. Isolated product typically met crystal form specifications and (E,Z)- bisantrene 2 isomer levels were typically less than 0.1%, with overall purity greater than 99%. The process is summarized in the scheme below.

[0198] The1H-NMR spectrum of a typical batch of (E,E)-bisantrene 1 produced according to the above scheme, recorded on 400MHz Bruker AVANCE NEO FT-NMR spectrometer using DMSO-d6 as solvent, is shown in Figure 17.13C-NMR, DEPT-135 and D2O exchange NMR spectra were also acquired (not shown). The1H and13C NMR chemical shifts are reported on the δ scale in ppm relative to TMS and DMSO-d6 (0.00 and 39.51) as internal reference standards, respectively. The DEPT-135 spectrum revealed the presence of methine groups as positive peaks and methylene groups as negative peaks, and the D2O exchange analysis confirmed exchangeable protons at δ 8.84 and 13.37. The NMR chemical shift assignments are as follows:Table 10 – NMR assignments for (E,E)-bisantrene1Refer to the structural formula for numbering.2 1H-1H coupling constant. s-singlet, m-multiplet, and br-broad.

[0199] Differential Scanning Calorimetric analysis of an exemplary batch of (E,E)- bisantrene was carried out on a TA DSC Q200. The thermogram was recorded under a nitrogen atmosphere at a heating rate of 10ºC / min. The thermogram, shown in Figure 18, displayed an endotherm with onset temperatures at 101.04ºC, 209.55ºC, 223.07ºC and 236.19ºC and end point temperatures at 119.87ºC, 218.45ºC, 228.90ºC and 240.29ºC, respectively. Thermogravimetric analysis was carried out on a TGA TA Q500 instrument. The thermogram, recorded under a nitrogen atmosphere at a heating rate of 10ºC / min (Figure 19), shows a weight loss of 8.31%, indicating that this particular form of bisantrene is a hydrate.

[0200] An X-ray powder diffraction analysis of an exemplary batch of bisantrene dihydrochloride carried out on a PANalaytical X’Pert3Powder diffractometer equipped with vertical goniometer in θ / θ geometry produced the pattern shown in Figure 20. The Copper Kα (λ=1.5406Ǻ) radiation was used and the sample was scanned between 3-45 degrees 2θ. The diffractogram indicates that the compound is crystalline in nature. Major peaks at 2θ of about 9.25⁰ and 14.02⁰, with minor peaks at 2θ of about 15.9⁰, 16.5⁰, 20.8⁰, 22.2⁰, 22.9⁰, 23.2⁰, 24.5⁰, 25.7⁰, 26.0⁰, 26.3⁰ and 28.3⁰ were observed.

[0201] These results confirm the structure of 9,10-(E,E)-(2-(4,5-dihydro-1H- imidazol-2-yl)hydrazinylidene)methyl)anthracene dihydrochloride (i.e. (E,E)-bisantrene dihydrochloride) in the desired crystalline form. ADVANTAGES OF THE INVENTION

[0202] The present invention is directed to formulations of bisantrene, particularly bisantrene dihydrochloride, and methods for their use, wherein the formulations are suitable for intravenous administration into peripheral veins, as well as methods for preparation of such formulations. The formulations of the present invention eliminate the need to administer bisantrene by central venous catheter administration. Administration of bisantrene by central venous catheter is, however, still possible using the formulations produced by the methods of the present invention. The formulations produced by the methods of the present invention, especially those comprising an α- hydroxy acid, improve the blood solubility of bisantrene, particularly bisantrene dihydrochloride; these formulations enable the administration of bisantrene through more commonly practiced peripheral vein infusions with a low risk of phlebitis or other significant side effects caused by bisantrene precipitation during peripheral vein infusion. The ability to administer therapeutically effective dosages of bisantrene without causing side effects such as phlebitis increases the utility of the drug for the treatment of malignancies and other conditions.

[0203] Compositions produced by methods according to the present invention can be administered to treat a range of malignancies, and can be used together with other anti-neoplastic drugs; they can also be used to treat other diseases and conditions. By administering bisantrene in compositions produced according to methods of the present invention, a major toxicity (i.e. phlebitis) associated with bisantrene is significantly reduced allowing for broader use, better drug delivery and better therapeutic outcomes.

[0204] Methods for preparation of bisantrene-containing compositions according to the present invention possess industrial applicability as methods for preparation of a pharmaceutical composition.

[0205] Where methods are referred to, the methods of the present invention provide specific method steps that are more than general applications of laws of nature and require that those practicing the method steps employ steps other than those conventionally known in the art, in addition to the specific applications of laws of nature recited or implied in the claims, and thus confine the scope of the claims to the specific applications recited therein. In some contexts, these claims are directed to new formulations of an existing drug, including methods for preparing such new formulations.

[0206] The inventions illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” and similar language shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the future shown and described or any portion thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of these inventions. This includes the generic description of each invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised materials specifically resided therein. Moreover, when the term “comprising” is used herein as a transitional phrase in claims, the term “comprising” also includes both “consisting essentially of” and “consisting of” unless the narrower terms are specifically excluded.

[0207] It is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the invention should, therefore,be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patents and patent publications, are incorporated herein by reference.

Claims

Claims:

1. A pharmaceutical composition in one or more dosage units comprising: (a) bisantrene; (b) a cyclodextrin; and (c) an ^-hydroxy acid.

2. The pharmaceutical composition of claim 1 wherein the bisantrene is in salt form or in free base form.

3. The pharmaceutical composition of claim 2 wherein the bisantrene is bisantrene dihydrochloride.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the cyclodextrin is selected from the group consisting of ^-cyclodextrin, ^- cyclodextrin, ^-cyclodextrin, a sulfobutyl ether ^-cyclodextrin (SBECD), 2-hydroxypropyl- ^-cyclodextrin (HPBCD), 2-hydroxypropyl-^-cyclodextrin (HPGCD), and a randomly methylated ^-cyclodextrin (RMBCD).

5. The pharmaceutical composition of claim 4 wherein the cyclodextrin is selected from the group consisting of ^-cyclodextrin, an SBECD, and HPBCD, preferably an SBECD.

6. The pharmaceutical composition of claim 5 wherein the cyclodextrin is an SBECD comprising from 1 to 7 sulfobutyl ether moieties, optionally from 1 to 2 sulfobutyl ether moieties, optionally from 3 to 4 sulfobutyl ether moieties, or from 5 to 7 sulfobutyl ether moieties.

7. The pharmaceutical composition of claim 6 wherein the sulfonic acid groups of the SBECD sulfobutyl ether moieties are in acid form, salt form, or a mixture of acid form and salt form.

8. The pharmaceutical composition of claim 7 wherein the counterion of the sulfonic acid groups of the SBECD sulfobutyl ether moieties in salt form is selected from the group consisting of Li+, Na+, K+, and ammonium.

9. The pharmaceutical composition of any one of claims 1 to 8 which comprises an ^-hydroxy acid.

10. The pharmaceutical composition of claim 9 wherein the ^-hydroxy acid is selected from the group consisting of glycolic acid, lactic acid, mandelic acid, tartaric acid, malic acid, and citric acid.

11. The pharmaceutical composition of claim 10 wherein the ^-hydroxy acid is selected from the group consisting of lactic acid, tartaric acid, and malic acid, preferably tartaric acid, more preferably L-tartaric acid.

12. The pharmaceutical composition of any one of claims 1 to 11 wherein the molar ratio of the bisantrene to the cyclodextrin is from about 1:1 to about 1:6, more preferably from about 1:1.4 to about 1:5, more preferably from about 1:2.5 to about 1:4.5., more preferably about 1:

3.

13. The pharmaceutical composition of any one of claims 1 to 12 wherein the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:5, optionally from about 1:0.75 to about 1:1.

25.

14. The pharmaceutical composition of any one of claims 1 to 13 wherein the composition is a solution, a suspension, a lyophilized powder or cake, or a spray-dried powder, wherein the lyophilized powder or cake or the spray-dried powder is suitable for reconstitution with water prior to use.

15. The pharmaceutical composition of claim 14 wherein the pH of the solution, the pH of the suspension, the pH of the lyophilized powder or cake on reconstitution with water, or the pH of the spray-dried powder on reconstitution with water is from about 5.0 to about 6.

0.

16. The composition of any one of claims 1 to 15 wherein greater than 60% of the bisantrene is (E,E)-bisantrene or a pharmaceutically acceptable salt thereof.

17. The composition of claim 16 wherein greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%, or greater than 99% of the bisantrene is (E,E)-bisantrene or a pharmaceutically acceptable salt thereof.

18. The composition of any one of claims 1 to 17 wherein the form of bisantrene used for incorporation into the pharmaceutical composition is the dihydrochloride salt in crystalline form having the following properties: (a) a weight loss of up to 10% up to 150^C in thermogravimetric analysis (TGA);(b) a sharp endothermic peak at between 95^C and 115^C in differential scanning calorimetry (DSC); and (c) predominant peaks at 2^ of about 9.3^ and 14.0^ in X-ray powder diffraction (XRPD).

19. A method for preparing a pharmaceutical composition of bisantrene wherein the pharmaceutical composition comprises: (i) bisantrene; (ii) a cyclodextrin; and (iii) an ^-hydroxy acid, wherein the method comprises the steps of: (a) preparing a stock solution of a cyclodextrin and an ^-hydroxy acid in water; (b) combining a stock solution of bisantrene with the stock solution of the cyclodextrin and the ^-hydroxy acid from step (a) to produce a combined stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid; and (c) adjusting the pH of the combined stock solution of the bisantrene, the cyclodextrin, and the ^-hydroxy acid from step (b).

20. The method of claim 19 wherein the stock solution of a cyclodextrin and an ^-hydroxy acid in water is prepared by: a) preparing a stock solution of a cyclodextrin in water and dissolving the ^-hydroxy acid into the stock solution of the cyclodextrin; b) preparing a stock solution of an ^-hydroxy acid in water and dissolving the cyclodextrin into the stock solution of the ^-hydroxy acid; or c) combining a cyclodextrin in solid form with an ^-hydroxy acid in solid form and then dissolving the resulting solid mixture in water.

21. The method of claim 19 or claim 20 which further comprises a step of sterile filtration of the pH-adjusted combined stock solution.

22. The method of claim 21 wherein the step of sterile filtration comprises filtration through one or more filters, at least one of which has a filtration cutoff of about 0.2 ^m.

23. The method of any one of claims 19 to 22 wherein the pharmaceutical composition is placed in vials.

24. The method of claim 23 wherein the vials are silanized or unsilanized amber glass.

25. The method of any one of claims 19 to 24 further comprising a step of lyophilizing the combined stock solution.

26. The method of claim 25 wherein the step of lyophilizing the combined stock solution is performed in vials.

27. The method of any one of claims 19 to 26 wherein the cyclodextrin concentration in the stock solution of cyclodextrin and ^-hydroxy acid is from about 0.9 g / mL to about 1.3 g / mL, preferably from about 1 g / mL to about 1.2 g / mL.

28. The method of any one of claims 19 to 27 wherein the bisantrene is in salt form, preferably wherein the bisantrene salt is bisantrene dihydrochloride.

29. The method of any one of claims 19 to 28 wherein the stock solution of the bisantrene is prepared at a concentration of from about 25 mg / mL to about 50 mg / mL, preferably from about 30 mg / mL to about 40 mg / mL, wherein the concentration of bisantrene is determined as the free base.

30. The method of any one of claims 19 to 29 wherein the cyclodextrin is selected from the group consisting of ^-cyclodextrin, ^-cyclodextrin, ^-cyclodextrin, a sulfobutyl ether ^-cyclodextrin (SBECD), 2-hydroxypropyl-^-cyclodextrin (HPBCD), 2- hydroxypropyl-^-cyclodextrin (HPGCD), a randomly methylated ^-cyclodextrin (RMBCD), and derivatives and analogs thereof.

31. The method of claim 30 wherein the cyclodextrin is selected from the group consisting of ^-cyclodextrin, an SBECD, and HPBCD, preferably wherein the cyclodextrin is an SBECD.

32. The method of claim 31 wherein the cyclodextrin is an SBECD comprising from 1 to 7 sulfobutyl ether moieties, optionally from 1 to 2 sulfobutyl ether moieties, from 3 to 4 sulfobutyl ether moieties, or from 5 to 7 sulfobutyl ether moieties.

33. The method of claim 32, wherein the sulfonic acid groups of the SBECD sulfobutyl ether moieties are in acid form, salt form, or a mixture of acid form and salt form.

34. The method of claim 33 wherein, when one or more of the sulfonic acid groups of the SBECD sulfobutyl ether moieties are in salt form, and thecounterions of the sulfobutyl ether moieties that are in salt form are selected from the group consisting of Li+, Na+, K+, NH4+.

35. The method of any one of claims 19 to 34 wherein the ^-hydroxy acid is selected from the group consisting of glycolic acid, lactic acid, mandelic acid, tartaric acid, malic acid, citric acid, and derivatives and analogs thereof.

36. The method of claim 35 wherein the ^-hydroxy acid is selected from the group consisting of lactic acid, tartaric acid, and malic acid.

37. The method of claim 36 wherein the ^-hydroxy acid is tartaric acid, preferablyL-tartaric acid.

38. The method of any one of claims 19 to 37 wherein the molar ratio of the bisantrene to the cyclodextrin is from about 1:1 to about 1:6, preferably from about 1:2.5 to about 1:4.

5.

39. The method of any one of claims 19 to 38 wherein the molar ratio of the bisantrene to the ^-hydroxy acid is from about 1:0.5 to about 1:5, preferably from about 1:0.75 to about 1:1.

25.

40. The method of any one of claims 19 to 39 wherein the pH of the combined stock solution including the bisantrene, the cyclodextrin, and the ^-hydroxy acid is adjusted to between about 2.5 and about 6.

0.

41. The method of any one of claims 19 to 40 wherein the combined stock solution is lyophilized and the pH of the lyophilized powder or cake on reconstitution with water is from about 2.5 to about 6.

0.

42. A method for producing a pharmaceutical composition comprising bisantrene dihydrochloride,L-tartaric acid, and SBECD, wherein the method comprises the steps of: (a) preparing a solution of theL-tartaric acid and the SBECD in water for injection; (b) adding the bisantrene dihydrochloride to the solution of (a); (c) adjusting the pH of the solution from step (b) with aqueous sodium hydroxide to a pH of about 5.5; and (d) filtering the solution of step (c) through a 0.22-^m filter.

43. The method of claim 42 further comprising the step of filling the filtered solution into vials.

44. The method of claim 43 further comprising the step of freezing and lyophilizing the solution in the vials.

45. The method of any one of claims 42 to 44 wherein the bisantrene dihydrochloride added in step (b) is in solid form.

46. The method of any one of claims 44 to 44 wherein the bisantrene dihydrochloride added in step (b) is in a solution in water for injection.

47. The method of any one of claims 42 to 46 wherein the aqueous sodium hydroxide used to adjust the pH in step (c) is about 1 M.

48. The method of any one of claims 42 to 47 wherein the method further comprises a step of stirring the pH-adjusted solution of step (c) for about an additional 10 minutes at room temperature prior to filtration.

49. The method of any one of claims 42 to 48 wherein the ratio of SBECD to bisantrene is 3:

1.

50. The method of any one of claims 42 to 48 wherein the ratio of SBECD to bisantrene is 4.5:1, wherein the method comprises the additional step of adding additional water for injection to adjust the concentrations of the bisantrene dihydrochloride, the L-tartaric acid, and the SBECD after pH adjustment and prior to filtration.

51. The method of claim 50 wherein the method further comprises a step of stirring the solution of step (a) for about 10 minutes at room temperature, sonicating for about 10 minutes at room temperature, and stirring the solution again for about 10 minutes at room temperature prior to adding the bisantrene dihydrochloride.

52. A method for producing a pharmaceutical composition comprising bisantrene dihydrochloride, L-tartaric acid, and SBECD wherein the ratio of SBECD to bisantrene is 3:1, wherein the method comprises the steps of: (a) preparing a solution of theL-tartaric acid and the SBECD in water for injection; (b) adjusting the pH of the solution of (a) with an aqueous solution of sodium hydroxide to about 5.3 to about 5.7;(c) adding the bisantrene dihydrochloride to the solution of (b); (d) adjusting the pH of the solution including the bisantrene dihydrochloride from step (c) with aqueous sodium hydroxide to a pH of about 5.3; (e) adding additional water for injection to adjust the concentrations of the bisantrene dihydrochloride, the L-tartaric acid, and the SBECD; and (f) filtering the solution of step (e) through a 0.22-^m filter.

53. The method of claim 52 further comprising the step of filling the filtered solution into vials.

54. The method of claim 53 further comprising the step of freezing and lyophilizing the solution in the vials.

55. The method of any one of claims 52 to 54 wherein the bisantrene dihydrochloride added in step (b) is in solid form.

56. The method of any one of claims 52 to 54 wherein the bisantrene dihydrochloride added in step (b) is in a solution in water for injection.

57. The method of any one of claims 52 to 56 wherein the aqueous sodium hydroxide used to adjust the pH in step (b) is about 2 M.

58. The method of any one of claims 52 to 57 wherein the aqueous sodium hydroxide used to adjust the pH in step (d) is about 0.5 M.

59. The method of any one of claims 52 to 58 wherein the method further comprises a step of stirring the solution of step (a) for about 10 minutes at room temperature, sonicating for about 10 minutes at room temperature, and stirring again for about 10 minutes.

60. The method of any one of claims 52 to 59 wherein the method further comprises a step of stirring the solution of step (e) for about 10 minutes at room temperature.

61. The method of any one of claims 52 to 60 wherein the pH at steps (b) and (d) is adjusted to 5.

3.

62. The method of any one of claims 52 to 60 wherein the pH at steps (b) and (d) is adjusted to 5.

5.

63. The method of any one of claims 52 to 60 wherein the pH at steps (b) and (d) is adjusted to 5.7.

64. The method of any one of claims 19 to 63 wherein the method produces a pharmaceutical composition in which greater than 60% of the bisantrene is (E,E)-bisantrene or a pharmaceutically acceptable salt thereof.

65. The method of claim 64 wherein greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%, or greater than 99% of the bisantrene is (E,E)-bisantrene or a pharmaceutically acceptable salt thereof.

66. The method of any one of claims 19 to 65 wherein the form of bisantrene used for producing the pharmaceutical composition is the dihydrochloride salt in crystalline form having the following properties: (a) a weight loss of up to 10% up to 150^C in thermogravimetric analysis (TGA); (b) a sharp endothermic peak at between 95^C and 115^C in differential scanning calorimetry (DSC); and (c) predominant peaks at 2^ of about 9.3^ and 14.0^ in X-ray powder diffraction (XRPD).

67. A pharmaceutical composition of bisantrene comprising bisantrene, a cyclodextrin and an ^-hydroxy acid, produced by a method according to any one of claims 19 to 66.

68. A method for treating a disease or condition treatable by administration of bisantrene comprising administering a pharmaceutical composition according to any one of claims 1 to 18 or 67, wherein the pharmaceutical composition is administered by infusion to the patient to treat the disease or condition treatable by administration of bisantrene.

69. The method of claim 68 wherein the condition treatable by administration of bisantrene is a malignancy, optionally selected from the group consisting of: breast cancer; acute myelocytic leukemia; acute lymphocytic leukemia of childhood; myelodysplastic syndrome; chronic myelocytic leukemia; chronic lymphocytic leukemia; Hodgkin’s lymphoma; non-Hodgkin’s lymphoma; mycosis fungoides; prostate cancer; lung small-cell carcinoma; lung non-small-cell carcinoma; glioblastoma; neuroblastoma; a malignancy characterized by overexpressed topoisomerase II; a malignancy characterized by an overexpressed and / or mutated tyrosine kinase selectedfrom the group consisting of EGFR and HER2 / ErbB2, MET, KIT, a FGFR-family receptor tyrosine kinase, IR, FLT3, PDGFRA, ALK, RET, and a TRK-family receptor tyrosine kinase; ovarian cancer; renal cancer; melanoma; gastric cancer; adrenal cancer; head and neck cancer; hepatocellular cancer; hypernephroma; bladder cancer; myeloma; and localized polyp stage colon cancer.

70. The method of claim 68 wherein the condition treatable by administration of bisantrene is a non-malignant disease or condition, optionally selected from the group consisting of immunodeficiency, obesity, Type 2 diabetes, and metabolic syndrome.

71. A method for reducing cardiotoxicity or for cardioprotective activity comprising administering a pharmaceutical composition according to any one of claims 1 to 18 or 67, wherein the pharmaceutical composition is administered by infusion to the patient to treat the disease or condition treatable by administration of bisantrene.

72. The method of any one of claims 68 to 71 wherein the method comprises the steps of: (a) reconstituting the contents of one or more vials comprising the pharmaceutical composition using sterile water for injection; (b) diluting the reconstituted contents of a portion of or all of the one or more vials from step (a) into an intravenous infusion vehicle; and (c) administering the reconstituted and diluted pharmaceutical composition from step (b) into a patient with cancer to treat the disease or condition treatable by administration of bisantrene, wherein administration is performed by infusion into a vein.

73. The method of any one of claims 68 to 72 wherein the pharmaceutical composition is a solution, a suspension, a lyophilized powder or cake, or a spray-dried powder, wherein the lyophilized powder or cake or the spray-dried powder is suitable for reconstitution with water prior to use.

74. The method of claim 72 wherein the intravenous infusion vehicle is selected from the group consisting of 5% dextrose in water, lactated Ringer’s solution, and Sorenson’s buffer.

75. The method of claim 74 wherein the intravenous infusion vehicle is 5% dextrose in water.

76. The method of any one of claims 68 to 75 wherein the dosage received by the patient is from about 5 mg / m2to about 500 mg / m2body surface area.

77. The method of any one of claims 68 to 76 wherein the pharmaceutical composition is administered intravenously and the volume of the intravenous infusion solution is between 50 mL and 1 L.

78. The method of claim 77 wherein the duration of the infusion is from about 0.1 hours to about 2.5 hours.

79. The method of any one of claims 68 to 77 wherein greater than 60% of the bisantrene is (E,E)-bisantrene or a pharmaceutically acceptable salt thereof.

80. The method of claim 79 wherein greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%, or greater than 99% of the bisantrene is (E,E)-bisantrene or a pharmaceutically acceptable salt thereof.

81. The method of any one of claims 68 to 80 wherein the form of bisantrene used for producing the pharmaceutical composition is the dihydrochloride salt in crystalline form having the following properties: (a) a weight loss of up to 10% up to 150^C in thermogravimetric analysis (TGA); (b) a sharp endothermic peak at between 95^C and 115^C in differential scanning calorimetry (DSC); and (c) predominant peaks at 2^ of about 9.3^ and 14.0^ in X-ray powder diffraction (XRPD).

82. Use of: (i) a therapeutically effective quantity of bisantrene; (ii) a cyclodextrin; and (iii) an ^-hydroxy acid for the manufacture of a medicament for infusion to a patient for treating a disease or condition treatable by administration of bisantrene.

83. The use of claim 82 wherein said disease or condition is cancer.

84. A pharmaceutical composition comprising: (i) a therapeutically effective quantity of bisantrene; (ii) a cyclodextrin; and (iii) an ^-hydroxy acid for treatment of a disease or condition treatable by administration of bisantrene.

85. The pharmaceutical composition for use according to claim 84 wherein said disease or condition is cancer.

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