WO2019073296A1

WO2019073296A1 - Liposomal formulations of bisantrene or derivatives or analogs thereof

Info

Publication number: WO2019073296A1
Application number: PCT/IB2018/001266
Authority: WO
Inventors: John Rothman
Original assignee: Race Oncology Ltd
Current assignee: Race Oncology Ltd
Priority date: 2017-10-13
Filing date: 2018-10-09
Publication date: 2019-04-18
Anticipated expiration: 2020-04-13

Abstract

La présente invention concerne des compositions comprenant, dans un liposome, du bisantrène ou un dérivé ou analogue de celui-ci, ainsi que des compositions comprenant, dans un liposome, du bisantrène ou un dérivé ou analogue de celui-ci et un antimétabolite analogue de la pyrimidine tel que la cytarabine, ainsi que des procédés d'utilisation de ces compositions, en particulier dans le traitement de malignités. Les compositions améliorent la stabilité et la biodisponibilité du bisantrène ou du dérivé ou analogue de celui-ci et réduisent la probabilité d'apparition et la gravité d'effets secondaires, tels qu'une phlébite et d'autres complications touchant le système circulatoire, de l'administration du bisantrène ou du dérivé ou analogue de celui-ci et fournissent également une manière efficace d'administrer au moins deux agents antinéoplasiques, lorsque l'antimétabolite analogue de la pyrimidine est inclus dans la composition, dans des quantités thérapeutiquement efficaces pour le traitement d'un certain nombre de types de malignités.The present invention relates to compositions comprising, in a liposome, bisantrene or a derivative or analog thereof, as well as compositions comprising, in a liposome, bisantrene or a derivative or analog thereof and an antimetabolite analogous thereto. pyrimidine such as cytarabine, as well as methods of using these compositions, particularly in the treatment of malignancies. The compositions enhance the stability and bioavailability of bisantrene or derivative or analog thereof and reduce the likelihood of occurrence and the severity of side effects, such as phlebitis and other circulatory system complications, administration of bisantrene or derivative or analog thereof and also provide an effective way of administering at least two antineoplastic agents, when the pyrimidine analog antimetabolite is included in the composition, in therapeutically effective amounts for treatment of a number of types of malignancies.

Description

LIPOSOMAL FORMULATIONS OF BISANTRENE OR DERIVATIVES OR ANALOGS

THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Application Serial No. 62/572,047 by Dr. John Rothman, entitled "Liposomal

Formulations of Bisantrene or Derivatives or Analogs Thereof," and filed on October 13, 2018, the contents of which are incorporated in their entirety by this reference.

FIELD OF THE INVENTION

[0002] This invention is directed to liposomal formulations of bisantrene or derivatives or analogs thereof, particularly combination liposomal formulations of bisantrene together with cytarabine or another pyrimidine nucleoside analog.

BACKGROUND OF THE INVENTION

[0003] The search for and identification of cures for many life-threatening diseases that plague humans still remains an empirical and sometimes serendipitous process. While many advances have been made from basic scientific research to improvements in practical patient management, there still remains tremendous frustration in the rational and successful discovery of useful therapies particularly for life-threatening diseases such as cancer, inflammatory conditions, infection, and other conditions. [0004] Since the "War on Cancer" began in the early 1970's by the United States National Cancer Institute (NCI) of the National Institutes of Health (NIH), a wide variety of strategies and programs have been created and implemented to prevent, diagnose, treat and cure cancer. One of the oldest and arguably most successful programs has been the synthesis and screening of small chemical entities (<1500 MW) for biological activity against cancer. This program was organized to improve and streamline the progression of events from chemical synthesis and biological screening to preclinical studies for the logical progression into human clinical trials with the hope of finding cures for the many types of life-threatening malignant tumors. The synthesis and screening of hundreds of thousands of chemical compounds from academic and industrial sources, in addition to the screening of natural products and extracts from prokaryotes, invertebrate animals, plant collections, and other sources from all over the world has been and continues to be a major approach for the identification of novel lead structures as potential new and useful medicines. This is in addition to other programs including biotherapeutics designed to stimulate the human immune system with vaccines, therapeutic antibodies, cytokines, lymphokines, inhibitors of tumor blood vessel development (angiogenesis) or gene and antisense therapies to alter the genetic make-up of cancer cells, and other biological response modifiers.

[0005] The work supported by the NCI, other governmental agencies both domestic and foreign in academic or industrial research and development laboratories has resulted in an extraordinary body of biological, chemical and clinical information. In addition, large chemical libraries have been created, as well as highly characterized in vitro and in vivo biological screening systems that have been successfully used.

However, from the tens of billions of dollars spent over the past thirty years supporting these programs both preclinical^ and clinically, only a small number of compounds have been identified or discovered that have resulted in the successful development of useful therapeutic products. Nevertheless, the biological systems both in vitro and in vivo and the "decision trees" used to warrant further animal studies leading to clinical studies have been validated. These programs, biological models, clinical trial protocols, and other information developed by this work remain critical for the discovery and development of any new therapeutic agent.

[0006] Unfortunately, many of the compounds that have successfully met the preclinical testing and federal regulatory requirements for clinical evaluation were either unsuccessful or disappointing in human clinical trials. Many compounds were found to have untoward or idiosyncratic side-effects that were discovered during human clinical Phase I dose-escalation studies used to determine the maximum tolerated dose (MTD) and side-effect profile. In some cases, these toxicities or the magnitude of their toxicity were not identified or predicted in preclinical toxicology studies. In other cases, chemical agents where in vitro and in vivo studies suggested a potentially unique activity against a particular tumor type, molecular target or biological pathway were not successful in human Phase II clinical trials where specific examination of particular cancer indications/types were evaluated in government sanctioned (e.g., U.S. FDA), IRB approved clinical trials. In addition, there are those cases where potential new agents were evaluated in randomized Phase III clinical trials where a significant clinical benefit could not be demonstrated; such cases have also been the cause of great frustration and disappointment. Finally, a number of compounds have reached commercialization but their ultimate clinical utility has been limited by poor efficacy as monotherapy (<25% response rates) and untoward dose-limiting side-effects (Grade III and IV) (e.g., myelosuppression, neurotoxicity, cardiotoxicity, gastrointestinal toxicities, or other significant side effects).

[0007] In many cases, after the great time and expense of developing and moving an investigational compound into human clinical trials and where clinical failure has occurred, the tendency has been to return to the laboratory to create a better analog, look for agents with different structures but potentially related mechanisms of action, or try other modifications of the drug. In some cases, efforts have been made to try additional Phase I or II clinical trials in an attempt to make some improvement with the side-effect profile or therapeutic effect in selected patients or cancer indications. In many of those cases, the results did not realize a significant enough improvement to warrant further clinical development toward product registration. Even for

commercialized products, their ultimate use is still limited by suboptimal performance.

[0008] With so few therapeutics approved for cancer patients and the realization that cancer is a collection of diseases with a multitude of etiologies and that a patient's response and survival from therapeutic intervention is complex with many factors playing a role in the success or failure of treatment including disease indication, stage of invasion and metastatic spread, patient gender, age, health conditions, previous therapies or other illnesses, genetic markers that can either promote or retard

therapeutic efficacy, and other factors, the opportunity for cures in the near term remains elusive. Moreover, the incidence of cancer continues to rise with an

approximate 4% increase predicted for 2003 in the United States by the American Cancer Society such that over 1.3 million new cancer cases are estimated. In addition, with advances in diagnosis such as mammography for breast cancer and PSA tests for prostate cancer, more patients are being diagnosed at a younger age. For difficult to treat cancers, a patient's treatment options are often exhausted quickly resulting in a desperate need for additional treatment regimens. Even for the most limited of patient populations, any additional treatment opportunities would be of considerable value. This invention focuses on inventive compositions and methods for improving the therapeutic benefit of suboptimally administered chemical compounds including bisantrene and derivatives and analogs thereof.

[0009] Relevant literature includes Foye, W.O., "Cancer Chemotherapeutic Agents," American Chemical Society, 1995, and Dorr, R.T., and Von Hoff, D.D., "Cancer Chemotherapy Handbook," Appleton and Lange, 1994.

[0010] Bisantrene, generally employed as the dihydrochloride, is an unusual agent with direct cytotoxic action as well as genomic and immunologic methods of action. The chemical name for bisantrene dihydrochloride is 9, 10- anthracenedicarboxaldehyde-bis [(4, 5-dihydro-1 H-imidazole-2-yl) hydrazine]

dihydrochloride, and it was originally classed as an anthracycline chemotherapeutic agent. These are drugs with planar structures based around a resonant aromatic ring structure that intercalates within the helices of DNA and disrupt various functions, including replication, presumably due to a strong inhibitory effect on the enzyme topoisomerase II. It was found that, like other anthracyclines, it could kill tumor cells in donogenic assays and intercalate with DNA, where it inhibits both DNA and RNA synthesis. The primary chemotherapeutic mechanism for bisantrene is its preferential binding to A-T rich regions where it effects changes to supercoiling and initiates strand breaks in association with DNA associated proteins. This results from the inhibition of the enzyme topoisomerase II, which relaxes DNA coiling during replication. It was found that while inactive orally, intravenously (i.v.), intraperitoneally (i.p.), or

subcutaneously (s.c), the drug was effective in cancer models using colon 26, Lewis lung, Ridgway osteosarcoma, B16, Lieberman plasma cell, P388 or L1210 cancer cells. Activity in donogenic assays from 684 patients 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. Importantly, a lack of cross resistance with Adriamycin and mitoxantrone was found.

[0011] However, bisantrene dihydrochloride has a number of toxicities. Toxicity studies in dogs and monkeys revealed that at high doses leukopenia, anorexia, diarrhea, injection site necrosis, enterocolitis, muscle degeneration, and pulmonary edema were observed. Although anthracyclines, have limited therapeutic utility due to their propensity to cause cardiac toxicity, the toxicity of bisantrene was observed to be less than that of any other agent in the anthracycline class.

[0012] Because of its lack of aqueous solubility at physiologic pH bisantrene precipitates in the body have been observed in studies of rabbits and calves.

Deposition of drug into the tissues has been associated with phlebitis. Its lack of aqueous solubility has limited its bioavailability.

[0013] Bisantrene is normally administered intravenously. However, the intravenous administration of bisantrene has been associated with severe local venous toxicity. Various alternatives have been tried to minimize this toxicity. In one

alternative, bisantrene doses have been infused via central venous access devices over 1 hour. In another alternative, bisantrene has been infused through peripheral veins over 2 hours, and has been "piggybacked" into a running dextrose infusion in an attempt to lessen delayed swelling in the arm used for infusion. In yet another alternative, to reduce venous irritation, hyperpigmentation, drug extravasation, and anaphylactoid reactions, patients have been given hydrocortisone (50 mg IV) and the antihistamine diphenhydramine (50 mg IM) immediately prior to bisantrene. Resultant nausea is frequently controlled with anti-emetic agents.

[0014] The nucleoside analog cytarabine has been used for the treatment of acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, and non-Hodgkin's lymphoma. It is an antimetabolite that can be incorporated into DNA in rapidly proliferating cells but then blocks DNA synthesis. It also inhibits both DNA and RNA polymerases and nucleotide reductases.

[0015] Liposomal formulations including a combination of the anthracycline derivative daunorubicin and cytarabine have been developed. Daunorubicin is an intercalating agent that inhibits topoisomerase II. Such formulations are disclosed in United States Patent No. 8,022,279 to Mayer et al.

[0016] However, there is a need for improved formulations of bisantrene that reduce toxicity, improve bioavailability, and prevent venous damage, extravasation of the drug, and phlebitis. Additionally, there is a need for liposomal formulations including both bisantrene or derivatives or analogs thereof and a nucleoside analog such as cytarabine.

SUMMARY OF THE INVENTION

[0017] One aspect of the present invention is a liposomal formulation including bisantrene or a derivative or analog thereof that is useful for achieving a drug retention and a sustained drug release for the bisantrene or the derivative or analog thereof. The formulations are useful for treatment of neoplastic diseases and conditions including, but not limited to, acute myeloid leukemia, acute lymphocytic leukemia, and chronic myelogenous leukemia.

[0018] In this aspect of the present invention, the composition comprises:

(1 ) a therapeutically effective quantity of bisantrene or a derivative or analog thereof; and (2) a liposome encapsulating the therapeutically effective quantity of bisantrene or the derivative or analog thereof.

[0019] In one alternative, the composition comprises bisantrene. Typically, the bisantrene is bisantrene dihydrochloride.

[0020] In another alternative, the composition comprises a derivative or analog of bisantrene. The derivative or analog of bisantrene can be selected from the group consisting of:

(a) the bisantrene analog of Formula (II)

the bisantrene analog of Formula (III)

the bisantrene analog of Formula (IV)

(IV); the bisantrene analog of Formula (V)

(V);

(e) the bisantrene analog of Formula (VI)

(VI); the bisantrene analog of Formula (VII)

(VII); the bisantrene analog of Formula (VIII)

(VIII);

(h) the bisantrene analog anthracen-9-ylmethylene- methoxyethoxymethylsulfanyl]-5-pyridin-3-yl-[1 ,2,4]triazol-4-amine;

(i) the bisantrene analog of Formula (X)

(X); the bisantrene analog of Formula (XI)

wherein Ri and R3 are the same or different and are hydrogen, C1 -C6 alkyi, -C(0)-Rs, wherein R5 is hydrogen, C1 -C6 alkyi, phenyl, mono-substituted phenyl (wherein the substituent can be ortho, meta, or para and is fluoro, nitro, C1 -C6 alkyi, C1-C3 aikoxy, or cyano), pentafluorophenyl, naphthyl, furanyl,

CH HCOOC(CH₃)3_*— CHNHfc, ~CH₂CH₂COOH,

OC(CH₃b, — CHiQCH , ~<CH₂hCOOH,

(CH₂)2S0₃H or — CH₂N®— (CH₃)3Cie));

O O O

II II II

^•P(OH)_2>— P(OC₂H5)₂, ~~P(0 >2*

-SO3H; wherein only one of Ri and R3 may be hydrogen or C1 -C6 alkyi; R2 and R₄ are the same or different and are: hydrogen, Ci -C₄ alkyi or -C(0)-R6, where R6 is hydrogen, C1 -C6 alkyi, phenyl, mono-substituted phenyl (wherein the substituent may be in the ortho, meta, or para position and is fluoro, nitro, C1 -C6 alkyi, C1-C3 aikoxy, or cyano), pentafluorophenyl, naphthyl, furanyl, or -ChbOChh; wherein the compounds can have the schematic structure B(Q)n, wherein B is the residue formed by removal of a hydrogen atom from one or more basic nitrogen atoms of an amine, amidine, guanidine, isourea, isothiourea, or biguanide-containing pharmaceutically active compound, and Q is hydrogen or A, wherein A is

R'O O

P

/

R'O such that R' and R" are the same or different and are R (where R is C1-C6 alkyl, aryl, aralkyl, heteroalkyl, NC-CH2CH2-,

CI3C-CH2-, or R7OCH2CH2-, where R₇ is hydrogen or C1-C6 alkyl, hydrogen, or pharmaceutically acceptable cation or R' and R" are linked to form a -CH2CH2 or a

group, and n is an integer representing the number of primary or secondary basic nitrogen atoms in the compound such that at least one Q is A;

(n) the bisantrene analog 9, 10-bis[(2- hydroxyethyl)iminomethyl]anthracene;

(o) the bisantrene analog 9, 10-bis{[2-(-2- hydroxyethylamino)ethyl]iminomethyl}anthracene;

(p) the bisantrene analog 9, 10-bis{[2-(-2- hydroxyethylamino)ethyl]iminomethyl}anthracene; (q) the bisantrene analog 9, 10-bis{[2-(morpholin-4- yl)ethyl]iminomethyl}anthracene;

(r) the bisantrene analog 9, 10-bis[(2- hydroxyethyl)aminomethyl]anthracene;

(s) the bisantrene analog 9, 10-bis{[2-(2- hydroxyethylamino)ethyl]aminomethyl}anthracene tetrahydrochloride;

(t) the bisantrene analog 9, 10-bis{[2-(piperazin-1 - yl)ethyl]aminomethyl}anthracene hexahydrochloride;

(u) the bisantrene analog 9, 10- bis{[2-(morpholin-4- yl)ethyl]aminomethyl}anthracene tetrahydrochloride;

(v) N,N'-bis[2-(dimethylamino)ethyl]-9, 10-anthracene- bis(methylamine);

(w) N,N'-bis(1 -ethyl-3-piperidinyl)-9, 10-anthracene-bis(methylamine); and

(x) derivatives and salt forms of the compounds of (a)-(w).

[0021] In another alternative, the derivative or analog of bisantrene is a derivative of bisantrene selected from the group consisting of:

(a) a derivative of bisantrene in which at least one of the hydrogen atoms bound to the carbon atoms that are directly bound to the tricyclic aromatic nucleus is replaced with lower alkyl;

(b) a derivative of bisantrene in which at least one of the hydrogen atoms in the N=NH moiety is replaced with lower alkyl; and

(c) a derivative of bisantrene in which at least one of the hydrogen atoms bound to the nitrogens of the five-membered rings are replaced with lower alkyl.

[0022] Preferably, the composition comprises bisantrene, such as bisantrene dihydrochloride.

[0023] Typically, the liposomes comprise at least one lipid selected from the group consisting of a phosphatidylcholine lipid, a phosphatidylglycerol lipid, a sterol, an ether lipid, a phosphatidic acid, a phosphonate, a ceramide, a ceramide analog, a sphingosine, a sphingosine analog, a serine-containing lipid, a hydrophilic polymer-lipid conjugate, phosphatidylglycerol, phosphatidylinositol, and a negatively charged lipid having a hydrophilic portion and a hydrophobic portion with a neutral non-zwitterionic moiety attached to the hydrophilic portion of the lipid. Preferably, the liposomes comprise a phosphatidylcholine lipid such as diastearoylphosphatidylcholine.

Preferably, the liposomes also comprise a phosphatidylglycerol lipid such as

distearoylphosphatidylglycerol. Preferably, the liposomes also comprise a sterol such as cholesterol. Typically, the liposomes comprise diastearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol. Typically, the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.5 to about 7.5 of distearoylphosphatidylcholine, about 1.5 to about 2.5 of distearoylphosphatidylglycerol, and about 0.8 to 1 .2 of cholesterol. Preferably, the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.8 to about 7.2 of distearoylphosphatidylcholine, about 1 .8 to about 2.2 of distearoylphosphatidylglycerol, and about 0.9 to 1 .1 of cholesterol. More preferably, the molar ratio of the distearoylphosphatidylcholine, the

distearoylphosphatidylglycerol, and the cholesterol is about 7:2:1 . Typically, the liposomes have a diameter of less than about 300 nm. Preferably, the liposomes have a diameter of less than 200 nm. Typically, the liposomes have an intraliposomal osmolality of 500 mOSM/kg or less.

[0024] Another aspect of the present invention is a method for treating a malignancy comprising the step of administering a therapeutically effective quantity of a composition according to the present invention as described above to treat the malignancy. Typically, the malignancy is selected from the group consisting of breast cancer, ovarian cancer, renal cancer, small-cell lung cancer, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myelocytic leukemia, melanoma, gastric cancer, adrenal cancer, head and neck cancer, hepatocellular cancer, hypernephroma, bladder cancer, acute leukemias of childhood, chronic lymphocytic leukemia, prostate cancer, glioblastoma, and myeloma. When the malignancy is breast cancer, the breast cancer can be selected from the group consisting of refractory breast cancer, triple-negative breast cancer, and breast cancer characterized by overexpressed Her-2-neu. When the malignancy is an acute leukemia of childhood, the acute leukemia of childhood can be selected from the group consisting of acute myelocytic leukemia (AML) and acute lymphocytic leukemia (ALL) of childhood. When the malignancy is prostate cancer, the prostate cancer can be androgen-resistant prostate cancer. When the malignancy is glioblastoma, the glioblastoma can be glioblastoma that is resistant to one or both of the following therapeutic agents:

temozolomide (Temodar) or bevacizumab (Avastin), or is characterized by EGFR Variant III. Alternatively, the malignancy can be a malignancy that is characterized by overexpressed topoisomerase II or overexpressed and/or mutated EGFR.

[0025] Suitable liposomal compositions for use in methods according to the present invention are as described above.

[0026] Typically, the liposomal composition is administered parenterally, such as intravenously or intraperitoneally. Typically, the liposomal composition is administered in a pharmaceutical formulation suitable for administration.

[0027] In one alternative, the method further comprises administration of a therapeutically effective quantity of an additional therapeutic agent. For example, the malignancy is breast cancer, and the additional therapeutic agent can be selected from the group consisting of tamoxifen, anastrozole, letrozole, cyclophosphamide, docetaxel, paclitaxel, methotrexate, fluorouracil, and trastuzumab. In another example, the malignancy is ovarian cancer, and the additional therapeutic agent can be selected from the group consisting of: a platinum-containing antineoplastic drugs selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin,

phenanthriplatin, picoplatin, and satraplatin; paclitaxel; topotecan; gemcitabine;

etoposide; and bleomycin. In yet another example, the malignancy is renal cancer, and the additional therapeutic agent can be selected from the group consisting of

everolimus, torisel, nexavar, sunitinib, axitinib, inferferon, interleukin-2, pazopanib, sorafenib, nivolumab, cabozanitib, and levanitib. In yet another example, the

malignancy is small-cell lung cancer, and the additional therapeutic agent can be selected from the group consisting of cyclophosphamide, cisplatin, etoposide, vincristine, paclitaxel, and carboplatin. In yet another example, the malignancy is non- small-cell lung cancer, and the additional therapeutic agent can be selected from the group consisting of cisplatin, erlotinib, gefitinib, afatinib, crizotinib, bevacizumab, carboplatin, paclitaxel, nivolumab, and pembrolizumab. In yet another example, the malignancy is Hodgkin's lymphoma, and the additional therapeutic agent can be selected from the group consisting of mechlorethamine, vincristine, prednisone, procarbazine, bleomycin, vinblastine, dacarbazine, etoposide, and cyclophosphamide. In yet another example, the malignancy is non-Hodgkin's lymphoma, and the additional therapeutic agent can be selected from the group consisting of cyclophosphamide, vincristine, and prednisone. In yet another example, the malignancy is acute myelocytic leukemia, and the additional therapeutic agent can be selected from the group consisting of cytarabine, fludarabine, all-frans-retinoic acid, interleukin-2, and arsenic trioxide. In yet another example, the malignancy is melanoma, and the additional therapeutic agent can be selected from the group consisting of temozolomide, dacarbazine, interferon, interleukin-2, ipilimumab, pembrolizumab, nivolumab, vemurafenib, dabrafenib, and trametinib. In yet another example, the malignancy is gastric cancer, and the additional therapeutic agent can be selected from the group consisting of 5-fluorouracil, capecitabine, carmustine, semustine, mitomycin C, cisplatin, taxotere, and trastuzumab. In yet another alternative, the malignancy is adrenal cancer, and the additional therapeutic agent can be selected from the group consisting of mitotane, cisplatin, etoposide, and streptozotocin. In yet another alternative, the malignancy is head and neck cancer, and the additional therapeutic agent can be selected from the group consisting of paclitaxel, carboplatin, cetuximab, docetaxel, cisplatin, and 5-fluorouracil. In yet another alternative, the malignancy is hepatocellular cancer, and the additional therapeutic agent can be selected from the group consisting of tamoxifen, octreoside, synthetic retinoids, cisplatin, 5-fluorouracil, interferon, taxol, and sorafenib. In yet another alternative, the malignancy is hypernephroma, and the additional therapeutic agent can be selected from the group consisting of nivolumab, everolimus, sorafenib, axitinib, lenvatinib, temsirolimus, sunitinib, pazopanib, interleukin- 2, cabozanitib, bevacizumab, interferon a, ipilimumab, atezolizumab, varilumab, durvalumab, tremelimumab, and avelumab. In yet another alternative, the malignancy is bladder cancer, and the additional therapeutic agent can be selected from the group consisting of cisplatin, 5-fluorouracil, mitomycin C, gemcitabine, methotrexate, vinblastine, carboplatin, paclitaxel, docetaxel, ifosfamide, and pemetrexed. In yet another alternative, the malignancy is acute myelocytic leukemia of childhood, and the additional therapeutic agent can be selected from the group consisting of methotrexate, nelarabine, asparaginase, blinatumomab, cyclophosphamide, clofarabine, cytarabine, dasatinib, methotrexate, imatinib, pomatinib, vincristine, 6-mercaptopurine,

pegaspargase, and prednisone. In yet another alternative, the malignancy is acute lymphocytic leukemia, and the additional therapeutic agent can be selected from the group consisting of asparaginase, vincristine, dexamethasone, methotrexate, 6- mercaptopurine, cytarabine, hydrocortisone, 6-thioguanine, prednisone, etoposide, cyclophosphamide, mitoxantrone, and teniposide. In yet another alternative, the malignancy is chronic lymphocytic leukemia, and the additional therapeutic agent can be selected from the group consisting of fludarabine, cyclophosphamide, rituximab, vincristine, prednisolone, bendamustine, alemtuzumab, ofatumumab, obinutuzumab, ibrutinib, idelalisib, and venetoclax. In yet another alternative, the malignancy is prostate cancer, and the additional therapeutic agent can be selected from the group consisting of temozolomide, docetaxel, cabazitaxel, bevacizumab, thalidomide, prednisone, sipuleucel-T, abiraterone, and enzalutamide. In yet another alternative, the malignancy is glioblastoma, and the additional therapeutic agent can be selected from the group consisting of temozolomide and bevacizumab. In yet another alternative, the malignancy is myeloma and the additional therapeutic agent can be selected from the group consisting of bortezomib, lenalidomide, dexamethasone, melphalan, prednisone, thalidomide, and cyclophosphamide.

[0028] Another aspect of the present invention is a liposomal formulation including both bisantrene or a derivative or analog thereof and a nucleoside analog such as cytarabine that is useful for achieving a drug retention and a sustained drug release for each of the two therapeutic agents. The formulations are useful for treatment of neoplastic diseases and conditions including, but not limited to, acute myeloid leukemia, acute lymphocytic leukemia, and chronic myelogenous leukemia. [0029] In this aspect of the present invention, the composition comprises:

(1 ) a therapeutically effective quantity of bisantrene or a derivative or analog thereof;

(2) a therapeutically effective quantity of a pyrimidine analog antimetabolite; and

(3) a liposome encapsulating both the therapeutically effective quantity of bisantrene or the derivative or analog thereof and the pyrimidine analog

antimetabolite.

[0030] The pyrimidine analog antimetabolite is typically selected from the group consisting of cytarabine, 5-azacytidine, gemcitabine, floxuridine, 5-fluorouracil, capecitabine, 6-azauracil, troxacitabine, thiarabine, sapacitabine, CNDAC (2'-cyano-2'- deoxy-1 -β-D-arabinofuranosylcytosine), 2'-deoxy-2'-methylidenecytidine, 2'-deoxy-2'- fluoromethylidenecytidine, 2'-deoxy-2'-methylidene-5-fluorocytidine, 2'-deoxy-2',2'- difluorocytidine, and 2'-C-cyano-2'-deoxy- -arabinofuranosylcytosine. Preferably, the pyrimidine analog antimetabolite is selected from the group consisting of cytarabine, 5- azacytidine, gemcitabine, floxuridine, 5-fluorouracil, capecitabine, and 6-azauracil. More preferably, the pyrimidine analog antimetabolite is cytarabine.

[0031] Bisantrene derivatives and analogs suitable for use in the composition are as described above. Preferably, the bisantrene or derivative or analog thereof is bisantrene, such as bisantrene dihydrochloride.

[0032] A preferred composition according to the present invention comprises bisantrene and cytarabine.

[0033] Typically, the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 25: 1 to about 1 : 1. Preferably, the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 10: 1 to about 3: 1. More preferably, the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is about 5:1 .

[0034] Lipids that comprise the liposomes are as described above. [0035] This alternative of compositions according to the present invention can be used in a method for treating a malignancy as described above. Suitable methods and malignancies to be treated are as described above. Additional therapeutic agents as described above can also be used, with the proviso that a pyrimidine analog antimetabolite used in the composition is not generally used as an additional therapeutic agent.

DETAILED DESCRIPTION OF THE INVENTION

[0036] One aspect of the invention is a composition of matter comprising: (i) a therapeutically effective quantity of bisantrene or a derivative or analog thereof; and (ii) a liposome encapsulating both the therapeutically effective quantity of bisantrene or the derivative or analog thereof.

[0037] I. Bisantrene and Derivatives and Analogs Thereof

[0038] Bisantrene and derivatives or analogs suitable for use in a composition of matter according to the present invention are described below.

[0039] The structure of bisantrene dihydrochloride is shown in Formula (I), below.

(I).

[0040] Bisantrene is a tricyclic aromatic compound with the chemical name, 9, 10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1 H-imidazol-2-yl)hydrazine] dihydrochloride. The molecular formula is C22H22N8 · 2HCI and the molecular weight, 471 .4. The alkylimidazole side chains are very basic and, at physiologic pH, are positively charged. This is believed to facilitate electrostatic attractions to negatively charged ribose phosphate groups in DNA.

[0041] Bisantrene has shown antitumor activity in murine tumor models including P-388 leukemia and B-16 melanoma (R.V. Citarella et al., "Anti-Tumor Activity of CL-216942: 9, 10-Anthracenedicarboxaldehyde bis (4,5-dihydro-1 H-imidazol-2- yl)hydrazone)]dihydrochloride (Abstract #23) in Abstracts of the 20^th Interscience

Conference on Antimicrobial Agents and Chemotherapy (Bethesda, Md., American Society for Microbiology 1980)). Human tumor cells that were sensitive to bisantrene as assessed by in vitro colony-forming assays include breast cancer, ovarian cancer, renal cancer, small cell and non-small cell lung cancer, lymphoma, acute myelogenous leukemia, melanoma, gastric cancer, adrenal cancer, and head and neck cancer (D.D. Von Hoff et al, "Activity of 9, 10-Anthracenedicarboxaldehyde bis[( 4,5-dihydro-1 H- imidazol-2-yl)hydrazine]dihydrochloride (CL216,942) in a human tumor cloning system," Cancer Chemother. Pharmacol. 6: 141 -144 (1981 ) ("Von Hoff et al. (1981 a)"). In phase I clinical trials bisantrene showed activity in hepatocellular cancer and hypernephroma (one patient each) (D.D. Von Hoff et al., Phase I Clinical Investigation of 10- Anthracenedicarboxaldehyde bis[( 4,5-dihydro-1 H-imidazol-2- yl)hydrazine]dihydrochloride (CL216,942)," Cancer Res. 31 18-3121 (1981 ) ("Von Hoff et al. (1981 b)") and in lymphoma, myeloma, melanoma, renal cancer, and tumors of the bladder and lung (D.S. Alberts et al., "Phase I Clinical Investigation of 9, 10- Anthracenedicarboxaldehyde bis[(4,5-dihydro-1 H-imidazol-2-yl)hydrazone]

Dihydrochloride with Correlative in Vitro Human Tumor Clonogenic Assay," Cancer Res. 42: 1 170-1 175 (1982)). Phase I activity was also observed in two other hypernephroma patients (R. J . Spiegel et al., "Phase I Clinical Trial of 9, 10-Anthracene

Dicarboxaldehyde (Bisantrene) Administered in a Five-Day Schedule, "Cancer Res. 42: 354-358 (1982)). Bisantrene was inactive in human colon cancer tested in vitro or in vivo (M.C. Perry et al. "Phase II trial of bisantrene in advanced colorectal cancer: A cancer and leukemia group B study," Cancer Treat. Rep. 66: 1997-1998 (1982); Von Hoff et al. (1981 a); Von Hoff et al. (1981 b). It was also inactive in refractory malignant melanoma (D.S. Alberts et al., "Phase II Evaluation of Bisantrene Hydrochloride in Refractory Malignant Melanoma," Invest. New Drugs 5: 289-292 (1987)).

[0042] In Phase II clinical trials, bisantrene was active in patients with metastatic breast cancer (H.-Y. Yap et al., "Bisantrene, an Active New Drug in the Treatment of Metastatic Breast Cancer," Cancer Res. 43: 1402-1404 (1983)). Partial response rates were observed in heavily pretreated patients with metastatic breast cancer. However, the study was terminated because of significant local toxicity observed.

[0043] The mechanism of action for bisantrene has been studied. Bisantrene has been shown to induce altered DNA supercoiling indicative of DNA intercalation (G.T. Bowden et al., "Comparative Molecular Pharmacology in Leukemic LI210 cells of the Anthracene Anticancer Drugs Mitoxantrone and Bisantrene, Cancer Res. 45: 4915- 4920 (1985)). In L-1210 leukemia cells bisantrene was also shown to induce protein- associated DNA strand breaks typical of drug-induced inhibition of DNA topoisomerase II enzymes (Bowden et al., 1985). Both cytotoxicity and the DNA strand breaks appear to be reduced in hypoxic conditions (C.U. Ludwig et al., "Reduced Bisantrene-lnduced Cytotoxicity and Protein-Associated DNA Strand Breaks Under Hypoxic Condition," Cancer Treat. Rep. 68: 367-372 (1984)). The noncovalent binding of bisantrene to DNA appears to comprise two types of interactions: (1 ) intercalation of the planar anthracene moiety between DNA base pairs, and (2) electrostatic binding between negatively charged ribose phosphates of DNA and positively charged basic nitrogens on the alkyl side chains of the drug. This is reflected in the biphasic DNA dissociation curves for bisantrene in calf thymus DNA in vitro (W.O. Foye et al., "DNA-Binding Abilities of Bisguanylhydrazones of anthracene-9, 10-dicarboxaldehyde," Anti-Cancer Drug Design 1 : 65-71 (1986)).

[0044] In one alternative, bisantrene vials have been reconstituted with 2 to 5 ml_ of Sterile Water for Injection, USP, and then diluted with approximately 0.1 to 0.5 mg/ml_ in D5W (5% dextrose in water). Bisantrene is incompatible with saline and unstable in light (G. Powis et al., "Pharmacokinetic Study of ADAH in Humans and Sensitivity of ADAH to Light" (Abstract #C-74)," ASCO Proc. 1 : 19 (1982). [0045] Maximally tolerated doses in several bisantrene phase I schedules include: (1 ) 200 mg/m² weekly x3 (150 mg/mg² for patients with poor bone marrow reserve (e.g., those patients who have received radiotherapy or extensive

chemotherapy regimens) (Alberts et al. (1982), supra); (2) 150 mg/m² weekly x 3 (repeat every 4-5 week) (B.-S. Yap et al., "Phase I Clinical Evaluation of 9, 10- Anthracenedicarboxaldehyde[bis(4,5-dihydro-1 /-/-imidazol-2- yl)hydrazone]dihydrochloride (Bisantrene)," Cancer Treat. Rep. 66: 1517-1520 (1982)) (3) 260 mg/m² monthly (every 3-4 week) (240 mg/mg² for patients with poor bone marrow reserve (e.g., those patients who have received radiotherapy or extensive chemotherapy regimens) (Von Hoff et al., 1981 b); and (4) 80 mg/m² daily x 5 (repeat every 4 week) (R. J . Spiegel et al. (1982), supra).

[0046] More than 95% of bisantrene is bound to plasma proteins and the drug has a long terminal plasma half-life. There appeared to be three phases of elimination: an initial distributive phase of 6 minutes, a beta phase of approximately 1 .5 hours, and a final gamma elimination phase of 23 to 54 hours (Alberts et al. (1983), supra). Typical areas under the plasma concentration x time curve are 4.4 to 5.7 mg h/mL following intravenous doses of 260 to 340 mg/m², respectively (Alberts et al. 1983, supra). Less than 7% of a bisantrene dose is excreted in the urine and the majority of the drug is eliminated by the hepatobiliary route. The drug may be metabolized to some extent in vivo. In vitro bisantrene is a substrate for hepatic microsomal enzymes but specific metabolites have not been identified. Preclinical drug distribution studies showed that the tissues with the highest concentration (in descending order) are kidney, liver, gallbladder, spleen, lung, and heart. Brain levels were extremely low. The drug did distribute to lymph nodes and bone marrow (W.H. Wu & G. Nicolau, "Disposition and Metabolic Profile of a New Antitumor Agent, CL 216,942 (Bisantrene) in Laboratory Animals," Cancer Treat Rep. 66: 1 173-1 185 (1982)).

[0047] The major dose-limiting toxic effect of bisantrene is leukopenia (Von Hoff et al. 1981 b; Alberts et al. 1982, supra; Spiegel et al. 1982, supra; Yap et al 1982, supra)). On a schedule of every 3 to 4 weeks, the nadir for myelosuppression was 9 days with recovery by 19 days (Von Hoff et al. 1981 b). Thrombocytopenia was mild although bisantrene can also inhibit platelet aggregation (M.E. Rybak et al., "The Effects of Bisantrene on Human Platelets," Invest. New Drugs 4: 1 19-125 (1986)). Anemia and cumulative myelosuppressive toxic effects were not encountered with this drug.

[0048] In addition to myelosuppression, bisantrene produced severe phlebitis along peripheral veins used for drug infusion (Von Hoff et al. 1981 b; Alberts et al. 1982). This results 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 1981 b). Severe arm swelling, hyperpigmented veins, and punctate perivenous orange discolorations have been occasionally observed following bisantrene infusions given through 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 which physically decomposes bisantrene (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)).

[0049] Up to 10% of patients experienced anaphylactoid reactions following a bisantrene infusion (J.W. Myers et al., "Anaphylactoid Reactions Associated with

Bisantrene Infusions," Invest. New Drugs 1 : 85-88 (1983)). Symptoms included chills, chest pain, shortness of breath, flushing, and pruritus. These effects may be caused by drug-induced histamine release. Hypotension is also reported with bisantrene, and prolongation of the infusion was recommended to reduce this complication (Von Hoff et al., 1981 b). In addition, a few patients experienced diaphoresis and palpitations, usually near the end of a bisantrene infusion (Von Hoff et al., 1981 b). The drug was not cardiotoxic in animals and use in the clinic has confirmed less cardiotoxicity than other agents in its class. No patients experienced electrocardiographic changes while receiving the drug and radioangiocardiographic monitoring demonstrated no decrease in ejection fraction or any other significant change in cardiac function (J.W. Myers et al., "Radioangiocardiographic Monitoring in Patients Receiving Bisantrene," Am. J. Clin. Oncol. 7: 129-130 (1984)). [0050] Bisantrene has been reported to produce very little nausea or vomiting. Alopecia (hair loss) is also less intense with bisantrene compared with doxorubicin (J.D. Cowan et al., "Randomized Trial of Doxorubicin, Bisantrene, and Mitoxantrone in

Advanced Breast Cancer: A Southwest Oncology Group Study," J. Nat'l Cancer Inst. 83: 1077-1084 (1991 )). However, bisantrene can produce a mild fever in some patients and malaise may be particularly common. This was reported by up to one-half of patients studied (Yap et al. (1982), supra).

[0051] Various formulations suitable for use in the administration of 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 of matter 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 of matter 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 salts of a phosphoramidic acid. United States Patent No. 5, 1 16,827 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 includes 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.

[0052] The present application, therefore, provides improved formulations for the use of bisantrene and analogs or derivatives thereof together with nucleoside analogs for the treatment of malignancies while avoiding the side effects described above and improving the therapeutic efficacy of the drug. The present application also provides methods for administration of formulations according to the present invention for the treatment of malignancies and other diseases and conditions as described below.

[0053] As detailed above, in addition to direct antineoplastic effects related to the activity of bisantrene as a DNA intercalator, bisantrene also possesses other

mechanisms of action, including immunopotentiation. These mechanisms are described in: (i) N.R. West et al., "Tumor-Infiltrating Lymphocytes Predict Response to Anthracycline-Based Chemotherapy in Estrogen-Resistant Breast Cancer," Breast Cane. Res. 13: R126 (2011 ), which concludes that the level of tumor-infiltrating lymphocytes is correlated with a response to the administration of anthracycline-based agents; the markers associated with tumor-infiltrating lymphocytes (TIL) include CD19, CD3D, CD48, GZMB, LCK, MS4A1, PRF1, and SELL; (ii) L. Zitvogel et al.,

"Immunological Aspects of Cancer Chemotherapy," Nature Rev. Immunol. 8: 59-73 (2008), which states that DNA damage, such as that produced by intercalating agents such as bisantrene, induces the expression of NKG2D ligands on tumor cells in an ATM-dependent and CHK1 -dependent (but p53-independent) manner; NKG2D is an activating receptor that is involved in tumor immunosurveillance by NK cells, NKT cells, γδ T cells and resting (in mice) and/or activated (in humans) CD8⁺ T cells, and also states that anthracycline-based agents may act as immunostimulators, particularly in combination with IL-12; such agents also promote HMGB1 release and activate T cells;

(iii) D.V. Krysko et al., "TLR2 and TLR9 Are Sensors of Apoptosis in a Mouse Model of Doxorubicin-lnduced Acute Inflammation," Cell Death Different. 18: 1316-1325 (201 1 ), which states that anthracycline-based antibiotics induce an immunogenic form of apoptosis that has immunostimulatory properties mediated by MyD88, TLR2, and TLR9;

(iv) C. Ferraro et al., "Anthracyclines Trigger Apoptosis of Both G0-G1 and Cycling Peripheral Blood Lymphocytes and Induce Massive Deletion of Mature T and B Cells," Cancer Res. 60: 1901 -1907 (2000), which stated that anthracyclines induce apoptosis and ceramide production, as well as activate caspase-3 in resting and cycling cells; the apoptosis induced is independent from CD95-L/CD95 and TNF/TNF-R; and (v) K. Lee et al., "Anthracycline Chemotherapy Inhibits HIF-1 Transcriptional Activity and Tumor- Induced Mobilization of Circulating Angiogenic Cells," Proc. Natl. Acad. Sci USA 106: 2353-2358 (2009), which provides another antineoplastic mechanism for anthracycline- based antibiotics, namely inhibition of HIF-1 mediated gene transcription, which, in turn, inhibits transcription of VEGF required for angiogenesis; HIF-1 also activates

transcription of genes encoding glucose transporter GLUT1 and hexokinases HK1 and HK2, which are required for the high level of glucose uptake and phosphorylation that is observed in metastatic cancer cells, and pyruvate dehydrogenase kinase 1 (PDK1 ), which shunts pyruvate away from the mitochondria, thereby increasing lactate

production; patients with HIF-1 a overexpression based on immunohistochemical results were suggested to be good candidates for treatment with anthracycline-based

antibiotics.

[0054] Among the types of cancer for which a response to bisantrene has been seen are bladder carcinoma, multiple myeloma, lung adenocarcinoma, melanoma, and renal cell carcinoma (Alberts et al. (1982), supra), as well as breast cancer (Bowden et al. (1985), supra) and acute myelogenous leukemia, especially relapsed or refractory acute myeloid leukemia (A. Spadea et al., "Bisantrene in Relapsed and Refractory Myelogenous Leukemia," Leukemia Lymphoma 9: 217-220 (1993)). [0055] Bisantrene has been reported as activating tumor-cytostatic macrophages (B.S. Wang et al., "Activation of Tumor-Cytostatic Macrophages with the Antitumor Agent 9, 10-Anthracenedicarboxaldehyde Bis[(4,5-dihydro-1 H-imidazole-2- yl)hydrazine Dihydrochloride (Bisantrene)," Cancer Res. 44: 2363-2367 (1984)). The minimal effective in vivo dose of bisantrene appeared to be 25 mg/kg, with peak activation being achieved at doses of 50 to 100 mg/kg. A number of macrophage activators are known, including Bacillus Calmette-Guerin, Corynebacterium parvum, endotoxins, muramyl dipeptide, pl:pC copolymer, pyran copolymer, lymphokines, Adriamycin, cyclophosphamide, and mitomycin C. The efficacy of bisantrene in allogeneic macrophage transplants and with supernatants of macrophages activated by bisantrene has been shown in B.S. Wang et al., "Immunotherapy of a Murine

Lymphoma by Adoptive Transfer of Syngeneic Macrophages Activated by Bisantrene," Cancer Res. 46: 503-506 (1986). Specifically, the active cells were obtained from peritoneal exudate. Bisantrene-activated macrophages were shown to be highly cytostatic to tumor cells. Repeated treatments with activated macrophages were shown to be more effective in protecting animals inoculated with tumors. This represents immunotherapy by adoptive transfer of immunocompetent cells. Culture supernatants of activated macrophages were also found to have antiproliferative effects on tumor cells, indicating that a cytostatic factor or factors were produced by these macrophages. (B.S. Wang et al., "Activation of Tumor-Cytostatic Macrophages with the Antitumor Agent 9, 10-Anthracenedicarboxaldehyde Bis[(4,5-dihydro-1 H-imidazole-2-yl)hydrazine] Dihydrochloride (Bisantrene)," Cancer Res. 44: 2363-2367 (1984)).

[0056] Bisantrene and analogs thereof have been reported as inhibiting telomerase activity, especially by stabilizing G-quadruplex DNA structures as disclosed in M. Folini et al., "Remarkable Interference with Telomeric Function by a G-Quadruplex Selective Bisantrene Regioisomer," Biochem. Pharmacol. 79: 1781 -1790 (2010). The bisantrene analogs used are those of Formulas (II), (III), (IV), (V), (VI), (VII), and (VIII):



(VIII).

[0057] Telomerase is a ribonucleoprotein reverse transcriptase responsible for maintenance of telomere length. Its expression is associated with cell immortalization and tumorigenesis since it is expressed in most human tumor cells but is not active in most normal somatic cells. Telomerase machinery inhibitors have been evaluated as potential anticancer agents, including nucleotide analogs such as 7-deaza-2'- deoxyguanosine, BIBR1532 (2-[[(E)-3-naphthalen-2-ylbut-2-enoyl]amino]benzoic acid), antisense oligonucleotides, imetelstat sodium, and other agents. For such agents, a number of different pathways are involved in inhibition of telomerase activity. Generally, inhibition of telomerase activity results in cellular senescence or apoptosis in a time- dependent manner that correlates with the initial telomere length in the cells in which telomerase is inhibited. When telomere architecture collapses or is disrupted, a signaling cascade comparable to that produced by DNA damage is activated and cell cycle arrest (accelerated senescence) or cell death through apoptosis is induced.

[0058] Telomerase substrates are the telomeres, double-stranded DNA portions with a 3' protruding overhang (100-200 bases long), formed by a repeating noncoding sequence (TTAGGG (SEQ ID NO: 1 ) in humans). In analogy to other G-rich

sequences, the single-stranded portion can fold into a structure called G-quadruplex. These folding results of overlapping planar regions were identified by four Hoogsteen- paired guanines. Hoogsteen base-pairing is between the N7 position of the purine base as a hydrogen-bond acceptor and the C6 amino group of the pyrimidine base as a donor. By recognizing and stabilizing this abnormal DNA base-pairing arrangement, selected ligands impair telomere-telomerase interaction thus interfering with the telomere elongation step catalyzed by the enzyme. Additionally, they can displace the telomere binding proteins (i.e., TRF2 and hPOT1 ) involved in telomere capping, thereby allowing recognition of the free terminal sequence as a DNA damage region. Several compounds able to interact with and stabilize G-quadruplex structures formed by G-rich single-stranded overhangs of telomeres have been identified, including anthraquinones, fluorenones, acridines, triazine, cationic porphyrins, and perylenes, as well as other compounds. These compounds share a general consensus structural motif based on a large flat aromatic surface linked to protonatable side chains. DNA binding occurs mainly through stacking on a terminal G-tetrad, whereas side chains contribute to the stability of the complex by hydrophobic/ionic interactions into the DNA grooves.

[0059] Since similar basic features characterize intercalation and base stacking, the scaffolds of classical intercalating agents are commonly used as starting structures to produce G-quadruplex recognition. Literature data have proven that, by working on the number, the length and the position of the charged side chains bound to a

"classical" intercalator, it is possible to preferentially direct drug binding towards G- quadruplex forms. Indeed, such an approach has led to the identification of effective G- quadruplex binders such as the tri-substituted acridine BRAC019 (N,N'-(9-{[4- (dimethylamino)phenyl]amino}acridine-3,6-diyl)bis(3-pyrrolidin-1 -ylpropanamide) trihydroch!oride) and the 2,6 or 2,7 bis-substituted amido-anthraquinones. These binders are characterized by poor cytotoxicity and are able to induce a reduction in telomere length upon long-term drug exposure. Bisantrene shares the structural "consensus motif" characteristic of effective G-quadruplex binders.

[0060] At least two side chains with amine groups protonatable at physiological pH are required for G-quadruplex binding. This includes bisantrene. Bisantrene is believed to intercalate between adjacent base pairs of double-stranded DNA through π- π stacking, with side chains located in either groove (threading mode), which grants affinity constants well above 10⁶ M^"1 under physiological conditions. For the analogs described above, the fact that the most efficient G-quadruplex binders are substituted on two distinct aromatic rings with side chains pointing in opposite directions with reference to the long axis of the aromatic system likely suggests formation of additional specific interactions between the 4,5-dihydro-1 H-imidazol-2-yl hydrazone groups and the G-quadruplex structure.

[0061] At least one of the bisantrene analogs, Formula (III), has the ability to act both at the telomerase level, by interfering with substrate recognition (hence

suppressing its catalytic activity), and at the telomere level, by modifying its structural organization. This compound affects telomere function not only in telomerase- expressing cells but also in ALT-positive cell lines, since it consistently provokes a DNA damage response, as evidenced by the formation of γΗ2ΑΧ foci that partially co-localize at the telomere, in agreement with results reported for telomestatin. For this compound, such a DNA damage response, together with the absence of apoptosis and the induction of cell cycle impairment (mainly G2M phase arrest), suggest a drug-mediated activation of a senescence pathway.

[0062] Additional bisantrene analogs have been described in T.P. Wunz et al., "New Antitumor Agents Containing the Anthracene Nucleus," J. Med. Chem. 30: 1313- 1321 (1987), including N,N'-bis[2-(dimethylamino)ethyl]-9, 10-anthracene- bis(methylamine) and N,N'-bis(1 -ethyl-3-piperidinyl)-9, 10-anthracene-bis(methylamine). [0063] Another bisantrene analog is the compound known as HL-37 and described in S.Q. Xie et al., "Anti-Tumour Effects of HL-37, a Novel Anthracene

Derivative, In-Vivo and In-Vitro," J. Pharm. Pharmacol. 60:213-219 (2008). HL-37 is anthracen-9-ylmethylene-[2-methoxyethoxymethylsulfanyl]-5-pyridin-3-yl-[1 ,2,4]triazol-4- amine and has the structure shown below as Formula (IX):

(IX).

[0064] Other bisantrene analogs and derivatives are known in the art, including the bisantrene analogs disclosed in J. A. Elliott et al., "Interaction of Bisantrene Anti- Cancer Agents with DNA: Footprinting, Structural Requirements for DNA Unwinding, Kinetics and Mechanism of Binding and Correlation of Structural and Kinetic

Parameters with Anti-Cancer Activity," Anticancer Drug Pis. 3: 271 -282 (1989). In C. Sissi et al., "DNA-Binding Preferences of Bisantrene Analogs: Relevance to the

Sequence Specificity of Drug-Mediated Topoisomerase II Poisoning," Mol. Pharmacol. 54: 1036-1045 (1998) discloses additional analogs, including an aza-bioisostere that can be considered a bisantrene-amsacrine hybrid. Still other bisantrene analogs and derivatives are disclosed in G. Zagotto et al., "Synthesis, DNA-Damaging and Cytotoxic Properties of Novel Topoisomerase ll-Directed Bisantrene Analogues," Bioorg. Med. Chem. Lett. 20: 121 -126 (1998). T.L. Fields et al., "The Synthesis of Heterocyclic Analogs of Bisantrene," J. Heterocyclic Chem. 25: 1917-1918 (1988) discloses bisguanylhydrazones of anthracene-9,10-dicarboxaldehyde as bisantrene analogs.

Bisantrene-amsacrine hybrids are also disclosed in G. Capranico et al., "Mapping Drug Interactions at the Covalent Topoisomerase ll-DNA Complex by Bisantrene/Amsacrine Congeners," J. Biol. Chem. 273: 12732-12739 (1998). These compounds are depicted below as Formulas (X), (XI), (XII), and (XIII):

[0065] Additional derivatives and analogs of bisantrene include the

diphosphoramidic and monophosphoramidic derivatives of bisantrene, disclosed in United States Patent No. 4,900,838 to Murdock and United States Patent No. 5,212, 191 to Murdock et al. These compounds are compounds of Formula (XIV):

(XIV) wherein Ri and R3 are the same or different and are hydrogen, C1-C6 alkyl, -C(0)-Rs, wherein R5 is hydrogen, C1-C6 alkyl, phenyl, mono-substituted phenyl (wherein the substituent can be ortho, meta, or para and is fiuoro, nitro, C1-C6 alkyl, C1-C3 alkoxy, or cyano), pentafluorophenyl, naphthyl, furanyl,

— OCfCH₃>3,— CH2OCH3,— {CH₂)3COOH_f

-(C¾)2S0₃H or -CH₂N«~(CHj)3Cie));

0 0 o

!l ii I!

-P<OH)2, -P(OC₂Hs)2, -P(0~

-SO3H; wherein only one of Ri and R3 may be hydrogen or C1-C6 alkyl; R2 and R₄ are the same or different and are: hydrogen, Ci-C₄ alkyl or -C(0)-R6, where R6 is hydrogen, C1-C6 alkyl, phenyl, mono-substituted phenyl (wherein the substituent may be in the ortho, meta, or para position and is fluoro, nitro, C1-C6 alkyl, C1-C3 alkoxy, or cyano), pentafluorophenyl, naphthyl, furanyl, or -ChbOChh. The compounds can have the schematic structure B(Q)n, wherein B is the residue formed by removal of a hydrogen atom from one or more basic nitrogen atoms of an amine, amidine, guanidine, isourea, isothiourea, or biguanide-containing pharmaceutically active compound, and Q is hydrogen or A, wherein A is

R'O O

\ll

P'

R"0 such that R' and R" are the same or different and are R (where R is C1-C6 alkyl, aryl, aralkyl, heteroalkyl, NC-CH2CH2-,

group, and n is an integer representing the number of primary or secondary basic nitrogen atoms in the compound such that at least one Q is A.

[0066] Additional bisantrene analogs are disclosed in M. Kozurkova et al., "DNA Binding Properties and Evaluation of Cytotoxic Activity of 9, 10-Bis-N-Substituted (Aminomethyl)anthracenes," Int. J. Biol. Macromol. 41 : 415-422 (2007). These compounds include 9, 10-bis[(2-hydroxyethyl)iminomethyl]anthracene; 9, 10-bis{[2-(-2- hydroxyethylamino)ethyl]iminomethyl}anthracene; 9, 10-bis{[2-(morpholin-4- yl)ethyl]iminomethyl}anthracene; 9, 10-bis[(2-hydroxyethyl)aminomethyl]anthracene; 9, 10-bis{[2-(2-hydroxyethylamino)ethyl]aminomethyl}anthracene tetrahydrochloride; 9, 10-bis{[2-(piperazin-1 -yl)ethyl]aminomethyl}anthracene hexahydrochloride; and 9, 10- bis{[2-(morpholin-4-yl)ethyl]aminomethyl}anthracene tetrahydrochloride.

[0067] As used herein, the term "derivative" as applied to bisantrene refers to a compound that has the same carbon skeleton as bisantrene, including the tricyclic aromatic nucleus and the two side chains attached to the tricyclic aromatic nucleus but has one or more substituents as described below that replace at least one hydrogen present in bisantrene with another moiety. As used herein, the term "analog" as applied to bisantrene applies to a compound related structurally to bisantrene but alters one or more of the tricyclic aromatic nucleus or one or more of the side chains, for example, by replacing one or more carbons in the tricyclic aromatic nucleus with nitrogens or by removing or moving one or both of the side chains. Some analogs are described above; others are known to one of skill in the art.

[0068] Derivatives of bisantrene include, but are not limited to: (1 ) derivatives of bisantrene in which at least one of the hydrogen atoms bound to the carbon atoms that are directly bound to the tricyclic aromatic nucleus is replaced with lower alkyl; (2) derivatives of bisantrene in which at least one of the hydrogen atoms in the N=NH moiety is replaced with lower alkyl; or (3) derivatives of bisantrene in which at least one of the hydrogen atoms bound to the nitrogens of the five-membered rings are replaced with lower alkyl. Other derivatives of bisantrene are described below. [0069] Analogs of bisantrene include, but are not limited to compounds described above as Formulas (ll)-(XIV), as well as additional compounds described above and their derivatives.

[0070] As described above, and as detailed more generally below, derivatives and analogs of bisantrene can be optionally substituted with one or more groups that do not substantially affect the pharmacological activity of the derivative or analog. These groups are generally known in the art. Definitions for a number of common groups that can be used as optional substituents are provided below; however, the omission of any group from these definitions cannot be taken to mean that such a group cannot be used as an optional substituent as long as the chemical and pharmacological requirements for an optional substituent are satisfied.

[0071] As used herein, the term "alkyl" refers to an unbranched, branched, or cyclic saturated hydrocarbyl residue, or a combination thereof, of from 1 to 12 carbon atoms that can be optionally substituted; the alkyl residues contain only C and H when unsubstituted. Typically, the unbranched or branched saturated hydrocarbyl residue is from 1 to 6 carbon atoms, which is referred to herein as "lower alkyl." When the alkyl residue is cyclic and includes a ring, it is understood that the hydrocarbyl residue includes at least three carbon atoms, which is the minimum number to form a ring. As used herein, the term "alkenyl" refers to an unbranched, branched or cyclic hydrocarbyl residue having one or more carbon-carbon double bonds. As used herein, the term "alkynyl" refers to an unbranched, branched, or cyclic hydrocarbyl residue having one or more carbon-carbon triple bonds; the residue can also include one or more double bonds. With respect to the use of "alkenyl" or "alkynyl," the presence of multiple double bonds cannot produce an aromatic ring. As used herein, the terms "hydroxyalkyl," "hydroxyalkenyl," and "hydroxyalkynyl," respectively, refer to an alkyl, alkenyl, or alkynyl group including one or more hydroxyl groups as substituents; as detailed below, further substituents can be optionally included. As used herein, the term "aryl" refers to a monocyclic or fused bicyclic moiety having the well-known characteristics of aromaticity; examples include phenyl and naphthyl, which can be optionally substituted. As used herein, the term "hydroxyaryl" refers to an aryl group including one or more hydroxyl groups as substituents; as further detailed below, further substituents can be optionally included. As used herein, the term "heteroaryl" refers to monocyclic or fused bicylic ring systems that have the characteristics of aromaticity and include one or more

heteroatoms selected from 0, S, and N. The inclusion of a heteroatom permits aromaticity in 5-membered rings as well as in 6-membered rings. Typical

heteroaromatic systems include monocyclic C5-C6 heteroaromatic groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, triazolyl, triazinyl, tetrazolyl, tetrazinyl, and imidazolyl, as well as the fused bicyclic moieties formed by fusing one of these monocyclic heteroaromatic groups with a phenyl ring or with any of the heteroaromatic monocyclic groups to form a Cs-C-io bicyclic group such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, pyrazolylpyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and other ring systems known in the art. Any monocyclic or fused ring bicyclic system that has the characteristics of aromaticity in terms of delocalized electron distribution throughout the ring system is included in this definition. This definition also includes bicyclic groups where at least the ring that is directly attached to the remainder of the molecule has the characteristics of aromaticity, including the delocalized electron distribution that is characteristic of aromaticity. Typically, the ring systems contain 5 to 12 ring member atoms and up to four heteroatoms, wherein the heteroatoms are selected from the group consisting of N, 0, and S. Frequently, the monocyclic heteroaryls contain 5 to 6 ring members and up to three heteroatoms selected from the group consisting of N, 0, and S; frequently, the bicyclic heteroaryls contain 8 to 10 ring members and up to four heteroatoms selected from the group consisting of N, 0, and S. The number and placement of heteroatoms in heteroaryl ring structures is in

accordance with the well-known limitations of aromaticity and stability, where stability requires the heteroaromatic group to be stable enough to be exposed to water at physiological temperatures without rapid degradation. As used herein, the term

"hydroxheteroaryl" refers to a heteroaryl group including one or more hydroxyl groups as substituents; as further detailed below, further substituents can be optionally included. As used herein, the terms "haloaryl" and "haloheteroaryl" refer to aryl and heteroaryl groups, respectively, substituted with at least one halo group, where "halo" refers to a halogen selected from the group consisting of fluorine, chlorine, bromine, and iodine, typically, the halogen is selected from the group consisting of chlorine, bromine, and iodine; as detailed below, further substituents can be optionally included. As used herein, the terms "haloalkyl," "haloalkenyl," and "haloalkynyl" refer to alkyl, alkenyl, and alkynyl groups, respectively, substituted with at least one halo group, where "halo" refers to a halogen selected from the group consisting of fluorine, chlorine, bromine, and iodine, typically, the halogen is selected from the group consisting of chlorine, bromine, and iodine; as detailed below, further substituents can be optionally included.

[0072] As used herein, the term "optionally substituted" indicates that the particular group or groups referred to as optionally substituted may have no non- hydrogen substituents, or the group or groups may have one or more non-hydrogen substituents consistent with the chemistry and pharmacological activity of the resulting molecule. If not otherwise specified, the total number of such substituents that may be present is equal to the total number of hydrogen atoms present on the unsubstituted form of the group being described; fewer than the maximum number of such

substituents may be present. Where an optional substituent is attached via a double bond, such as a carbonyl oxygen (C=0), the group takes up two available valences on the carbon atom to which the optional substituent is attached, so the total number of substituents that may be included is reduced according to the number of available valences. As used herein, the term "substituted," whether used as part of "optionally substituted" or otherwise, when used to modify a specific group, moiety, or radical, means that one or more hydrogen atoms are, each, independently of each other, replaced with the same or different substituent or substituents.

[0073] Substituent groups useful for substituting saturated carbon atoms in the specified group, moiety, or radical include, but are not limited to,— Z^a, =0,— OZ^b,— SZ^b, =S^",— NZ^CZ^C, =NZ^b, =N— OZ^b, trihalomethyl,— CF₃,— CN,— OCN,— SCN,—NO, — NO₂, =N₂,— N₃,— S(O)₂Z^b,— S(O)₂NZ^b,— S(O₂)O^",— S(O₂)OZ^b,— OS(O₂)OZ^b,— OS(O₂)O^",— OS(O₂)OZ^b,— P(O)(O-)₂,— P(O)(OZ^b)(O^"),— P(O)(OZ^b)(OZ^b),— C(O)Z^b, — C(S)Z^b,— C(NZ^b)Z^b,— C(O)O^",— C(O)OZ^b,— C(S)OZ^b,— C(O)NZ^cZ^c,— C(NZ^b)NZ^cZ^c,— OC(0)Z^b,— OC(S)Z^b,—00(0)0^",— OC(O)OZ^b,— OC(S)OZ^b,— NZ^bC(O)Z^b,— NZ^bC(S)Z^b,— NZ^bC(O)O^",— NZ^bC(O)OZ^b,— NZ^bC(S)OZ^b, —

NZ^bC(O)NZ^cZ^c,— NZ^bC(NZ^b)Z^b,— NZ^bC(NZ^b)NZ^cZ^c, wherein Z^a is selected from the group consisting of alkyl, cycloalkyl, heteroalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl; each Z^b is independently hydrogen or Z^a; and each Z^c is independently Z^b or, alternatively, the two Z^c's may be taken together with the nitrogen atom to which they are bonded to form a 4-, 5-, 6-, or 7-membered cycloheteroalkyl ring structure which may optionally include from 1 to 4 of the same or different heteroatoms selected from the group consisting of N, O, and S. As specific examples,— NZ^CZ^C is meant to include— NH2,— NH-alkyl,— N-pyrrolidinyl, and— N-morpholinyl, but is not limited to those specific alternatives and includes other alternatives known in the art. Similarly, as another specific example, a substituted alkyl is meant to include— alkylene-O-alkyl,— alkylene-heteroaryl,— alkylene-cycloheteroaryl,— alkylene- C(O)OZ^b,— alkylene-C(O)NZ^bZ^b, and— CH^ CH₂— C(O)-CH₃, but is not limited to those specific alternatives and includes other alternatives known in the art. The one or more substituent groups, together with the atoms to which they are bonded, may form a cyclic ring, including, but not limited to, cycloalkyl and cycloheteroalkyl.

[0074] Similarly, substituent groups useful for substituting unsaturated carbon atoms in the specified group, moiety, or radical include, but are not limited to,— Z^a, halo,— O-,— OZ^b,— SZ^b,— S^",— NZ^CZ^C, trihalomethyl,— CF₃,— CN,— OCN,— SCN, —NO,— NO₂,— N₃,— S(O)₂Z^b,— S(O₂)O-,— S(O₂)OZ^b,— OS(O₂)OZ^b,— OS(O₂)O^",— P(O)(O-)₂,— P(O)(OZ^b)(O^"),— P(O)(OZ^b)(OZ^b),— C(O)Z^b,— C(S)Z^b,— C(NZ^b)Z^b,— C(O)O^",— C(O)OZ^b,— C(S)OZ^b,— C(O)NZ^cZ^c,— C(NZ^b)NZ^cZ^c,— OC(O)Z^b,— OC(S)Z^b, — OC(O)O^",— OC(O)OZ^b,— OC(S)OZ^b,— NZ^bC(O)OZ^b,— NZ^bC(S)OZ^b,—

NZ^bC(O)NZ^cZ^c,— NZ^bC(NZ^b)Z^b, and— NZ^bC(NZ^b)NZ^cZ^c, wherein Z^a, Z^b, and Z^c are as defined above.

[0075] Similarly, substituent groups useful for substituting nitrogen atoms in heteroalkyl and cycloheteroalkyl groups include, but are not limited to,— Z^a, halo,— O^", — OZ^b,— SZ^b,— S^",— NZ^CZ^C, trihalomethyl,— CF₃,— CN,—OCN,—SCN,—NO,— NO₂,— S(O)₂Z^b,— S(O₂)O^",— S(O₂)OZ^b,— OS(O₂)OZ^b,— OS(O₂)O^",— P(O)(O )₂,— P(0)(OZ^b)(0-),— P(0)(OZ^b)(OZ^b),— C(0)Z^b,— C(S)Z^b,— C(NZ^b)Z^b,— C(0)OZ^b,— C(S)OZ^b,— C(0)NZ^cZ^c,— C(NZ^b)NZ^cZ^c,— OC(0)Z^b,— OC(S)Z^b,— OC(0)OZ^b,— OC(S)OZ^b,— NZ^bC(0)Z^b,— NZ^bC(S)Z^b,— NZ^bC(0)OZ^b,— NZ^bC(S)OZ^b,—

NZ^bC(0)NZ^cZ^c,— NZ^bC(NZ^b)Z^b, and— NZ^bC(NZ^b)NZ^cZ^c, wherein Z^a, Z^b, and Z^c are as defined above.

[0076] The compounds described herein may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers such as E and Z), enantiomers or diastereomers. The invention includes each of the isolated stereoisomeric forms (such as the

enantiomerically pure isomers, the E and Z isomers, and other stereoisomeric forms) as well as mixtures of stereoisomers in varying degrees of chiral purity or percentage of E and Z, including racemic mixtures, mixtures of diastereomers, and mixtures of E and Z isomers. Accordingly, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated compounds including the

stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan. The invention includes each of the isolated stereoisomeric forms as well as mixtures of stereoisomers in varying degrees of chiral purity, including racemic mixtures. It also encompasses the various diastereomers. Other structures may appear to depict a specific isomer, but that is merely for convenience, and is not intended to limit the invention to the depicted isomer. When the chemical name does not specify the isomeric form of the compound, it denotes any one of the possible isomeric forms or mixtures of those isomeric forms of the compound.

[0077] The compounds may also exist in several tautomeric forms, and the depiction herein of one tautomer is for convenience only, and is also understood to encompass other tautomers of the form shown. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds. The term "tautomer" as used herein refers to isomers that change into one another with great ease so that they can exist together in equilibrium. For example, ketone and enol are two tautomeric forms of one compound.

[0078] 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 of the invention, with one or more solvent molecules. 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 salt and/or prodrug of the present compound may also exist in a solvate form. The solvate is typically formed via hydration which is either part of the preparation of the present compound or through natural absorption of moisture by the anhydrous compound of the present invention.

[0079] As used herein, the term "ester" means any ester of a present compound in which any of the -COOH functions of the molecule is replaced by a -COOR function, in which the R moiety of the ester is any carbon-containing group which forms a stable ester moiety, including but not limited to alkyl, alkenyl, alkynyl, cycloalkyl,

cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and substituted derivatives thereof. The hydrolyzable esters of the present compounds are the compounds whose carboxyls are present in the form of hydrolyzable ester groups. That is, these esters are pharmaceutically acceptable and can be hydrolyzed to the corresponding carboxyl acid in vivo.

[0080] In addition to the substituents described above, alkyl, alkenyl and alkynyl groups can alternatively or in addition be substituted by C-i-Cs acyl, C2-C8 heteroacyl, C6-C10 aryl, C3-C8 cycloalkyl, C3-C8 heterocyclyl, or C5-C10 heteroaryl, each of which can be optionally substituted. Also, in addition, when two groups capable of forming a ring having 5 to 8 ring members are present on the same or adjacent atoms, the two groups can optionally be taken together with the atom or atoms in the substituent groups to which they are attached to form such a ring. [0081] "Heteroalkyl," "heteroalkenyl," and "heteroalkynyl" and the like are defined similarly to the corresponding hydrocarbyl (alkyl, alkenyl and alkynyl) groups, but the 'hetero' terms refer to groups that contain 1 -3 0, S or N heteroatoms or combinations thereof within the backbone residue; thus at least one carbon atom of a corresponding alkyl, alkenyl, or alkynyl group is replaced by one of the specified heteroatoms to form, respectively, a heteroalkyl, heteroalkenyl, or heteroalkynyl group. For reasons of chemical stability, it is also understood that, unless otherwise specified, such groups do not include more than two contiguous heteroatoms except where an oxo group is present on N or S as in a nitro or sulfonyl group.

[0082] While "alkyl" as used herein includes cycloalkyl and cycloalkylalkyl groups, the term "cycloalkyl" may be used herein to describe a carbocyclic non-aromatic group that is connected via a ring carbon atom, and "cycloalkylalkyl" may be used to describe a carbocyclic non-aromatic group that is connected to the molecule through an alkyl linker.

[0083] Similarly, "heterocyclyl" may be used to describe a non-aromatic cyclic group that contains at least one heteroatom (typically selected from N, 0 and S) as a ring member and that is connected to the molecule via a ring atom, which may be C (carbon-linked) or N (nitrogen-linked); and "heterocyclylalkyl" may be used to describe such a group that is connected to another molecule through a linker. The heterocyclyl can be fully saturated or partially saturated, but non-aromatic. The sizes and

substituents that are suitable for the cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl groups are the same as those described above for alkyl groups. The heterocyclyl groups typically contain 1 , 2 or 3 heteroatoms, selected from N, 0 and S as ring members; and the N or S can be substituted with the groups commonly found on these atoms in heterocyclic systems. As used herein, these terms also include rings that contain a double bond or two double bonds, as long as the ring that is attached is not aromatic. The substituted cycloalkyl and heterocyclyl groups also include cycloalkyl or heterocyclic rings fused to an aromatic ring or heteroaromatic ring, provided the point of attachment of the group is to the cycloalkyl or heterocyclyl ring rather than to the aromatic/heteroaromatic ring. [0084] As used herein, "acyl" encompasses groups comprising an alkyl, alkenyl, alkynyl, aryl or arylalkyi radical attached at one of the two available valence positions of a carbonyl carbon atom, and heteroacyl refers to the corresponding groups wherein at least one carbon other than the carbonyl carbon has been replaced by a heteroatom chosen from N, 0 and S.

[0085] Acyl and heteroacyl groups are bonded to any group or molecule to which they are attached through the open valence of the carbonyl carbon atom.

Typically, they are C-i-Cs acyl groups, which include formyl, acetyl, pivaloyl, and benzoyl, and C2-C8 heteroacyl groups, which include methoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl.

[0086] Similarly, "arylalkyi" and "heteroarylalkyl" refer to aromatic and

heteroaromatic ring systems which are bonded to their attachment point through a linking group such as an alkylene, including substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic linkers. Typically the linker is C-i-Cs alkyl. These linkers may also include a carbonyl group, thus making them able to provide substituents as an acyl or heteroacyl moiety. An aryl or heteroaryl ring in an arylalkyi or heteroarylalkyl group may be substituted with the same substituents described above for aryl groups. Preferably, an arylalkyi group includes a phenyl ring optionally substituted with the groups defined above for aryl groups and a C1-C4 alkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl groups or heteroalkyl groups, where the alkyl or heteroalkyl groups can optionally cyclize to form a ring such as cyclopropane, dioxolane, or oxacyclopentane. Similarly, a heteroarylalkyl group preferably includes a C5-C6 monocyclic heteroaryl group that is optionally substituted with the groups described above as substituents typical on aryl groups and a C1-C4 alkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl groups or heteroalkyl groups, or it includes an optionally substituted phenyl ring or C5-C6 monocyclic heteroaryl and a C1-C4 heteroalkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl or heteroalkyl groups, where the alkyl or heteroalkyl groups can optionally cyclize to form a ring such as cyclopropane, dioxolane, or oxacyclopentane. [0087] Where an arylalkyl or heteroarylalkyl group is described as optionally substituted, the substituents may be on either the alkyl or heteroalkyi portion or on the aryl or heteroaryl portion of the group. The substituents optionally present on the alkyl or heteroalkyi portion are the same as those described above for alkyl groups generally; the substituents optionally present on the aryl or heteroaryl portion are the same as those described above for aryl groups generally.

[0088] "Arylalkyl" groups as used herein are hydrocarbyl groups if they are unsubstituted, and are described by the total number of carbon atoms in the ring and alkylene or similar linker. Thus a benzyl group is a C7-arylalkyl group, and phenylethyl is a C8-arylalkyl.

[0089] "Heteroarylalkyl" as described above refers to a moiety comprising an aryl group that is attached through a linking group, and differs from "arylalkyl" in that at least one ring atom of the aryl moiety or one atom in the linking group is a heteroatom selected from N, O and S. The heteroarylalkyl groups are described herein according to the total number of atoms in the ring and linker combined, and they include aryl groups linked through a heteroalkyi linker; heteroaryl groups linked through a hydrocarbyl linker such as an alkylene; and heteroaryl groups linked through a heteroalkyi linker. Thus, for example, C7-heteroarylalkyl would include pyridylmethyl, phenoxy, and N- pyrrolylmethoxy.

[0090] "Alkylene" as used herein refers to a divalent hydrocarbyl group; because it is divalent, it can link two other groups together. Typically it refers to— (CH2)n— where n is 1 -8 and preferably n is 1 -4, though where specified, an alkylene can also be substituted by other groups, and can be of other lengths, and the open valences need not be at opposite ends of a chain. The general term "alkylene" encompasses more specific examples such as "ethylene," wherein n is 2, "propylene," wherein n is 3, and "butylene," wherein n is 4. The hydrocarbyl groups of the alkylene can be optionally substituted as described above.

[0091] In general, any alkyl, alkenyl, alkynyl, acyl, or aryl or arylalkyl group that is contained in a substituent may itself optionally be substituted by additional substituents. The nature of these substituents is similar to those recited with regard to the primary substituents themselves if the substituents are not otherwise described.

[0092] "Amino" as used herein refers to— NH2, but where an amino is described as "substituted" or "optionally substituted", the term includes NR'R" wherein each R' and R" is independently H, or is an alkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl group, and each of the alkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl groups is optionally substituted with the substituents described herein as suitable for the corresponding group; the R' and R" groups and the nitrogen atom to which they are attached can optionally form a 3- to 8-membered ring which may be saturated, unsaturated or aromatic and which contains 1 -3 heteroatoms independently selected from N, 0 and S as ring members, and which is optionally substituted with the substituents described as suitable for alkyl groups or, if NR'R" is an aromatic group, it is optionally substituted with the substituents described as typical for heteroaryl groups.

[0093] As used herein, the term "carbocycle," "carbocyclyl," or "carbocyclic" refers to a cyclic ring containing only carbon atoms in the ring, whereas the term

"heterocycle" or "heterocyclic" refers to a ring comprising a heteroatom. The carbocyclyl can be fully saturated or partially saturated, but non-aromatic. For example, the general term "carbocyclyl" encompasses cycloalkyl. The carbocyclic and heterocyclic structures encompass compounds having monocyclic, bicyclic or multiple ring systems; and such systems may mix aromatic, heterocyclic, and carbocyclic rings. Mixed ring systems are described according to the ring that is attached to the rest of the compound being described.

[0094] As used herein, the term "heteroatom" refers to any atom that is not carbon or hydrogen, such as nitrogen, oxygen or sulfur, although, in some contexts, "heteroatom" can refer to phosphorus, selenium, or other atoms other than carbon or hydrogen. When it is part of the backbone or skeleton of a chain or ring, a heteroatom must be at least divalent, and will typically be selected from N, O, P, and S.

[0095] As used herein, the term "alkanoyl" refers to an alkyl group covalently linked to a carbonyl (C=0) group. The term "lower alkanoyl" refers to an alkanoyl group in which the alkyl portion of the alkanoyl group is C1-C6. The alkyl portion of the alkanoyl group can be optionally substituted as described above. The term

"alkylcarbonyl" can alternatively be used. Similarly, the terms "alkenylcarbonyl" and "alkynylcarbonyl" refer to an alkenyl or alkynyl group, respectively, linked to a carbonyl group.

[0096] As used herein, the term "alkoxy" refers to an alkyl group covalently linked to an oxygen atom; the alkyl group can be considered as replacing the hydrogen atom of a hydroxyl group. The term "lower alkoxy" refers to an alkoxy group in which the alkyl portion of the alkoxy group is C1-C6. The alkyl portion of the alkoxy group can be optionally substituted as described above. As used herein, the term "haloalkoxy" refers to an alkoxy group in which the alkyl portion is substituted with one or more halo groups.

[0097] As used herein, the term "sulfo" refers to a sulfonic acid (— SO3H) substituent.

[0098] As used herein, the term "sulfamoyl" refers to a substituent with the structure— S(02)NH2, wherein the nitrogen of the NH2 portion of the group can be optionally substituted as described above.

[0099] As used herein, the term "carboxyl" refers to a group of the structure— C(0₂)H.

[0100] As used herein, the term "carbamyl" refers to a group of the structure— C(02)NH2, wherein the nitrogen of the NH2 portion of the group can be optionally substituted as described above.

[0101] As used herein, the terms "monoalkylaminoalkyl" and "dialkylaminoalkyl" refer to groups of the structure— Alki-NH-Alk₂ and — Alki-N(Alk₂)(Alk3), wherein Alki, Alk2, and Alk3 refer to alkyl groups as described above.

[0102] As used herein, the term "alkylsulfonyl" refers to a group of the structure — S(0)2-Alk wherein Alk refers to an alkyl group as described above. The terms

"alkenylsulfonyl" and "alkynylsulfonyl" refer analogously to sulfonyl groups covalently bound to alkenyl and alkynyl groups, respectively. The term "arylsulfonyl" refers to a group of the structure— S(0)2-Ar wherein Ar refers to an aryl group as described above. The term "aryloxyalkylsulfonyl" refers to a group of the structure— S(0)2-Alk-0-Ar, where Alk is an alkyi group as described above and Ar is an aryl group as described above. The term "arylalkylsulfonyl" refers to a group of the structure— S(0)2-AlkAr, where Alk is an alkyi group as described above and Ar is an aryl group as described above.

[0103] As used herein, the term "alkyloxycarbonyl" refers to an ester substituent including an alkyi group wherein the carbonyl carbon is the point of attachment to the molecule. An example is ethoxycarbonyl, which is ChhChbOCiO)— . Similarly, the terms "alkenyloxycarbonyl," "alkynyloxycarbonyl," and "cycloalkylcarbonyl" refer to similar ester substituents including an alkenyl group, alkenyl group, or cycloalkyl group respectively. Similarly, the term "aryloxycarbonyl" refers to an ester substituent including an aryl group wherein the carbonyl carbon is the point of attachment to the molecule. Similarly, the term "aryloxyalkylcarbonyl" refers to an ester substituent including an alkyi group wherein the alkyi group is itself substituted by an aryloxy group.

[0104] Other combinations of substituents are known in the art and, are described, for example, in United States Patent No. 8,344, 162 to Jung et al. For example, the term "thiocarbonyl" and combinations of substituents including

"thiocarbonyl" include a carbonyl group in which a double-bonded sulfur replaces the normal double-bonded oxygen in the group. The term "alkylidene" and similar terminology refer to an alkyi group, alkenyl group, alkynyl group, or cycloalkyl group, as specified, that has two hydrogen atoms removed from a single carbon atom so that the group is double-bonded to the remainder of the structure.

[0105] Accordingly, methods and compositions according to the present invention encompass bisantrene derivatives and analogs including one or more optional substituents as defined above, provided that the optionally substituted bisantrene derivative or analog possesses substantially equivalent pharmacological activity to bisantrene as defined in terms of either or both topoisomerase II inhibition and DNA intercalation. Methods for determination of topoisomerase II inhibition are known in the art and are described, for example, in A. Constantinou et al., "Novobiocin- and Phorbol- 12-Myristate-13-Acetate-lnduced Differentiation of Human Leukemia Cells Associates with a Reduction in Topoisomerase II Activity," Cancer Res. 49: 1 1 10-1 1 17 (1989). Methods for determination of DNA intercalation are known in the art and are described, for example, in H. Zipper et al., "Investigations on DNA Intercalation and Surface

Binding by SYBR Green I, Its Structure Determination and Methodological Implications," Nucl. Acids. Res. 32(12): e103 (2004).

[0106] Other analogs and derivatives are known in the art, including further derivatives and salt forms of the compounds described above.

[0107] In general, the preferred alternative for bisantrene or a derivative or analog thereof is bisantrene, particularly bisantrene dihydrochloride.

[0108] II. Liposomes

[0109] Liposomes suitable for incorporating the bisantrene or derivative or analog thereof and, in other alternatives as described below, the pyrimidine analog antimetabolite, in a composition according to the present invention can be prepared by techniques well known in the art, including those described in A.S. Janoff, ed.,

Liposomes: Rational Design (1999, Marcel Dekker, Inc., New York). The use of liposomes to administer antineoplastic agents is disclosed in United States Patent No. 8,750,810 to Okada et al. and in United States Patent No. 9,717,686 to Yang et al.

Suitable liposomes include, but are not necessarily limited to, large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs), and interdigitating fusion liposomes. The liposomes can include a phosphatidylcholine lipid, such as distearylphosphatidylcholine. The liposomes can also include a phosphatidylglycerol lipid, such as

distearoylphosphatidylglycerol. The liposomes can also include a sterol such as cholesterol. Liposomes according to the present invention can also include other types of lipids, including, but not limited to, ether lipids, phosphatidic acid, phosphonates, ceramides, ceramide analogs, sphingosines, sphingosine analogs, and serine- containing lipids. Liposomes according to the present invention can also include surface stabilizing hydrophilic polymer-lipid conjugates such as polyethylene glycol- DSPE, to enhance circulation longevity. The incorporation of negatively charged lipids such as phosphatidylglycerol (PG) and phosphatidylinositol (PI) may also be added to liposome formulations to increase the circulation longevity of the carrier. These lipids may be employed to replace hydrophilic polymer-lipid conjugates as surface stabilizing agents.

[0110] Typically, liposomes according to the present invention have a diameter of less than about 300 nm. Preferably, liposomes according to the present invention have a diameter of less than about 200 nm.

[0111] Various methods may be utilized to encapsulate active agents in liposomes. One method that can be used is encapsulation. Encapsulation includes covalent or non-covalent association of an agent with the lipid-based delivery vehicle. For example, this can be by interaction of the agent with the outer layer or layers of the liposome or entrapment of an agent within the liposome, equilibrium being achieved between different portions of the liposome. Thus encapsulation of an agent can be by association of the agent by interaction with the bilayer of the liposomes through covalent or non-covalent interaction with the lipid components or entrapment in the aqueous interior of the liposome, or in equilibrium between the internal aqueous phase and the bilayer. The term "loading" or equivalent terminology is used herein to refer to the act of encapsulating one or more agents into a delivery vehicle. Techniques for encapsulation are dependent on the nature of the delivery vehicles. For example, therapeutic agents may be loaded into liposomes using both passive and active loading methods. Passive methods of encapsulating active agents in liposomes involve encapsulating the agent during the preparation of the liposomes. This includes a passive entrapment method described by Bangham et al. (J. Mol. Biol. (1965) 12:238). This technique results in the formation of multilamellar vesicles (MLVs) where the aqueous phase containing the agent of interest is put into contact with a film of dried vesicle-forming lipids deposited on the walls of a reaction vessel. Upon agitation by mechanical means, swelling of the lipids will occur and multilamellar vesicles (MLV) will form that can be converted to large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs) upon extrusion.

Another suitable method of passive encapsulation includes an ether injection technique described by Deamer and Bangham (Biochim. Biophys. Acta (1976) 443:629) involving dissolving vesicle-forming lipids in ether and, instead of first evaporating the ether to form a thin film on a surface, this film is thereafter put into contact with an aqueous phase to be encapsulated, the ether solution is directly injected into the aqueous phase and the ether is evaporated afterwards, whereby liposomes with encapsulated agents are obtained. Another technique is the Reverse Phase Evaporation technique as described by Szoka and Paphadjopoulos (Proc. Natl. Acad. Sci. (1978) 75:4194), in which a solution of lipids in a water insoluble organic solvent is emulsified in an aqueous carrier phase and the organic solvent is subsequently removed under reduced pressure. In addition, another suitable method of passive encapsulation involves passive equilibration after the formation of liposomes. This process involves incubating preformed liposomes under altered or non-ambient (based on temperature, pressure, or other factors) conditions and adding a therapeutic agent (e.g., the bisantrene or the derivative or analog thereof and the pyrimidine analog antimetabolite as described in the alternative below) to the exterior of the liposomes. The therapeutic agents then equilibrate into the interior of the liposomes, across the liposomal membrane. The liposomes are then returned to ambient conditions and unencapsulated therapeutic agents, if present, are removed via dialysis or another suitable method. Other methods of passive entrapment that may be used include subjecting liposomes to successive dehydration and rehydration treatment, or freezing and thawing. Dehydration is carried out by evaporation or freeze-drying. This technique is disclosed by Kirby et al.,

Biotechnology (1984) 979-984. Also, Shew and Deamer (Biochim. Biophys. Acta (1985) 816: 1 -8) describe a method wherein liposomes prepared by sonication are mixed in aqueous solution with the solute to be encapsulated, and the mixture is dried under nitrogen in a rotating flask. Upon rehydration, large liposomes are produced in which a significant fraction of the solute has been encapsulated. Active methods of encapsulation include the pH gradient loading technique described in U.S. Pat. Nos. 5,616,341 , 5,736, 155, and 5,785,987 and active metal-loading. One method of pH gradient loading is the citrate-base loading method utilizing citrate as the internal buffer at a pH of 4.0 and a neutral exterior buffer. Other methods employed to establish and maintain a pH gradient across a liposome involve the use of an ionophore that can insert into the liposome membrane and transport ions across membranes in exchange for protons (see U.S. Pat. No. 5,837,282). A recent technique utilizing transition metals to drive the uptake of drugs into liposomes via complexation in the absence of an ionophore may also be used. This technique relies on the formation of a drug-metal complex rather than the establishment of a pH gradient to drive uptake of drug. Other procedures for generation of liposomes are known in the art, including, but not limited to, lipid film/hydration, reverse phase evaporation, detergent dialysis, freeze/thaw, homogenization, solvent dilution, and extrusion.

[0112] For pH gradient loading, in order to create a pH gradient, the original external medium can be replaced by a new external medium having a different pH value. The replacement of the external medium can be accomplished by various techniques, such as, by passing the lipid vesicle preparation through a gel filtration column, e.g., a Sephadex G-50 column, which has been equilibrated with the new medium, or by centrifugation, dialysis, or related techniques. The internal medium may be either acidic or basic with respect to the external medium. After establishment of a pH gradient, a pH gradient loadable agent is added to the mixture and encapsulation of the agent in the liposome occurs as described above. A preferred method of pH gradient loading is the citrate-based loading method utilizing citrate as the internal buffer at a pH of 2-6 and a neutral external buffer. Various methods are known in the art for establishing and maintaining a pH gradient across a liposome. This may involve the use of ionophores that can insert into the liposome membrane and transport ions across membranes in exchange for protons. Compounds encapsulated in the interior of the liposome that are able to shuttle protons across the liposomal membrane and thus set up a pH gradient may also be utilized. These compounds comprise an ionizable moiety that is neutral when deprotonated and charged when protonated. The neutral deprotonated form (which is in equilibrium with the protonated form) is able to cross the liposome membrane and thus leave a proton behind in the interior of the liposome and thereby cause a decrease in the pH of the interior by making the interior more acidic. Examples of such compounds include methylammonium chloride, methylammonium sulfate, or ethylenediammonium sulfate. Internal loading buffers that are able to establish a basic internal pH can also be utilized. In this case, the neutral form is protonated such that protons are shuttled out of the liposome interior to establish a basic interior. An example of such a compound is calcium acetate.

[0113] In some alternatives, particularly as described below, two or more agents may be loaded into a liposome using the same active loading methods or may involve the use of different active loading methods. These alternatives are not required when only the bisantrene or the derivative or analog of bisantrene is to be loaded into the liposome. For instance, metal complexation loading may be utilized to actively load multiple agents or may be coupled with another active loading technique, such as pH gradient loading. Metal-based active loading typically uses liposomes with passively encapsulated metal ions (with or without passively loaded therapeutic agents). Various salts of metal ions are used, presuming that the salt is pharmaceutically acceptable and soluble in an aqueous solution. Actively loaded agents are selected based on being capable of forming a complex with a metal ion and thus being retained when so complexed within the liposome, yet capable of loading into a liposome when not complexed to metal ions. Agents that are capable of coordinating with a metal typically comprise coordination sites such as amines, carbonyl groups, ethers, ketones, acyl groups, acetylenes, olefins, thiols, hydroxyl or halide groups or other suitable groups capable of donating electrons to the metal ion thereby forming a complex with the metal ion. Uptake of an agent may be established by incubation of the mixture at a suitable temperature after addition of the agent to the external medium. Depending on the composition of the liposome, temperature and pH of the internal medium, and the chemical nature of the agent, uptake of the agent may occur over a time period of minutes or hours. Methods of determining whether coordination occurs between an agent and a metal within a liposome include spectrophotometric analysis and other conventional techniques known in the art.

[0114] Passive encapsulation of two or more agents is possible for many drug combinations. Again, this alternative is not required when only the bisantrene or the derivative or analog of bisantrene is to be loaded into the liposome, but can be used when both the bisantrene or the derivative or analog of bisantrene and the pyrimidine analog antimetabolite are to be loaded into the liposome. This approach is limited by the solubility of the drugs in aqueous buffer solutions and the large percentage of drug that is not trapped within the delivery system. The loading may be improved by co- lyophilizing the drugs with the lipid sample and rehydrating in the minimal volume allowed to solubilize the drugs. The solubility may be improved by varying the pH of the buffer, increasing temperature or addition or removal of salts from the buffer.

[0115] Furthermore, liposome loading efficiency and retention properties using metal-based procedures carried out in the absence of an ionophore in the liposome are dependent on the metal employed and the lipid composition of the liposome. By selecting lipid composition and a metal, loading or retention properties can be tailored to achieve a desired loading or release of a selected agent from a liposome.

[0116] Formation of liposomes requires the presence of "vesicle-forming lipids" which are amphipathic lipids capable of either forming or being incorporated into a bilayer structure. The latter term includes lipids that are capable of forming a bilayer by themselves or when in combination with another lipid or lipids. An amphipathic lipid is incorporated into a lipid bilayer by having its hydrophobic moiety in contact with the interior, hydrophobic region of the membrane bilayer and its polar head moiety oriented toward an outer, polar surface of the membrane. Hydrophilicity may arise from the presence of functional groups such as hydroxyl, phosphato, carboxyl, sulfato, amino or sulfhydryl groups which are polar or substantially polar, including charged groups.

Hydrophobicity results from the presence of a long chain of aliphatic hydrocarbon groups which are substantially nonpolar. The vesicle-forming lipids included in the liposomes of the invention will typically comprise at least one acyl group with a chain length of at least 16 carbon atoms. In one alternative, phospholipids used as vesicle forming components include dipalmitoyl phosphatidylcholine (DPPC) and distearoyl phosphatidylcholine (DSPC). DPPC is a common saturated chain (Cie) phospholipid with a bilayer phase transition temperature of 41 .5° C. Liposomes containing DPPC and other lipids that have a similar or higher transition temperature, and that mix ideally with DPPC (such as 1 ,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1 -glycerol)] (DPPG) (Tc=41 .5° C) and 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (T_c=55.1 ° C)) have been studied. In one alternative, the liposomes of the present invention have a phase transition temperature greater than 38° C; this can be accomplished by employing components which confer this property. The ultimate transition temperature will depend on the acyl chain length as well as the degree of unsaturation of the acyl groups. Including unsaturation in the chain lowers the transition temperature so that in the event the acyl groups are unsaturated, acyl groups containing 18 carbons or 20 carbons or more can be used. Liposomes may also be prepared such that the liquid crystalline transition temperature is greater than 45° C. Vesicle-forming lipids making up the liposome are phospholipids such as phosphatidylcholine (PC), phosphatidyl (PA) or phosphatidylethanolamine (PE), containing two saturated fatty acids, while the acyl chains are preferably stearoyl (18:0), nonadecanoyl (19:0), arachidoyl (20:0),

heniecosanoyl (21 :0), behenoyl (22:0), tricosanoyl (23:0), lignoceroyl (24:0) or cerotoyl (26:0); other lipids can also be incorporated.

[0117] A heterodisperse suspension of liposomes formed by methods described above may be "size reduced" using conventional techniques to produce liposomes within a desired size range and reduced polydispersity. Conventional size-reduction techniques include but are not limited to sonication, homogenization and extrusion. In extrusion methods a variety of membrane pore sizes are available to produce liposomes in various size ranges. A drawback of this technique for low-cholesterol liposomes is the tendency for large vesicles to deform and thus pass through narrow extrusion filters when extruded under standard temperatures (i.e., above the vesicle phase transition temperature, which is a temperature above the phase transition temperature of the highest melting lipid in the lipid-based delivery vehicle). The result is a suspension with increased heterogeneity containing oversized vesicles. A further drawback of this technique is the inability to filter sterilize the resultant suspension. In one embodiment, a heterodisperse suspension of MLVs is extruded at least once at the higher

temperature and then at least once at a temperature below the vesicle phase transition temperature, thus overcoming the difficulties in size-reducing gel-phase liposomes to levels that are sufficient for filter sterilization by reducing the number of excessively large liposomes that inadvertently pass through the extrusion filter at high temperatures. [0118] In some alternatives of compositions according to the present invention, the liposomes are low-cholesterol liposomes. The incorporation of less than 20 mol % cholesterol in liposomes can allow for retention of drugs not optimally retained when liposomes are prepared with greater than 20 mol % cholesterol. Additionally, liposomes prepared with less than 20 mol % cholesterol display narrow phase transition

temperatures, a property that may be exploited for the preparation of liposomes that release encapsulated agents due to the application of heat (thermosensitive liposomes). Liposomes may also be prepared with surface stabilizing hydrophilic polymer-lipid conjugates such as polyethylene glycol-DSPE, to enhance circulation longevity. The incorporation of negatively charged lipids such as phosphatidylglycerol (PG) and phosphatidylinositol (PI) may also be added to liposome formulations to increase the circulation longevity of the carrier. These lipids may be employed to replace hydrophilic polymer-lipid conjugates as surface stabilizing agents. Cholesterol-free liposomes containing PG or PI to prevent aggregation may be prepared, thereby increasing the blood residence time of the carrier.

[0119] Suitable proportions of lipids used in liposomes according to the present invention are as described below. In particular, the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof can be from about 25: 1 to about 1 : 1 . Preferably, the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 10: 1 to about 3: 1. More preferably, the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is about 5: 1 .

[0120] In one alternative, the liposomes can include a negatively charged lipid having a hydrophilic portion and a hydrophobic portion with a neutral non-zwitterionic moiety attached to the hydrophilic portion of the lipid. The negatively charged lipid is typically a phospholipid or a sphingophospholipid. Preferably, the lipid is a

phospholipid; i.e., a glycerol to which two acyl groups are attached and wherein the third hydroxyl is coupled to a phosphate. The non-zwitterionic moiety is attached to this negatively charged lipid, preferably to the phosphate group. Preferably, the non- zwitterionic moiety is neutral such that the net negative charge on a lipid used in this invention is due solely to the negative charge of the lipid component. The non- zwitterionic moiety may comprise functional groups that impart a desired hydrophilicity to the lipid, such groups being selected from alcohols, ketones, carboxylic acids, ethers and amines. A preferred non-zwitterionic moiety is a short-chain alcohol such as glycerol, or a cyclic alcohol, such as inositol, or a polyalkylene oxide such as PEG. In this alternative, typically, the liposomes comprise at least 10% of such negatively charged lipids having a hydrophilic portion and a hydrophobic portion with a neutral non- zwitterionic moiety attached to the hydrophilic portion of the lipid.

[0121] In some alternatives, the liposomes can have an intraliposomal osmolality of 500 mOSM/kg or less.

[0122] In one alternative, the liposome comprises distearoylphosphatidylcholine, distearoylphosphatidylglycerol and cholesterol. Typically, in this alternative, the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.5 to about 7.5 of distearoylphosphatidylcholine, about 1 .5 to about 2.5 of distearoylphosphatidylglycerol, and about 0.8 to 1 .2 of cholesterol.

Preferably, in this alternative, the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.8 to about 7.2 of distearoylphosphatidylcholine, about 1 .8 to about 2.2 of distearoylphosphatidylglycerol, and about 0.9 to 1 .1 of cholesterol. More preferably, in this alternative, the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is about 7:2: 1 .

[0123] Liposomes suitable for use as part of compositions according to the present invention are disclosed in the following United States Patents: 7,850,990 to Tardi et al.; 8,022,279 to Mayer et al.; 8,431 ,806 to Mayer et al.; 8,518,437 to Tardi et al.; and 9,271 ,931 to Tardi et al., and also in the following United States Patent

Application Publications: 2003/0147944 by Mayer et al.; 2003/0147945 by Tardi et al.; 2005/01 18249 by Webb et al.; 2005/01 18250 by Tardi et al.; 2006/0216341 by Tardi et al.; 2006/0240090 by Mayer et al.; 2008/0107722 by Tardi et al.; 2009/0098196 by Tardi et al.; 2009/0148506 by Dicko et al.; 2010/0303895 by Louie et al.; 201 1/0002982 by Tardi et al.; and 201 1/0223241 by Tardi et al. [0124] Another aspect of the invention is a composition of matter comprising: (i) a therapeutically effective quantity of bisantrene or a derivative or analog thereof; (ii) a therapeutically effective quantity of a pyrimidine analog antimetabolite; and (iii) a liposome encapsulating both the therapeutically effective quantity of bisantrene or the derivative or analog thereof and the pyrimidine analog antimetabolite.

[0125] In this aspect of the invention, the bisantrene or derivative or analog thereof is as described above. The liposome is also as described above, with the limitation that the liposome encapsulates both the therapeutically effective quantity of bisantrene or the derivative or analog thereof and the pyrimidine analog antimetabolite.

[0126] III. Pyrimidine Analog Antimetabolites

[0127] Pyrimidine analog antimetabolites that can be included in this alternative according to compositions according to the present invention include cytarabine, 5- azacytidine, gemcitabine, decitabine, fluoropyrimidines, 2'-deoxy-2'- methylidenecytidines, and analogs and derivatives as described below, as well as other pyrimidine analog antimetabolites as described below. The pyrimidine analog antimetabolite is typically selected from the group consisting of cytarabine, 5- azacytidine, gemcitabine, floxuridine, 5-fluorouracil, capecitabine, 6-azauracil, troxacitabine, thiarabine, sapacitabine, CNDAC (2'-cyano-2'-deoxy-1 -β-D- arabinofuranosylcytosine), 2'-deoxy-2'-methylidenecytidine, 2'-deoxy-2'- fluoromethylidenecytidine, 2'-deoxy-2'-methylidene-5-fluorocytidine, 2'-deoxy-2',2'- difluorocytidine, and 2'-C-cyano-2'-deoxy- -arabinofuranosylcytosine. Preferably, the pyrimidine analog antimetabolite is selected from the group consisting of cytarabine, 5- azacytidine, gemcitabine, floxuridine, 5-fluorouracil, capecitabine, and 6-azauracil. More preferably, the pyrimidine analog antimetabolite is cytarabine.

[0128] Cytarabine has the structure shown as Formula (N-l):

(N-l).

[0129] Cytarabine (4-amino-1 -[(2R,3S,4R,5R)-3,4-dihydroxy-5- (hydroxymethyl)oxolan-2-yl] pyrimidin-2-one) is a pyrimidine nucleoside antimetabolite. The compound is an analog of 2'-deoxycytidine with the 2'-hydroxyl in a position trans to the 3'-hydroxyl of the sugar moiety; therefore, the sugar moiety is an arabinose moiety rather than a ribose moiety. Cytarabine must be activated via conversion of the 5'-monophosphate nucleotide (AraCMP) to terminate strand synthesis. AraCMP is then able to react with nucleotide kinases to form diphosphate and triphosphate nucleotides (AraCDP and AraCTP). The incorporation of cytarabine into DNA is S-phase specific. Cytarabine inhibits DNA synthesis and kills cells, particularly rapidly dividing cells. It also inhibits DNA and RNA polymerases, as well as nucleotide reductases. Resistance to cytarabine can develop. Cytarabine can be rapidly deaminated by the enzyme cytidine deaminase in serum into the inactive uracil derivative. AraCMP is also deaminated by deoxycytidylate deaminase, leading to the inactive uridine-5'- monophosphate analog. Additionally, AraCTP is also a substrate for SAMDH1 , which hydrolyzes 2'-deoxynucleoside-5'-triphosphates (dNTPs) into 2'-deoxynucleosides and inorganic triphosphate products. Cytarabine can exhibit toxicity, particularly

myelosuppression; cytarabine can produce leukopenia, thrombocytopenia, and anemia with megaloblastic changes. Gastrointestinal disturbances, fever, conjunctivitis, pneumonitis, hepatic dysfunction, dermatitis, and neurotoxicity have also been noted, particularly at higher dosages.

[0130] The nucleoside analog 5-azacytidine has the structure shown as Formula

(N-ll):

(N-ll).

[0131] The analog 5-azacytidine (4-amino-1 -p-D-ribofuranosyl-1 ,3,5-triazin- 2(1 /-/)-one) inhibits DNA methyltransferase, causing hypomethylation of DNA, and also is incorporated directly into both RNA and DNA resulting in cell death; 5-azacytidine is incorporated into RNA more frequently than into DNA. The analog 5-azacytidine is used in the treatment of myelodysplastic syndrome and acute myeloid leukemia. It has a number of side effects, including anemia, neutropenia, thrombocytopenia,

hepatotoxicity, and renal toxicity.

[0132] The nucleoside analog gemcitabine has the structure shown as Formula

(N-lll):

(N-lll).

[0133] Gemcitabine is 4-amino-1 -(2-deoxy-2,2-difluoro- -D-eryi/?ro- pentofuranosyl)pyrimidin-2(1 /-/)-one. Gemcitabine is used for treating pancreatic and in combination with cisplatin for advanced or metastatic bladder cancer and advanced or metastatic non-small cell lung cancer, as well as in various combinations for ovarian cancer or breast cancer. Gemcitabine is hydrophilic and is transported into cells via molecular transporters for nucleosides, particularly SLC29A1 SLC28A1 , and SLC28A3. Gemcitabine is then phosphorylated to gemcitabine monophosphate

(dFdGMP) and then eventually to gemcitabine triphosphate. Gemcitabine then is incorporated into DNA in place of cytidine. When gemcitabine is incorporated into DNA it allows a native, or normal, nucleoside base to be added next to it. This leads to "masked chain termination" as gemcitabine is a "faulty" base, but due to its neighboring native nucleoside it eludes the cell's normal repair system (base-excision repair). Thus, incorporation of gemcitabine into the cell's DNA creates an irreparable error that leads to inhibition of further DNA synthesis, and thereby leading to cell death. Gemcitabine, like many other inhibitors of DNA replication, can inhibit bone marrow function and cause anemia, neutropenia, and thrombocytopenia. It also may have other side effects, particularly affecting the respiratory system.

[0134] Floxuridine is another nucleoside analog. Floxuridine has the structure shown as Formula ( -IV):

(N-IV).

[0135] Floxuridine is 5-fluoro-1 -[4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2- yl]-1 /-/-pyrimidine-2,4-dione. Floxuridine is used for the treatment of colorectal cancer, kidney cancer, and stomach cancer. Floxuridine is converted in vivo to 5-fluorouracil, which interferes with both DNA and RNA synthesis; 5-fluorouracil also inhibits uracil riboside phosphorylase, which prevents the utilization of preformed uracil in RNA synthesis. The side effects of floxuridine are similar to the side effects of other pyrimidine analog antimetabolites.

[0136] The product of metabolism of floxuridine, 5-fluorouracil, is itself used as an antimetabolite; the structure of 5-fluorouracil is shown as Formula (N-V):

(N-V).

[0137] The compound 5-fluorouracil is used to treat esophageal cancer, colon cancer, stomach cancer, pancreatic cancer, breast cancer, and cervical cancer, among others. It has the lUPAC name of 5-fluoro-1 H,3H-pyrimidine-2,4-dione and blocks the action of thymidylate synthase, inhibiting DNA synthesis. The side effects of 5- fluorouracil are generally similar to the side effects of other pyrimidine antimetabolites.

[0138] Capecitabine is another pyrimidine antimetabolite. Capecitabine has the structure shown as Formula (N-VI):

(N-VI).

[0139] Capecitabine is used to treat breast cancer, gastric cancer, and

colorectal cancer. Its lUPAC name is pentyl [1 -(3,4-dihydroxy-5-methyltetrahydrofuran- 2-yl)-5-fluoro-2-oxo-1 H-pyrimidin-4-yl]carbamate. Capecitabine is converted to 5- fluorouracil in vivo. The side effects of capecitabine administration are similar to those of 5-fluorouracil.

[0140] Other pyrimidine antimetabolites include 6-azauracil and the additional pyridine antimetabolites disclosed in W.B. Parker, "Enzymology of Purine and

Pyrimidine Antimetabolites Used in the Treatment of Cancer," Chem. Rev. 109: 2880- 2893 (2009), including the following additional agents: troxacitabine, thiarabine, and sapacitabine.

[0141] The compound 6-azauracil has the structure shown in Formula (N-VII):

(N-VII), and has the lUPAC name 1 ,2,4-triazine-3,5(2/-/,4/-/)-dione.

[0142] Troxacitabine has the structure shown in Formula (N-VII I):

(N-VIII), and has the lUPAC name 4-amino-1 -[(2S,4S)-2-(hydroxymethyl)-1 ,3-dioxolan-4- yl]pyrimidin-2(1 H)-one.

[0143] Thiarabine has the structure shown in Formula (N-IX):

(N-IX), and has the lUPAC name 4-amino-1 -[(2R,3S,4S,5R)-3,4-dihydroxy-5- (hydroxymethyl)thiolan-2-yl]pyrimidin-2-one.

[0144] Sapacitabine has the structure shown in Formula (N-X):

(N-X), and has the lUPAC name 1 -(2-cyano-2-deoxy-p-D-arabinofuranosyl)-4- (palmitoylamino)pyrimidin-2(1 /-/)-one. It is a prodrug of CNDAC (2'-cyano-2'-deoxy-1 -β- D-arabinofuranosylcytosine).

[0145] CNDAC (2'-cyano-2'-deoxy-1 - -D-arabinofuranosylcytosine) has the structure shown in Formula -XI):

(N-XI), and has the lUPAC name (2R,3S,4S,5R)-2-(4-amino-2-oxopyrimidin-1 -yl)-4-hydroxy-5- (hydroxymethyl)oxolane-3-carbonitrile.

[0146] Still other pyrimidine analog antimetabolites are known in the art, including 2'-deoxy-2'-methylidenecytidine compounds as disclosed in United States Patent No. 5,776,488 to Mori et al., including 2'-deoxy-2'-methylidenecytidine, 2'-deoxy- 2'-fluoromethylidenecytidine, 2'-deoxy-2'-methylidene-5-fluorocytidine, 2'-deoxy-2',2'- difluorocytidine, and 2'-C-cyano-2'-deoxy- -arabinofuranosylcytosine. Additional pyrimidine analog antimetabolites are also known in the art. [0147] Liposomes suitable for incorporating the bisantrene or derivative or analog thereof and the pyrimidine analog antimetabolite in this aspect of the invention can be prepared by techniques well known in the art and described above.

[0148] IV. Administration and Use of Compositions According to the Invention

[0149] Compositions according to the present invention as described above can be administered to treat malignancies, including, but not limited to, breast cancer, ovarian cancer, renal cancer, small-cell lung cancer, non-small cell lung cancer,

Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myelocytic leukemia, melanoma, gastric cancer, adrenal cancer, head and neck cancer, hepatocellular cancer, hypernephroma, bladder cancer, acute leukemias of childhood, chronic lymphocytic leukemia, prostate cancer, glioblastoma, and myeloma. A method of treating a malignancy according to the present invention comprises administration of a

therapeutically effective quantity of a composition according to the present invention as described above. In one alternative, the composition comprises a therapeutically effective quantity of bisantrene or a derivative or analog thereof as described above. In another alternative, the composition comprises a therapeutically effective quantity of bisantrene or a derivative or analog thereof and a pyrimidine analog antimetabolite as described above.

[0150] When the cancer is breast cancer, the breast cancer can be selected from the group consisting of refractory breast cancer, triple-negative breast cancer, and breast cancer characterized by overexpressed Her-2-neu. When the cancer is an acute leukemia of childhood, the acute leukemia of childhood can be selected from the group consisting of acute myelocytic leukemia (AML) and acute lymphocytic leukemia (ALL) of childhood. When the cancer is prostate cancer, the prostate cancer can be androgen- resistant prostate cancer. When the cancer is glioblastoma, the glioblastoma can be glioblastoma that is resistant to one or both of the following therapeutic agents:

temozolomide (Temodar) or bevacizumab (Avastin), or is characterized by EGFR Variant III. Alternatively, the cancer can be a malignancy characterized by

overexpressed topoisomerase II or overexpressed and/or mutated EGFR. [0151] Compositions according to the present invention can also be used to treat other hyperproliferative diseases, including myelodysplastic syndrome and mycosis fungoides.

[0152] Compositions according to the present invention can be administered to humans or to socially or economically important non-human species such as dogs, cats, pigs, sheep, goats, rabbits, cattle, or horses. Unless specifically stated, methods as described below are not limited to treatment of humans.

[0153] For treatment of human ailments, a qualified physician will determine how the compositions according to the present invention should be utilized with respect to dose, schedule and route of administration using established protocols. Such

applications may also utilize dose escalation should agents encapsulated in delivery vehicle compositions of the present invention exhibit reduced toxicity to healthy tissues of the subject.

[0154] Preferably, compositions according to the present invention are

administered parenterally, i.e., intraarterially, intravenously, intraperitoneally,

subcutaneously, or intramuscularly. More preferably, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus or infusional injection. For example, see Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4, 145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871 ; Schneider, U.S. Pat. No. 4,224, 179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.

[0155] In other methods, compositions according to the present invention can be contacted with the target tissue by direct application of the preparation to the tissue. The application may be made by topical, "open," or "closed" procedures. The term "topical," as used herein, means the direct application of the multi-drug preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like. "Open" procedures are those procedures that include incising the skin of a patient and directly visualizing the underlying tissue to which the

pharmaceutical preparations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approach to the target tissue. "Closed" procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin. For example, compositions according to the present invention may be administered to the peritoneum by needle lavage. Alternatively, compositions according to the present invention may be administered through endoscopic devices.

[0156] Compositions according to the present invention can be prepared according to standard techniques and may comprise water, buffered water, 0.9% saline, 0.3% glycine, 5% dextrose, iso-osmotic sucrose solutions and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate

physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like. For example, suitable carriers can include water, buffered water, 5% dextrose, 0.4% saline or 0.9% saline, 0.3% glycine, and proteins for enhanced stability, including albumin, lipoproteins, glycoproteins, or globulin in order to produce a pharmaceutical composition suitable for administration by the selected route of administration as described above. Additionally, the delivery vehicle suspension may include lipid-protective agents which protect lipids against free- radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as a-tocopherol, ascorbyl palmitate, and water-soluble iron-specific chelators, such as ferrioxamine, are suitable. Additionally, for administration, compositions according to the present invention may further contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like. Leucovorin may also be administered with compositions of the invention through standard techniques to enhance the life span of administered fluoropyrimidines when a composition according to the present invention includes a fluoropyrimidine.

[0157] The concentration of delivery vehicles in the pharmaceutical formulations can vary widely, such as from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, and the like, in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. Alternatively, delivery vehicles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration. For diagnosis, the amount of delivery vehicles administered will depend upon the particular label used, the disease state being diagnosed and the judgment of the clinician.

[0158] Preferably, pharmaceutical compositions according to the present invention are administered intravenously. However, other routes of administration, particularly parenteral administration, such as intraperitoneal administration, can alternatively be used. Dosage for the delivery vehicle formulations will depend on the ratio of drug to lipid and the administrating physician's opinion based on age, weight, and condition of the patient as well as other drugs being administered and

pharmacokinetic considerations such as kidney and liver function.

[0159] As used herein, terminology such as "treat," "treating," or "treatment" or equivalent terminology refer to both therapeutic treatment and prophylactic or

preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the growth, development, or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. The results of treatment can be determined by methods known in the art, such as determination of reduction of pain as measured by reduction of requirement for administration of opiates or other pain medication, determination of reduction of tumor burden, determination of restoration of function as determined by an improvement in the Karnofsky Performance Score, or other methods known in the art. The use of terms such as "treat" or "treatment" is not to be understood as implying a cure for any disease or condition.

[0160] Therapeutic activity of compositions according to the present invention may be measured after administration into an animal model. Preferably, the animal model comprises a tumor although delivery vehicle compositions may be administered to animal models of other diseases. Rodent species such as mice and rats of either inbred, outbred, or hybrid origin including immunocompetent and immunocompromised, as well as knockout, or transgenic models may be used. Models can consist of solid or non-solid tumors implanted as cell suspensions, bries or tumor fragments in either subcutaneous, intravenous, intraperitoneal, intramuscular, intrathecal, or orthotopic regions. Tumors may also be established via the application or administration of tumorigenic/carcinogenic agents or may be allowed to arise spontaneously in

appropriate genetically engineered animal models. Tumor types can consist of tumors of ectodermal, mesodermal, or endodermal origin such as carcinomas, sarcomas, melanomas, gliomas, leukemias and lymphomas. In one alternative, mouse models of tumors are employed. Human xenograft solid tumors grown in immune compromised mice may be utilized and selected on the basis of defined genetics and growth attributes. Tumor cells utilized in these experiments can be genetically manipulated or selected to express preferable properties and are injected into mice. Once the tumors have grown to a palpable (measurable) size, delivery vehicle compositions can be administered, preferably intravenously, and their effects on tumor growth are monitored. Intended therapeutic treatments can consist of single bolus or push administrations or multiple or continuous administrations over several days or weeks and by any

appropriate route such as by the oral, nasal, subcutaneous, intravenous, intraperitoneal, intrathecal, intratumoral, or other appropriate routes using syringes, tablets, liquids, and pumps (such as osmotic). Dose and schedule dependency may be evaluated in order to determine the maximum anti-tumor activity that can be achieved.

[0161] Various methods of determining therapeutic activity in animal models comprising a tumor may be utilized. This includes solid tumor model evaluation methods and non-solid tumor model evaluation methods. Solid tumor model evaluation methods include measurement of tumor volume (mass), tumor weight inhibition (TWI %), tumor growth delay (T-C), tumor regression, cell kill and clonogenic assays. Tumor volume measurements are determined from vernier caliper measurements of

perpendicular length and width measurements (height measurements can often be obtained as well). Tumor volume (ml_) or mass (g) is calculated from: volume=(length x width²/2; or volume=^6 x (length x width x height). Data is plotted with respect to time. Tumor weight inhibition (TWI %) is determined by measuring the mean tumor weight of a treated group divided by the mean tumor weight of a control group, minus 1 x 100 at a defined time point. Tumor growth delay (T-C) is measured as the median time in days for a treated group (T) to reach an arbitrarily determined tumor size (for example, 300 mg) minus median time in days for the control group to reach the same tumor size. Tumor regression as a result of treatment may also be used as a means of evaluating a tumor model. Results are expressed as reductions in tumor size (mass) over time. Cell kill methods of solid tumor model evaluation can involve measuring tumors repeatedly by calipers until all exceed a predetermined size (e.g., 200 mg). The tumor growth and tumor doubling time can then be evaluated. Log-io cell kill parameters can be calculated by: logio cell kill/dose=(T-C)/((3.32)(T_d)(No. of doses)) log™ cell kill (total)=(T- C)/((3.32(T_d)) log™ cell kill (net)=((T-C)-(duration of R_x))/((3.32(T_d)) where: (T-C)=tumor growth delay Td=Tumor doubling time.

[0162] Clonogenicity assays express the effectiveness of therapy. These assays include excision assays and characterization of cell suspensions from solid tumors. Excision assays, used to assess what fraction of cells, in a suspension prepared from tumors, have unlimited proliferative potential (i.e., are clonogenic). Three types of excision assays are: (i) TD50, or endpoint dilution assays, which determine the number of cells required to produce tumor takes from inocula in vivo; (ii) in vivo colony assays, which assess the ability of individual cells to form nodules (colonies) in, for example, the lung; and (iii) in vitro colony assay, which test the ability of individual cells to grow into colonies either in liquid media, when colonies form on the plastic or glass surface of culture dishes, or in semisolid media such as agar, in which the colonies form in suspension.

[0163] Characterization of cell suspensions from solid tumors are required for in vitro and in vivo clonogenic assays, flow-cytometric measurements, and for numerous biochemical and molecular analyses performed on a per cell basis. Preparation is by a number of methods such as enzymatic, mechanical, chemical, combinations thereof, and surface activity agents. Evaluations could include, cell yield, cell morphology, tumor cell clonogenicity, retention of biochemical or molecular characteristics. Non- solid tumor model evaluation methods include measurement of increase in life span (ILS %), tumor growth delay (T-C), long-term survivors (cures). Increase in life-span (ILS %) measures the percentage increase in life-span of treated groups versus control or untreated groups. Tumor growth delay (T-C) measures median time in days for treated (T) group survival minus median time in days for control (C) group survival. Long-term survivors (cures) measures treatment groups that survive up to and beyond 3-times the survival times of untreated or control groups. Methods of determining therapeutic activity in humans afflicted with cancer include measurements of survival and surrogate endpoints. The time at which survival is reasonably evaluated depends on the tumor in question. By way of example, survival rates for patients with low-grade lymphomas may be examined at 5 or 10 years post diagnosis, whereas the survival or patients having aggressive diseases such as advanced non-small cell lung cancer may be best evaluated at 6 or 12 months post diagnosis. Methods of determining

therapeutic activity using surrogate endpoints includes measuring complete response (CR), partial response (PR), progression-free survival (PFS), time-to-progression (TTP) or duration of response (DOR), plasma and urine markers, enzyme inhibition and/or receptor status, changes in gene expression and quality of life (QOL). A complete response means the disappearance of all known sites of disease without the development of any new disease for a period of time appropriate for the tumor type being treated. Assessments are based on a variety of examinations such as those stated above. Partial response is at least a 50% decrease in the sum of the products of the bidimensional measurement of all lesions with no new disease appearing for a period of time appropriate for the tumor type being treated. Assessments are based on a variety of examinations (CT scan, MRI, ultrasound, PET scan, bone scan, physical examination) of patients. Progression-free Survival (PFS): Duration from treatment in which a patient survives and there is no growth of existing tumor nor appearance of new tumor masses. PFS may be expressed as either the duration of time or as the proportion of patients who are surviving and progression-free at a given time after diagnosis. Time-to-progression (TTP) or duration of response (DOR) refer to the duration of time from treatment to a progression of tumor growth, measured either as an increase in size of existing tumor masses or the appearance of new tumor masses. Plasma and urine markers include measuring markers such as, but not limited to, the following markers: prostate specific antigen (PSA) and carcinoembryonic antigen (CEA). Enzyme inhibition and/or receptor status can include assessment of growth factor receptors such as, but not limited to, tyrosine kinase receptors, EGF receptor, PDGF receptor, Her-1 and Her-2 receptors, or assessment of enzymes such as, but not limited to, integrin-linked kinases, protein kinases and the like. Changes in gene expression include serial analysis of gene expression (genomics) and changes in protein

expression (proteomics). Quality of Life (QOL) include methods such as the EORTC QLQ-C30 scoring method that evaluates yields scores for five functional scales

(physical, role, cognitive, social, and emotional), three symptom scales (nausea, pain, and fatigue), and a global health and quality of life scale. The measure also yields single-item ratings of additional symptoms commonly reported by cancer patients (dyspnea, appetite loss, sleep disturbance, constipation, and diarrhea) as well as the perceived financial impact of the disease and its treatment.

[0164] Methods according to the present invention can further comprise administration of a therapeutically effective quantity of an additional therapeutic agent to treat the disease, disorder, or condition, in particular, a malignancy as described above. [0165] When the disease, disorder, or condition is breast cancer, the additional therapeutic agent can be selected from the group consisting of tamoxifen, anastrozole, letrozole, cyclophosphamide, docetaxel, paditaxel, methotrexate, fluorouracil, and trastuzumab. When the disease, disorder, or condition is ovarian cancer, the additional agent can be selected from the group consisting of: platinum-containing antineoplastic drugs such as cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin, phenanthriplatin, picoplatin, and satraplatin; paditaxel; topotecan; gemcitabine; etoposide; and

bleomycin. When the disease, disorder, or condition is renal cancer, the additional agent can be selected from the group consisting of everolimus, torisel, nexavar, sunitinib, axitinib, inferferon, interleukin-2, pazopanib, sorafenib, nivolumab, cabozanitib, and levanitib. When the disease, disorder, or condition is small-cell lung cancer, the additional agent can be selected from the group consisting of cyclophosphamide, cisplatin, etoposide, vincristine, paditaxel, and carboplatin. When the disease, disorder, or condition is non-small-cell lung cancer, the additional agent can be selected from the group consisting of cisplatin, erlotinib, gefitinib, afatinib, crizotinib, bevacizumab, carboplatin, paditaxel, nivolumab, and pembrolizumab. When the disease, disorder, or condition is Hodgkin's lymphoma, the additional agent can be selected from the group consisting of mechlorethamine, vincristine, prednisone, procarbazine, bleomycin, vinblastine, dacarbazine, etoposide, and cyclophosphamide. When the disease, disorder, or condition is non-Hodgkin's lymphoma, the additional agent can be selected from the group consisting of cyclophosphamide, vincristine, and prednisone. When the disease, disorder, or condition is acute myelocytic leukemia, the additional agent can be selected from the group consisting of cytarabine (provided that the liposomal

composition does not include cytarabine), fludarabine, all-frans-retinoic acid, interleukin- 2, and arsenic trioxide. When the disease, disorder, or condition is melanoma, the additional agent can be selected from the group consisting of temozolomide,

dacarbazine, interferon, interleukin-2, ipilimumab, pembrolizumab, nivolumab, vemurafenib, dabrafenib, and trametinib. When the disease, disorder, or condition is gastric cancer, the additional agent can be selected from the group consisting of 5- fluorouracil (provided that the liposomal composition does not include 5-fluorouracil), capecitabine (provided that the liposomal composition does not include capecitabine), carmustine, semustine, mitomycin C, cisplatin, taxotere, and trastuzumab. When the disease, disorder, or condition is adrenal cancer, the additional agent can be selected from the group consisting of mitotane, cisplatin, etoposide, and streptozotocin. When the disease, disorder, or condition is head and neck cancer, the additional agent can be selected from the group consisting of paclitaxel, carboplatin, cetuximab, docetaxel, cisplatin, and 5-fluorouracil (provided that the liposomal composition does not include 5- fluorouracil). When the disease, disorder, or condition is hepatocellular cancer, the additional agent can be selected from the group consisting of tamoxifen, octreoside, synthetic retinoids, cisplatin, 5-fluorouracil (provided that the liposomal composition does not include 5-fluorouracil), interferon, taxol, and sorafenib. When the disease, disorder, or condition is hypernephroma, the additional agent can be selected from the group consisting of nivolumab, everolimus, sorafenib, axitinib, lenvatinib, temsirolimus, sunitinib, pazopanib, interleukin-2, cabozanitib, bevacizumab, interferon a, ipilimumab, atezolizumab, varilumab, durvalumab, tremelimumab, and avelumab. When the disease, disorder, or condition is bladder cancer, the additional agent can be selected from the group consisting of cisplatin, 5-fluorouracil (provided that the liposomal composition does not include 5-fluorouracil), mitomycin C, gemcitabine (provided that the liposomal composition does not include gemcitabine), methotrexate, vinblastine, carboplatin, paclitaxel, docetaxel, ifosfamide, and pemetrexed. When the disease, disorder, or condition is acute myelocytic leukemia of childhood, the additional agent can be selected from the group consisting of methotrexate, nelarabine, asparaginase, blinatumomab, cyclophosphamide, clofarabine, cytarabine (provided that the liposomal composition does not include cytarabine), dasatinib, methotrexate, imatinib, pomatinib, vincristine, 6-mercaptopurine, pegaspargase, and prednisone. When the disease, disorder, or condition is acute lymphocytic leukemia of childhood, the additional agent can be selected from the group consisting of asparaginase, vincristine, dexamethasone, methotrexate, 6-mercaptopurine, cytarabine (provided that the liposomal composition does not include cytarabine), hydrocortisone, 6-thioguanine, prednisone, etoposide, cyclophosphamide, mitoxantrone, and teniposide. When the disease, disorder, or condition is chronic lymphocytic leukemia, the additional agent can be selected from the group consisting of fludarabine, cyclophosphamide, rituximab, vincristine, prednisolone, bendamustine, alemtuzumab, ofatumumab, obinutuzumab, ibrutinib, idelalisib, and venetoclax. When the disease, disorder, or condition is prostate cancer, the additional agent can be selected from the group consisting of temozolomide, docetaxel, cabazitaxel, bevacizumab, thalidomide, prednisone, sipuleucel-T, abiraterone, and enzalutamide. When the disease, disorder, or condition is glioblastoma, the additional agent can be selected from the group consisting of temozolomide and bevacizumab. When the disease, disorder, or condition is myeloma, the additional agent can selected from the group consisting of bortezomib, lenalidomide, dexamethasone, melphalan, prednisone, thalidomide, and cyclophosphamide.

[0166] Methods for administration of the additional agents are known in the art. Typically, they are administered in a separate pharmaceutical composition according to methods well known in the art, with suitable carriers, excipients, and other components as generally used in the art, although, in some cases, in which the additional agent or agents are compatible with a liposome composition according to the present invention as described above, they may be administered in the liposome composition.

ADVANTAGES OF THE INVENTION

[0167] The present inventions provides compositions employing liposomes for improved administration of bisantrene or derivatives or analogs of bisantrene together with pyrimidine analog antimetabolites such as cytarabine, providing improved stability and bioavailability and reduced side effects, particularly reduced occurrence of phlebitis and other circulatory system complications, as well as methods for administration of bisantrene or derivatives or analogs of bisantrene and pyrimidine analog antimetabolites for the treatment of hyperproliferative diseases such as cancer employing liposomes.

[0168] Methods according to the present invention possess industrial

applicability for the preparation of a medicament for the treatment of a number of diseases and conditions, especially hyperproliferative diseases, and compositions according to the present invention possess industrial applicability as pharmaceutical compositions.

[0169] 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 ways of using an existing drug.

[0170] 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," etc. 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 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 transitional phrase

"comprising" is included in claim language in which the term "comprising" is followed by one or more elements, the claim is to be interpreted as encompassing alternatives in which "comprising" is replaced by "consisting essentially of" or "consisting of" unless the alternatives of "consisting essentially of" or "consisting of" are expressly excluded.

[0171] In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. It is also to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of 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 patent publications, are incorporated herein by reference.

Claims

What is claimed is:

1 . A composition of matter comprising:

(a) a therapeutically effective quantity of bisantrene or a derivative or analog thereof; and

(b) a liposome encapsulating the therapeutically effective quantity of bisantrene or the derivative or analog thereof.

2. The composition of claim 1 wherein the composition comprises bisantrene.

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

4. The composition of claim 1 wherein the composition comprises a derivative or analog of bisantrene.

5. The composition of claim 4 wherein the derivative or analog of bisantrene is selected from the group consisting of:

(a) the analog of Formula (II)

the bisantrene analog of Formula (III)

(in);

(C) the bisantrene analog of Formula (IV)

(IV);

(d) the bisantrene analog of Formula (V)

(V); the bisantrene analog of Formula (VI)

(VI); the bisantrene analog of Formula (VII)

(VII); the bisantrene analog of Formula (VIII)

(VIII);

(i) the bisantrene analog of Formula (X)

the bisantrene analog of Formula (XIII)

(XIII);

(m) bisantrene analogs of Formula (XIV)

(XIV), wherein Ri and R3 are the same or different and are hydrogen, C1-C6 alkyi, -C(0)-Rs, wherein R5 is hydrogen, C1-C6 alkyi, phenyl, mono-substituted phenyl (wherein the substituent can be ortho, meta, or para and is fluoro, nitro, C1-C6 alkyi, C1-C3 alkoxy, or cyano), pentafluorophenyl, naphthyl, furanyl,

CH3 CH3

I I

CH HCO⁽X CH3)3,—€ΗΝ¾— CHiCHiCOOH,

OC(CH₃)3,— CH2OCH3, ~{CH₂)₃C0OH, COOH

(CH₂)2 0 H or— CHaN®— (€Η3)3θθ))_;

-SO3H; wherein only one of Ri and R3 may be hydrogen or C1-C6 alkyi; R2 and R₄ are the same or different and are: hydrogen, Ci-C₄ alkyi or -C(0)-R6, where R6 is hydrogen, C1-C6 alkyi, phenyl, mono-substituted phenyl (wherein the substituent may be in the ortho, meta, or para position and is fluoro, nitro, C1-C6 alkyi, C1-C3 alkoxy, or cyano), pentafluorophenyl, naphthyl, furanyl, or -ChbOChh; wherein the compounds can have the schematic structure B(Q)n, wherein B is the residue formed by removal of a hydrogen atom from one or more basic nitrogen atoms of an amine, amidine, guanidine, isourea, isothiourea, or biguanide-containing pharmaceutically active compound, and Q is hydrogen or A, wherein A is

R'O O

\li

CI3C-CH2-, or R7OCH2CH2-, where R₇ is hydrogen or C1 -C6 alkyl, hydrogen, or pharmaceutically acceptable cation or R' and R" are linked to form a -CH2CH2 or a

(n) the bisantrene analog 9, 10-bis[(2- hydroxyethyl)iminomethyl]anthracene;

(p) the bisantrene analog 9, 10-bis{[2-(-2- hydroxyethylamino)ethyl]iminomethyl}anthracene;

(q) the bisantrene analog 9, 10-bis{[2-(morpholin-4- yl)ethyl]iminomethyl}anthracene;

(r) the bisantrene analog 9, 10-bis[(2- hydroxyethyl)aminomethyl]anthracene; (s) the bisantrene analog 9, 10-bis{[2-(2- hydroxyethylamino)ethyl]aminomethyl}anthracene tetrahydrochloride;

(v) N,N'-bis[2-(dimethylamino)ethyl]-9, 10-anthracene- bis(methylamine);

(w) N,N'-bis(1 -ethyl-3-piperidinyl)-9, 10-anthracene-bis(methylamine); and

(x) derivatives and salt forms of the compounds of (a)-(w).

6. The composition of claim 4 wherein the derivative or analog of bisantrene is a derivative of bisantrene selected from the group consisting of:

7. The composition of claim 1 wherein the liposomes comprise at least one lipid selected from the group consisting of a phosphatidylcholine lipid, a

phosphatidylglycerol lipid, a sterol, an ether lipid, a phosphatidic acid, a phosphonate, a ceramide, a ceramide analog, a sphingosine, a sphingosine analog, a serine-containing lipid, a hydrophilic polymer-lipid conjugate, phosphatidylglycerol, phosphatidylinositol, and a negatively charged lipid having a hydrophilic portion and a hydrophobic portion with a neutral non-zwitterionic moiety attached to the hydrophilic portion of the lipid.

8. The composition of claim 7 wherein the liposomes comprise a phosphatidylcholine lipid.

9. The composition of claim 8 wherein the phosphatidylcholine lipid is diastearoylphosphatidylcholine.

10. The composition of claim 7 wherein the liposomes comprise a phosphatidylglycerol lipid.

1 1 . The composition of claim 10 wherein the phosphatidylglycerol lipid is distearoylphosphatidylglycerol.

12 The composition of claim 7 wherein the liposomes comprise a sterol.

13 The composition of claim 12 wherein the sterol is cholesterol.

14 The composition of claim 7 wherein the liposomes comprise diastearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol.

15. The composition of claim 14 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.5 to about 7.5 of distearoylphosphatidylcholine, about 1.5 to about 2.5 of distearoylphosphatidylglycerol, and about 0.8 to 1 .2 of cholesterol.

16. The composition of claim 15 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.8 to about 7.2 of distearoylphosphatidylcholine, about 1.8 to about 2.2 of distearoylphosphatidylglycerol, and about 0.9 to 1 .1 of cholesterol.

17. The composition of claim 16 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is about 7:2: 1 .

18. The composition of claim 7 wherein the liposomes have a diameter of less than about 300 nm.

19. The composition of claim 18 wherein the liposomes have a diameter of less than about 200 nm.

20. The composition of claim 7 wherein the liposomes have an intraliposomal osmolality of 500 mOSM/kg or less.

21 . The composition of claim 14 wherein the composition comprises bisantrene, diastearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol.

22. A method for treating a malignancy comprising the step of administering a therapeutically effective quantity of the composition of claim 1 to treat the malignancy.

23. The method of claim 22 wherein the malignancy is selected from the group consisting of breast cancer, ovarian cancer, renal cancer, small-cell lung cancer, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myelocytic leukemia, melanoma, gastric cancer, adrenal cancer, head and neck cancer, hepatocellular cancer, hypernephroma, bladder cancer, acute leukemias of childhood, chronic lymphocytic leukemia, prostate cancer, glioblastoma, and myeloma.

24. The method of claim 23 wherein the malignancy is breast cancer and wherein the breast cancer is selected from the group consisting of refractory breast cancer, triple-negative breast cancer, and breast cancer characterized by

overexpressed Her-2-neu.

25. The method of claim 23 wherein the malignancy is an acute leukemia of childhood and wherein the acute leukemia of childhood is selected from the group consisting of acute myelocytic leukemia (AML) and acute lymphocytic leukemia (ALL) of childhood.

26. The method of claim 23 wherein the malignancy is prostate cancer and wherein the prostate cancer is androgen-resistant prostate cancer.

27. The method of claim 23 wherein the malignancy is a malignancy that is characterized by overexpressed topoisomerase II or overexpressed and/or mutated EGFR.

28. The method of claim 22 wherein the composition comprises bisantrene.

29. The method of claim 28 wherein the bisantrene is bisantrene dihydrochloride.

30. The method of claim 22 wherein the composition comprises a derivative or analog of bisantrene.

31 . The method of claim 30 wherein the derivative or analog of bisantrene is selected from the group consisting of:

(a) the analog of Formula (II)

• 2HCI

(IV); the bisantrene analog of Formula (V)

(V);

(e) the bisantrene analog of Formula (VI)

(VI); the bisantrene analog of Formula (VII)

(VII); the bisantrene analog of Formula (VIII)

(VIII);

(i) the bisantrene analog of Formula (X)

(X); the bisantrene analog of Formula (XI)

( i); the bisantrene analog of Formula (XII)

(XII); the bisantrene analog of Formula (XIII)

(XIII);

(m) bisantrene analogs of Formula (XIV)

(XIV), wherein Ri and R3 are the same or different and are hydrogen, C1-C6 alkyl, -C(0)-Rs, wherein R5 is hydrogen, C1-C6 alkyl, phenyl, mono-substituted phenyl (wherein the substituent can be ortho, meta, or para and is fluoro, nitro, C1-C6 alkyi, C1-C3 aikoxy, or cyano), pentafluorophenyl, naphthyl, furanyl,

CHNHCOOC{CH3)3»— CHNH2,— CH2CH2COOH,

— OC(CH3)₃,— CH2OCH3,— {CHahCOOH_*

(CH₂)2S03H or— CH₂N®~ (€Η3)3θθ))_;

-SO3H; wherein only one of Ri and R3 may be hydrogen or C1-C6 alkyi; R2 and R₄ are the same or different and are: hydrogen, Ci-C₄ alkyi or -C(0)-R6, where R6 is hydrogen, C1-C6 alkyi, phenyl, mono-substituted phenyl (wherein the substituent may be in the ortho, meta, or para position and is fluoro, nitro, C1-C6 alkyi, C1-C3 aikoxy, or cyano), pentafluorophenyl, naphthyl, furanyl, or -ChbOChh; wherein the compounds can have the schematic structure B(Q)n, wherein B is the residue formed by removal of a hydrogen atom from one or more basic nitrogen atoms of an amine, amidine, guanidine, isourea, isothiourea, or biguanide-containing pharmaceutically active compound, and Q is hydrogen or A, wherein A is

R'O O

\li

P—

/

CI3C-CH2-, or R7OCH2CH2-, where R₇ is hydrogen or C1-C6 alkyl, hydrogen, or a pharmaceutically acceptable cation or R' and R" are linked to form a -CH2CH2- group or a

(n) the bisantrene analog 9, 10-bis[(2- hydroxyethyl)iminomethyl]anthracene;

(q) the bisantrene analog 9, 10-bis{[2-(morpholin-4- yl)ethyl]iminomethyl}anthracene; (r) the bisantrene analog 9, 10-bis[(2- hydroxyethyl)aminomethyl]anthracene;

(v) N,N'-bis[2-(dimethylamino)ethyl]-9, 10-anthracene- bis(methylamine);

(w) N,N'-bis(1 -ethyl-3-piperidinyl)-9, 10-anthracene-bis(methylamine); and

(x) derivatives and salt forms of the compounds of (a)-(w).

32. The method of claim 30 wherein the derivative or analog of bisantrene is a derivative of bisantrene selected from the group consisting of:

33. The method of claim 22 wherein the liposomes comprise at least one lipid selected from the group consisting of a phosphatidylcholine lipid, a

34. The method of claim 33 wherein the liposomes comprise a phosphatidylcholine lipid.

35. The method of claim 34 wherein the phosphatidylcholine lipid is diastearoylphosphatidylcholine.

36. The method of claim 33 wherein the liposomes comprise a phosphatidylglycerol lipid.

37. The method of claim 36 wherein the phosphatidylglycerol lipid is distearoylphosphatidylglycerol.

38. The method of claim 33 wherein the liposomes comprise a sterol.

39. The method of claim 38 wherein the sterol is cholesterol.

40. The method of claim 33 wherein the liposomes comprise diastearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol.

41 . The method of claim 40 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.5 to about 7.5 of distearoylphosphatidylcholine, about 1.5 to about 2.5 of distearoylphosphatidylglycerol, and about 0.8 to 1 .2 of cholesterol.

42. The method of claim 41 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.8 to about 7.2 of distearoylphosphatidylcholine, about 1.8 to about 2.2 of distearoylphosphatidylglycerol, and about 0.9 to 1 .1 of cholesterol.

43. The method of claim 42 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is about 7:2: 1 .

44. The method of claim 22 wherein the liposomes have a diameter of less than about 300 nm.

45. The method of claim 44 wherein the liposomes have a diameter of less than about 200 nm.

46. The method of claim 22 wherein the liposomes have an intraliposomal osmolality of 500 mOSM/kg or less.

47. The method of claim 22 wherein the composition comprises bisantrene, diastearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol.

48. The method of claim 22 wherein the composition is administered parenterally.

49. The method of claim 48 wherein the composition is administered intravenously or intraperitoneally.

50. The method of claim 22 wherein the composition is administered in a pharmaceutical formulation suitable for administration.

51 . The method of claim 22 wherein the method further comprises administration of a therapeutically effective quantity of an additional therapeutic agent.

52. The method of claim 51 wherein the malignancy is breast cancer and wherein the additional therapeutic agent is selected from the group consisting of tamoxifen, anastrozole, letrozole, cyclophosphamide, docetaxel, paclitaxel,

methotrexate, fluorouracil, and trastuzumab.

53. The method of claim 51 wherein the malignancy is ovarian cancer and wherein the additional therapeutic agent is selected from the group consisting of: a platinum-containing antineoplastic drug selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin, phenanthriplatin, picoplatin, and satraplatin; paclitaxel; topotecan; gemcitabine; etoposide; and bleomycin.

54. The method of claim 51 wherein the malignancy is renal cancer and wherein the additional therapeutic agent is selected from the group consisting of everolimus, torisel, nexavar, sunitinib, axitinib, inferferon, interleukin-2, pazopanib, sorafenib, nivolumab, cabozanitib, and levanitib.

55. The method of claim 51 wherein the malignancy is small-cell lung cancer and wherein the additional therapeutic agent is selected from the group consisting of cyclophosphamide, cisplatin, etoposide, vincristine, paclitaxel, and carboplatin.

56. The method of claim 51 wherein the malignancy is non-small-cell lung cancer and wherein the additional therapeutic agent is selected from the group consisting of cisplatin, erlotinib, gefitinib, afatinib, crizotinib, bevacizumab, carboplatin, paditaxel, nivolumab, and pembrolizumab.

57. The method of claim 51 wherein the malignancy is Hodgkin's lymphoma and wherein the additional therapeutic agent is selected from the group consisting of mechlorethamine, vincristine, prednisone, procarbazine, bleomycin, vinblastine, dacarbazine, etoposide, and cyclophosphamide.

58. The method of claim 51 wherein the malignancy is non-Hodgkin's lymphoma and wherein the additional therapeutic agent is selected from the group consisting of cyclophosphamide, vincristine, and prednisone.

59. The method of claim 51 wherein the malignancy is acute myelocytic leukemia and wherein the additional therapeutic agent is selected from the group consisting of cytarabine, fludarabine, all-frans-retinoic acid, interleukin-2, and arsenic trioxide.

60. The method of claim 51 wherein the malignancy is melanoma and wherein the additional therapeutic agent is selected from the group consisting of temozolomide, dacarbazine, interferon, interleukin-2, ipilimumab, pembrolizumab, nivolumab, vemurafenib, dabrafenib, and trametinib.

61 . The method of claim 51 wherein the malignancy is adrenal cancer and wherein the additional therapeutic agent is selected from the group consisting of mitotane, cisplatin, etoposide, and streptozotocin.

62. The method of claim 51 wherein the malignancy is head and neck cancer and wherein the additional therapeutic agent is selected from the group consisting of paditaxel, carboplatin, cetuximab, docetaxel, cisplatin, and 5-fluorouracil.

63. The method of claim 51 wherein the malignancy is hepatocellular cancer and wherein the additional therapeutic agent is selected from the group consisting of tamoxifen, octreoside, synthetic retinoids, cisplatin, 5-fluorouracil, interferon, taxol, and sorafenib.

64. The method of claim 51 wherein the malignancy is hypernephroma and wherein the additional therapeutic agent is selected from the group consisting of nivolumab, everolimus, sorafenib, axitinib, lenvatinib, temsirolimus, sunitinib, pazopanib, interleukin-2, cabozanitib, bevacizumab, interferon a, ipilimumab, atezolizumab, varilumab, durvalumab, tremelimumab, and avelumab.

65. The method of claim 51 wherein the malignancy is bladder cancer and wherein the additional therapeutic agent is selected from the group consisting of cisplatin, 5-fluorouracil, mitomycin C, gemcitabine, methotrexate, vinblastine, carboplatin, paclitaxel, docetaxel, ifosfamide, and pemetrexed.

66. The method of claim 51 wherein the malignancy is acute myelocytic leukemia of childhood and wherein the additional therapeutic agent is selected from the group consisting of methotrexate, nelarabine, asparaginase, blinatumomab,

cyclophosphamide, clofarabine, cytarabine, dasatinib, methotrexate, imatinib, pomatinib, vincristine, 6-mercaptopurine, pegaspargase, and prednisone.

67. The method of claim 51 wherein the malignancy is acute

lymphocytic leukemia and wherein the additional therapeutic agent is selected from the group consisting of asparaginase, vincristine, dexamethasone, methotrexate, 6- mercaptopurine, cytarabine, hydrocortisone, 6-thioguanine, prednisone, etoposide, cyclophosphamide, mitoxantrone, and teniposide.

68. The method of claim 51 wherein the malignancy is chronic lymphocytic leukemia and wherein the additional therapeutic agent is selected from the group consisting of fludarabine, cyclophosphamide, rituximab, vincristine, prednisolone, bendamustine, alemtuzumab, ofatumumab, obinutuzumab, ibrutinib, idelalisib, and venetoclax.

69. The method of claim 51 wherein the malignancy is prostate cancer and wherein the additional therapeutic agent is selected from the group consisting of temozolomide, docetaxel, cabazitaxel, bevacizumab, thalidomide, prednisone, sipuleucel-T, abiraterone, and enzalutamide.

70. The method of claim 51 wherein the malignancy is glioblastoma and wherein the additional therapeutic agent is selected from the group consisting of temozolomide and bevacizumab.

71 . The method of claim 51 wherein the malignancy is myeloma and wherein the therapeutic agent is selected from the group consisting of bortezomib, lenalidomide, dexamethasone, melphalan, prednisone, thalidomide, and cyclophosphamide.

72. A composition of matter comprising:

(a) a therapeutically effective quantity of bisantrene or a derivative or analog thereof;

(b) a therapeutically effective quantity of a pyrimidine analog antimetabolite; and

(c) a liposome encapsulating both the therapeutically effective quantity of bisantrene or the derivative or analog thereof and the pyrimidine analog antimetabolite.

73. The composition of claim 72 wherein the composition comprises bisantrene.

74. The composition of claim 73 wherein the bisantrene is bisantrene dihydrochloride.

75. The composition of claim 72 wherein the composition comprises a derivative or analog of bisantrene.

76. The composition of claim 75 wherein the derivative or analog of bisantrene is selected from the group consisting of:

(a) the analog of Formula (II)

the bisantrene analog of Formula (III)

(in);

(C) the bisantrene analog of Formula (IV)

(IV);

(d) the bisantrene analog of Formula (V)

(V); the bisantrene analog of Formula (VI)

(VI); the bisantrene analog of Formula (VII)

(VII); the bisantrene analog of Formula (VIII)

(VIII);

(i) the bisantrene analog of Formula (X)

the bisantrene analog of Formula (XIII)

(XIII);

(m) bisantrene analogs of Formula (XIV)

CH3 CH3

I I

CH HCO⁽X CH3)3,—€ΗΝ¾— CHiCHiCOOH,

OC(CH₃)3,— CH2OCH3, ~{CH₂)₃C0OH,

(CH₂)2 0 H or— CHaN®— (€Η3)3θθ))_;

R'O O

\li

(n) the bisantrene analog 9, 10-bis[(2- hydroxyethyl)iminomethyl]anthracene;

(v) N,N'-bis[2-(dimethylamino)ethyl]-9, 10-anthracene- bis(methylamine);

(w) N,N'-bis(1 -ethyl-3-piperidinyl)-9, 10-anthracene-bis(methylamine); and

(x) derivatives and salt forms of the compounds of (a)-(w).

77. The composition of claim 75 wherein the derivative or analog of bisantrene is a derivative of bisantrene selected from the group consisting of:

78. The composition of claim 72 wherein the pyrimidine analog antimetabolite is selected from the group consisting of cytarabine, 5-azacytidine, gemcitabine, floxuridine, 5-fluorouracil, capecitabine, 6-azauracil, troxacitabine, thiarabine, sapacitabine, CNDAC (2'-cyano-2'-deoxy-1 - -D-arabinofuranosylcytosine), 2'-deoxy-2'-methylidenecytidine, 2'-deoxy-2'-fluoromethylidenecytidine, 2'-deoxy-2'- methylidene-5-fluorocytidine, 2'-deoxy-2',2'-difluorocytidine, and 2'-C-cyano-2'-deoxy- - arabinofuranosylcytosine.

79. The composition of claim 78 wherein the pyrimidine analog antimetabolite is selected from the group consisting of cytarabine, 5-azacytidine, gemcitabine, floxuridine, 5-fluorouracil, capecitabine, and 6-azauracil.

80. The composition of claim 79 wherein the pyrimidine analog antimetabolite is cytarabine.

81 . The composition of claim 80 wherein the composition comprises bisantrene and cytarabine.

82. The composition of claim 72 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 25: 1 to about 1 : 1 .

83. The composition of claim 82 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 10: 1 to about 3: 1 .

84. The composition of claim 83 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is about 5:1 .

85. The composition of claim 81 wherein the ratio of the cytarabine and the bisantrene is from about 25: 1 to about 1 : 1 .

86. The composition of claim 85 wherein the ratio of the cytarabine and the bisantrene is from about 10: 1 to about 3: 1 .

87. The composition of claim 86 wherein the ratio of the cytarabine and the bisantrene is about 5: 1 .

88. The composition of claim 72 wherein the liposomes comprise at least one lipid selected from the group consisting of a phosphatidylcholine lipid, a phosphatidylglycerol lipid, a sterol, an ether lipid, a phosphatidic acid, a phosphonate, a ceramide, a ceramide analog, a sphingosine, a sphingosine analog, a serine-containing lipid, a hydrophilic polymer-lipid conjugate, phosphatidylglycerol, phosphatidylinositol, and a negatively charged lipid having a hydrophilic portion and a hydrophobic portion with a neutral non-zwitterionic moiety attached to the hydrophilic portion of the lipid.

89. The composition of claim 88 wherein the liposomes comprise a phosphatidylcholine lipid.

90. The composition of claim 89 wherein the phosphatidylcholine lipid is diastearoylphosphatidylcholine.

91 . The composition of claim 88 wherein the liposomes comprise a phosphatidylglycerol lipid.

92. The composition of claim 91 wherein the phosphatidylglycerol lipid is distearoylphosphatidylglycerol.

93. The composition of claim 88 wherein the liposomes comprise a sterol.

94. The composition of claim 93 wherein the sterol is cholesterol.

95. The composition of claim 88 wherein the liposomes comprise diastearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol.

96. The composition of claim 95 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.5 to about 7.5 of distearoylphosphatidylcholine, about 1.5 to about 2.5 of distearoylphosphatidylglycerol, and about 0.8 to 1 .2 of cholesterol.

97. The composition of claim 96 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.8 to about 7.2 of distearoylphosphatidylcholine, about 1.8 to about 2.2 of distearoylphosphatidylglycerol, and about 0.9 to 1 .1 of cholesterol.

98. The composition of claim 97 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is about 7:2: 1 .

99. The composition of claim 88 wherein the liposomes have a diameter of less than about 300 nm.

100. The composition of claim 99 wherein the liposomes have a diameter of less than about 200 nm.

101 . The composition of claim 88 wherein the liposomes have an intraliposomal osmolality of 500 mOSM/kg or less.

102. The composition of claim 98 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 25: 1 to about 1 : 1 .

103. The composition of claim 102 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 10: 1 to about 3: 1 .

104. The composition of claim 103 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is about 5:1 .

105. The composition of claim 98 wherein the composition comprises cytarabine and bisantrene.

106. The composition of claim 105 wherein the ratio of the cytarabine and the bisantrene is from about 25: 1 to about 1 : 1 .

107. The composition of claim 106 wherein the ratio of the cytarabine and the bisantrene is from about 10: 1 to about 3: 1 .

108. The composition of claim 107 wherein the ratio of the cytarabine and the bisantrene is about 5: 1.

109. A method for treating a malignancy comprising the step of administering a therapeutically effective quantity of the composition of claim 72 to treat the malignancy.

1 10. The method of claim 109 wherein the malignancy is selected from the group consisting of breast cancer, ovarian cancer, renal cancer, small-cell lung cancer, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myelocytic leukemia, melanoma, gastric cancer, adrenal cancer, head and neck cancer, hepatocellular cancer, hypernephroma, bladder cancer, acute leukemias of childhood, chronic lymphocytic leukemia, prostate cancer, glioblastoma, and myeloma.

1 1 1 . The method of claim 1 10 wherein the malignancy is breast cancer and wherein the breast cancer is selected from the group consisting of refractory breast cancer, triple-negative breast cancer, and breast cancer characterized by

overexpressed Her-2-neu.

1 12. The method of claim 1 10 wherein the malignancy is an acute leukemia of childhood and wherein the acute leukemia of childhood is selected from the group consisting of acute myelocytic leukemia (AML) and acute lymphocytic leukemia (ALL) of childhood.

1 13. The method of claim 1 10 wherein the malignancy is prostate cancer and wherein the prostate cancer is androgen-resistant prostate cancer.

1 14. The method of claim 1 10 wherein the malignancy is glioblastoma and wherein the glioblastoma is glioblastoma that is resistant to one or both of the following therapeutic agents: temozolomide (Temodar) or bevacizumab (Avastin), or is characterized by EGFR Variant III.

1 15. The method of claim 1 10 wherein the malignancy is a malignancy that is characterized by overexpressed topoisomerase II or overexpressed and/or mutated EGFR.

1 16. The method of claim 109 wherein the composition comprises bisantrene.

1 17. The method of claim 1 16 wherein the bisantrene is bisantrene dihydrochloride.

1 18. The method of claim 109 wherein the composition comprises a derivative or analog of bisantrene.

1 19. The method of claim 1 18 wherein the derivative or analog of bisantrene is selected from the group consisting of:

(a) the analog of Formula (II)

the bisantrene analog of Formula (III)

(in);

(C) the bisantrene analog of Formula (IV)

(IV);

(d) the bisantrene analog of Formula (V)

(V); the bisantrene analog of Formula (VI)

(VI); the bisantrene analog of Formula (VII)

(VII); the bisantrene analog of Formula (VIII)

(VIII);

(i) the bisantrene analog of Formula (X)

the bisantrene analog of Formula (XIII)

(XIII);

(m) bisantrene analogs of Formula (XIV)

CH3 CH3

I I

CH HCO⁽X CH3)3,—€ΗΝ¾— CHiCHiCOOH,

OC(CH₃)3,— CH2OCH3, ~{CH₂)₃C0OH, COOH

(CH₂)2 0 H or— CHaN®— (€Η3)3θθ))_;

R'O O

\li

(n) the bisantrene analog 9, 10-bis[(2- hydroxyethyl)iminomethyl]anthracene;

(v) N,N'-bis[2-(dimethylamino)ethyl]-9, 10-anthracene- bis(methylamine);

(w) N,N'-bis(1 -ethyl-3-piperidinyl)-9, 10-anthracene-bis(methylamine); and

(x) derivatives and salt forms of the compounds of (a)-(w).

120. The method of claim 1 18 wherein the derivative or analog of bisantrene is a derivative of bisantrene selected from the group consisting of:

121 . The method of claim 109 wherein the pyrimidine analog antimetabolite is selected from the group consisting of cytarabine, 5-azacytidine, gemcitabine, floxuridine, 5-fluorouracil, capecitabine, 6-azauracil, troxacitabine, thiarabine, sapacitabine, CNDAC (2'-cyano-2'-deoxy-1 - -D-arabinofuranosylcytosine), 2'-deoxy-2'-methylidenecytidine, 2'-deoxy-2'-fluoromethylidenecytidine, 2'-deoxy-2'- methylidene-5-fluorocytidine, 2'-deoxy-2',2'-difluorocytidine, and 2'-C-cyano-2'-deoxy- - arabinofuranosylcytosine.

122. The method of claim 121 wherein the pyrimidine analog antimetabolite is selected from the group consisting of cytarabine, 5-azacytidine, gemcitabine, floxuridine, 5-fluorouracil, capecitabine, and 6-azauracil.

123. The method of claim 122 wherein the pyrimidine analog antimetabolite is cytarabine.

124. The method of claim 123 wherein the composition comprises bisantrene and cytarabine.

125. The method of claim 109 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 25: 1 to about 1 :1 .

126. The method of claim 125 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 10: 1 to about 3:1 .

127. The method of claim 126 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is about 5: 1 .

128. The method of claim 124 wherein the ratio of the cytarabine and the bisantrene is from about 25: 1 to about 1 :1 .

129. The method of claim 128 wherein the ratio of the cytarabine and the bisantrene is from about 10: 1 to about 3: 1 .

130. The method of claim 129 wherein the ratio of the cytarabine and the bisantrene is about 5: 1 .

131 . The method of claim 109 wherein the liposomes comprise at least one lipid selected from the group consisting of a phosphatidylcholine lipid, a

132. The method of claim 131 wherein the liposomes comprise a phosphatidylcholine lipid.

133. The method of claim 132 wherein the phosphatidylcholine lipid is diastearoylphosphatidylcholine.

134. The method of claim 131 wherein the liposomes comprise a phosphatidylglycerol lipid.

135. The method of claim 134 wherein the phosphatidylglycerol lipid is distearoylphosphatidylglycerol.

136. The method of claim 131 wherein the liposomes comprise a sterol.

137. The method of claim 136 wherein the sterol is cholesterol.

138. The method of claim 131 wherein the liposomes comprise diastearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol.

139. The method of claim 138 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.5 to about 7.5 of distearoylphosphatidylcholine, about 1.5 to about 2.5 of distearoylphosphatidylglycerol, and about 0.8 to 1 .2 of cholesterol.

140. The method of claim 139 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is from about 6.8 to about 7.2 of distearoylphosphatidylcholine, about 1.8 to about 2.2 of distearoylphosphatidylglycerol, and about 0.9 to 1 .1 of cholesterol.

141 . The method of claim 140 wherein the molar ratio of the distearoylphosphatidylcholine, the distearoylphosphatidylglycerol, and the cholesterol is about 7:2: 1 .

142. The method of claim 131 wherein the liposomes have a diameter of less than about 300 nm.

143. The method of claim 142 wherein the liposomes have a diameter of less than about 200 nm.

144. The method of claim 109 wherein the liposomes have an

intraliposomal osmolality of 500 mOSM/kg or less.

145. The method of claim 141 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 25: 1 to about 1 :1 .

146. The method of claim 145 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is from about 10: 1 to about 3:1 .

147. The method of claim 146 wherein the ratio of the pyrimidine analog antimetabolite and the bisantrene or derivative or analog thereof is about 5: 1 .

148. The method of claim 141 wherein the composition comprises cytarabine and bisantrene.

149. The method of claim 148 wherein the ratio of the cytarabine and the bisantrene is from about 25: 1 to about 1 :1 .

150. The method of claim 149 wherein the ratio of the cytarabine and the bisantrene is from about 10: 1 to about 3: 1 .

151 . The method of claim 150 wherein the ratio of the cytarabine and the bisantrene is about 5: 1 .

152. The method of claim 109 wherein the composition is administered parenterally.

153. The method of claim 152 wherein the composition is administered intravenously or intraperitoneally.

154. The method of claim 109 wherein the composition is administered in a pharmaceutical formulation suitable for administration.

155. The method of claim 109 wherein the method further comprises administration of a therapeutically effective quantity of an additional therapeutic agent.

156. The method of claim 155 wherein the malignancy is breast cancer and wherein the additional therapeutic agent is selected from the group consisting of tamoxifen, anastrozole, letrozole, cyclophosphamide, docetaxel, paclitaxel,

methotrexate, fluorouracil, and trastuzumab.

157. The method of claim 155 wherein the malignancy is ovarian cancer and wherein the additional therapeutic agent is selected from the group consisting of: a platinum-containing antineoplastic drug selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin, phenanthriplatin, picoplatin, and satraplatin; paclitaxel; topotecan; gemcitabine (provided that the composition does not comprise gemcitabine); etoposide; and bleomycin.

158. The method of claim 155 wherein the malignancy is renal cancer and wherein the additional therapeutic agent is selected from the group consisting of everolimus, torisel, nexavar, sunitinib, axitinib, inferferon, interleukin-2, pazopanib, sorafenib, nivolumab, cabozanitib, and levanitib.

159. The method of claim 155 wherein the malignancy is small-cell lung cancer and wherein the additional therapeutic agent is selected from the group consisting of cyclophosphamide, cisplatin, etoposide, vincristine, paclitaxel, and carboplatin.

160. The method of claim 155 wherein the malignancy is non-small-cell lung cancer and wherein the additional therapeutic agent is selected from the group consisting of cisplatin, erlotinib, gefitinib, afatinib, crizotinib, bevacizumab, carboplatin, paclitaxel, nivolumab, and pembrolizumab.

161 . The method of claim 155 wherein the malignancy is Hodgkin's lymphoma and wherein the additional therapeutic agent is selected from the group consisting of mechlorethamine, vincristine, prednisone, procarbazine, bleomycin, vinblastine, dacarbazine, etoposide, and cyclophosphamide.

162. The method of claim 155 wherein the malignancy is non-Hodgkin's lymphoma and wherein the additional therapeutic agent is selected from the group consisting of cyclophosphamide, vincristine, and prednisone.

163. The method of claim 155 wherein the malignancy is acute myelocytic leukemia and wherein the additional therapeutic agent is selected from the group consisting of cytarabine (provided that the composition does not comprise cytarabine), fludarabine, all-frans-retinoic acid, interleukin-2, and arsenic trioxide.

164. The method of claim 155 wherein the malignancy is melanoma and wherein the additional therapeutic agent is selected from the group consisting of temozolomide, dacarbazine, interferon, interleukin-2, ipilimumab, pembrolizumab, nivolumab, vemurafenib, dabrafenib, and trametinib.

165. The method of claim 155 wherein the malignancy is gastric cancer and wherein the additional therapeutic agent is selected from the group consisting of 5- fluorouracil (provided that the composition does not comprise 5-fluorouracil),

capecitabine (provided that the composition does not comprise capecitabine), carmustine, semustine, mitomycin C, cisplatin, taxotere, and trastuzumab.

166. The method of claim 155 wherein the malignancy is adrenal cancer and wherein the additional therapeutic agent is selected from the group consisting of mitotane, cisplatin, etoposide, and streptozotocin.

167. The method of claim 155 wherein the malignancy is head and neck cancer and wherein the additional therapeutic agent is selected from the group consisting of paclitaxel, carboplatin, cetuximab, docetaxel, cisplatin, and 5-fluorouracil (provided that the composition does not comprise 5-fluorouracil).

168. The method of claim 155 wherein the malignancy is hepatocellular cancer and wherein the additional therapeutic agent is selected from the group consisting of tamoxifen, octreoside, synthetic retinoids, cisplatin, 5-fluorouracil (provided that the composition does not comprise 5-fluorouracil), interferon, taxol, and sorafenib.

169. The method of claim 155 wherein the malignancy is

hypernephroma and wherein the additional therapeutic agent is selected from the group consisting of nivolumab, everolimus, sorafenib, axitinib, lenvatinib, temsirolimus, sunitinib, pazopanib, interleukin-2, cabozanitib, bevacizumab, interferon a, ipilimumab, atezolizumab, varilumab, durvalumab, tremelimumab, and avelumab.

170. The method of claim 155 wherein the malignancy is bladder cancer and wherein the additional therapeutic agent is selected from the group consisting of cisplatin, 5-fluorouracil (provided that the composition does not comprise 5-fluorouracil), mitomycin C, gemcitabine (provided that the composition does not comprise

gemcitabine), methotrexate, vinblastine, carboplatin, paclitaxel, docetaxel, ifosfamide, and pemetrexed.

171 . The method of claim 155 wherein the malignancy is acute myelocytic leukemia of childhood and wherein the additional therapeutic agent is selected from the group consisting of methotrexate, nelarabine, asparaginase, blinatumomab, cyclophosphamide, clofarabine, cytarabine (provided that the composition does not comprise cytarabine), dasatinib, methotrexate, imatinib, pomatinib, vincristine, 6-mercaptopurine, pegaspargase, and prednisone.

172. The method of claim 155 wherein the malignancy is acute lymphocytic leukemia and wherein the additional therapeutic agent is selected from the group consisting of asparaginase, vincristine, dexamethasone, methotrexate, 6- mercaptopurine, cytarabine (provided that the composition does not comprise

cytarabine), hydrocortisone, 6-thioguanine, prednisone, etoposide, cyclophosphamide, mitoxantrone, and teniposide.

173. The method of claim 155 wherein the malignancy is chronic lymphocytic leukemia and wherein the additional therapeutic agent is selected from the group consisting of fludarabine, cyclophosphamide, rituximab, vincristine, prednisolone, bendamustine, alemtuzumab, ofatumumab, obinutuzumab, ibrutinib, idelalisib, and venetoclax.

174. The method of claim 155 wherein the malignancy is prostate cancer and wherein the additional therapeutic agent is selected from the group consisting of temozolomide, docetaxel, cabazitaxel, bevacizumab, thalidomide, prednisone, sipuleucel-T, abiraterone, and enzalutamide.

175. The method of claim 155 wherein the malignancy is glioblastoma and wherein the additional therapeutic agent is selected from the group consisting of temozolomide and bevacizumab.

176. The method of claim 155 wherein the malignancy is myeloma and wherein the therapeutic agent is selected from the group consisting of bortezomib, lenalidomide, dexamethasone, melphalan, prednisone, thalidomide, and

cyclophosphamide.