DESCRIPTION

COMBINATION OF CHEMOTHERAPEUTIC COMPOUNDS FOR TREATING

CANCER

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates generally to the fields of cancer and chemopharmaceuticals. More particularly, it concerns the combined use of substituted anthracycline compounds and alkylating agents to treat cancer.

B. Description of Related Art Cancer is a leading cause of death in most countries, and the result of billions of dollars in healthcare expense around the world. Through great effort, significant advances have been made in treating cancer, primarily due to the development of radiation and chemotherapy-based treatments. Unfortunately, a common problem is tumor cell resistance to radiation and chemotherapeutic drugs. For example, NSCLC accounts for at least 80% of the cases of lung cancer, but patients with NSCLC are generally unresponsive to chemotherapy (Doyle, 1993). This phenomenon, called pleiotropic drug resistance or multi-drug resistance (MDR), may account for much of the drug resistance that occurs in previously treated cancer patients.

One of the traditional ways to attempt to circumvent this problem of drug resistance has been combination chemotherapy. Combination chemotherapy uses the differing mechanisms of action and cytotoxic potentials of multiple drugs. Commonly used chemotherapeutic agents are classified by their mode of action, origin, or structure, although some drugs do not fit clearly into any single group. The categories include alkylating agents, anti-metabolites, antibiotics, alkaloids, and miscellaneous agents (including hormones). Agents in the different categories have different sites of action. Although combination chemotherapy has been useful in some cases, often times cancer cells becomes resistant to the combination of chemotherapeutic agents, thereby reducing the beneficial effects of a particular combination.

SUMMARY OF THE INVENTION

The present invention overcomes the deficiencies in the art by providing compositions and corresponding methods of treating hyperproliferative diseases such as cancer in a subject comprising the combined use of an alkylating agent and a substituted anthracycline compound. This combination provides a synergistic effect leading to the effective treatment of a variety of different cancers.

In one aspect of the present invention there is disclosed a method of treating cancer in a subject comprising administering to the subject in need of the treatment a therapeutically effective amount of an alkylating agent and a substituted anthracycline compound having the formula:

wherein R ¹ is an alkyl chain, a (-COCH ₂ R ¹³ ) group, or a C(OH)- CH ₂ R ¹³ ) group, wherein R ¹³ is: a hydrogen (-H) group; a hydroxyl group (-OH); a methoxy group (-OCH ₃ ); an alkoxy group having 1-20 carbon atoms; an alkyl group having 1-20 carbon atoms; an aryl group having 1-20 carbon atoms; a fatty acyl group having the general structure -O-CO(CH ₂ ) _k CH ₃ , wherein k = an integer from 1-20; or a fatty acyl group having the general structure -O-CO- (CH ₂ ) _L (CH=CH) _m (CH ₂ ) _n CH ₃ , -O-CO-(CH ₂ ) _n -CH ₂ NH ₂ , or -OCO-(CH ₂ ) _n -CO ₂ H, wherein L is an integer between 1-3, m is an integer between 1-6, and n is an integer between 1-9; R ² and R ³ are independently a hydrogen (-H), a hydroxyl group (-OH), or a methoxy group (-OCH ₃ ); R ⁴ is a hydrogen (-H) group, a methoxy group (-OCH ₃ ), a hydroxyl group (-OH), or a halide; Y and Y are independently a double bonded oxygen, sulfur, or nitrogen atom; Z is a hydrogen (-H) group, a hydroxy (-OH) group, a -CO ₂ H group, or a -CO ₂ R ¹⁹ group; R ⁵ and R ⁶

are independently a hydrogen (-H) group, a hydroxy (-OH) group, a halide, -OR ¹⁹ , -SH, - SR ¹⁹ , -NH ₂ , -NHR ¹⁹ , -N(R ¹⁹ ) ₂ , or -CH ₃ ; R ⁹ is a hydrogen (-H) group, -CH ₃ , an alkyl group, an aryl group, CH ₂ OH, or CH ₂ F; R ¹⁰ , R ¹¹ and R ¹² are independently a hydrogen (-H) group, a hydroxy (-OH) group, a halide, -OR ¹⁹ ; -SH; -S R ¹⁹ ; -NH ₂ ; -NHR ¹⁹ ; -N(R ¹⁹ ) ₂ or -CH ₃ ; R ¹⁹ is an alkyl chain, an alkylating moiety, a cycloalkyl chain, a cyclic ring, a hydrogen or a chain(R) such as -OCO-(CH ₂ ) _n -CH ₂ NH ₂ ; or OCO-(CHa) _n -CO ₂ H and its salts; one of R ⁷ and R ^s is a -

H; and one of R ⁷ and R ⁸ is a X-alkyl-aromatic-ring ( — XAAR) substituent, wherein A is an alkyl group and AR is an unsubstituted phenyl ring, a substituted phenyl ring, a substituted five-member ring, a heteroaromatic five-member ring, or a six-member ring having the structure:

wherein, R ¹ -R ¹ are independently a hydrogen (-H) group, a hydroxyl (-OH) group, a methoxy (-OCH ₃ ) group, a nitro (-NO ₂ ) group, an amine (-NH ₂ ) group, a halide, an alkoxy group having 1-20 carbon atoms, an alkyl group having 1-20 carbon atoms, an aryl group having 1-20 carbon atoms, an alkyl-amino group, an alkyl-thio group, a cyano group (CN, SCN), an -CO ₂ H group, or an -CO ₂ R ¹⁹ group; X is a -O, -N or -S, -SO, or -SO ₂ group; and A is (CH ₂ ) _n where n = 0-10; wherein if R ⁷ is a XAAR substituent, R ⁸ is not, and if R ⁸ is a XAAR substituent, R ⁷ is not.

In certain aspects, the alkylating agent is temozolomide. The substituted anthracycline can have the formula:

The method can further include the human subject previously failing one or more anti- glioma therapies. The alkylating agent and the substituted anthracycline compound can be administered in a treatment regimen comprising at least one cycle of the alkylating agent and the substituted anthracycline compound. The treatment regimen can include administering the alkylating agent to the subject multiple times within the treatment cycle. The treatment regimen can include administering the substituted anthracycline compound to the subject multiple times within the treatment cycle. The treatment regimen can include administering the alkylating agent to the subject prior to administering the substituted anthracylcine compound within the treatment cycle. The treatment regimen can include administering the alkylating agent to the subject after administering the substituted anthracylcine compound within the treatment cycle. The treatment regimen can include administering the alkylating agent to the subject prior to and after administering the substituted anthracylcine compound within the treatment cycle. The treatment regimen can include administering the substituted anthracylcine compound to the subject prior to and after administering the alkylating agent within the treatment cycle. The treatment regimen can include administering the alkylating agent at about the same time as administering the substituted anthracycline compound to the subject. The cycle can be repeated at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, or more week intervals for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more cycles. In other aspects, the treatment regimen includes administering temozolomide at a dose from 75 mg/m2/day to 150 mg/m2/day for 5 days (days 1 to 5 of the cycle),

combined with WP744 at a dose from 5 mg/m2/day to 15 mg/m2/day for 3 days (during days 1 to 5 of the cycle), followed by 14 to 23 days of recovery. In certain embodiments, the cycle is repeated at 4 to 6 week intervals for a total of 6 to 8 cycles. The alkylating agent and/or the substituted anthracycline compound can be modified after the first cycle. The subject can be evaluated for neurotoxicity and/or ototoxicity during or after the first cycle. The subject can be evaluated for blood count and platelet count during and/or after each cycle. Administering can be through dietary administration, oral administration, or via intravenous injection. The oral administration can be in the form of a pill or a liquid. The intravenous injection can be in the form of a mixture containing an injectable vehicle. The alkylating agent can be administered orally and the substituted anthracylcine compound can be administered intravenously. The alkylating agent and the substituted anthracycline compound can be included in the same composition. The subject can have previously received radiation therapy. The subject can have previously received chemotherapy treatment in addition to the alkylating agent and the substituted anthracycline compound.

In other aspects, there is disclosed a method of treating cancer comprising administering to a subject with cancer a therapeutically effective amount of temozolomide and a substituted anthracycline compound having the formula:

Also disclosed is a method of inhibiting the progression of cancer comprising administering to a subject with cancer a therapeutically effective amount of an alkylating agent and a substituted anthracycline compound to inhibit the progression of cancer. The

memoα can be further defined as inhibiting the vascularization, growth or spread of the cancer.

In another aspect, there is disclosed a method for inhibiting cancer development comprising (a) identifying a subject at risk of developing cancer and (b) administering to said subject a dose of an alkylating agent and a substituted anthracycline compound effective to inhibit the development of the cancer. The subject can have a familial history of cancer or has been exposed to a carcinogenic environment.

In yet another embodiment, there is disclosed a method of extending the life of a subject having cancer comprising administering to the subject a therapeutically effective amount of an alkylating agent and a substituted anthracycline compound.

Also disclosed is a method of enhancing the effects of temozolomide on cancer comprising administering to a subject having cancer an amount of a substituted anthracycline compound sufficient to enhance the effects of temozolomide on the cancer.

There is also disclosed a method of enhancing the effects of the substituted anthracycline compound of claim 1 on cancer comprising administering to a subject having cancer an amount of temozolomide sufficient to enhance the effects of the substituted anthracycline compound on the cancer.

Also disclosed is a method for inhibiting cancer recurrence comprising administering to a subject previously having cancer a dose of an alkylating agent and a substituted anthracycline compound effective to inhibit the development of the cancer. In other aspects, there is disclosed a pharmaceutically acceptable composition comprising an alkylating agent and a substituted anthracycline compound. The alkylating agent can be temozolomide.

A kit comprising an alkylating agent and a substituted anthracycline compound is also contemplated. The alkylating agent is temozolomide.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

The term "about" or "approximately" are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

The term "effective," as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.

The terms "patient" or "subject" can include an animal. Preferred animals are mammals, including but not limited to humans, pigs, cats, dogs, rodents, horses, cattle, sheep, goats and cows. Preferred patients and subjects are humans.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the examples, while indicating specific embodiments of the invention, are given by way of illustration only. Additionally, it is contemplated that changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 Ex Vivo Clonogenic Assay in AML Patient Samples. The upper panels represent the number of blast colonies formed in each of three patients. Data are depicted in terms of reduction of absolute numbers of AML colonies. The lower graphs represent the %

inhibition of blast colony growth. Closed squares and solid rectangles represent WP744, while open circles and open rectangles represent doxorubicin. Each data point represents the mean value of duplicate and triplicate measurements +/- SD.

FIG. 2 Survival of Animals Treated with WP744 in a Mouse Model of Glioma. U87GBM cells were implanted intracranially in nude (Nu-Nu) mice and allowed to grow for five days. Animals were then treated with vehicle (PBS) or WP744 10 mg/kg administered intravenously A second dose of 5 mg/kg RTA 744 was administered 10 days after the first dose.

FIG. 3 Survival of Animals Receiving Temozolomide +/- WP744 in a Mouse Model of Glioma. Groups of 6 mice each received vehicle control (PBS, QDx5 i.p.), temozolomide (7.5 mg/kg QDx5 i.p.), or temozolomide (7.5mg/kg QDx5 i.p.) plus RTA 744 (5 mg/kg, QDx5 i.p.). Median survival in this model was 30 days for the control group, compared with 40 days for animals receiving temozolomide alone and 48 days for animals receiving the combination of temozolomide plus RTA 744. FIG. 4. Mean Plasma Concentration vs. Time Curve after Intravenous

Administration of WP744 to Mice. WP744 administered as an intravenous bolus injection to CD-I mice, given at a dose of 20 mg/kg. Each open circle represents data corresponding to the mean of 5 mice sampled per time point. The solid line represents the pharmacokinetic model fit of the data. FIG. 5. Tissue Distribution of RTA 744 in Plasma and Brain of CD-I Mice after

Intravenous Administration (20 mg/kg).

FIG. 6. WP744 Distribution in a Murine Glioma Model. Male tumor-bearing Nu/Nu mice (U87 xenograft) were given WP744 at 20 mg/kg as an i.v. bolus. Blood, tumor, and contralateral brain samples were harvested at 0.5, 1, and 2 lirs post dose and WP744 was extracted from plasma (using a solid phase extraction method) and from tissues (using a liquid: liquid extraction method).

FIG. 7. Protein binding of WP744 in mouse, rat, dog, and human plasma using ultrafiltration. WP744 was dissolved in animal plasmas (mouse, rat, dog, and human) to a final concentration of lOOO ng/mL. Ami con Centrifree columns were loaded with 1 ml of treated plasma, placed in fixed-angle centrifuge and centrifuged at 2000 x g for 30 minutes. Aliquots (125 ml) were removed from protein-free filtrate, protein-rich plasma, and unfiltered plasma, then analyzed for WP744 content.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Drug resistance is a major obstacle in the treatment of hyperproliferative diseases such as cancer. Clinical experience shows that some cancers demonstrate selective sensitivity to certain drugs but resistance to others. Treatment decisions, however, are typically made empirically using a trial-and-error approach. A need exists for new methods and therapeutic compositions that can overcome drug resistance and/or enhance the effectiveness of other anti-cancer agents.

The present invention overcome the deficiencies in the art by providing methods and compositions for treating hyperproliferative diseases in a subject by administering a combination of an alkylating agent and a substituted anthracycline compound having the formula as described in this specification to a subject in need of such treatment. This combination has a synergistic effect which can lead to more efficient and efficacious cancer treatment regimens. These and other aspects of the present invention are described in further detail in the following sections. A. Hyperproliferative Diseases

In certain aspects, the present invention can be used to treatment of hyperproliferative diseases including, but not limited to, cancer. A hyperproliferative disease is any disease or condition which has, as part of its pathology, an abnormal increase in cell number. Included in such diseases are benign conditions such as benign prostatic hypertrophy and ovarian cysts. Also included are premalignant lesions, such as squamous hyperplasia. At the other end of the spectrum of hyperproliferative diseases are cancers. A hyperproliferative disease can involve cells of any cell type. The hyperproliferative disease may or may not be associated with an increase in size of individual cells compared to normal cells.

Another type of hyperproliferative disease is a hyperproliferative lesion, a lesion characterized by an abnormal increase in the number of cells. This increase in the number of cells may or may not be associated with an increase in size of the lesion. Examples of hyperproliferative lesions that are contemplated for treatment include benign tumors and premalignant lesions. Examples include, but are not limited to, squamous cell hyperplastic lesions, premalignant epithelial lesions, psoriatic lesions, cutaneous warts, periungual warts, anogenital warts, epidermdysplasia verruciformis, intraepithelial neoplastic lesions, focal epithelial hyperplasia, conjunctival papilloma, conjunctival carcinoma, or squamous carcinoma lesion. The lesion can involve cells of any cell type. Examples include keratinocytes, epithelial cells, skin cells, and mucosal cells.

1. Cancer

"Cancer" includes a tissue of uncontrolled growth or proliferation of cells, such as a tumor. Cancer develops through the accumulation of genetic alterations (Fearon and

Vogelstein, 1990) and gains a growth advantage over normal surrounding cells. The genetic transformation of normal cells to neoplastic cells occurs through a series of progressive steps.

Genetic progression models have been studied in some cancers, such as head and neck cancer

(Califano et al, 1996). Examples of cancers that can be treated with the present invention include, but are not limited to, breast cancer, lung cancer, prostate cancer, ovarian cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head & neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, leukemia, and brain cancer.

2. Brain Cancer

Brain cancer is of particular interest in the context of the present invention. Brain cancers include brain tumors (gliomas) which are typically any intracranial tumor created by abnormal or uncontrolled cell division, normally either found in the brain itself (e.g., neurons, glia cells (astrocytes, oligodendrocytes, ependymal cells), lymphatic tissue, blood vessels), in the cranial nerves (myelin-producing Schwann cells), in the brain envelopes (meninges), skull, pituitary and pineal glands, or spread from cancers primarily located in other organs {e.g., metastatic tumors). Gliomas are a diverse group of brain tumors that arise from the normal "glial" cells of the brain. Gliomas have specific signs and symptoms that are primarily related to the location of the glioma. The temporal lobe gliomas, for example, may cause epilepsy, difficulty with speech or loss of memory. The frontal lobe gliomas may cause behavioral changes, weakness of the arms or legs or difficulty with speech. The occipital gliomas may cause loss of vision. The parietal gliomas may cause loss of spatial orientation, diminished sensation on the opposite side of the body, or inability to recognize once familiar objects or persons.

The most important determinant of survival for gliomas is the "grade" of the glioma. The low-grade gliomas have a protracted natural history, while the high grade gliomas (anaplastic astrocytoma and glioblastoma multiforme) are much more difficult to successfully treat, hi this classification, astrocytomas and glioblastomas represent different grades of malignancy of the same tumor. Grade I tumors, typically slow growing, are characterized by most cells having normal characteristics, and few mitotic features. Endothelial proliferation is absent. Grade II tumors, previously designated "astroblastomas," are characterized by an

increased number of cells with polymorphic nuclei in mitoses. There is no clear line of demarcation from normal tissue. Grade III tumors represent anaplastic astrocytomas and Grade IV tumors represent the typical glioblastoma multiforme, characterized by cellular pleomorphism, vascular proliferation, mitoses, and multinucleated giant cells. The current morphologically-based tumor classifications often mix cell lineage features with tumor growth characteristics. However, there are two general classifications — the anaplastic glioma strata and glioblastomas. The former is comprised of various gliomas including anaplastic astrocytomas, anaplastic oligoastrocytomas, anaplastic oligodendrogliomas, malignant glioma, anaplastic gliomas non-specified, and anaplastic ependymoma.

The anaplastic gliomas are intermediate grade infiltrative gliomas - classified between low (localized, slow growing) and glioblastoma multiforme (rapidly growing and highly invasive). Anaplastic astrocytomas (AA) are tumors that arise from brain cells called astrocytes and/or their precursors. Astrocytes are support cells of the central nervous system. The majority of astrocytic tumors in children are low-grade, whereas the majority in adults are high-grade. These tumors can occur anywhere in the brain and spinal cord.

Oligodendrogliomas are gliomas derived from oligodendrocytes and/or their precursors. Oligodendrocytes that have a role in the structure and function of myelinated neurons in the brain. Anaplastic oligodendroglioma (AO) are more aggressive than oligodendrogliomas, but are also more sensitive to chemotherapy than are anaplastic astrocytomas. A high rate of response to the use of PCV (procarbazine, CCNU, vincristine) chemotherapy has led to the common use of PCV chemotherapy prior to radiation therapy, following irradiation, and/or at tumor recurrence and progression. Another glioma appears as histologic mixture of both oligodendroglioma and astrocytoma tumor forms and is called oligoastrocytoma. While oligoastrocytoma can be low-grade, the majority of the mixed oligoastrocytomas are anaplastic oligoastrocytomas (AOA).

The last glioma subgroup are ependymomas. One subtype of malignant ependymomas is the anaplastic ependymoma (AE); these tumors arise from ependymal cells and/or their precursors that line the cerebrospinal fluid passageways, called ventricles. These tumors are classified as either supratentorial (in the top part of the head) or infratentorial (in the back of the head).

Metastatic brain cancer also presents a serious medical challenge. Certain primary cancers arising outside the CNS, such as breast and lung cancer, tend to metastasize to the brain. Brain metastasis is a frequent cause of mortality in these patients. Unfortunately, very

few agents have proven effective in treating CNS metastatic disease. The anthracycliiies of the current invention, because they have shown activity in preclinical models of breast cancer and other non-CNS cancers, and against drug-resistant cancer cell lines, are useful in treating metastatic CNS cancer, particularly in combination with temozolomide. The present invention overcomes previous deficiencies in treating hyperproliferative diseases such as cancer. The inventor has discovered that the combination of an alkylating agent with a substituted anthracycline compound has a synergistic treatment effect. Non- limiting examples of the alkylating agents and substituted anthracycline compounds that can be used in the context of the present invention are described in the following sections. B. Alkylating Agents

Alkylating agents include compounds that directly interact with genomic DNA to prevent the cancer cell from proliferating. These compounds typically have the ability to add an alkyl group(s) to electronegative groups. They can stop tumor growth by cross-linking guanine nucelobases in DNA double-helix strands. This prevents DNA strands from uncoiling and separating, thereby preventing DNA replication and cell division. Non-limiting examples of aklylating agents that can be used in the context of the present invention include temozolomide, busulfan, chlorambucil, cisplatin, cyclophosphamide (Cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan.

In certain aspects, the preferred alkylating agent is temozolomide (3,4-dihydro-3- methyl-4-oxoimidazo[5,l-d]-as-tetrazine-8-carboxamide). Temozolomide has been shown to treat brain tumors. It is derived from dacarbazine and was first synthesised in 1984. The chemical formula for temozolomide is C ₆ H ₆ N ₆ O ₂ (CAS # 85622-93-1) and its corresponding structure is:

Temozolomide Temozolomide is typically administered orally once a day for 5 days in a 28-day cycle and is used to treat patients with malignant glioma. It has been shown to have high bioavailability and can cross the blood-brain barrier where it is spontaneously hydrolysed to

its active form (3-methyl-(triazen-l-yl)imidazole-4-carboxamide (MTIC)). Temozolomide is sold by Schering-Plough under the brand name TEMOD AR ^® in 5mg, 20mg, lOOmg, and 250mg capsules.

C. Substituted Anthracycline Compounds

Another aspect of the present invention includes using substituted anthracycline compounds in combination with alkylating agents to treat a variety of different cancers. Non- limiting examples of substituted anthracycline compounds, and methods of using and making the same, are described in U.S. Patent 6,673,907 to Priebe et al. ('907 Patent), the disclosure of which is incorporated by reference in its entirety. In certain aspects, the '907 Patent describes two main classes of substituted anthracycline compounds; one bearing modified substituents at the C-3' sugar moiety and the other bearing modifications at the C-4' sugar moiety.

For instance, the substituted anthracycline compound can have the following generic formula:

wherein R ¹ can include any suitable group or combination of groups that form but are not limited to a nucleic acid intercalator or binding compound and a topoisomerase inhibitor, including but not limited to, an alkyl chain, a ( — COCH ₂ R ¹³ ) group, or a C(OH)- CH ₂ R ¹³ ) group. R ¹³ can be a hydrogen (-H) group, a hydroxyl group (-OH), a methoxy group (-OCH ₃ ), an alkoxy group having 1-20 carbon atoms, an alkyl group having 1-20 carbon atoms, an aryl group having 1-20 carbon atoms, a fatty acyl group having the general structure -O-CO(CH ₂ )i _c CH ₃ , wherein k = an integer from 1-20, or a fatty acyl group having the general

structure -O-CO-(CH ₂ ) _L (CH=CH) _m (CH ₂ ) _n CH ₃ , -O-CO-(CH ₂ ) _n -CH ₂ NH ₂ , or -OCO-(CH ₂ ) _n - CO ₂ H, wherein L is an integer between 1-3, m is an integer between 1-6, and n is an integer between 1-9. R ² and R ³ can independently be ahydrogen (-H), a hydroxyl (-OH) group, or a methoxy (-OCH ₃ ) group. R ⁴ can be a hydrogen (-H) group, a methoxy group (-OCH ₃ ), a hydroxyl group (-OH), or a halide. Y ¹ and Y ² can independently be a double bonded oxygen, sulfur, or nitrogen atom. Z can be a hydrogen (-H) group, a hydroxy (-OH) group, a -CO ₂ H group, or a -CO ₂ R ¹⁹ group. R ⁹ can be a hydrogen (-H) group, -CH ₃ , an alkyl group, an aryl group, CH ₂ OH, or CH ₂ F. R ¹⁰ , R ¹¹ and R ¹² can independently be a hydrogen (-H) group, a hydroxy (-OH) group, a halide, -OR ¹⁹ ; -SH; -S R ¹⁹ ; -NH ₂ ; -NHR ¹⁹ ; -N(R ¹⁹ ) ₂ or -CH ₃ . R ¹⁹ can be an alkyl chain, an alkylating moiety, a cycloalkyl chain, a cyclic ring, or a hydrogen.

1. Modified substituents at the C-3' sugar moiety

As noted above, in certain aspects the substituted anthracycline compounds include modified substituents at the C-3' sugar moiety. This is exemplified in the above generic structure when R ⁶ and R ⁷ can independently be a hydrogen (-H) group, a hydroxy (-OH) group, a halide, -OR ¹⁹ , -SH, -SR ¹⁹ , -NH ₂ , -NHR ¹⁹ , -N(R ¹⁹ ) ₂ , or -CH ₃ , and R ⁷ can additionally be a saccharide. One of R ⁵ and R ⁶ can be a -H and one of R ⁵ and R ⁶ can be an X-alkyl- aromatic-ring ( — XAAR) substituent, wherein A can be an alkyl group and AR can be an unsubstituted phenyl ring, a substituted phenyl ring, a substituted five-member ring, a heteroaromatic five-member ring, or a six-member ring having the structure:

wherein R ¹⁴ -R ¹⁸ can independently a hydrogen (-H) group, a hydroxyl (-OH) group, a methoxy (-OCH ₃ ) group, a nitro (-NO ₂ ) group, an amine (-NH ₂ ) group, a halide, an alkoxy group having 1-20 carbon atoms, an alkyl group having 1-20 carbon atoms, an aryl group having 1-20 carbon atoms, an alkyl-amino group, an alkyl-thio group, a cyano group (CN, SCN), an -CO ₂ H group, or an -CO ₂ R ¹⁹ group. X can be an -O, -N or -S, -SO, or -SO ₂ group.

A can be (CH ₂ ) _n where n = 0-10. In certain aspects, if R ⁵ is a XAAR substituent, R ⁶ is not, and if R ⁶ is a XAAR substituent, R ⁵ is not. Non-limiting examples of substituted anthracycline compounds that are modified at the C-3' sugar moiety are described in the '907 Patent at Figures 2-16 as WP831, WP791, WP790, WP787, WP786, WP785, WP784, WP780, WP778, WP775, WP774, WP758, WP757, WP756, WP755.

2. Modified substituents at the C-4' sugar moiety

In other aspects of the present invention, the substituted anthracycline compounds include modified substituents at the C-4' sugar moiety. This is exemplified in the above generic structure when R ⁵ and R ⁶ can independently be a hydrogen (-H) group, a hydroxy (- OH) group, a halide, -OR ¹⁹ , -SH, -SR ¹⁹ , -NH ₂ , -NHR ¹⁹ , -N(R ¹⁹ ) ₂ , or -CH ₃ , and R ⁵ can additionally be a saccharide. One of R ⁶ and R ⁷ can be a -H and one of R ⁶ and R ⁷ can be an X- alkyl-aromatic-ring ( — XAAR) substituent, wherein A can be an alkyl group and AR can be an unsubstituted phenyl ring, a substituted phenyl ring, a substituted five-member ring, a heteroaromatic five-member ring, or a six-member ring having the structure:

wherein R ¹⁴ -R ¹⁸ can independently a hydrogen (-H) group, a hydroxyl (-OH) group, a methoxy (-OCH ₃ ) group, a nitro (-NO ₂ ) group, an amine (-NH ₂ ) group, a halide, an alkoxy group having 1-20 carbon atoms, an alkyl group having 1-20 carbon atoms, an aryl group having 1-20 carbon atoms, an alkyl-amino group, an alkyl-thio group, a cyano group (CN, SCN), an -CO ₂ H group, or an -CO ₂ R ¹⁹ group. X can be an -O, -N or -S, -SO, or -SO ₂ group. A can be (CH ₂ ) _n where n = 0-10. In certain aspects, if R is a XAAR substituent, R is not, and if R ⁸ is a XAAR substituent, R ⁷ is not. Non-limiting examples of substituted anthracycline compounds that are modified at the C-4' sugar moiety are described in the '907 Patent at Figures 17-25 as WP799, WP797, WP794, WP783, WP750, WP744, WP727 and WP571.

In certain embodiments of the present invention, the preferred substituted anthracycline compound that can be used with the alkylating agents is WP744 which has the following generic structure:

The chemical name for the above molecule is 4'-O-Benzyl doxorubicin hydrochloride or 7-O- (3-amino-4-O-benzyl-2,3 ,6-trideoxy-α-L-lyxo-hexopyranosyl) adriamycinone hydrochloride. Its molecular formula is C ₃₄ H ₃₆ ClNO ₁₁ , and molecular weight is 670.1 g/mol. The mechanism for action of this compound involves intercalation, disruption of topoisomerase II activity, and free radical formation (Denesi et ah, 2002; Faderl et ah, 2001). WP744 can cross the blood brain barrier in amounts sufficient to have a therapeutic effect. This characteristic makes WP744 especially useful for treating brain cancers and preventing metastases. Non-limiting data shows that this compound has potent cytotoxic activity with some selectivity for cancer cells vs. normal fibroblasts. It is more potent than doxorubicin, with a similar spectrum of activity. WP744 demonstrates excellent in vivo activity in brain tumors, extending survival in a rigorous U87 orthotopic mouse model of glioma. Additionally, activity was demonstrated in the U87 flank tumor model of glioma, the mouse L1210 leukemia model and the mouse MDA-MB293 breast cancer xenograft model. Further non-limiting data shows that WP744 can surprisingly and unexpectedly enhance the effects alkylating agents such as temozolomide.

D. Treatment Protocol

A therapeutic protocol using alkylating agents and substituted anthracycline compounds in the treatment of cancer is also contemplated. For example, the alkylating agents can be administered prior to, at the same time, or after the substituted anthracycline compounds are administered to the subject within a single cycle. However, it is contemplated that other orders will provide similar results. For example, alkylating agent is "A" and substituted anthracycline compound is "B", the following orders can be used:

AJBIA B/A/B AJAIB B/A/A AJBIAJB

BIAJBIA A/A/AB B/A/A/A A/B/A/A A/A/B/A In addition, it is contemplated that each alkylating agent/substituted anthracycline compound can be repeated one, two, three, four, five six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more times. In certain cases, the dosages of the alkylating agent and/or substituted anthracycline compound can be adjusted within each cycle or for each new cycle. The following is one example of a particular treatment protocol. A cycle constitutes 3 to 4 weeks. Temozolomide is administered orally on days 1 through 5 (150 mglrn^ per day).

WP 744 is administered i.v. (10 mg/m2 per day) on days 1 through 3. The cycle ends on day 21 to 28, and is then repeated for a total of 6 to 8 cycles, with breaks for late nadir times and failure of blood counts to return to an acceptable level within the allotted 14 day period after completion of a treatment cycle.

In certain non-limiting aspects, a subject can be evaluated by neurological examination during the study for neurological changes considered to be independent of tumor and graded using NCI Common Toxicity Criteria (neurotoxicity). Aside from baseline audiometric testing, repeat audiometric testing for ototoxicity can be performed at the physician's discretion for patients who had evidence of hearing loss or progression of hearing loss by neurological examination. In addition, blood counts can be performed biweekly, and serum creatinine, alkaline phosphatase, bilirubin and alanine amino-transferase tests can be performed before each cycle.

E. Combination Therapies In order to increase the effectiveness of a treatment with the compounds of the present invention, it may be desirable to combine these compounds other cancer treatments. Non- limiting examples of additional treatments that can be used in the context of the present

invention include the administration of other chemotherapeutic compounds, radiotherapy, immunotherapy, and surgery.

Non-limiting examples of combination chemotherapies include cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorabicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inliibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, Temazolomide (an aqueous foπn of DTIC), or any analog or derivative variant of the foregoing. As for radiotherapy, non-limiting examples include the use of γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays can range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes can also vary, and often times depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionucleotide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

Surgery can include resection in which all or part of cancerous or other relevant tissue is physically removed, excised, and/or destroyed. Tumor resection includes physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery), laparascopic surgery and harmonic scalpel surgery.

F. Modifications and Equivalents

It is also contemplated that modifications can be made to the alkylating agents and substituted anthracycline compounds of the present invention. Non-limiting examples of such modifications include the addition or removal of lower alkanes such as methyl, ethyl, propyl, or substituted lower alkanes such as hydroxymethyl or aminomethyl groups; carboxyl groups and carbonyl groups; hydroxyls; nitro, amino, amide, and azo groups; sulfate, sulfonate, sulfono, sulfhydryl, sulfonyl, sulfoxido, phosphate, phosphono, phosphoryl groups, and lialide substituents. Additional modifications can include an addition or a deletion of one or more atoms of the atomic framework, for example, substitution of an ethyl by a propyl; substitution of a phenyl by a larger or smaller aromatic group. Alternatively, in a cyclic or bicyclic structure, hetero atoms such as N, S, or O can be substituted into the structure instead of a carbon atom.

Known and unknown equivalents to the compounds discussed throughout this specification can be used with the compositions and methods of the present invention. The equivalents can be used as substitutes for the specific compounds, agents, and active components. The equivalents can also be used to add to the methods and compositions of the present invention. A person of ordinary skill in the art would be able to recognize and identify acceptable known and unknown equivalents to the specific compounds, agents, and active ingredients without undue experimentation. G. Compositions and Routes of Administration

One embodiment of this invention includes methods of treating or preventing cancer by administering to a subject a combination of an alkylating agent and a substituted anthracycline compound as described throughout this specification. The following subsections provide non-limiting examples of pharmaceutical compositions and routes of administration.

1. Compositions

Compositions of the present invention can include an alkylating agent or a substituted anthracycline compound, or both. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human. The preparation of a pharmaceutical composition(s) will be known to those of skill in

the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990.

"Therapeutically effective amounts" include amounts effective to produce a beneficial result in the recipient subject. For instance, an effective amount can include an amount sufficient to detectably and/or repeatedly ameliorate, reduce, minimize or limit the extent of the disease or its symptoms. More rigorous definitions may apply, including elimination, eradication or cure of disease. Such amounts may be initially determined by reviewing the published literature, by conducting in vitro tests, or by conducting metabolic studies in healthy experimental animals. Before use in a clinical setting, it may be beneficial to conduct confirmatory studies in an animal model, preferably a widely accepted animal model of the particular disease to be treated. Preferred animal models for use in certain embodiments are rodent models, which are preferred because they are economical to use and, particularly, because the results gained are widely accepted as predictive of clinical value.

Non limiting examples of "pharmaceutically acceptable carrier" include solvents, dispersion media, coatings, surfactants, antioxidants, preservatives {e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combination s thereof, as would be known to one of ordinary skill in the art (Remington's, 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The actual dosage amount of active ingredients of the present invention {e.g., alkylating agents and substituted anthracycline compounds) administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration can determine the concentration of ingredient in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50

micrograni/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 miciOgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

Alternatively, a subject may be given 1 x 10" ⁵ , 10" ⁶ , 10" ⁶ , 10" ⁷ , 10" ⁸ , 10" ⁹ , 10 ^"10 ,

10" H, 10' 12 M of a substance (or any range derivable therein), of an active ingredient, in a volume of 0.1 1, 1.0 1, 10 1, 100 1, 1 ml, 5 ml, 10 ml, 20 ml, 25 ml, 50 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, or more (or any range derivable therein). Active ingredients may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times over a course of 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years on a regular or as needed basis.

The composition can also include various antioxidants to retard oxidation of one or more active ingredients. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The compositions of the present invention can include different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.

The compositions may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium,

calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

In certain embodiments, the compositions are prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. Examples of carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

In certain aspects, an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. Binders can include, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof. Excipients can include, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or com binations thereof. Disintegrating agenta can include, for example, corn starch, potato starch, alginic acid or combinations thereof. Lubricants can include, for example, magnesium stearate. Sweetening agents can include, for example, sucrose, lactose, saccharin or combinations thereof. Flavoring agents can include, for example, peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.

Additional formulations which are suitable for other modes of administration include suppositories. Suppositories include solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.

Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area. 2. Routes of Administration

The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrauterinely, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g.. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (Remington's, 1990).

H. Kits

The inventor also contemplates the use of a kits in certain aspects of the present invention. For example, any of the compositions, compounds, agents, or ingredients

described in this specification may be included in a kit. In a non-limiting example, a kit can include an alkylating agent or a substituted anthracycline compound, or both. The alkylating agent and/or the substituted anthracycliiie compound can be included into separate compositions or the same composition. Where there is more than one component in the kit (they may be packaged together), the kit also will generally contain a second, third or other additional containers into which the additional components may be separately placed. The kits of the present invention also can include a container housing the components in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the. desired bottles, dispensers, or packages are retained.

A kit can also include instructions for employing the kit components as well the use of any other compositions, compounds, agents, ingredients, or objects not included in the kit. Instructions may include variations that can be implemented. For example, the instructions can include an explanation of how to apply, use, and maintain the products or compositions.

EXAMPLES

The following examples are included to demonstrate certain non-limiting aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 In Vitro Pharmacology Studies

In Vitro Cytotoxicity: WP744 is more potent than doxorubicin in a panel of human tumor cell lines (Table 1). Human tumor cells were plated on Day 0 in 96-well plates with each condition replicated six times. Cells were treated with 10-fold serial dilutions (1 μM to 0.1 nM) of WP744 or doxorubicin on Days 1 and 3. Control cells were treated with vehicle (DMSO) alone. The cells were fixed on Day 5 and stained with sulforhodamine B. Fractional survival was determined by dividing the average absorbance (A ₄₉₂ ) value of test wells by control wells. The fractional survival values were plotted against the log [drug concentration] to determine the IC _5O value for each drug and cell line (Table 1). In the broad

panel of solid tumor cell lines tested, WP744 was more potent than doxorubicin at inhibiting cell growth.

Table 1. IC ₅₀ Values for WP744 and Doxorubicin in Various Human Tumor Cell Lines

Cell Line Tumor Type WP744 IC ₅₀ (nM) Doxorubicin IC ₅₀ (nM)

MCF-7 Breast 0.72 4.02

NCI-H522 Lung 3.40 8.32

A549 Lung 1.07 4.68

NCI-H23 Lung 1.73 2.61

SW480 Colon 2.99 8.26

HT-29 Colon 3.66 23.40

AsPC-1 Pancreas 5.62 80.50

BxPC-3 Pancreas 4.05 15.70

Capan-1 Pancreas 5.30 30.75

OVCAR-3 Ovarian 5.31 11.53

Ex Vivo Cytotoxicity in AML Bone Marrow Cells: The cytotoxic potential of WP744 was further examined in bone marrow cells obtained from three patients with AML. After fractionation of adherent cells and depletion of T lymphocytes, remaining cells were cultured for clonogenic assay with WP744 and doxorubicin at concentrations ranging from 0.05 to 0.5 μg/mL. Both WP744 and doxorubicin consistently inhibited proliferation of AML blast colony- forming cells in a dose-dependent manner (FIG. 1). Patient samples (particularly from patients #2 and #3) were substantially more sensitive to RTA 744 than to doxorubicin.

In Vitro Selectivity: WP744 caused DNA fragmentation in CEM leukemia cells at one-tenth the concentration needed by doxorubicin to induce the same degree of damage. In contrast, WP744 requires a 10-fold higher concentration to cause similar damage in normal fibroblasts (Table 2).

Table 2. Apoptotic Fragmentation Induced by WP744 in CEM Leukemia Cells versus Doxorubicin and in Normal WI38 Fibroblasts

WP744 Doxorubicin WP744

% Fragmented DNA % Fragmented DNA % Total DNA

Concentration (μM) CEM Leukemia CEM Leukemia WI38 Fibroblasts

0 0 0 0 0.05 9.5

0.1 31.0 0.2

0.5 50.6 13.9 6.5

1 28.5

2 42.3 41.1 6.5 5 42A ; 55J

Note: Values are fragmented DNA expressed as percentage of total DNA, as determined by quantitative apoptotic fragmentation assay of CEM leukemic cells incubated with drugs for 24 hrs. Data Source: Faderl S, Estrov Z, Kantarjian HM, et al: WPl 44, a novel anthracycline with enhanced proapoptotic and antileukemic activity. Anticancer Res 21:3777-84, 2001

EXAMPLE 2 In Vivo Pharmacology Studies of WP744 Alone and In Combination With

Temozolomide

Mouse Glioma Orthotopic Model: Two studies of WP744 as a single agent and one study of WP744 in combination with temozolomide were conducted in an orthotopic mouse model of glioblastoma multiforme. In this model, nude mice were seeded with U87GBM cells by direct intracerebral injection. This places the tumors in the natural setting and requires compounds to cross the blood-brain barrier in order to be effective. Tumors are allowed to develop over 5 days prior to treatment.

Treatment with WP744 extended survival by 33% compared to vehicle controls (FIG. 2). Similar results were seen in a second study testing different dosing regimens in this same model. WP744 was administered using 7 mg/kg 5 days on 4 days off, 17.5 mg/kg twice weekly. Compared to controls, survival was extended significantly by both regimens.

A third study demonstrated that the combination of WP744 with temozolomide produced better survival than vehicle or temozolomide alone (FIG. 3). Median survival increased 60% over controls for the combination group, compared with 33% for animals receiving temozolomide alone. Additionally, one animal in the combination therapy group survived to day 78, whereas control and temozolomide-treated animals were all dead by days 32 and 45, respectively.

Mouse U87 Flank Tumor Model: The in vivo activity of WP744 in brain tumors was further characterized using a flank tumor model of glioma. Mice (Nu-Nu) were implanted

with U87 tumor xenografts by subcutaneous injections into the flank and then treated with WP744 or temozolomide intraperitoneally as indicated in Table 3. WP744 demonstrated a consistent, dose-dependent effect, inhibiting tumor growth by as much as 65%.

Table 3. TGI for WP744 or Temozolomide Treatment - U87 Flank Tumor

Total

Group Treatment (Dose) Dosing Days Dose Max Weight Change TGI

(mg/kg)

1 Vehicle Control 1-5 3.9% 0.00%

2 WP744 (7 mg/kg) 1-5 and 10-14 70 -20.8% 65.69%

3 WP744 (15 mg/kg) 1 , 5, 9 and 13 60 -14.0% 65.11 %

4 WP744 (17.5 mg/kg) 1 , 6 and 11 52.5 -15.4% 58.52%

5 Temozolomide (137 mg/kg) 1-5 685 -15.3% 81.75%

Note: 8 animals were treated per group. Treatment was started when the average tumor size was ~ 72 mg.

MDA-MB-231 Breast Cancer Model: To further characterize the in vivo activity of WP744 and compare it to the standard agent doxorubicin, WP744 was evaluated in the MDA- MB-231 human breast cancer xenograft mouse model. WP744 and doxorubicin were administered i.p. to 7 animals per group on the schedules listed in Table 4. Treatment was started when the average tumor size was approximately 40 mg. WP744 exhibited a dose- dependent anti-tumor effect in this relatively treatment-resistant model (Table 4). The doxorubicin group performed slightly better than the WP 744 15 mg/kg group; however, this difference was not statistically significant and there was one animal death in this group compared with none in the WP744-treated groups.

Table 4. Tumor Weights after Treatment with WP744 or Doxorubicin in the MDA-MB-231 Breast Cancer Model

EXAMPLE 3 Preclinical Pharmacokinetics

Preclinical pharmacokinetic studies were performed in CD-I mice. Blood samples (0.5 ml) were collected in heparinized tubes from groups of five mice at selected time points

up to 72 hours following drug administration. Based on these results, the intravenous pharmacokinetics of WP744 in the mouse are best described using a two compartment model (FIG. 4). Summary data are presented in Table 5.

Table 5. Summary of Intravenous Pharmacokinetics in the Mouse

WP744 ^λ Doxorubicin ² ' ³

Dose (mg/kg) 20 10

Cmax (ng/mL) 907.4 + 173.9 950

AUC ₀ -O ₀ (ng*hr/mL) 2741 ± 237

V _d (L/kg) 22.0 ± 4.2 15.2 (10)

Ti/2α (hr) 1.08 ± 0.25 0.067 (67)

Ti/ ₂ β (hr) 12.6 ± 1.7 7.3 (24.6)

CL (L/hr/kg) 7.30 ± 0.63 4.08 (11.0)

Values are expressed as the mean ± SD

^{2 2} VVaalluueess aarree eexxpprreesssseedd aass tthhee mmeeaann aanndd & coefficient of variation (CV)

³ Data taken from Gustafson et. al. 2002

EXAMPLE 4 Preclinical Distribution and Metabolism

In Vivo Biodistribution: The tissue distribution of WP744 after intravenous administration was studied in CD-I mice. WP744 crossed the blood-brain barrier and reached high concentrations in brain tissue at 1 hour following a single i.v. bolus injection (FIG. 5). Mean maximum brain concentration at 1 hour was 244.3 ng/g compared to a plasma concentration of 374.1 ng/mL. The distribution of WP744 into brain tissue after intravenous administration was also studied in a murine glioma model (FIG. 6). WP744 sequestered preferentially in the tumor tissue versus brain tissue. At 1 hour following administration of WP744, the concentration of WP744 was approximately 10-fold higher in the tumor tissue than contralateral brain tissue.

In Vitro Protein Binding: Ultrafiltration methods were employed to assess the relative in vitro plasma protein binding affinity of WP744. Protein binding varied between species with the relative order of binding: human > rat > dog > mouse (FIG. 7).

Metabolic Profile of WP744: In vitro drug metabolism studies of WP744 were conducted in the murine S9 liver fraction model. After the activity of the test system was confirmed, WP744 was added to the samples and reactions were stopped with acetonitrile at 4, 7, and 24 hour time points. Extracted samples were subjected to HPLC analysis and UV detection using a photodiode array detector. Spectra extracted from these analyses

demonstrated the time dependent formation of a more hydrophilic metabolite, which elutes from this system 3 minutes prior to the elution of the precursor parent compound WP744.

To identify and further characterize the WP744 metabolite, quadrupolar time-of-flight mass spectrometry was employed. Results from this analysis illustrate a +2 reduction of WP744 indicating that the principal metabolite, as with doxorubicin, is most probably a two- electron reduction of the side chain carbonyl group to a secondary alcohol, yielding a 4'-O- benzyl doxorubicinol.

EXAMPLE 5 Clinical Pharmacokinetics

The pharmacokinetic profile of WP744 injection in humans is being ascertained in a current Phase 1 dose escalation clinical trial. In this trial, WP744 injection is administered to patients as a daily 2-hour intravenous infusion for three consecutive days. Blood samples are taken at pre-specified time points on days 1 through 5 and analyzed for RTA 744 concentration. Data are available for four patients treated with WP744. Based on these four patients, the half-life of WP744 is greater than 24 hours (Table 6).

Table 6. Pharmacokinetics of WP744 Injection in Humans

Parameter Patient 101 Patient 102 Patient 103 Patient 104

Dose Level 1.2 mg/m ² /day x 2.4 mg/m ² /day x 2.4 mg/m ² /day x 2.4 mg/m ² /day x

3 days 3 days 3 days 3 days

C _max (ng/ml) 1.50, 1.39, 1.70 3.42, 3.60, 4.24 2.66, 3.41 , 6.15 5.75, 4.03, 8.69

Clearance 39 106 37.3 27.6

(L/hr/m ² )

Ti _/2 β (hrs) 33.4 21.7 46.7 38.2

Vss (Um ² ) 1550 2817 2287 1366

EXAMPLE 6

Preclinical Toxicology Studies

WP744 has been studied in a number of preclinical toxicology studies, including IND- directed GLP Toxicology studies in rats and dogs. Based on these studies, target organs of toxicity appear to be the bone marrow, lymphoid organs, digestive tract, and heart. Although WP744 has been shown to cross the blood-brain barrier, no signs of neurotoxicity have been observed during preclinical studies in any species, and histopathology revealed no primary drug-related effects on the central nervous system. The overall pattern of toxicity of WP744 appears consistent with that of other anthracyclines, including doxorubicin and epirubicin.

Single Dose Toxicity Studies: Acute toxicity studies of a single intravenous dose of WP744 were conducted in CD-I mice and Fisher 344 and Sprague-Dawley rats. Findings from these studies are summarized in Table 7.

Table 7

Multiple Dose Toxicity Studies: WP744 has been studied in multiple dose toxicity studies in CD-I and nu/nu mice, Sprague-Dawley rats, and Beagle dogs. 28-day GLP Toxicology studies of WP744 administered intravenously for three consecutive days were completed in Sprague-Dawley rats and beagle dogs. Findings from these studies are summarized in Table 8.

Table 8

EXAMPLE 7 Myocardial Toxicity Study

Due to the relationship between members of the anthracycline class and cardiac toxicity, a myocardial toxicity study of WP744 with doxorubicin serving as a positive control was perfomed. Fifteen mice (CDl, female) per group were treated with vehicle, doxorubicin (1, 2, or 4 mg/kg), or WP744 (1, 2, or 4 mg/kg). All treatments were administered as 2 doses per week for 2 weeks, followed by a 2 week observation period, then 2 doses additional per week for 3 weeks, followed by a 4 week observation period prior to sacrifice and necropsy.

Cardiotoxic effects of treatment were evaluated for severity and extent according to criteria established by Bertazzoli for quantifying the cardiotoxicity of doxorubicin (Bertazzoli et al, Cancer Treat Rep. 1979 Nov-Dec;63(ll-12):1877-83). In this system, the Bertazzoli score is the product of the severity and extent scores (Table 9), allowing comparison across groups.

Table 9 Calculation of Bertazzoli Score

Severityjrf Toxicity X Extent of Toxicity

1 Sarcoplasmic microvacuolization and/or 0 No lesions inclusions and interstitial cellular o.ε > < 10 single altered myocytes in the whole edema. heart section

2 Same as 1 + sarcoplasmic 1 Scattered single altered myocytes macrovacuolizations or atrophia, 2 Scattered small groups of altered myocytes necrosis, fibrosis, endocardial lesions 3 Widely spread small groups of altered and thrombi. myocytes

4 Confluent groups of altered myocytes

5 Most cells damaged

In this study, no animals died or had significant gross lesions upon necropsy. Cardiotoxicity findings are summarized in Table 10. From these results, it is inferred that cumulative doses of WP744 produced less cardiotoxicity than equivalent doses of doxorubicin.

EXAMPLE 8

Comparitive Toxicology of Doxorubicin, Epirubicin, and WP744

Based upon data from preclinical studies conducted with WP744, this compound has a similar spectrum of toxicity compared to other members _^ of the anthracycline class, most notably doxorubicin and epirubicin. A comparison of toxicology data for RTA 744 and Summary Basis of Approval documentation for Adriamycin® (doxorubicin) and Ellence®

(epirubicin) indicates that WP744 has a preclinical toxicology profile that is quite similar to these other agents, with the exception being increased tolerability in rodents.

The single dose LD ₅₀ for WP744 is higher than that for doxorubicin and epirubicin in both mice and rats (56 vs. 15.9 and 23 mg/kg in mice and 15-20 vs. 8.7 and 14-15 in rats). In dogs, the LD ₅₀ is closer among the agents, with WP744 having the lowest value (1.5-2.25 vs.

2.6 and 2). AU data expressed in mg/kg. Schedule for all studies was a single bolus injection except for the WP744 dog study, in which drug was administered on a daily x 3 schedule.

EXAMPLE 9 Phase 1 Dose-Escalation Study A Phase 1 dose-escalation study (Protocol RTA744-C-0401) is currently underway to evaluate the tolerability and antitumor activity of WP744 Injection given intravenously for three consecutive days of a three-week cycle in adult patients with recurrent or refractory primary brain tumors (glioblastoma multiforme, anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic mixed oligo-astrocytoma, or gliosarcoma). This study is designed to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities of WP744 when administered in this patient population. Additionally, the study is assessing the safety, tolerability, and pharmacokinetic profile of WP744. MRI imaging at baseline and after even-numbered cycles is also being conducted to provide a preliminary indicator of activity. Four patients have been treated in this trial at two dose levels, for a total of 11 cycles.

Patients have received up to four cycles of therapy.

EXAMPLE 10

Human Treatment with C-3' or C-4' Substituted Anthracycline Compounds in Combination With Alkylating Agents This example describes a protocol to facilitate the treatment of cancer using C-3' or C-

4' substituted anthracycline compounds (e.g. WP744) in combination with alkylating agents (e.g., temozolomide). For illustrative purposes only, WP744 will be used as the C-4' substituted anthracycline compound and temozolomide as the alkylating agent. However, it is contemplated that all substituted anthracycline compounds and alkylating agents described in this specification can be used.

A cancer patient presenting, for example, an MDR cancer is treated using the following protocol. Patients may, but need not, have received previous chemo- radio- or gene therapeutic treatments. Optimally, the patient exhibits adequate bone marrow function (e.g., peripheral absolute granulocyte count of > 2,000/mm3 and platelet count of 100, 000/mm3,

adequate liver function (bilirubin 1.5mg/dl) and adequate renal function (creatinine 1.5mg/dl)).

Exemplary Protocol for the Treatment of Multi-Drug Resistant Cancer: Composition(s) of the present invention can be administered orally or parenterally in dosage unit foπnulations containing standard, well known, non-toxic physiologically acceptable carriers, adjuvants, and vehicles as desired. The term parenteral encompasses subcutaneous injections, intravenous, intramuscular, intra-arterial injection, or infusion techniques. WP744 and temozolomide can be delivered to the patient before, after, or concurrently with the other anti-cancer agents or other cancer therapies. A non-limiting treatment course can include about six doses of each of WP744 and temozolomide delivered over a 7- to 21 -day period. Upon election by the clinician, the regimen may be continued six doses every three weeks or on a less frequent (monthly, bimonthly, quarterly etc.) basis. Of course, these are only exemplary times for treatment, and the skilled practitioner will readily recognize that many other time-courses are possible. A major challenge in clinical oncology is that many cancers are multi-drug resistant.

One goal has been to find ways to improve the efficacy of chemotherapy, hi the context of the present invention, the combination of WP744 and temozolomide have a surprising cytotoxicity against cancers. To kill MDR cancer cells using the methods and compositions described in the present invention, one will generally contact a target cell with WP744 and temozolomide in amounts effective to kill or inhibit the proliferation of the cell.

In certain embodiments, it is contemplated that one would contact the cell with agent(s) of the present invention about every 6 hours to about every one week. In some situations, however, it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, 7 or more) to several weeks (1, 2, 3, 4, 5, 6, 7, or more) lapse between respective administrations. Regional delivery of WP744 and temozolomide is an efficient method for delivering a therapeutically effective dose to counteract the clinical disease. Likewise, the chemotherapy may be directed to a particular affected region. Alternatively, systemic delivery of active agents may be appropriate.

The therapeutic composition of the present invention can be administered to the patient directly at the site of the tumor. This is in essence a topical treatment of the surface of the cancer. The volume of the composition should usually be sufficient to ensure that the tumor is contacted by WP744 and temozolomide. hi one embodiment, administration simply entails injection of therapeutic composition(s) into the tumor, hi another embodiment, a

catheter is inserted into the site of the tumor, and the cavity may be continuously perfused for a desired period of time.

Clinical responses may be defined by acceptable measure. For example, a complete response may be defined by the disappearance of all measurable disease for at least a month, whereas a partial response may be defined by a 50% or greater reduction of the sum of the products of perpendicular diameters of all evaluable tumor nodules or at least one month with no tumor sites showing enlargement. Similarly, a mixed response may be defined by a reduction of the product of perpendicular diameters of all measurable lesions by 50% or greater, with progression in one or more sites. Of course, the above-described treatment regimes may be altered in accordance with the knowledge gained from clinical trials such as those described above. Those of skill in the art are able to take the information disclosed in this specification and optimize treatment regimes based on the clinical trials described in the specification.

EXAMPLE 11 Human Treatment with C-3' or C-4' Substituted Anthracycline Compounds in

Combination With Alkylating Agents

This example explains the development of human treatment protocols using C-3' or C- 4' substituted anthracycline compounds (e.g. WP744) in combination with alkylating agents (e.g., temozolomide). For illustrative purposes only, WP744 will be used as the C-4' substituted anthracycline compound and temozolomide as the alkylating agent. However, it is contemplated that all substituted anthracycline compounds and alkylating agents described in this specification can be used.

These compounds are of use in the clinical treatment of various MDR cancers in which transformed or cancerous cells play a role. Such treatment is a particularly useful tool in anti-tumor therapy, for example, in treating patients with brain, ovarian, breast and lung cancers that are resistant to conventional chemotherapeutic regimens. The various elements of conducting a clinical trial, including patient treatment and monitoring, is known to those of skill in the art in light of the present disclosure. The following information is being presented as a general guideline for use in establishing substituted anthracyclines drugs made by the use of this invention, in clinical trials.

Patients with human brain cancer, metastatic breast and/or epithelial ovarian carcinoma, colon cancer leukemia, or sarcoma are chosen for clinical study. Measurable disease is not required, however the patient must have easily accessible pleural effusion and/or ascites. Further the patients must carry tumors that express MDR phenotype. In an exemplary

clinical protocol, patients may undergo placement of a Tenckhoff catheter, or other suitable device, in the pleural or peritoneal cavity and undergo serial sampling of pleural/peritoneal effusion. Typically, one will wish to determine the absence of known loculation of the pleural or peritoneal cavity, creatinine levels that are below 2 mg/dl, and bilirubin levels that are below 2 mg/dl. The patient should exhibit a normal coagulation profile.

In regard to WP744 and temozolomide administration, a Tencldioff catheter, or alternative device, may be placed in the pleural cavity or in the peritoneal cavity, unless such a device is already in place from prior surgery. A sample of pleural or peritoneal fluid can be obtained, so that baseline cellularity, cytology, LDH, and appropriate markers in the fluid (CEA, CAl 5-3, CA 125, pi 85) and in the cells (ElA, pi 85) may be assessed and recorded.

In the same procedure, WP744 and temozolomide can be administered. The administration may be in the pleural/peritoneal cavity, directly into the tumor, or in a systemic manner. The starting dose may be 0.5mg/kg body weight. Three patients may be treated at each dose level in the absence of grade > 3 toxicity. Dose escalation may be done by 100% increments (0.5mg, lmg, 2mg, 4mg) until drug related Grade II toxicity is detected. Thereafter, dose escalation may proceed by 25% increments. The administered dose may be fractionated equally into two infusions, separated by 6 hours if the combined endotoxin levels determined for the lot of bisanthracycline exceed 5EU/kg for any given patient.

WP744 and temozolomide can be administered over a short infusion time or at a steady rate of infusion over a 7- to 21 -day period. The infusion given at any dose level is dependent upon the toxicity achieved after each. Hence, if Grade II toxicity was reached after any single infusion, or at a particular period of time for a steady rate infusion, further doses should be withheld or the steady rate infusion stopped unless toxicity improves. Increasing doses of WP744 and/or temozolomide in combination with other an anti-cancer drugs or therapies can be administered to groups of patients until approximately 60% of patients show unacceptable Grade III or IV toxicity in any category. Doses that are 2/3 of this value could be defined as the safe dose.

Physical examination, tumor measurements, and laboratory tests can be performed before treatment and at intervals of about 3-4 weeks later. Laboratory studies can include CBC, differential and platelet count, urinalysis, SMA- 12- 100 (liver and renal function tests), coagulation profile, and any other appropriate chemistry studies to determine the extent of disease, or determine the cause of existing symptoms. Also appropriate biological markers in serum can be monitored, (e.g., CEA, CA 15-3, pi 85 for breast cancer, and CA 125, pi 85 for ovarian cancer).

To monitor disease course and evaluate the anti-tumor responses, it is contemplated that the patients can be examined for appropriate tumor markers every 4 weeks, if initially abnormal, with twice weeldy CBC, differential and platelet count for the 4 weeks. Then, if no myelosuppression has been observed, weekly. If any patient has prolonged myelosuppression, a bone marrow examination is advised to rule out the possibility of tumor invasion of the marrow as the cause of pancytopenia. Coagulation profile shall be obtained every 4 weeks. An SMA- 12- 100 shall be performed weeldy. Pleural/peritoneal effusion may be sampled 72 hours after the first dose, weekly therXX Xeafter for the first two courses, then every 4 weeks until progression or off study. Cellularity, cytology, LDH, and appropriate markers in the fluid (CEA, CA15-3, CA 125, pl85) and in the cells (pl85) may be assessed. An example of an evaluation profile is shown in Table 11. When measurable disease is present, tumor measurements are to be recorded every 4 weeks. Appropriate radiological studies should be repeated every 8 weeks to evaluate tumor response. Spirometry and DLCO may be repeated 4 and 8 weeks after initiation of therapy and at the time study participation ends. A urinalysis may be performed every 4 weeks.

Table 11. Evaluations Before and During Therapy

EVALUATIONS PRE- TWICE WEEKLY EVERY 4 EVERY 8 STUDY WEEKLY WEEKS WEEKS

History X X

Physical X. X

Tumor Measurements X X

CBC X X ¹ X

Differential X X ¹ X

Platelet Count X X ¹ X

SMAl 2-100 (SGPT, X X Alkaline Phosphatase, Bilirubin, Alb/Total Protein) Coagulation Profile X X

Serum Tumor markers X X ³ (CEA, CAl 5-3, CA-125, Her-2/neu) Urinalysis X X

X-rays: chest X ⁴ others X Pleural/Peritoneal Fluids: X ⁵ X (cellularity, cytology, LDH,

EVALUATIONS PRE- TWICE WEEKLY EVERY 4 EVERY 8 STUDY WEEKLY WEEKS WEEKS tumor markers, ElA, HER- 2/neu) Spirometry and DLCO X X ⁶ X ⁶

For the first 4 weeks, then weekly, if no myelosuppression is observed.

² As indicated by the patient's condition.

³ Repeated every 4 weeks if initially abnormal.

For patients with pleural effusion, chest X-rays may be performed at 72 hours after first dose, then prior to each treatment administration.

⁵ Fluids may be assessed 72 hours after the first dose, weekly for the first two courses and then every 4 weeks thereafter.

⁶ Four and eight weeks after initiation of therapy.

All of the compositions and/or methods disclosed and claimed in this specification can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

U.S. Patent No. 6,673,907

Bertazzoli et al, Cancer Treat Rep. 1979 Nov-Dec; 63(11-12):1877-83.

Danesi R, Fogli S, German A, et al. Phaπnacokinetic-pharmacodynamic relationships of the anthracycline anticancer drugs. Clin. Pharmacokinet. 41:431-44, 2002.

Faderl S, Estrov Z, Kantarjian HM, et al. WP744, a novel anthracycline with enhanced proapoptotic and antileukemic activity. Anticancer Res. 21:3777-84, 2001.

Gustafson DL, Rastatter JC, Colombo T, et al. Doxorubicin pharmacokinetics: macromolecule binding, metabolism, and excretion in the context of a physiologic model. J Pharm. Sci. 91:1488-501, 2002.