WO2009027838A2 - Kinamycin f for cancer treatment - Google Patents

Abstract

Disclosed are methods relating to the use of kinamycin F as an anti-cancer agent. Cytotoxic methods induced by kinamycin F include DNA and protein damage and topoisomerase inhibition. As set forth herein, the anti-cancer effects of kinamycin F may be attributed to, for example, DNA binding, DNA intercalation, topoisomerase Ila decatenation activity inhibition and/or production of radical species by kinamycin F.

Description

DESCRIPTION KINAMYCIN F FOR CANCER TREATMENT

BACKGROUND OF THE INVENTION

This application claims priority to U.S. Provisional Patent Application Serial No. 60/911,764 filed April 13, 2007, the entire contents of which are expressly incorporated herein by reference.

1. Field of the Invention

The present invention relates generally to the field of cancer treatment. More particularly, it concerns the cytotoxic effects of kinamycin F exerted via, for example, DNA intercalation, topoisomerase Ilα inhibition, and/or reductive or peroxidative activation to produce DNA- and/or protein-damaging species.

2. Description of Related Art

The bacterial metabolites kinamycins A, B, C and D were isolated in 1970 from Streptomyces murayamaensis and were shown to exhibit good activity against Gram positive, but not Gram negative bacteria (Omura et al, 1971). Kinamycins have been classified as type II polyketides and are used to treat infections (Hata et al , 1971; Omura et al, 1971). The genes for most of the biosynthesis of kinamycin from S. murayamaensis have been cloned and heterologously expressed. See, e.g., Gould, et al., 1998. Kinamycins A and C are highly cytotoxic to cancer cells (Hasinoff et al., 2006).

The kinamycins contain a diazo group, which is unusual for a natural product. The kinamycins were initially assigned an N-cyanocarbazole structure. However, two separate research groups independently determined that the structures assigned to the kinamycins were incorrect and that the kinamycins are in fact derivatives of diazobenzo[b]fluorene, rather than of N-cyanobenzo[b]carbazole (Mithani et al., 1994; Gould et al., 1994). The deacetylated form of the kinamycins, kinamycin F (FIG. 1), though it was first prepared by chemical deacetylation of kinamycin C (Omura et al, 1973), has been detected more recently as a metabolite in culture broths of S. murayamaensis (Seaton and Gould, 1989). Due to their unique structures, members of the kinamycin family may offer chemotherapeutic benefits that supersede effects presently found with other chemotherapeutics in the art. SUMMARY OF THE INVENTION

The present invention provides for the use of kinamycin F as an anti-cancer agent. In studying the means by which kinamycin F exerts its cytotoxic effects, the present inventors have found that kinamycin F is indeed a good candidate for an anti- cancer therapeutic. Kinamycin F's cytotoxic mechanisms likely involve inhibition of topoisomerase Ilα, DNA binding and intercalation, and/or DNA- and protein-induced damage via the production of radical species. Indeed, kinamycin F was found to be an unexpectedly stronger topoisomerase Ilα inhibitor than either kinamycin A or kinamycin C. In certain embodiments, the radical-mediated DNA- and protein- damaging effects of kinamycin F render this agent particularly useful in treating tumors that are intolerant of oxidative stress.

Accordingly, one aspect of the present invention contemplates a method of damaging DNA, damaging a protein, and/or inhibiting topoisomerase Ilα comprising administering kinamycin F to a cell. Damage to DNA and damage to a protein may be measured using conventional methods known in the art, some of which are described below. For example, measurements of nicking of DNA may be used to measure DNA damage. To assess whether a protein has been damaged, one may assay that protein before and after subjecting it to conditions thought to induce damage. Differences in activity of the protein may indicate whether the protein has been damaged or not. Another method is described in U.S. Patent No. 5,273,886, involving monitoring of isoaspartate, a major product of protein degradation.

The administration may take place via any method known to those of skill in the art. In certain embodiments, the administration may be in vivo. The in vivo administration may be to a mammal, such as a human. The in vivo administration may be to a human who has cancer. The cancer may be any type of cancer known to those of skill in the art. Non-limiting examples of cancers include leukemia, Ehrlich ascites carcinoma, breast, cervical, ovarian, prostate, brain, lung, colon, pancreatic, multiple myeloma, lymphoma, bone, and head and neck cancer. In certain embodiments, the method of damaging DNA, damaging a protein, and/or inhibiting topoisomerase Ilα comprising administering kinamycin F to a cell may be further defined as a method of killing or inhibiting a cancer cell in the human. In certain embodiments, the method of damaging DNA, damaging a protein, and/or inhibiting topoisomerase Ilα comprising administering kinamycin F to a cell may take place in vitro. In certain embodiments, the method of damaging a DNA, damaging a protein, and/or inhibiting topoisomerase Ilα comprising administering kinamycin F to a cell may comprise production of a radical of kinamycin F. The radical may be a semiquinone radical or a phenoxyl radical. Methods described herein may further comprise reactive oxygen species (ROS) production.

In certain embodiments, the present invention contemplates a method of damaging a DNA and/or a protein comprising administering kinamycin F to a cell. The damage may, in certain embodiments, be iron-dependent, hydrogen peroxide- dependent, and/or hydroxyl radical-dependent. In any method of the present invention, kinamycin F may bind to DNA. The binding to DNA may be by the intercalation of kinamycin F into DNA. DNA damage may, in certain embodiments, be caused by the nicking of DNA by kinamycin. Measurements of DNA nicking are known to those of skill in the art, and are described herein. In particular embodiments, the present invention contemplates a method of damaging a protein comprising administering kinamycin F to a cell. The protein may be any protein known to those of skill in the art. In certain embodiments, the protein comprises one or more sulfur atoms, such as sulfhydryl groups (-SH). In certain embodiments, the protein is a topoisomerase, such as topoisomerase Ilα. In certain embodiments, the present invention contemplates a method of inhibiting topoisomerase Ilα. Topoisomerase Ilα may be inhibited about or at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or more, or any range derivable therein. In particular embodiments, methods of inhibiting the decatenation activity of topoisomerase Ilα comprising administering an effective amount of kinamycin F to a cell (in vitro) or subject (in vivo) are contemplated.

In other general aspects, the present invention contemplates a method of killing or inhibiting the growth of a cancer cell comprising administering kinamycin F to the cell. The cancer cell may be that of any type known to those of skill in the art. The cancer cell may be in vivo or in vitro. The method may comprise production of a radical of kinamycin F, such as a semiquinone radical or a phenoxyl radical. The radical of kinamycin F may damage the DNA and/or a protein of the cancer cell. The damage may be iron-dependent, hydrogen peroxide-dependent, or hydroxyl radical- dependent. In certain embodiments of this method, kinamycin F may bind to DNA. Such binding may be via intercalation. In certain embodiments, DNA damage may occur via the nicking of DNA by kinamycin F. In certain embodiments comprising a method of killing or inhibiting the growth of a cancer cell comprising administering kinamycin F to the cell, kinamycin F may inhibit the decatenation activity of topoisomerase Ilα in the cancer cell. In this or any other method of the present invention, the cancer cell may be comprised in a tumor. The cancer cell may be intolerant of oxidative stress.

Other general aspects of the present invention contemplate a method of treating a tumor in a subject, comprising administering a therapeutically effective amount of kinamycin F to the subject, such as a mammal (e.g., a human, rodent, or pig). In this or any other method of the present invention, the cancer cell or the tumor may be intolerant of oxidative stress.

Certain embodiments of the present invention contemplate a method of treating cancer in a subject, comprising administering a therapeutically effective amount of kinamycin F to a subject. The subject may be a mammal, such as a human.

In this or any method of the present invention, kinamycin F is comprised in a pharmaceutically acceptable composition, such as with a pharmaceutically acceptable carrier. Such compositions are described herein. In certain embodiments, a second treatment may be administered to the subject. The second treatment may be, for example, chemotherapy, radiation or gene therapy. Kits comprising kinamycin F are also contemplated by the present invention.

An "anti-cancer" agent is capable of negatively affecting cancer in a subject, for example, by killing one or more cancer cells, inducing apoptosis in one or more cancer cells, reducing the growth rate of one or more cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or one or more cancer cells, promoting an immune response against one or more cancer cells or a tumor, preventing or inhibiting the progression of a cancer, or increasing the lifespan of a subject with a cancer. Anti-cancer agents are well-known in the art and include, for example, chemotherapy agents (chemotherapy), such as DNA intercalators, radiotherapy agents (radiotherapy), a surgical procedure, immune therapy agents (immunotherapy), genetic therapy agents (gene therapy), reo viral therapy, hormonal therapy, other biological agents (biotherapy), and/or alternative therapies.

To kill a cell in accordance with the present invention, one would generally contact the cell with kinamycin F in amount effective to kill the cell. The term "in an amount effective to kill the cell" means that the amount of kinamycin F is sufficient so that, when administered to a cell, cell death is induced. A number of in vitro parameters may be used to determine the effect produced by the compositions and methods of the present invention. These parameters include, for example, the observation of net cell numbers before and after exposure to the compositions described herein.

The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which kinamycin F is administered or delivered to a target cell or are placed in direct juxtaposition with the target cell. The terms

"administered" and "delivered" are used interchangeably with "contacted" and "exposed."

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

"Treatment" and "treating" as used herein refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a subject {e.g., a mammal, such as a human) having cancer may be subjected to a treatment comprising administration of kinamycin F. The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well- being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, a therapeutically effective amount of kinamycin F may be administered to a subject having a cancerous tumor, such that the tumor shrinks. It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.

Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.

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 alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device and/or method being employed to determine the value.

As used herein the specification, "a" or "an" may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

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 specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various 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 FIGURES

The following figures 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. Structure of kinamycin F.

FIG. 2. Kinamycin F reacts with the thiols GSH and DTT. FIG. 2A: The kinamycin spectrum was recorded prior to addition of 5 mM GSH (0 h, dashed line) and the spectral changes occurring in the 20 μM kinamycin F solution are plotted at 1 h (dotted line), 2, 3, 4 h (solid lines) and 5 h (thick line) after the addition of 5 rnM GSH. FIG. 2B: Absorbance-time traces at 330, 420 and 464 nm obtained from the complete spectral data are plotted. FIG. 2C: The kinamycin spectrum was recorded prior to addition of 40 μM DTT (0 h, dashed line) and the spectral changes occurring in the 20 μM kinamycin F solution are plotted 3 (solid line) and 9 (thick line) min after the addition of 40 μM DTT. FIG. 2D: Absorbance-time traces at 339, 456 and 559 nm obtained from the complete spectral data are plotted. The reactions were carried out in 20 mM Tris buffer (pH 7.6) at 37°C.

FIG. 3. Kinamycin F binds to DNA as shown by absorbance changes upon the addition of DNA and by the fluorescence ethidium bromide displacement assay.

FIG. 3 A: Changes in absorbance of kinamycin F (40 μM) at 430 nm were measured upon the addition of small volumes of calf thymus DNA. The assay mixture contained

10 mM Tris (pH 7.5) at 20⁰C. FIG. 3B: Changes in the fluorescence at 590 nm of ethidium bromide bound to calf thymus DNA are plotted as a function of the kinamycin F concentration. The assay mixture contained calf thymus DNA (16 μg/ml) and ethidium bromide (1 μM) at a temperature of 25°C. The solid curved lines are from spline and quadratic fits, respectively, to the data.

FIG. 4. Kinamycin F docks into DNA. The highest scoring structure of kinamycin F in the equatorial conformation (ball-and-stick structure) is shown docked into DNA (stick structure). The H-atoms are not shown for clarity. The complex forms both base stacking and H-bonding interactions (green dotted lines) with hydroxyl groups on C 1 and C2 of the 6-membered D ring. The DNA structure is a doxorubicin/DNA x-ray structure (1DA9 from the Protein Data Bank) in which two doxorubicin molecules are bound to a 6-base pair piece of DNA. Only the first 3 base pairs of the DNA are shown in the figure for clarity. One doxorubicin was removed and kinamycin F was docked into its place with the genetic algorithm docking program GOLD.

FIG. 5. GSH potentiates kinamycin F-induced DNA nicking. FIG. 5A: This fluorescent image of the ethidium bromide-stained gel shows that when GSH (5 mM) was added to reaction mixtures containing various amounts of kinamycin F, the amount of DNA nicking was increased. In the absence of GSH a slight increase in nicking was seen at 400 μM kinamycin F. FIG. 5B: In this image it is shown that deferoxamine (100 μM) and DMSO (0.5% (v/v)) pre-treatment of the DNA reduced the amount of DNA nicking induced by kinamycin F (200 μM) and GSH (5 mM). FIG. 5C: In this image it is shown that catalase (50 μg/ml) decreased the amount of DNA nicking, but boiled catalase (b, 50 μg/ml) did not. Catalase was added immediately prior to the kinamycin F (200 μM) and GSH (5 mM). All lanes contained 80 ng of pBR322 plasmid DNA, except for those with linear DNA which contained 80 ng of linear pBR322 plasmid DNA. Linear DNA was present in lane 7 and lanes 4 and 11 in FIGS. 5A and 5B, respectively. NC, nicked circular DNA; LIN, linear DNA; SC, supercoiled DNA. All incubations for the DNA nicking experiments were for 5 h at 37°C.

FIG. 6. Kinamycin F treatment produces DNA single strand breaks in K562 cells. The amount of DNA damage as a result of exposure to kinamycin F was determined using the Fast Micromethod DNA Single Strand Break assay. Unwinding of DNA in K562 cells was measured from the fluorescence of double-stranded DNA (dsDNA) remaining after exposure to various concentrations of kinamycin F. Error bars were from four replicate measurements. *** designates a significant difference (p < 0.001) compared to the untreated control value.

FIG. 7. Kinamycin F inhibits topoisomerase Ilα catalytic activity but is not a topoisomerase Ilα poison. FIG. 7A: Inhibitory effects of kinamycin F on the catalytic decatenation activity of topoisomerase Ilα. The solid line is a non-linear least squares fit to a 3-parameter logistic equation and yields an IC₅₀ of 0.85 ± 0.26 μM. FIG. 7B: Inhibition of growth of K562 (o) and K/VP.5 (•) cells by kinamycin F. Cells were treated with kinamycin F for 72 h prior to the assessment of growth inhibition by an MTS assay. The curved lines are non-linear least squares fits to logistic equations and yield ICso's of 0.33 ± 0.02 and 0.42 ± 0.10 μM, respectively for K562 (solid line) and K/VP.5 (dashed line) cells.

FIG. 8. Reduction of intracellular GSH levels in K562 cells increases the potency of kinamycin F. K562 cells were preincubated with (o) or without (•) 100 μM BSO for 48 h and then treated for 72 h with kinamycin F. The data shown is representative of 3 sets of paired experiments performed on separate days. The curved lines are non-linear least squares fits to 4-parameter logistic equations and yield IC₅₀'s of 2.4 ± 0.2 and 1.1 ± 0.10 μM, respectively for K562 cells not treated with BSO (solid line), or with BSO (dashed line). A paired t-test of three independent sets of experiments determined that the average decrease in IC₅₀ after BSO treatment was significant (p < 0.007). Error bars are standard errors from three replicates.

FIG. 9. Kinamycin F increases the oxidation of DCFH to DCF in K562 cells through a UOn-H₂O₂ dependent pathway. In the H₂O₂-treated cells, inhibition of intracellular catalase with the catalase inhibitor AT significantly (p < 0.001) increased the rate of DCF production, but did not significantly affect the rate of DCF production by kinamycin F. Kinamycin F treatment in the absence (p < 0.01) or presence (p < 0.05) of AT significantly increased the rate of DCF production compared to untreated control cells as did the H₂O₂ treatment (p < 0.001 for both AT treated and untreated). K562 cells were loaded with DCFH-DA for 20 min with or without prior treatment with AT. After obtaining a fluorescence baseline measurement for 10 min, cells were treated with either 5 μM kinamycin F or 200 μM H₂O₂ and the increase in the rate of DCF fluorescence produced was measured. Error bars were from four replicate measurements. Significant differences between treated and untreated samples are designated by ***, p < 0.001; **, p < 0.01; *, p < 0.05. Significant differences between treated samples and the corresponding treatment with AT are designated by fff, p < 0.001. NS is not significant. The results are averages from 4 wells.

FIG. 10. EPR spectra of free radicals produced by treatment of kinamycin F with either GSH or HRIVH₂O₂. FIG. 1OA: EPR spectrum under oxic conditions of kinamycin F (0.5 mM) treated with GSH (5 mM). The modulation amplitude was 2 G and the sweep width was 50 G. FIG. 1OB: EPR spectrum under oxic conditions of kinamycin F (0.5 mM) treated with H₂O₂ (25 μM) and HRP (100 μg/ml) at pH 6.5.

The modulation amplitude was 0.5 G and the sweep width was 10 G. For both spectra a total of 20 spectra were accumulated over 14 min and averaged. The spectra were recorded at 25°C.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is based on the finding that kinamycin F likely acts as a topoisomerase Ilα inhibitor, a DNA-intercalator, a DNA-damaging agent, and/or a protein-damaging agent. These cytotoxic effects of kinamycin F strongly implicate kinamycin F as an effective anti-cancer agent. A. The Present Invention

Kinamycin F was found to inhibit the growth of K562 cancer cells with a submicromolar IC₅₀ value (0.33 μM). In examining whether reductive and/or oxidative activation mechanisms were at play, it was found that kinamycin F can be reductively activated to a semiquinone free radical and peroxidatively activated to a phenoxyl free radical. More specifically, the experiments carried out herein show that kinamycin F can be reductively activated by cellular levels of GSH to produce reactive radical species and can also be peroxidatively activated by a peroxidase/FhCh system. These radical species likely contribute to cytotoxicity by damaging DNA and/or proteins. In addition, kinamycin F may also damage DNA by binding and nicking DNA in the presence of GSH, as shown herein. Further, the inventors have determined that kinamycin F is also a likely DNA intercalator. DNA intercalators are often used in chemotherapeutic treatment to inhibit DNA replication in cancer cells.

The present inventors also demonstrate that kinamycin F is a topoisomerase Ilα inhibitor, but does not appear to act as a topoisomerase Ilα poison.

Topoisomerase Ilα may be one of the proteins damaged by radical species produced by kinamycin F. Topoisomerase II is an isomerase enzyme that acts on the topology of DNA. The double-helical configuration of DNA strands makes them difficult to separate, and yet they must be separated by helicase proteins if other enzymes are to transcribe the sequences that encode proteins, or if chromosomes are to be replicated.

Topoisomerase Ilα cuts both strands, and passes an unbroken double strand through it then reanneals the cut strand — such activity is referred to as decatenation.

Mammalian topoisomerase II has been further classified as type Ilα and type Ilβ.

Drugs acting on topoisomerase II are divided into two main categories, topoisomerase II poisons and topoisomerase II catalytic inhibitors. The topoisomerase II poisons are associated with their ability to stabilize the enzyme -

DNA cleavable complex and shift the equilibrium of the catalytic cycle towards cleavage, thereby increasing the concentration of the transient protein-associated breaks in the genome (Froelich-Ammon and Osheroff, 1995). Topoisomerase II catalytic inhibitors interfere with the overall catalytic function of the enzyme. As the present inventors show, kinamycin F may inhibit the decatenation activity topoisomerase Ilα by reacting with critical sulfhydryl groups on the protein. It is therefore further postulated that kinamycin F does not exert it cytotoxicity primarily through the inhibition of topoisomerase II, but other interactions are also likely involved. Moreover, other proteins containing sulfur atoms, such as sulfyhydryl groups, may be susceptible to damage by kinamycin F.

Regarding the role of the diazo moiety of kinamycin F, the results herein indicate that it is possible that this normally relatively stable group becomes activated upon reduction to the semiquinone or the hydroquinone under the reducing conditions of the cell. It may be that reduction of the quinone makes the diazo group much more electrophilic and able to target DNA or critical proteins causing inhibition of cell growth. The fact that kinamycin F binds to DNA and was able to damage DNA in cells would also implicate DNA as the biological target of kinamycin F. It is, however, not possible to rule out that kinamycin F has some other target or targets that contain critical protein sulfhydryl groups (or some other nucleophilic protein group). As such, in certain embodiments, the present invention contemplates administering kinamycin F as a protein-damaging agent for cancer treatment. In view of the results presented herein, the present application contemplates, in certain embodiments, administration of kinamycin F to cancer cells (such as cancer cells comprised in a tumor) that are less able to withstand oxidative insult.

Cancer cells that are intolerant of oxidative insult (e.g., oxidative stress) may, in certain embodiments, be low in antioxidants or antioxidant enzymes. The phrase "low in antioxidants or antioxidant enzymes" refers to any amount of any antioxidant or any antioxidant enzyme in a cell that is lower than an expected amount of antioxidant or enzyme in a cell of the same type under normal conditions, as would be understood by one of ordinary skill in the art. Such antioxidants and antioxidant enzymes are known to those of skill in the art. Measurements of cellular amounts of antioxidants and antioxidant enzymes are also well-known in the art. Non-limiting examples of antioxidants include glutathione, vitamin E and vitamin C. Non- limiting examples of antioxidant enzymes include catalase, glutathione peroxidase and superoxide dismutase.

B. Pharmaceutical Preparations Certain of the methods set forth herein pertain to methods involving the administration of a pharmaceutically and/or therapeutically effective amount of kinamycin F for chemotherapeutic purposes. In certain embodiments, kinamycin F may be administered to kill tumor cells by any method that allows contact of the active ingredient with the agent's site of action in the tumor. Kinamycin F be administered by any conventional methods available for use in conjunction with pharmaceuticals, either as an individual therapeutically active ingredient or in a combination of therapeutically active ingredients. Kinamycin F may be administered alone, but will generally be administered with a pharmaceutically acceptable carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

Kinamycin F may be extensively purified and/or dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. Such methods are well-known in the art. The active compound will then generally be formulated for administration by any known route, such as parenteral administration. Methods of administration are discussed in greater detail below. Compositions of the present invention, such as aqueous compositions, will typically have an effective amount of kinamycin F to kill or slow the growth of cancer cells. Further the potential recognition of genes can be accomplished by the modification of kinamycin F with specific structures that allow for the recognition of specific parts of DNA. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

Moreover, it will be generally understood that kinamycin F can be provided in prodrug form, meaning that an environment to which a kinamycin F is exposed alters the prodrug into an active, or more active, form. For example, a compound comprising a hydroxy group may be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound. It is contemplated that the term "precursor" covers compounds that are considered "prodrugs."

1. Pharmaceutical Formulations and Routes for Administration to Subjects

Pharmaceutical compositions of the present invention comprise an effective amount of one or more candidate substances {e.g., kinamycin F) or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. 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, as appropriate. The preparation of a pharmaceutical composition that contains at least one candidate substance or additional active ingredient 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, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA or other comparable governmental agency. As used herein, "pharmaceutically acceptable carrier" includes any and all 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 combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, pp 1289-1329, 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The candidate substance may comprise 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 present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, via inhalation (e.g., aerosol inhalation), via injection, via infusion, via continuous infusion, via 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 foregoing as would be known to one of ordinary skill in the art

(see, for example, Remington's Pharmaceutical Sciences, 1990). In particular embodiments, the composition is administered to a subject using a drug delivery device. Any drug delivery device is contemplated for use in delivering a pharmaceutically effective amount of kinamycin F.

The actual dosage amount of a composition of the present invention administered to an animal patient 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 will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

The dose can be repeated as needed as determined by those of ordinary skill in the art. Thus, in some embodiments of the methods set forth herein, a single dose is contemplated. In other embodiments, two or more doses are contemplated. Where more than one dose is administered to a subject, the time interval between doses can be any time interval as determined by those of ordinary skill in the art. For example, the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval derivable within any of these recited ranges. In certain embodiments, it may be desirable to provide a continuous supply of a pharmaceutical composition to the patient. This could be accomplished by catheterization, followed by continuous administration of the therapeutic agent. The administration could be intra-operative or post-operative.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of kinamycin F. In other embodiments, kinamycin F 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 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/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. In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. 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 candidate substance 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. It may be preferable to include isotonic agents, such as, for example, sugars, sodium chloride, or combinations thereof. In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present invention. Such compositions are generally designed to be compatible with the target tissue type. In a non- limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in certain embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

In certain embodiments the candidate substance is 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. In certain embodiments, 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 embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, or combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the foregoing. 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 are 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. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are 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, certain methods of preparation may include 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. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

2. Combination Therapy In order to increase the effectiveness of kinamycin F, kinamycin F may be combined with traditional drugs. It is contemplated that this type of combination therapy may be used in vitro or in vivo. In a non-limiting example, an anti-cancer agent may be used in combination with kinamycin F to treat cancer {e.g., to inhibit growth of a cancer cell). For example, kinamycin F may be provided in a combined amount with an effective amount of an anti-cancer agent to reduce or block DNA replication in cancerous cells {e.g., tissues, tumors). This process may involve administering the agents at the same time or within a period of time wherein separate administration of the substances produces a desired therapeutic benefit. This may be achieved by contacting the cell, tissue, or organism with a single composition or pharmacological formulation that includes two or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes one agent and the other includes another.

The compounds of the present invention may precede, be co-current with and/or follow the other agents by intervals ranging from minutes to weeks. In embodiments where the agents are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) as the candidate substance. In other aspects, one or more agents may be administered within of from substantially simultaneously, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about 8 weeks or more, and any range derivable therein, prior to and/or after administering the candidate substance.

Various combination regimens of the agents may be employed. Non-limiting examples of such combinations are shown below, wherein kinamycin F is "A" and a second agent, such as an anti-cancer agent, is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

C. Examples

The following examples are included to demonstrate preferred embodiments 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, and thus can be considered to constitute preferred modes for its practice. 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.

List of Abbreviations

AT, 3-amino-l,2,4-triazole; BSO, buthionine sulfoximine; DCF, dichlorofluorescein, oxidized form; DCFH, 2',7'-dichlorodihydrofluorescein; DCFH- DA, 2',7'-dichlorodihydrofluorescein diacetate; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; GSH, glutathione; DTT, dithiothreitol; H2O2, hydrogen peroxide; HBSS, Hank's Balanced Salt Solution; HRP, horseradish peroxidase; ICso, 50% inhibitory concentration; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium; ROS, reactive oxygen species; SDS sodium dodecyl sulfate; SE, standard error of the mean; Tm, DNA melting temperature.

Materials and methods

Unless otherwise indicated, all chemicals were from Sigma-Aldrich (Oakville,

ON, Canada). Thioglo-1 was from EMD Biosciences (Mississauga, Canada), kDNA was obtained from TopoGEN (Columbus, OH) and pBR322 plasmid DNA was obtained from MBI Fermentas (Burlington, ON, Canada). One-way analysis of variance was used to identify significant differences between treatment groups and controls. The degree of significance between groups was compared using a Newman- Keuls post-hoc test. Errors quoted are standard errors obtained from non-linear-least- squares analysis (SigmaPlot, SyStat, Point Richmond, CA).

EXAMPLE 1 Preparation of Kinamycin F

The producing strain, S. murayamaensis, was obtained from the American Type Culture Collection (Manassas, VA) and kinamycin D was isolated as described (Seaton and Gould, 1989; Cone et al, 1989). Sodium methoxide (0.1 mM) in methanol was added to a solution of kinamycin D in methanol and stirred for 1 h at room temperature. The reaction mixture was then added dropwise to a suspension of H-ion exchange resin (Baker CGC-271 weak acid resin, J. T. Baker, North York, Canada) in methanol. After stirring for 5 min, the ion exchange resin was removed by vacuum filtration and washed with a small amount of methanol. Removal of the solvent from the filtrate gave a solid residue which was chromatographed on silica gel with elution by acetone to provide kinamycin F. Stock kinamycin F solutions were prepared in either DMSO or methanol, as indicated. When methanol was used it was evaporated with a flow of nitrogen after it was dispensed. EXAMPLE 2

Kinetics of the Reaction of Kinamycin F with GSH and DTT

To study the mechanistic behavior of kinamycin F, the reaction of GSH or DTT with kinamycin F was followed spectrophotometrically by repeated spectral scanning on a Cary 1 double beam spectrometer (Varian, Mississauga, Canada) with a thermostated cell compartment. The reaction was initiated by adding a small volume of stock GSH or DTT to 700 μl of freshly prepared 20 μM kinamycin F and 20 mM Tris, pH 7.6, in a semi-micro quartz cell at 37°C. The reaction mixtures also contained 5% (v/v) DMSO, part of which was from the stock kinamycin F. Results: It is well known that both diazonium groups (Price and Tsunawa, 1963) and quinones (Bolton et ah, 2000) react with thiol compounds. Therefore, the reaction of kinamycin F with both GSH and DTT was examined spectrophotometrically. The reaction with GSH (FIG. 2A) was spectrally complex. The reaction of kinamycin F with GSH was characterized by a small fast {tin < 1 min) initial drop in absorbance at 464 nm (FIG. 2B), which was then followed by much slower increases in absorbance in both the UV and visible regions (FIG. 2A and 2B). While maximal changes in absorbance at 464 nm and 420 nm occurred 5 h after addition of GSH, at 330 nm a larger slow increase in absorbance was initially observed which was followed by a subsequent slow decrease in absorbance over the next 1O h. The shape of the absorbance-time plots and the lack of a single isosbestic point in FIG. 2A indicates that reaction with GSH produces more than one product in a complex series of reactions. The reaction with DTT (FIG. 2C) was spectrally less complex than with GSH (FIG. 2A). As shown by the spectral changes in FIG. 2C and 2D, kinamycin F reacted quickly (tm ~ 4.5 min; calculated from a fit of the 339 nm data to a 3- parameter first-order exponential decay function) with DTT (pH 7.6). After this initial fast reaction the absorbance values remained nearly constant for the next 4 h (FIG. 2D).

In view of these results, kinamycin F reacts with physiological levels of GSH (and with DTT) in a complex series of reactions indicating multiple products (FIG. 2). Kinamycin F is a tetrasubstituted quinone which are often relatively unreactive in nucleophilic Michael addition reactions due to its fully substituted vinylogous positions (Paz and Tomasz, 2001). However, the tetra-substituted quinone mitomycin A, in its activation to a reactive electrophile, has been postulated to react with monothiols to produce multiple products, primarily through a reductive mechanism characterized by a transient addition of the thiol to mitomycin A in a Michael addition reaction (Paz and Tomasz, 2001). Unlike the quinone system in the mitomycins, however, the kinamycins quinone system (FIG. 1) is fused on one side to a benzenoid aromatic ring (ring A) and on the other to a fϊve-membered ring (ring C). Ring C would have aromatic character by virtue of a cyclopentadienyl anion contribution to the diazonium ion-like resonance structure. As such, this system should be relatively resistant towards nucleophilic conjugate nucleophilic addition at the carbon in the quinone. The fact that depletion of GSH in K562 cells using BSO, an inhibitor of GSH synthesis, decreased the cytotoxicity of kinamycin F (FIG. 8) also suggests a role for cellular GSH in the cytotoxicity of kinamycin F.

EXAMPLE 3

Cell Culture and Growth Inhibition Assays

Experiments were performed to assess the effect of kinamycin F on the growth of certain cells. Human leukemia K562 cells, obtained from the American Type

Culture Collection, and KTVP.5 cells (a 26-fold etoposide-resistant K562-derived cell line with decreased levels of topoisomerase Ilα protein and mRNA) (Ritke et al.,

1993; Ritke et al., 1994) were maintained as suspension cultures in Dulbecco's modified Eagle's medium (Invitrogen, Burlington, Canada) containing 4 mM L- glutamine and supplemented with 20 mM HEPES, 10% fetal calf serum (Invitrogen),

100 units/ml penicillin G, and 100 μg/ml streptomycin in an atmosphere of 5% CCh and 95% air at 37°C (pH 7.4).

For the measurement of cytotoxicity, K562 and K/VP.5 cells in exponential growth were harvested and seeded at 6000 cells/well in 96-well plates (100 μl/well). Twenty-four h later, cells were treated with vehicle or various concentrations of kinamycin F and allowed to grow an additional 72 h. For assessment of the effects of lowering GSH levels on kinamycin F cytotoxicity, K562 cells in exponential growth were seeded at 1500 cells/well in 96-well plates (100 μl/well). Twenty-four h later cells were treated in the presence or absence of BSO (100 μM) and allowed to grow an additional 48 h. Cells were then treated with vehicle or various concentrations of kinamycin F and allowed to grow an additional 72 h. Kinamycin F was dissolved in DMSO and the final concentration of DMSO did not exceed 0.5% (v/v), which was an amount that was shown not to affect cytotoxicity. The BSO treatment was shown not to cause any measurable cytotoxicity. After treatment, cells were assayed with the MTS CellTiter 96 Aqueous One Solution Cell Proliferation assay (Promega, Madison, WI). The spectrophotometric 96-well plate cell growth inhibition assay measures the ability of the cells to enzymatically reduce MTS. Three replicates were measured at each drug concentration and the /Cjo-values for growth inhibition were measured by fitting the absorbance-drug concentration data to a three- or four-parameter logistic equation as described (Hasinoff et al., 1997).

Results: Kinamycin F contains a diazo, a paraquinone, and phenolic functional groups, all of which may contribute to its cytotoxicity either directly or after activation. The present inventors found that kinamycin F had an /Cso-value for 72 -h growth inhibition in K562 cells of 0.33 μM (FIG. 7B), a value which was close to that found for kinamycin A and kinamycin C of 0.31 and 0.37 μM, respectively (Hasinoff et al., 2006). (K562 cells are a cell line of human erythroleukemia cells.) Thus, kinamycin F is a potent inhibitor of cancer cell growth.

Because the results of FIG. 2 indicated that kinamycin F reacted with GSH, whether depletion of intracellular GSH affected the cytotoxicity of kinamycin F was investigated. To lower GSH levels, K562 cells were preincubated with 100 μM BSO, an inhibitor of the rate limiting GSH-synthesizing enzyme α-glutamylcysteine synthetase, for 48 h. As shown by the data in FIG. 8, this concentration of BSO was shown not to cause a measurable cytotoxicity. This was followed by a 72 h-exposure to various concentrations of kinamycin F, after which cell growth was measured with the MTS assay. As determined using a fluorescent GSH Thioglo-1 assay previously described (Kagan et al., 2001), cellular levels of GSH levels in K562 cells were reduced by 65% following BSO treatment. As shown in FIG. 8, kinamycin F inhibited cell growth with an /Cso value of 2.4 μM for untreated cells, and for the BSO-treated cells this value was reduced approximately 2-fold to 1.1 μM. A paired t- test of three separate paired /Cso determinations showed that this reduction was significant (p < 0.007), a result which suggests that GSH partially protected these cells from the growth inhibitory effects of kinamycin F. EXAMPLE 4

Kinamycin F Binds To and Intercalates With DNA Thermal Denaturation of DNA Assay and Spectrophotometric DNA Titration Assay

Ligand binding to DNA stabilizes the DNA double helix, causing an elevation in the DNA melting temperature (Tm). The Tm of sonicated calf thymus DNA (5 μg/ml) was measured in 10 mM Tris buffer (pH 7.5) in a Cary 1 (Varian) double beam spectrophotometer by measuring the absorbance increase at 260 nm upon the application of a temperature ramp of l°C/min as previously described (Liang et ah, 2006). The maximum of the first derivative of the absorbance-temperature curve was used to obtain the Tm. Doxorubicin (2 μM), which increases Tm by 13.2°C (Liang et ah, 2006) and is a strong DNA intercalator (Chaires et ah, 1996), was used as a positive control. The changes in the visible spectrum of kinamycin F upon the addition of small amounts of calf thymus DNA was recorded to determine whether the kinamycin F bound to DNA. The semi-micro spectrophotometer cell contained 40 μM of kinamycin F and 0.5% DMSO in buffer (10 mM Tris, pH 7.5) at 20⁰C.

Ethidium Bromide Displacement Assay

Ethidium bromide fluoresces strongly when bound to DNA, but only weakly fluoresces when free, and thus the displacement of ethidium bromide by kinamycin F was measured fluorometrically. The ethidium bromide displacement assay was carried out essentially as described (Jenkins, 1997). In a 96-well plate, calf thymus DNA (16 μg/ml), water and kinamycin F (pH 6.5) were incubated for 5 min in the dark at room temperature, after which time ethidium bromide (1 μM) was added. The fluorescence of the solutions was immediately measured in a Fluostar Galaxy (BMG, Durham, NC) fluorescence plate reader using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. The C so, which is the concentration of the drug which results in a 50% decrease in the fluorescence intensity of the ethidium bromide- DNA complex (Jenkins, 1997), was determined from a plot of fluorescence vs. kinamycin F concentration.

Computational Methodologies for the Docking of Kinamycin F in an X-ray Crystal Structure of DNA

The positioning of kinamycin F in DNA was examined using molecular modeling. All molecular modeling was done using SYBYL 7.2 on a Hewlett-Packard XW4100 PC workstation with a Redhat Enterprise 3 Linux operating system. All molecules except the DNA were built using SYBYL. Kinamycin F was docked into the doxorubicin binding site of a 6 base-pair x-ray crystal structure of two molecules of doxorubicin bound to double-stranded DNA, d(TGGCCA)/doxorubicin (world wide web at .rcsb.org/pdb/; PDB ID: 1DA9) (Leonard et al, 1993), using the genetic algorithm docking program GOLD version 3.1 (GOLD V. 3.1 Software) and the default GOLD parameters (Verdonk et al, 2003) as described (Liang et al, 2006). GOLDScore was used as the fitness function with flipping options of amide bonds, planar and pyramidal nitrogens, and internal hydrogen bonds being allowed. No early termination was allowed. The 1DA9 x-ray structure shows that the first and second base pairs buckle out to accommodate bound doxorubicin (Leonard et al, 1993). Thus, the 1DA9 X-ray structure of the doxorubicin-DNA complex was used for the docking experiments, rather than constructing DNA in SYBYL, because it was reasoned that this DNA structure would be a more realistic model for the binding of kinamycin F because of its structural similarity to doxorubicin.

Kinamycin F was first geometry optimized with the Tripos force field using a conjugate gradient with a convergence criterion of 0.01 kcal/mol and Gasteiger- Huckel charges and a distance-dependent dielectric constant. Two conformational isomers of kinamycin F that differed in their D-ring conformation were separately docked: one had an axial 3-methyl conformation, and the other had an equatorial 3- methyl conformation. The DNA structure was prepared by removing one of the bound doxorubicin molecules and removing all of the water molecules to avoid potential interference with the docking. Hydrogens were added to the DNA, and the SYBYL Biopolymer module was used to add Kollman-All charges to the DNA. Using atom H341 which is located around the middle of the DNA structure as the reference atom, the binding site was defined as being within 20 A of the reference atom. Kinamycin F minimized structures were docked into the DNA structure to obtain the top 20 scoring GOLDScore structures for each conformer. As a test of the docking procedure, doxorubicin was docked back into the DNA structure with a heavyatom root-mean-squared distance of 1.7 A compared to the X-ray structure (Leonard et al, 1993). Values of 2.0 A or less in the extensive GOLD test set are considered to be good (Verdonk et al, 2003). Results: Kinamycin F-induced damage to DNA through production of ROS or reductively activated kinamycin F species would be greatly enhanced if kinamycin F or its activated intermediates were able to bind to DNA. The ability of kinamycin F to bind to DNA was studied by measuring absorbance changes upon the addition of calf thymus DNA. Free kinamycin F has an absorbance maximum at 430 nm (FIG. 2A dashed line). As shown in FIG. 3A increasing concentrations (> 2 mM, base-pair concentration) of calf thymus DNA to kinamycin F (40 μM) caused a decrease in absorbance at the peak maximum of 430 nm. This result and the shift in the peak maximum from 430 to 454 nm indicated kinamycin F weakly bound to DNA. In the second experiment, kinamycin F-induced changes in DNA melting temperature Tm were studied. While the changes were relatively small, suggesting weak binding, kinamycin F (20 μM) increased the Tm of DNA from 68°C to 71⁰C. This value can be compared to the previously determined value for 2 μM doxorubicin, which increases Tmhy 13.2°C (Liang et al, 2006). In order to further confirm that kinamycin F bound to DNA, an ethidium bromide displacement assay was used (Jenkins, 1997). As shown in FIG. 3B, kinamycin F displaced ethidium bromide from DNA and caused a 50% reduction in fluorescence at a concentration of 105 μM (/Cso). The association constant for kinamycin F binding to DNA, Kn_PP, was calculated to be 9 x 10⁴ M^"1 from Kn_PP = KEΪBIC EtBr/C5o (Jenkins, 1997). This is relatively weak binding as it is some 300-fold weaker than the value of 29.6 x 10⁶ M^"1 for the well known DNA intercalator doxorubicin (Chaires et ah, 1996). The K&_PP value was calculated from the concentration of ethidium bromide (CEtBr) used in the assay of 1 μM; the concentration of kinamycin F that reduced the fluorescence of ethidium bromide by 50% (Cso) of 105 μM; and an association constant of ethidium bromide (ATEtBr) of 9.5 x 10⁶ M^"1 (Jenkins, 1997). However, a high degree of binding is not necessary to cause site-specific DNA damage if ROS or reductively activated kinamycin F species, as discussed other Examples herein, are produced while directly bound to DNA.

The molecular modeling docking program GOLD was used to determine whether kinamycin F could be docked into a doxorubicin-DNA x-ray structure

(Leonard et ah, 1993), and how it interacted with DNA. The structure of the equatorial conformation of kinamycin F docked into DNA is shown in FIG. 4. While both equatorial and axial conformations of kinamycin F docked well, they differed in their binding through an approximate 180° rotation about their long axis. As shown in FIG. 4, the intercalating interaction of equatorial kinamycin F with DNA is stabilized through hydrogen binding of the hydroxyl groups on Cl and C2 of the 6- membered D ring (FIG. 1) to DNA. EXAMPLE 5

Kinamycin F Induces Oxidation of DCFH to DCF

Fluorometric Assay of DCF Oxidation

DCFH-DA is a non-fluorescent compound that, when taken up by cells, is hydrolyzed by esterases to yield non-permeable DCFH (O'Malley et al., 2004). Cellular or exogenous oxidants are then able to oxidize reduced DCFH to the fluorescent DCF. 3-Amino-l,2,4-triazole (AT) can be used to inhibit cellular catalase to measure H2Ch-dependent oxidation of DCF (Maresca et al, 1992).

K562 cells were first incubated with either AT (20 mM) or an appropriate amount of HBSS in complete media for 30 min at 37°C. The cells were centrifuged (1000 g, 6 min) and rinsed twice in either HBSS or HBSS containing AT (20 mM). Cells were resuspended in HBSS, with or without AT, and incubated with 50 μM of DCFH-DA for 20 min at 37°C. Cells were washed twice (10 min incubation with each wash) in either HBSS or HBSS containing AT (20 mM). Following washes, cells were allowed to rest for 20 min at 37°C in HBSS or HBSS containing AT (20 mM). Cells (50,000) were placed into each well of a 96-well plate and the volume was adjusted to 50 μl with HBSS or HBSS containing AT (20 mM). The fluorescence was then recorded on a fluorescence plate reader for 10 min to obtain baseline data using an excitation wavelength of 485 nm and an emission wavelength of 520 nm at 30⁰C, after which, either the positive control H2O2 (200 μM) or kinamycin F (5 μM) was added.

Results: As shown in FIG. 9, treatment of K562 cells with kinamycin F (5 μM) significantly increased the rate of oxidation to DCF to nearly that produced by 200 μM H2O2. In order to determine if this oxidation was H2Ch-dependent, experiments were also done in which cells were pretreated with the catalase inhibitor AT. While AT treatment significantly increased the rate of oxidation to DCF of H2Ch-treated cells, there was no significant change in the rate of oxidation to DCF of kinamycin F- treated cells. This result indicates that kinamycin F did not measurably oxidize DCFH through a FhCh-dependent pathway.

While DCFH oxidation to fluorescent DCF is often used as a measure of reactive oxygen and nitrogen species production, it has also been shown that some drugs can directly oxidize DCFH (O'Malley et al., 2004). In order to determine if kinamycin F could directly oxidize DCF in vitro, it was determined that 20 μM kinamycin F in 20 mM Tris buffer (pH 8.0) increased the rate of oxidation of 0.5 μM

DCFH (prepared from the DCFH-DA ester by base hydrolysis as described (Brandt and Keston, 1965)) by 4.1 -fold in the presence of 100 μM deferoxamine (to chelate trace iron in the buffer), and 5.1 -fold in its absence (data not shown). This data indicates that kinamycin F oxidized DCFH through a direct reaction and, thus, it may have also increased the rate of oxidation to DCF through this mechanism in cells.

These results indicate that kinamycin F may be producing ROS. The kinamycin F-induced oxidation of DCFH in K562 cells was not H2Ch-dependent as the catalase inhibitor AT did not significantly change the rate of DCFH oxidation. Because in cell-free experiments kinamycin F was able to directly oxidize DCFH, it was not possible to determine if the kinamycin F-induced oxidation of DCFH in K562 cells observed was due to direct oxidation of DCFH or due to formation of some other activated kinamycin F-derived oxidizing species. It has been shown previously that other drugs can directly oxidize DCFH (O'Malley et al., 2004), which confounds the use of this widely used indicator of cellular oxidative stress. The lack of H2O2 dependence on kinamycin F-induced DCFH oxidation in K562 cells (FIG. 9) does not rule out H2O2 as a damaging cytotoxic species as H2θ2-dependent oxidation of DCFH may still be occurring, but at levels that are obscured by the direct oxidation of DCFH by kinamycin F.

EXAMPLE 6

Kinamycin F Induces DNA Nicking Damage pBR322 DNA Nicking Assay

Whether kinamycin F could damage DNA via nicking was investigated. The nicking of double-stranded closed circular plasmid pBR322 DNA by a mixture of kinamycin F and GSH was followed after incubation by separating the reaction products using ethidium bromide gel electrophoresis. The 10 μl reaction mixture contained 80 ng of pBR322 plasmid DNA, kinamycin F, and 5 niM GSH that had been titrated to pH 7.6. The order of addition was kinamycin F, DNA and then GSH. The reaction mixture was incubated at 37°C for 5 h after which time loading buffer (10 mM Tris pH 8, 60% sucrose, and 0.5% bromophenol blue) was added. Either deferoxamine (100 μM; to complex adventitious iron), or DMSO (0.5% (v/v); to trap hydroxyl radicals) was pre-incubated 30 min at 37°C with the DNA and the water used to make it up to volume. In the experiments in which catalase (50 μg/ml) was used to test for H2Ch-dependent DNA nicking, either untreated active catalase or catalase that had been boiled for 5 min to inactivate it, was added immediately prior to drug addition. The test solutions were added to the kinamycin F which had been deposited at the bottom of the reaction tube from a methanol stock solution and then dried. GSH (5 mM) was then added to start the reaction. The nicked pBR322 DNA was separated by electrophoresis (2 h at 8 V/cm) on a Tris acetate-EDTA agarose gel (1.2%, wt/v) containing ethidium bromide (0.5 μg/ml). The linear pBR322 DNA control was prepared enzymatically as described previously (Hasinoff et ah, 2005). The DNA in the gel was imaged by its fluorescence on an Alpha Innotech (San Leandro, CA) Fluorochem 8900 imaging system equipped with a 365 nm UV illuminator and a CCD camera.

Fast Micromethod DNA Single-Strand-Break Assay The Fast Micromethod DNA Single-Strand-Break assay, which is based upon the Fluorometric Analysis of DNA Unwinding assay (FADU assay), in which damaged DNA with alkaline-labile sites or single strand breaks undergoes an increased rate of unwinding under alkaline conditions, was performed essentially as described (Schroder et ah, 2006). Briefly, 35,000 exponentially growing cells were incubated at 37°C for 2 h in HBSS supplemented with 1.25 mM CaCh, 0.81 mM MgSθ4, and 4.17 mM NaHCCb (pH 7.4) in the absence or presence of kinamycin F. Control cells were treated with an equal volume of the vehicle (2% (v/v) DMSO). Following incubation, cells were centrifuged at 1000 g for 6 min and the supernatant was discarded. Cells were resuspended in 125 μl Tris-EDTA buffer (10 mM Tris- HCl, pH 7.4, 1 mM EDTA) and 25 μl of the suspension (280 cells/μl) was placed into 4 wells of a 96-well plate. A set of blanks containing 25 μl of Tris-EDTA buffer were also analyzed in the same manner. Lysing buffer (25 μl of 9 M urea, 0.2 M EDTA, 0.1% SDS, (pH 10.0)) supplemented with PicoGreen (Invitrogen) (20 μl of original stock dye/ml of lysing solution)) was gently added to each well. Lysing occurred in the dark at room temperature for 1 h. To initiate DNA unwinding, 250 μl of working 0.1 M NaOH solution was added to give a final pH of 12.4. Fluorescence measurements were immediately started in the fluorescence plate reader using an excitation wavelength of 485 nm and an emission wavelength of 520 nm at 25°C and continued for 40 min. All values were corrected with blank readings.

Results: It was previously reported that lomaiviticin A (He et al., 2001) and kinamycin D (Zeng et al., 2006) could cleave DNA under reducing conditions. However, in the lomaiviticin A report (He et al., 2001) the conditions were not given. And in the kinamycin D report (Zeng et al., 2006), the incubation that yielded 27% nicked DNA was carried out for a long time (2 d) and at high kinamycin D (1 mM) and DTT concentrations (1 M). Whether kinamycin F could cleave DNA under physiological conditions was investigated. As shown in FIG. 5 A, kinamycin F alone induced a small amount of nicking of pBR322 plasmid DNA after 5 h at 37°C, even in the absence of GSH (1.2- and 1.4- fold increase at 200 and 400 μM kinamycin F, respectively). DNA damage was greatly enhanced in the presence of physiologically relevant levels of GSH (5 mM) with 1.1-, 1.3-, 1.9-, 3.2-, 5.5-fold increases in nicked DNA observed at kinamycin F concentrations of 10, 50, 100, 200, 400 μM kinamycin F, respectively. At 400 μM kinamycin F in the presence of GSH, where the degree of nicking was very high, a small amount of linear DNA was observed (8.5% of the total compared to 5.1% for supercoiled DNA). This linear DNA produced from DNA double strand breaks, which appeared only at the highest kinamycin F concentration, likely occurred through an accumulation of a large number of DNA single strand breaks rather than through kinamycin F causing double strand breaks directly.

To elucidate the mechanism by which kinamycin F induced nicking in DNA, experiments were carried out in which either DMSO, catalase, or deferoxamine were added prior to the addition of either kinamycin F or GSH (FIGS. 5B and 5C). Under these experimental conditions, kinamycin F induced nicking was reduced from 3.2- fold over control when used alone to 1.0-, 0.9-, 1.2-fold over control with DMSO, catalase, or deferoxamine pretreatments, respectively. Boiling catalase eliminated its ability to inhibit kinamycin F-induced DNA damage (FIG. 5C).

Having determined that the kinamycin F/GSH system could nick pBR322

DNA as shown above, it was investigated whether kinamycin F could also damage DNA in cells. To this end, the Fast Micromethod DNA Single Strand Break assay was used, which determines the frequency of single-strand breaks and alkaline-labile sites (Schroder et ah, 2006). The unwinding of DNA strands under basic conditions was followed for 20 min, at which time the fluorescence of the remaining double stranded DNA was measured. As shown in FIG. 6 treatment of K562 cells for 2 h with kinamycin F resulted in a concentration-dependent reduction in double-stranded

DNA that achieved significance (p < 0.001) at 200 μM kinamycin F compared to untreated cells.

These results show that deferoxamine, an iron chelator; DMSO, a scavenger of hydroxyl radicals; and catalase, an enzyme that decomposes H₂O₂, all strongly inhibited kinamycin F/GSH-induced nicking (FIGS. 5B and 5C) suggesting that kinamycin F, in the presence of GSH, nicks DNA in an iron-, H₂O₂-, and hydroxyl radical-dependent manner. Hence, kinamycin F-induced ROS production may be, in part, responsible for DNA damage. Kinamycin F may not be directly producing hydroxyl radicals in a Fenton-type reaction but may be damaging DNA by high-valent iron-oxo species with hydroxyl radical-like reactivity (Rush and Koppenol, 1986). The Fast Micromethod DNA Single Strand Break assay, which determines the frequency of single-strand breaks and alkaline-labile sites (Schroder et ah, 2006), showed that 200 μM kinamycin F in a 2 h incubation also had the ability to damage the DNA in K562 cells (FIG. 6), a result that is consistent with the in vitro DNA damage results of FIG. 5.

EXAMPLE 7

Kinamycin F Inhibits Topoisomerase Ilα Catalytic Activity But Does Not Act As

A Topoisomerase Ilα Poison

Topoisomerase Ha kDNA Decatenation Inhibition Assay Topoisomerase is an isomerase enzyme that acts on the topology of DNA.

Otherwise identical loops of DNA having different numbers of twists are topoisomers, and cannot be interconverted by any process that does not involve the breaking of DNA strands. Topoisomerases catalyze and guide the unknotting of DNA. This unlinking activity is termed "decatenation." Many drugs operate through interference with the topoisomerases (Kornberg and Baker, 1991; Pommier et al, 1998).

The ability of kinamycin F to inhibit topoisomerase Ilα was determined via a spectrofluorometric decatenation assay as described (Hasinoff et al., 2006). Topoisomerase Ilα is able to decatenate the highly knotted circular kDNA resulting in smaller circles of DNA in an ATPdependent reaction. Each 20 μl reaction contained 0.5 mM ATP, 50 mM Tris (pH 8.0), 120 mM KCl, 10 mM MgCh, 30 μg/ml bovine serum albumin, 40 ng kDNA, kinamycin F (0.5 μl in DMSO), 15 ng of topoisomerase Ilα protein (the amount that gave approximately 80% decatenation) and 0.12 μM DTT. Full length human topoisomerase Ilα was obtained as previously described (Hasinoff et al., 2005). Control reactions containing 2.5% (v/v) DMSO were shown not to affect the activity of topoisomerase Ilα. The reactions were incubated at 37°C for 20 min, after which time the reactions were terminated by the addition of 12 μl of 250 mM Na₂EDTA. Samples were centrifuged at 8000 g, 25°C for 15 min, and 20 μl of supernatant was mixed with 180 μl of 600-fold diluted PicoGreen dye in a 96-well plate. Fluorescence, which was proportional to the amount of kDNA present, was measured in a fluorescence plate reader using an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Results: As shown in FIG. 7A, kinamycin F inhibited the decatenation activity of human topoisomerase Ilα with an ICso of 0.85 ± 0.26 μM (FIG. 7A), which is, surprisingly, 9- and 11 -fold more potent than either kinamycin A or kinamycin C, respectively (Hasinoff et al., 2006). To determine whether kinamycin F acts as a topoisomerase Ilα poison, a comparison of the growth inhibitory effects of kinamycin F on K562 and the K/VP.5 cell lines was used. The K/VP.5 cell line contains one- fifth the amount of topoisomerase Ilα compared to the parent K562 cell line (Ritke et al., 1993; Ritke et al., 1994), and can be a convenient test of whether a compound is a topoisomerase II poison (Hasinoff et al., 2005). Fewer DNA strand breaks will be produced in cells containing less topoisomerase Ilα and thus, topoisomerase Ilα poisons will be less potent towards the K/VP.5 cell line. As shown in FIG. 7B, the ICso for growth inhibition of K562 cells was 0.33 ± 0.02 μM while that of K/VP.5 cells was 0.42 ± 0.10 μM. Thus, the lack of cross resistance indicates that, while kinamycin F (like kinamycin A and kinamycin C (Hasinoff et al., 2005)) can inhibit the catalytic activity of topoisomerase Ilα, it likely did not act as a topoisomerase Ilα poison in a cellular context. It may be that kinamycin F inhibits topoisomerase Ilα by reacting with critical sulfhydryl groups on the protein. (Hasinoff et al. , 2006; Bender et al., 2006; Hasinoff et al., 2005). It is therefore further postulated that kinamycin F may not exert its cytotoxicity singularly through the inhibition of topoisomerase II.

EXAMPLE 8

EPR Spectroscopy of GSH- and HRP/H₂O₂-induced Kinamycin F Free Radicals

Electron Paramagnetic Resonance Spectroscopy

Because the results of FIG. 5C showed that kinamycin F reacted rapidly with GSH, the ability of GSH to react with kinamycin F to produce a free radical was examined by EPR spectroscopy. A freshly prepared 15 μl aliquot of kinamycin F in the reaction systems indicated was injected into an 8-cm length of gas-permeable Teflon tubing (Zeus Industrial Products, Raritan, NJ) which was then folded at both ends and inserted into a quartz EPR tube open at both ends, and placed in the EPR cavity as described (Barnabe et al., 2002). The EPR spectra were recorded with a Bruker (Milton, Canada) EMX EPR spectrometer. Pre-purifϊed grade thermostated (25°C) air or argon (400 1/h) was flowed continuously over the sample while the spectra were recorded. Recording of the first derivative EPR spectra was started approximately 2 min after the sample was prepared. A total of 20 spectra (42 s/scan) were recorded over 14 min and their signals were averaged. The instrument settings were microwave power 20 mW, modulation frequency 100 kHz, microwave frequency 9.25 GHz, modulation amplitude 0.5 or 2.0 G as indicated, time constant 0.02 s, 1024 data points/scan and a 10 or 50 G scan range as indicated. The kinamycin F (0.5 mM) was dissolved in DMSO, which was present in the final reaction mixture at 5% (v/v). For the GSH treatment the final GSH concentration was 2 mM (pH 7.6). For the HRfVH₂O₂ experiments the HRP concentration was 100 μg/ml and the H₂O₂ concentration was 25 μM. Results: As shown in FIG. 1OA, kinamycin F reacted with GSH to produce an air- stable free radical EPR signal at g ~ 2.0 which was likely a semiquinone free radical (Bolton et al., 2000). Control experiments showed that kinamycin F alone did not produce an EPR free radical signal. Because the EPR signal quickly disappeared when the reaction mixture was made hypoxic, this indicated that the excess GSH was able to directly reduce the kinamycin F semiquinone to the EPR-silent kinamycin F hydroquinone. The free radical semiquinone signal was seen under oxic conditions because the quinone Q and the hydroquinone QH2 likely underwent an equilibrium disproportionation reaction (Q + QH2 ^ 2Q^'~ + 2H⁺) to yield low steady-state levels of the semiquinone Q^°~ (Swallow, 1982; Ollinger and Brunmark, 1991). Semiquinones react very quickly with oxygen to produce the superoxide radical anion Ch--, which in turn dismutates to produce H2O2. Thus, the EPR results suggest that kinamycin F may, in part, also be growth inhibitory through the production of ROS.

Because kinamycin F also contains a phenol group investigations were undertaken to determine whether it could be oxidized by the HRP/H2O2 system to produce an phenoxyl radical (Kagan et al., 1999). As shown in FIG. 1OB the HRP/H2O2 system produced an EPR signal probably due to a phenoxyl radical with fine structure that likely arose from splitting due to A-ring H-atoms (FIG. 1). The EPR signal slowly increased with time over 40 min, unlike the radical produced with the reaction with GSH, which slowly decreased with time. Control experiments showed that H2O2 and kinamycin F alone did not produce an EPR free radical signal. The free radical seen from the treatment of kinamycin F with HRP/H2O2 was different from that produced by treatment with GSH, because when the modulation amplitude in the GSH experiment was decreased from 2.0 G to 0.5 G no detectable fine structure in its EPR spectrum was observed (data not shown), unlike that seen in FIG. 1OB. Phenoxyl radicals may oxidize and damage cellular thiols through H-atom abstraction, as has been shown for etoposide (Tyurina et al, 1995). Without wishing to be bound by theory, the inventors postulate that oxidative diazo activation may be at play. EXAMPLE 9 Treatment of Tumors with Kinamycin F

Treatment with kinamycin F may be similar to the treatment regimes of other drugs, such as DNA intercalators (e.g., anthracyclines and their derivatives). For example, standard treatment with doxorubicin is described in Remington 's Pharmaceutical Sciences as follows.

Doxorubicin is administered intravenously to adults at 60 to 75 mg/m² at 21- day intervals or 25 to 30 mg/m on each of 2 or 3 successive days repeated at 3- or 4- week intervals or 20 mg/m once a week. The lowest dose should be used in elderly patients, when there is prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs. The dose should be reduced by 50% if the serum bilirubin lies between 1.2 and 3 mg/dL and by 75% if above 3 mg/dL. The lifetime total dose should not exceed 550 mg/m² in patients with normal heart function and 400 mg/m² in patients with normal heart function and 400 mg/m on each of 3 consecutive days, repeated every 4 weeks. Prescribing limits are as with adults. It has been reported that a 96-hr continuous infusion is as effective as and much less toxic than the same dose given by bolus injections.

Of course, modifications of the treatment regimes due to the unique nature of kinamycin F are possible and well within the ability of one skilled in the art. Appropriate modifications may be ascertained, for example, by following the protocols in the following examples for in vivo testing and developments of human protocols.

EXAMPLE 10

In vivo Prevention of Tumor Development Using Kinamycin F

In an initial round of in vivo trials, a mouse model of human cancer with the histologic features and metastatic potential resembling tumors seen in humans (Katsumata et al., 1995) is used. The animals are treated with kinamycin F to determine the suppression of tumor development. These studies are based on the discovery of the present invention that kinamycin F has anti-cancer activity in cancer cells. Kinamycin F is tested in vivo for antitumor activity against murine leukemia

L1210, P388 and P388 resistant to doxorubicin. In conjunction with these studies, the acute and sub-acute toxicity is studied in mice (LDlO, LD50, LD90). In a more advanced phase of testing, the antitumor activity of kinamycin F against human xenografts is assessed and cardiotoxicity studies is performed in a rat or rabbit model.

Two groups of mice of a suitable cancer model are treated with doses of kinamycin F. Several combinations and concentrations of kinamycin F are tested. Control mice are treated with buffer only.

The effect of kinamycin F on the development of tumors is compared with the control group by examination of tumor size, and histopathologic examination (tissue is cut and stained with hematoxylin and eosin) of the relevant tissue. With the chemopreventive potential of kinamycin F, it is predicted that, unlike the control group of mice that develop tumors, the testing group of mice is resistant to tumor development.

EXAMPLE 11

Human Treatment with Kinamycin F

This example describes a protocol to facilitate the treatment of cancer using kinamycin F. A cancer patient presenting 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 (defined as peripheral absolute granulocyte count of > 2,000/mm³ and platelet count of 100, 000/mm³, adequate liver function (bilirubin 1.5 mg/dl) and adequate renal function (creatinine 1.5 mg/dl).

A composition of the present invention is typically administered orally or parenterally in dosage unit formulations containing standard, well known non-toxic physiologically acceptable carriers, adjuvants, and/or vehicles as desired. The term "parenteral" as used herein includes subcutaneous injections, intravenous, intramuscular, intra-arterial injection, or infusion techniques. Kinamycin F may be delivered to the patient before, after, or concurrently with any other anti-cancer agent(s), if desired. A typical treatment course may comprise about six doses 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.

To kill cancer cells using the methods and compositions described in the present invention, one will generally contact a target cell with kinamycin F. These compositions are provided in an amount 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 kinamycin F is an efficient method for delivering a therapeutically effective dose to counteract the clinical disease. Likewise, 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 may 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 kinamycin F.

In one embodiment, administration simply entails injection of the therapeutic composition into the tumor. In 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. 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 1 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 in Example 7. Those of skill in the art are able to take the information disclosed in this specification and optimize treatment regimes based on the results from the trials.

EXAMPLE 12

Clinical Trials of the Use of Kinamycin F in Treating Cancer

This example is concerned with the development of human treatment protocols using kinamycin F. These compounds are of use in the clinical treatment of various cancers in which transformed or cancerous cells play a role.

The various elements of conducting a clinical trial, including patient treatment and monitoring, are 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 studying kinamycin F in clinical trials. Patients with cancer, such as human 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. The patient may carry tumors that express a MDR (multi-drug resistant) 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. Baseline cellularity, cytology, LDH, and appropriate markers in the fluid (CEA, CA15-3, CA 125, pl85) and in the cells (ElA, pl85) may also be assessed and recorded. The patient should exhibit a normal coagulation profile.

In the same procedure, kinamycin F may 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 0.5 mg/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 (0.5 mg, 1 mg, 2 mg, 4 mg) until drug related grade 2 toxicity is detected. Thereafter, dose escalation may proceed by 25% increments. The administered dose may be fractionated equally into two infusions, separated by six hours, if the combined endotoxin levels determined for the lot of kinamycin F exceeds 5 EU/kg for any given patient.

Kinamycin F may be administered over a short infusion time or at a steady rate of infusion over a 7 to 21 day period. The kinamycin F infusion may be administered alone or in combination with, for example, another anti-cancer drug. 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 improved. Increasing doses of kinamycin

F in combination with an anti-cancer drug is 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 should, of course, be performed before treatment and at intervals of about 3-4 weeks later.

Laboratory studies should 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 should 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 should be examined for appropriate tumor markers every 4 weeks, if initially abnormal, with twice weekly CBC, differential and platelet count for the 4 weeks; then, if no myelosuppression has been observed, then weekly. If any patient has prolonged myelosuppression, bone marrow examination is advised to rule out the possibility of tumor invasion of the marrow as the cause of pancytopenia. A coagulation profile shall be obtained every 4 weeks. An SMA-12-100 shall be performed weekly. Pleural/peritoneal effusion may be sampled 72 hours after the first dose, weekly thereafter for the first two courses, then every 4 weeks until progression or off study. Cellularity, cytology, LDH and appropriate markers in the fluid (CEA, CAl 5- 3, CA 125, pl85) and in the cells (pl85) may be assessed. For an example of an evaluation profile, see Table 1. 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. An urinalysis may be performed every 4 weeks. 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 1 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.

Table 1 Evaluations Before and During Therapy

EVALUATIONS PRE- TWICE EVERY 4 EVERY 8

STUDY WEEKLY WEEKLY WEEKS WEEKS

History X X

Physical X X

Tumor Measurements X X

CBC X X¹ X

Differential X X¹ X

Platelet Count X X¹ X

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

Serum Tumor markers X X³ (CEA, CAl 5-3, CA- 125, Her-2/neu) Urinalysis X X

X-rays: chest X X⁴ others X X

Pleural/Peritoneal X X⁵ X Fluids: (cellularity, cytology, LDH, tumor markers, ElA, HER- 2/neu) Spirometry and DLCO X X⁶ X⁶

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

As indicated by the patient's condition.

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.

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

Four and eight weeks after initiation of therapy.

All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the 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 methods 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

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Claims

1. A method of damaging DNA, damaging a protein, and/or inhibiting topoisomerase Ilα comprising administering kinamycin F to a cell.

2. The method of claim 1 , wherein the method takes place in vitro.

3. The method of claim 1, wherein the method comprises production of a radical of kinamycin F.

4. The method of claim 3, wherein the kinamycin F radical is a semiquinone radical or a phenoxyl radical.

5. The method of claim 1 , further comprising ROS production.

6. The method of claim 1, further defined as a method of damaging DNA and/or a protein.

7. The method of claim 6, wherein the damage is iron-dependent.

8. The method of claim 6, wherein the damage is hydroxyl radical-dependent.

9. The method of claim 6, wherein kinamycin F binds to DNA.

10. The method of claim 9, wherein kinamycin F binds to the DNA via intercalation.

11. The method of claim 6, wherein DNA damage is caused by the nicking of DNA by kinamycin F.

12. The method of claim 6, further defined as a method of damaging a protein.

13. The method of claim 12, wherein the protein comprises one or more sulfhydryl groups.

14. The method of claim 12, wherein the protein is a topoisomerase.

15. The method of claim 14, wherein the topoisomerase is further defined as topoisomerase Ilα.

16. The method of claim 1 further defined as a method of inhibiting topoisomerase Ilα.

17. The method of claim 16, wherein kinamycin F inhibits the decatenation activity of topoisomerase Ilα.

18. The method of claim 1 , wherein the administration is in vivo.

19. The method of claim 18, wherein the administration is to a human.

20. The method of claim 19, wherein the human has cancer.

21. The method of claim 20, further defined as a method of killing or inhibiting a cancer cell in the human.

22. A method of killing or inhibiting the growth of a cancer cell comprising administering kinamycin F to the cell.

23. The method of claim 22, wherein the cell is in vivo.

24. The method of claim 22, wherein the cell is in vitro.

25. The method of claim 22, wherein the method comprises production of a radical of kinamycin F.

26. The method of claim 25, wherein the kinamycin F radical is a semiquinone radical or a phenoxyl radical.

27. The method of claim 22, further comprising ROS production.

28. The method of claim 25, wherein the radical of kinamycin F damages the DNA and/or a protein of the cancer cell.

29. The method of claim 28, wherein the damage is iron-dependent.

30. The method of claim 28, wherein the damage is hydroxyl radical-dependent.

31. The method of claim 28, wherein kinamycin F binds to the DNA.

32. The method of claim 31 , wherein kinamycin F binds to the DNA via intercalation.

33. The method of claim 28, wherein the DNA is nicked by kinamycin F.

34. The method of claim 22, wherein the kinamycin F inhibits the decatenation activity of topoisomerase Ilα in the cancer cell.

35. The method of claim 22, wherein the cancer cell is comprised in a tumor.

36. The method of claim 22, wherein the cancer cell is intolerant of oxidative stress.

37. A method of treating a tumor in a subject, comprising administering a therapeutically effective amount of kinamycin F to the subject.

38. The method of claim 37, wherein the subject is a mammal.

39. The method of claim 38, wherein the mammal is a human.

40. A method of treating cancer in a subject, comprising administering a therapeutically effective amount of kinamycin F to a subject.

41. The method of claim 40, wherein the subject is a mammal.

42. The method of claim 41 , wherein the mammal is a human.

43. The method of claim 40, wherein the kinamycin F is comprised in a pharmaceutically acceptable composition with a pharmaceutically acceptable carrier.

44. The method of claim 40, further defined as comprising administering a second treatment to the subject.