WO2011143593A1 - Conjugates of a lipoic acid derivative and anti-proliferation agent and medical uses thereof - Google Patents

Abstract

The invention provides conjugate compounds, pharmaceutical compositions, therapeutic methods, and kits for use in treating cell proliferation disorders, such as cancer. The conjugates comprise a redox-modulating compound, such as a lipoic acid derivative, chemically bonded to an anti-proliferation agent, such as gemcitabine.

Description

CONJUGATES OF A LIPOIC ACID DERIVATIVE AND ANTI-PROLIFERATION AGENT AND MEDICAL USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to United States Provisional Patent Application serial number 61/344,053, filed May 14, 2010, the contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The invention provides conjugate compounds, pharmaceutical compositions, therapeutic methods, and kits for use in treating cell proliferation disorders, such as cancer. The conjugates comprise a redox-modulating compound chemically bonded to an anti- proliferation agent, such as gemcitabine.

BACKGROUND

[0003] Cancer is a leading cause of death in many industrialized countries. Recent estimates are that 10 million Americans are currently living with cancer, and that 1.2 million Americans are newly diagnosed with cancer each year. Significant advances have been made in improving the diagnosis and treatment of cancer. However, current treatment options often suffer from severe adverse side effects and/or the treatments are not effective for all patients. For example, many clinically-accepted chemotherapeutic agents require the use of large doses of the chemotherapeutic agent that can induce profound damage to normal, proliferative host cells. Another problem associated with many chemotherapeutic treatments is that, in many tumor types, there is either inherent or acquired resistance to the therapy. In an attempt to address these and other shortcomings in current cancer therapy, researchers have investigated the mechanism of cancer cell origination and survival.

[0004] The published scientific literature does not describe a unifying theory for the origination of cancer cells. However, recent research has confirmed that cancer is a disease arising from a patient's own cells and tissue. It is also known that an individual patient may possess multiple tumor cell types, which may not be the same across patients with the same diagnosis or even in the same patient (with disease progression being a further compounding factor).

[0005] Further, it has been observed that the vast majority of fast-growth tumor cells exhibit profound genetic, biochemical, and histological differences with respect to nontransformed cells, including a markedly-modified energy metabolism in comparison to the tissue of origin. The most notorious and well-known energy metabolism alteration in tumor cells is an increased glycolytic capacity even in the presence of a high 0₂ concentration, a phenomenon known as the Warburg effect. Consequently, glycolysis is generally believed to be the main energy pathway in solid tumors.

[0006] Transition to Warburg metabolism requires shutting down the pyruvate

dehydrogenase (PDH) complex because PDH complex activity controls metabolic and malignant phenotype in cancer cells. See, J. Biol Chem 283:22700-8. In this transition, there is enhanced signaling by hypoxia-inducing factor (HIF) in cancer cells, which in turn induces the overexpression of pyruvate dehydrogenase kinase (PDK) 1, which is particularly effective in maintaining an inactive PDH complex. However, alterations in PDK1 observed in cancer may be due to changes in its concentration and also to changes in its activity, and possibly in its amino acid sequence, even between one tumor type or one patient to another. Additionally, PDK1 may form different complexes with various molecules associated with tumors, depending upon the tumor type presented. Recent studies suggest that forcing cancer cells into more aerobic metabolism suppresses tumor growth. Furthermore, PDH complex activation may lead to the enhanced production of reactive oxygen and nitrogen species (RONS), which may in turn lead to apoptosis. Thus, inhibition of PDK may be a potential target in generating apoptosis in tumors. However, to date, known PDK1 inhibitors cause only about 60% inhibition of this isozyme.

[0007] Scientific reports have described a direct correlation between tumor progression and the activities of the glycolytic enzymes hexokinase and phosphofructokinase (PFK) 1, which are substantially increased in fast-growth tumor cells. Accordingly, it has been postulated that tumor cells that exhibit deficiencies in their oxidative capacity are more malignant than those that have an active oxidative phosphorylation. No matter whether under hypoxic or aerobic conditions, then, cancer tissue's reliance on glycolysis is associated with increased malignancy. [0008] Features of the mechanism of cancer cell origination and survival have influenced the design of cancer treatments. For example, because administration of two anti-cancer agents can have a favorable pharmacological interaction (e.g., same target but different toxicities), combination therapy using multiple therapeutic agents has been investigated. In particular, certain personalized cancer combination treatments take advantage of differing mechanistic features of the combination therapy, under which a cocktail of drugs that affect different molecular targets is directed against multiple tumor cell types. Such combination therapies are designed to prevent the emergence of treatment resistant disease and to achieve an additive or synergistic level of tumor cell kill, thereby ensuring treatment efficacy by maximizing the types, and therefore the amount, of tumor cells killed during a course of treatment.

[0009] However, despite the efforts devoted to developing combination therapies to treat cancer, a barrier to major advancements in anti-cancer combination therapy has been a lack of understanding about the intersection of critical cell signaling pathways. For example, synergy might be induced through the effect of multiple drugs either on the same signal pathway, on parallel pathways, or even on seemingly divergent pathways. Overall, because the number of drug combinations is potentially limitless, it remains difficult to identify a priori a strategy for determining the most effective anti-cancer therapeutic combinations.

[0010] Accordingly, the need exists for new therapeutic agents that provide improved efficacy and/or reduced side effects for treating cancer. The present invention addresses this need and provides other related advantages.

SUMMARY

[0011] The invention provides conjugate compounds, pharmaceutical compositions, therapeutic methods, and kits for use in treating cancer and other medical disorders. The conjugates comprise a redox-modulating compound, such as a lipoic acid derivative, chemically bonded to an anti-proliferation agent, such as gemcitabine. The conjugates are contemplated to provide particular advantages in treating patients suffering from a solid tumor, such as pancreatic cancer, breast cancer, or lung cancer.

[0012] Accordingly, one aspect of the invention provides a compound represented by Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein the variables are as defined in the detailed description. Pharmaceutical compositions comprising a compound described herein and a pharmaceutically acceptable carrier are provided.

[0013] Another aspect of the invention provides a method for treating cancer in a patient. The method comprises administering to a patient in need thereof a therapeutically effective amount of a compound described herein, such as a compound of Formula I. In certain embodiments, the cancer is pancreatic cancer, lung cancer, breast cancer, ovarian cancer, a primary melanoma, a metastatic melanoma, liver cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer, or prostate cancer.

[0014] Another aspect of the invention provides medical kits containing compounds and/or pharmaceutical compositions described herein, along with instructions for using the kits to treat a medical disorder, such as cancer.

DETAILED DESCRIPTION

[0015] The invention provides conjugate compounds, pharmaceutical compositions, therapeutic methods, and kits for use in treating cancer and other medical disorders. The conjugates comprise a redox-modulating compound, such as a lipoic acid derivative, chemically bonded to an anti-proliferation agent, such as gemcitabine. The conjugates are contemplated to provide particular advantages in treating patients suffering from a solid tumor, such as pancreatic cancer, breast cancer, or lung cancer. The practice of the present invention employs, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, and biochemistry. Such techniques are explained in the literature, such as "Comprehensive Organic Synthesis" (B.M. Trost & I. Fleming, eds., 1991-1992); which is incorporated by reference. Various aspects of the invention are set forth below in sections; however, aspects of the invention described in one particular section are not to be limited to any particular section.

I. DEFINITIONS

[0016] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0017] The terms "a," "an" and "the" as used herein mean "one or more" and include the plural unless the context is inappropriate

[0018] The term "alkyl" is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., Q-C30 for straight chain, C₃-C₃₀ for branched chain), and alternatively, about 20 or fewer. Likewise, cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, 6 or 7 carbons in the ring structure.

[0019] The term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0020] The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group.

[0021] The terms "alkenyl" and "alkynyl" are art-recognized and refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

[0022] The term "alkylene" refers to a diradical of an alkyl group. An exemplary alkylene group is -CH₂CH₂-. The term "alkynylene" as used herein refers to an alkynyl diradical, such as -C≡C-. The term "alkenylene" as used herein refers to an alkenyl diradical, such as -CH- =CH-.

[0023] The term "aryl" is art-recognized and refers to a carbocyclic aromatic group.

Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like.

[0024] The "heteroaryl" is art-recognized and refers to aromatic groups that include at least one ring heteroatom. In certain instances, a heteroaryl group contains 1, 2, 3, or 4 ring heteroatoms. Representative examples of heteroaryl groups includes pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like.

[0025] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formulas:

wherein R⁵⁰, R⁵¹, R⁵² and R⁵³ each independently represent a hydrogen, an alkyl, an alkenyl, -(CH₂)_m-R⁶¹, or R⁵⁰ and R⁵¹, taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R⁶¹ represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In certain embodiments, only one of R⁵⁰ or R⁵¹ may be a carbonyl, e.g., R⁵⁰, R⁵¹ and the nitrogen together do not form an imide. In other embodiments, R⁵⁰ and R⁵¹ (and optionally R⁵²) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH₂)_m-R⁶¹. The symbol indicates a point of attachment.

[0026] Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)- isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

[0027] As used herein, the term "patient" refers to organisms to be treated by the methods of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines (horses), bovines (cattle), porcines, canines, felines, and the like), and most preferably includes humans.

[0028] As used herein, the term "treating" includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof. [0029] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0030] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers. The compositions also can include stabilizers and

preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Martin,

Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975].

[0031] As used herein, the term "pharmaceutically acceptable salt" refers to any

pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention which, upon administration to a subject, is capable of providing a compound of this invention or an active metabolite or residue thereof. As is known to those of skill in the art, "salts" of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p- sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene- 2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Examples of bases include, but are not limited to, alkali metals (e.g., sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides, ammonia, and compounds of formula NW₄ ⁺, wherein W is C_1-4 alkyl, and the like.

[0032] Further examples of salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate,

cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate,

flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Still other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄ ⁺ (wherein W is a C_1-4 alkyl group), and the like. [0033] In certain embodiments, the pharmaceutically acceptable salts are those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, palicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic. In certain other embodiments, the

pharmaceutically acceptable salts are alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of a carboxylic acid group.

[0034] For therapeutic use, salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0035] Throughout the description, where compositions and kits are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions and kits of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0036] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls. II. Conjugate Compounds

[0037] The invention provides conjugate compounds comprising a redox-modulating compound bonded to an anti-proliferation agent. Exemplary redox-modulating compounds and exemplary anti-proliferation agents that may be bonded together to form an conjugate compound are described below. The redox-modulating compound and anti-proliferation agent may be bonded together using chemical coupling techniques described in, for example,

"Comprehensive Organic Synthesis" (B.M. Trost & I. Fleming, eds., 1991-1992); Carey, F.A. and Sundberg, R. J. Advanced Organic Chemistry Part B: Reactions and Synthesis, 3 Ed.; Plenum Press: New York, 1990; and/or J. March, Advanced Organic Chemistry, McGraw Hill Book Company, New York, (1992, 4^th edition). A. Exemplary Redox-Modulating Compounds

[0038] The redox-modulating compound may be a naturally-occurring or synthetic redox- modulating compound, or an analog or derivative thereof. In certain embodiments, the redox- modulating compound is a naturally-occurring or synthetic alkyl fatty acid, such as octanoic acid, lipoic acid, or a derivative of a lipoic acid such as a compound disclosed in U.S. Patent Nos. 6,331,559 or 6,951,887, or U.S. Patent Application Nos. 12/105,096 or 12/105,100; the contents of each of which are hereby incorporated by reference.

[0039] In certain other embodiments, the redox-modulating compound is an alkyl fatty acid having the general formula:

or a derivative, congener, and salt thereof, wherein:

Ri and/or R₂ is aryl or aralkyl;

P 3 and/or R₄ is S, Se, O, N, aryl, or a metal;

P 5 is alkylene, alkenylene, or alkynylene, with a chain length of one to eighteen carbons;

R₆, is alkyl, alkenyl, alkynyl, aryl, -COOH, -OH, -COH, -NH₂OH, -CC1₃, -CF₃, -NH₂, an amino acid (such as glutamate), a carbohydrate, a nucleic acid, a lipid, and multimers thereof; and

wherein R_l5 R₂, R5, and/or R₆ may be phosphorylated.

[0040] The anti-proliferation agent may be bonded to variable R_l5 R₂, and/or R₆ through an ionic bond, such as a salt, a hydrophobic interaction, a covalent bond, or a covalent reversible or cleavable bond, such as an amide, ether, or ester linkage.

[0041] The alkyl fatty acid compound of Formula II may have asymmetric centers, and the R-isomer of a particular active compound may possess greater physiological activity than does the S-isomer. Consequently, all chiral, diastereomeric, and geometric isomeric forms of a compound embraced by Formula II are intended, unless the specific stereochemistry or isomer form is specifically indicated, and the active compound should be present either solely in its R- or S-isomer form, in racemic mixtures, or various ratios of the R- and S-isomers. Additionally, where carbohydrates are co-formulated, both D and L isomers of the carbohydrates are embraced. Furthermore, to the extent a compound embraced by this general structure may be metabolized within the cell or mitochondrion to form a metabolite, such metabolites are embraced by the invention.

[0042] Further, and more generally, in embodiments where the redox-modulating compound has a stereogenic center, the redox-modulating compound may be provided as the R-isomer. In certain other embodiments, where the redox-modulating compound has a stereogenic center, the redox-modulating compound may be provided as the S-isomer. In certain other

embodiments, where the redox-modulating compound has a stereogenic center, the redox- modulating compound may be provided as a racemic mixture of the R- and S-isomers.

[0043] In certain embodiments, the redox-modulating compound is metabolized (i.e., a metabolite of unnaturally-occurring or synthetic alkyl fatty acid, such as octanoic acid, lipoic acid, or a derivative of a lipoic acid such as a compound disclosed in U.S. Patent Nos.

6,331,559 or 6,951,887, or U.S. Patent Application Nos. 12/105,096 or 12/105,100, or an alkyl fatty acid having the general formula II, described herein above.)

[0044] In certain embodiments, the redox-modulating compound is conjugated to at least one solubility-enhancing polymer containing an activated linkage chemistry moiety. In certain embodiments, the solubility-enhancing polymer is polyethylene glycol. In certain

embodiments, conjugation occurs at R_l5 R₂, and/or R₆ of general formula (II) of the redox- modulating alkyl fatty acid. In certain embodiments, conjugation occurs through an ionic bond, such as a salt. In certain embodiments, conjugation occurs through a hydrophobic interaction. In certain embodiments, conjugation occurs through a covalent bond. In certain embodiments, the covalent bond is reversible. In certain embodiments, the covalent bond is cleavable, such as an amide, ether, or ester linkage.

[0045] In certain other embodiments, the redox-modulating compound is lipoic acid. Lipoic acid (6,8-dithiooctanoic acid) is a sulfur-containing antioxidant with metal-chelating and anti- glycation capabilities. Lipoic acid is the oxidized part of a redox pair, capable of being reduced to dihydrolipoic acid (DHLA). Unlike many antioxidants which are active only in either the lipid or the aqueous phase, lipoic acid is active in both lipid and aqueous phases. The anti- glycation capacity of lipoic acid combined with its capacity for hydrophobic binding enables lipoic acid to prevent glycosylation of albumin in the bloodstream. Lipoic acid is readily absorbed from the diet and is rapidly converted to DHLA by NADH or NADPH in most tissues. Additionally, both lipoic acid and DHLA are antioxidants capable of modulating intracellular signal transduction pathways which use RONS as signaling molecules.

[0046] It is uncertain whether lipoic acid is produced by cells or is an essential nutrient, as differences in intracellular concentration may exist between tissue types as well as between healthy and diseased cells or even between individuals within a species. Mitochondrial pumps or uptake mechanisms, including binding and transport chaperones, may be important in transporting lipoic acid to mitochondria. It is already known that the expression levels and stoichiometry of the subunits comprising many of the lipoic acid-utilizing enzymes, which are linked to energy metabolism as well as growth, development and differentiation, vary with diet and exercise as well as genetics.

[0047] The role of lipoic acid as a cofactor in the PDH complex of healthy cells has been studied. The PDH complex has a central E2 (dihydrolipoyl transacetylase) subunit core surrounded by the El (pyruvate dehydrogenase) and E3 (dihydrolipoyl dehydrogenase) subunits to form the complex; the analogous alpha-ketoglutarate dehydrogenase (a-KDH) and branched chain alpha-keto acid dehydrogenase (BCAKDH) complexes also use lipoic acid as a cofactor. In the gap between the El and E3 subunits, the lipoyl domain ferries intermediates between the active sites. The lipoyl domain itself is attached by a flexible linker to the E2 core. Upon formation of a hemithioacetal by the reaction of pyruvate and thiamine pyrophosphate, this anion attacks the SI of an oxidized lipoate species that is attached to a lysine residue.

Consequently, the lipoate S2 is displaced as a sulfide or sulfhydryl moiety, and subsequent collapse of the tetrahedral hemithioacetal ejects thiazole, releasing the TPP cofactor and generating a thioacetate on the SI of the lipoate. At this point, the lipoate-thioester

functionality is translocated into the E2 active site, where a transacylation reaction transfers the acetyl from the "swinging arm" of lipoate to the thiol of coenzyme A. This produces acetyl- CoA, which is released from the enzyme complex and subsequently enters the TCA cycle. The dihydrolipoate, still bound to a lysine residue of the complex, then migrates to the E3 active site, where it undergoes a flavin-mediated oxidation back to its lipoate resting state, producing FADH₂ (and ultimately NADH) and regenerating the lipoate back into a competent acyl acceptor.

[0048] Because lipoic acid is a cofactor of various dehydrogenase complexes which affect glycolysis, the TCA cycle, and branched-chain amino acid metabolism (e.g., the PDH, a-KDH, and BCAKDH complexes), it is expected that various lipoic acid derivatives may have the same effect in cancer cells.

[0049] In certain other embodiments, the redox-modulating compound comprises 6,8-bis- benzylthio-octanoic acid.

B. Exemplary Anti-Proliferation Agents

[0050] The anti-proliferation agent may be any substance that inhibits cellular proliferation. Types of exemplary anti-proliferation agents include drugs, hormones, vitamins, nutrients, substances, and the like, that are useful in treatment of a disease, condition, syndrome, or symptoms thereof, characterized by cellular hyperproliferation, including cancer. The anti- proliferation agents may be uncharged or charged, nonpolar or polar, natural or synthetic, and thus include small molecule organic compounds, lipophilic polypeptides, cytotoxins, oligonucleotides, cytotoxic antineoplastic agents, antimicrotubule agents, antimetabolites, hormones, and radioactive molecules. The term "oligonucleotides" includes both antisense oligonucleotides and sense oligonucleotides (e.g., nucleic acids conventionally known as vectors). Oligonucleotides may be "natural" or "modified" with regard to subunits or bonds between subunits.

[0051] In certain embodiments, the anti-proliferation agent is an anti-cancer agent. In certain embodiments, the anti-cancer agent is gemcitabine or cytarabine.

[0052] Gemcitabine has the chemical name 2'-deoxy-2',2'-difluoro-cytidine, and has been described in U.S. Patent Nos. 4,808,614 and 5,464,826. Gemcitabine is commercially available as the monohydrochloride salt and as the β-isomer thereof. The mechanism of action of gemcitabine is understood to be its replacement of cytidine during DNA replication, arresting tumor growth through the failure of attachment of new nucleosides which results in apoptosis, and in its irreversible inactivation of ribonucleotide reductase. The commercial formulation of gemcitabine hydrochloride is commonly used in patients previously treated with 5-fluorouracil and is indicated as first-line treatment for patients with locally advanced (nonresectable Stage II or Stage III) or metastatic (Stage IV) adenocarcinoma of the pancreas. Gemcitabine is also used in other carcinomas, including non-small cell lung cancer, pancreatic cancer, bladder cancer and breast cancer. It has been investigated for use in esophageal cancer, and is used experimentally in lymphomas and other tumor types.

[0053] Gemcitabine is a hydrophilic compound and its uptake into cells is largely dependent on the activity of human equilibrative nucleoside transporters (hENTs) and human

concentrative nucleoside transporters (hCNTs). While hENTs are capable of transporting pyrimidine and purine nucleosides both ways across the cell membrane and are widely distributed in most human cells, hCNTs only transport these substrates unidirectionally into the cell and are mainly expressed in hepatic, renal, and intestinal cells. Gemcitabine is

phosphorylated by deoxycytidine kinase and extensively and rapidly deaminated by cytidine deaminase in liver, kidney, and plasma to less cytotoxic metabolites. Because of this rapid deamination, the elimination half-life of gemcitabine is often approximately 10-30 minutes.

[0054] Cytarabine has the chemical name 1-beta-D-arabinofuranosylcytosine, and has been described in U.S. Patent No. 3,116,282. Cytarabine has been reported to interfere with DNA replication, both through steric hindrance caused by cytarabine's attached arabinose, which results in the inability of the molecule to rotate during DNA synthesis and through inhibition of DNA polymerase, which results in a decrease in DNA replication and repair. Cytarabine has been reported for use in the treatment of non-Hodgkin lymphoma and hematological malignancies such as acute myeloid leukemia and acute lymphocytic leukemia.

[0055] Cytarabine is a hydrophilic compound and its uptake into cells is largely dependent on the activity of human equilibrative nucleoside transporters (hENTs) and human

concentrative nucleoside transporters (hCNTs). While hENTs are capable of transporting pyrimidine and purine nucleosides both ways across the cell membrane and are widely distributed in most human cells, hCNTs only transport these substrates unidirectionally into the cell and are mainly expressed in hepatic, renal, and intestinal cells. Cytarabine is

phosphorylated by deoxycytidine kinase and extensively and rapidly deaminated by cytidine deaminase in liver, kidney, and plasma to less cytotoxic metabolites. Because of this rapid deamination, the elimination half-life of cytarabine is often approximately 10-30 minutes. C. Exemplary Conjugate Compounds

[0056] In certain embodiments, the conjugate is a compound represented by Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

R 1 and R 2 each represent independently hydrogen or Z;

A 1 and A 2 each represent independently Q-Cealkyl, aryl or aralkyl;

n is 1, 2, 3, or 4; and provided that there is only one occurrence of Z.

[0057] In certain embodiments, A 1 and A2 are aralkyl. In certain other embodiments, A 1 and A 2 are benzyl. In certain embodiments, n is 3. In certain embodiments, R 1 is Z, and R2 hydrogen. In certain other embodiments, R 2 is Z, and R 1 is hydrogen.

[0058] In certain embodiments, the compound is

pharmaceutically acceptable salt thereof. [0059] In certain other embodiments, the compound is

or a pharmaceutically acceptable salt thereof.

[0060] In certain other embodiments, the conjugate is a compound represented by Formula III:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

R 1 and R 2 each represent independently hydrogen or Z;

A 1 and A 2 each represent independently Q-Cealkyl, aryl or aralkyl;

n is 1, 2, 3, or 4; and provided that there is only one occurrence of Z.

[0061] In certain embodiments, A 1 and A2 are aralkyl. In certain other embodiments, A 1 and A 2 are benzyl. In certain embodiments, n is 3. In certain embodiments, R 1 is Z, and R2 is hydrogen. In certain other embodiments, R 2 is Z, and R 1 is hydrogen. [0062] In certain embodiments, the compound is

or a pharmaceutically acceptable salt thereof.

[0063] In certain other embodiments, the compound is

or a pharmaceutically acceptable salt thereof.

[0064] Compounds described herein may be formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

III. THERAPEUTIC APPLICATIONS

[0065] The invention provides methods for treating medical disorders using a conjugate compound described herein. The methods are contemplated to provide particular advantages in treating cancer. Various aspects of the therapeutic methods are described in detail below.

A. General Therapeutic Methods

[0066] The therapeutic methods described herein are particularly well-suited for treatment of diseases characterized by aberrant cellular metabolism, particularly cellular

hyperproliferation, such as cancer. Exemplary types of cancer contemplated to be treated include a carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, and blastoma. Other types of cancer contemplated for treatment include a primary melanoma, metastatic melanoma, lung cancer (e.g., non-small cell lung cancer), liver cancer, Hodgkin's lymphoma, non- Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer. Further exemplary types of cancer include a benign tumor, malignant solid tumor, hematological malignancy, and cancer-causing stem cells, such as those exhibiting the altered energy metabolism known in the art as the Warburg effect. [0067] Non-limiting examples of other disorders characterized by cellular hyperproliferation and contemplated for treatment include age-related macular degeneration; Crohn's disease; cirrhosis; chronic inflammatory-related disorders; diabetic retinopathy; granulomatosis;

immune hyperproliferation associated with organ or tissue transplantation; an

immunoproliferative disease or disorder (e.g., inflammatory bowel disease, psoriasis, rheumatoid arthritis, or systemic lupus erythematosus); vascular hyperproliferation secondary to retinal hypoxia; and vasculitis.

[0068] Accordingly, one aspect of the invention provides a method for treating a disorder characterized by cellular hyperproliferation in a patient, comprising administering to a patient in need thereof a therapeutically effective amount of a conjugate compound (such as a compound of Formula I or II described herein).

[0069] In certain embodiments, the invention provides a method for treating cancer in a patient, comprising administering to a patient in need thereof a therapeutically effective amount of a conjugate compound described herein (such as a compound of Formula I or II described herein). In certain embodiments, the cancer is a solid tumor or a hematological malignancy. In certain other embodiments the cancer is pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), breast cancer, ovarian cancer, a primary melanoma, a metastatic melanoma, liver cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer, or prostate cancer. In still other embodiments, the cancer is lung cancer or breast cancer. In yet other embodiments, the cancer is pancreatic cancer. In still other embodiments, the cancer is a lymphoma or leukemia.

[0070] In certain embodiments, the compound is administered by intravenous

administration.

[0071] In certain embodiments, the patient is a human.

B. Dosing Amounts & Treatment Cycles

[0072] Generally, the conjugate compound is delivered to the patient in an effective amount. When the medical disorder is cancer, it is preferred that conjugate compound is selectively and specifically delivered to and taken up by a tumor mass and the transformed cells within, and effectively concentrated within the mitochondria of transformed cells, thereby sparing healthy cells and tissue from the effects of the agents. [0073] As is understood, the effective amount of a conjugate compound may vary with the activity of the specific agent employed; the metabolic stability and length of action of that agent; the species, age, body weight, general health, dietary status, sex and diet of the subject; the mode and time of administration; rate of excretion; and extent of presentation and/or severity of the particular condition being treated. The precise dosage can be determined, may involve one or several administrations per day, and the dosage may be adjusted by the individual practitioner to achieve a desired effect. Preferably, the dosage amount of the agent(s) used should be sufficient to interact solely with tumor cells, leaving normal cells essentially unharmed.

[0074] For the purposes of illustration, the dosage amount of a conjugate compound may range from about 0.3 mg/m 2 to 2000 mg/m 2 , or preferably at about 60 mg/m 2. The dosage amount may be administered in a single dose or in the form of individual divided doses, such as from one to four or more times per day. In the event that the response in a subject is insufficient at a certain dose, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent of patient tolerance. Of course, the conjugate compound can be prepared in any amount desired up to the maximum amount that can be administered safely to a patient. For example, the amount of the conjugate compound in a unit dose pharmaceutical composition may range from less than 0.01 mg/mL to greater than 1000 mg/mL, preferably about 50 mg/mL.

[0075] Contemplated advantages of the conjugate compounds and therapeutic methods include (i) effective cancer therapy with minimal side effects for the patient, (ii) the active agent incurs the least possible cost and is capable of being stored for the longest possible period, (iii) can treat diseased cells which modulate tumor cell metabolism in such a way as to culminate in tumor cell death, and (iv) can be used in diseased cells which modulate the structure, function, activity, and/or expression level of the PDH, a-KDH, and/or BCAKDH complexes in such a way as to culminate in tumor cell death. IV. MEDICAL KITS

[0076] Another aspect of the invention provides medical kits containing conjugate compounds and/or pharmaceutical compositions described herein, along with instructions for using the kits to treat a disorder characterized by cellular hyperproliferation, such as cancer. In certain embodiments, the medical kit comprises (i) a conjugate compound described herein (such as a compound of Formula I or II described herein), and (ii) instructions for treating cancer using said conjugate compound.

V. PHARMACEUTICAL COMPOSITIONS

[0077] Conjugate compounds described herein may be formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In further embodiments, the conjugate compounds may be formulated as pharmaceutically-acceptable oils; liposomes; oil- water or lipid-oil- water emulsions or nanoemulsions; liquids; or salts and crystalline forms delivered in tablets or capsules. To facilitate such formulations, the conjugate compounds may be combined with a pharmaceutically-acceptable carrier or excipient therefor. Examples of pharmaceutically-acceptable carriers are well known in the art and include those conventionally used in pharmaceutical compositions, such as salts, lipids, buffers, chelating agents, flavorants, colorants, preservatives, absorption promoters to enhance bioavailability, antimicrobial agents, and combinations thereof, optionally in combination with other therapeutic ingredients.

[0078] As is appreciated, hydrophilic pharmaceutical agents may be rendered hydrophobic for inclusion in oils, liposomes, emulsions, or nanoemulsions by chemical or ionic conjugation to lipophilicity-enhancing moieties, such as but not limited to lipids, aromatic or alkyl chains, carbohydrates, peptides, or amino acids. Such formulations may also include conjugation of the at least one redox-modulating alkyl fatty acid with solubility-enhancing polymers containing an activated linkage chemistry moiety, such as polyethylene glycol (PEG). These conjugations may occur through an ionic bond, such as a salt; a hydrophobic interaction; or through a covalent bond or a covalent reversible or cleavable bond, such as an ester linkage.

[0079] As described in detail below, the pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

[0080] The phrase "therapeutically-effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.

[0081] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0082] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0083] Examples of pharmaceutically- acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0084] Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0085] Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0086] Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more

pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0087] In certain embodiments, the conjugate compound is administered by intraparenteral administration. In certain other embodiments, the conjugate compound is formulated for inhalational, oral, topical, transdermal, nasal, ocular, pulmonary, rectal, transmucosal, intravenous, intramuscular, subcutaneous, intraperitoneal, intrathoracic, intrapleural, intrauterine, intratumoral, or infusion methodologies or administration, or combinations of any thereof, in the form of aerosols, sprays, powders, gels, lotions, creams, suppositories, ointments, and the like. As indicated above, if such a formulation is desired, other additives known in the art may be included to impart the desired consistency and other properties to the formulation.

[0088] The description above describes multiple aspects and embodiments of the invention, including conjugate compounds, pharmaceutical compositions, therapeutic methods, and medical kits. The patent application specifically contemplates all combinations and

permutations of the aspects and embodiments. For example, the invention contemplates treating pancreatic cancer cancer in a human patient by administering a therapeutically effective amount a compound of Formula I, particularly the compound 6,8-bz^'s-benzylthio- octanoic acid-gemcitabine amide conjugate (BBOAG-amide).

EXAMPLES

[0089] The invention now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

EXAMPLE 1- SYNTHESIS OF 6,8-BIS-BENZYLTHIO-OCTANOIC ACID- GEMCITABINE CONJUGATES [0090] Part I: 6,8-5/s-benzylthio-octanoic acid (259.2 mg, 0.667 mmol, 1.0 eq., Mol. Wt. = 388.60 g/mol) was dissolved in dimethyl formamide (DMF, 5 mL) and treated with O-il- Azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 253.6 mg, 0.667 mmol, 1.0 eq., Mol. Wt. = 380.23 g/mol) and triethylamine (0.14 mL, 1 mmol, 1.5 eq.). This reaction mixture was stirred at room temperature for five minutes.

[0091] Part II: In a separate flask (hereinafter Flask II), gemcitabine-HCl (259.2 mg, 0.667 mmol, 1.0 eq., Formula. Wt. = 299.70 g/mol) was suspended in DMF (5 mL) and treated with triethylamine (0.1 mL, 0.667 mol, 1.0 eq). This mixture was stirred for five minutes at room temperature.

[0092] Part III: The contents of the flask containing the activated 6,8-bz^'s-benzylthio- octanoic acid (from Part I) were cannulated into Flask II (from Part II). The pH of the reaction mixture was ensured to be >7 by testing with wet pH paper. The reaction was allowed to proceed overnight at room temperature. Then, the solvent (DMF) was evaporated under reduced pressure at 44 °C to yield the crude product as an oil. The crude product was purified by preparative thin layer chromatography on silica gel: 2 x 1 mm thick plates, eluent: 8% methanol in dichloromethane. The two bands with the highest polarity (lowest Rf) other than the baseline were isolated and the products were recovered by separately eluting the scrapings with 10% methanol in ethyl acetate. These two products, which were verified by mass spectrometry (634 (M + 1), 650 (M + Na)), NMR (DMSO-d<5, at 400 mHz), NMR-COSY and D₂0-exchange experiments, corresponded to the ester and amide formed by the reaction of 6,8- bz^'s-benzylthio-octanoic acid with gemcitabine. The structure of each formed conjugate is provided below.

6,8-bzs-benzylthio-octanoic acid-gemcitabine ester conjugate (hereinafter "BBOAG-ester")

6,8-b/s-benzylthio-octanoic acid-gemcitabine amide conjugate (hereinafter "BBOAG-amide")

EXAMPLE 2- ALTERNATIVE SYNTHESIS OF 6,8-BIS-BENZYLTHIO-OCTANOIC ACID-GEMCITABINE AMIDE CONJUGATE

[0093] 6,8-5zs-benzylthio-octanoic acid-gemcitabine amide conjugate (BBOAG-amide) was prepared according to the procedures illustrated in Scheme 1.

Scheme 1.

[0094] Part I: 6,8-5/s-benzylthio-octanoic acid (660.2 mg, 1.7 mmol, 1.0 eq., Mol. Wt. = 388.60 g/mol) was dissolved in N,N-dimethylformamide (DMF, 3 mL) and treated with O- (Benzotriazol-l-yl)-N,N,N'N'-tetramethyluronium hexafluorophosphate (HBTU, 644.8 mg, 1.7 mmol, 1.0 eq., Mol. Wt. = 379.30) and diisopropylethyl amine (DIEA, 0.6 mL, 3.4 mmol, 2 eq.). This reaction mixture was stirred at room temperature for 2h.

[0095] Part II: In a separate flask, gemcitabine-HCl (500 mg, 1.7 mmol, 1.0 eq., Formula. Wt. = 299.70 g/mol), was suspended in pyridine (3 mL) and acetonitrile (1 mL). The flask was cooled in an ice bath and trimethylsilyl chloride (TMSC1, 0.423 mL, 3.4 mmol, 2.0 eq.) in acetonitrile (5 mL) was added slowly, dropwise. After the addition was complete, the reaction mixture was stirred at room temperature for 2h.

[0096] Part III: The solution in the flask containing the activated 6,8-bz^'s-benzylthio- octanoic acid from the first reaction (Part I) was added dropwise into the flask containing the bis-protected gemcitabine. Dimethylaminopyridine (207 mg, 1.7 mmol, 1.0 eq., M. W. = 122.17) was added, and the reaction mixture was stirred at 70 °C for 48h. Ethanol (5 mL) and water (5 niL) was added, and the reaction mixture was stirred at 45 °C overnight. Volatile solvents were removed at 45 °C, under reduced pressure, and the residue was partitioned between ethyl acetate (100 mL) and water (50 mL). The water phase was re-extracted with ethyl acetate (3 x 50 mL). The combined ethyl acetate layers were washed with 0.1N HC1, water and dried (Na₂S0₄). Evaporation of the solvent gave the crude compound, which was purified by column chromatography on silica gel (using dichloromethane to 8% methanol in dichloromethane as eluent) to provide 880 mg of the title compound (81.7% yield). Spectral properties for the title compound are as follows: M.S. (ESI negative mode): 633 (M-l); M.S. (ESI positive mode): 634 (M + 1); 1H-NMR (CDC1₃) δ: 1.2 - 1.37 (m, 3H), 1.43 (m, 2H), 1.53 (m, 2H), 1.71 (m, 2H), 2.35 (t, 2H), 2.48 (m, 2H), 2.56 (t, IH), 3.63 (d, 4H), 3.88 (m, IH), 4.02 (m, 2H), 4.45 (m, IH), 6.2 (m, IH), 7.17 - 7.22 (m, 2H), 7.23 - 7.3 (m, 10H), 7.45 (d, IH), 8.01 (d, IH), 8.88 (broad, IH), ¹H-coupled- ¹⁹F NMR (CDC1₃) δ: -117.82.

EXAMPLES- /V VITRO ANTI- CANCER ACTIVITY OF 6,8-BIS-BENZYLTHIO- OCTANOIC ACID-GEMCITABINE CONJUGATES [0097] Conjugate compounds prepared in Example 1 were evaluated for in vitro cell killing towards BXPC3 human pancreatic cancer cells and H460 non small lung carcinoma cells. Experimental procedures and results are described below.

Materials and Methods

Materials

[0098] Materials were obtained through normal distribution channels from the manufacturer stated. Materials used include: (i) Costar opaque-walled plate, Corning Costar Corporation, Cambridge, MA, cat. no. 3917, Fisher Scientific cat no.07-200-628; (ii) FLUOstar OPTIMA, BMG LABTECH, Offenburg, Germany; (iii) CellTiter Glo® (CTG) Luminescent Cell Viability Assay, Promega, Fisher Scientific cat no. PR-G7573; (iv) RPMI 1640 Tissue culture medium, Mediatech, Fisher Scientific cat. no. MT-10040-CV; (v) Fetal Bovine Serum (FBS), Fisher Scientific cat. no. MTT35011CV; and (vi) Penicillin and Steptomycin, Fisher Scientific cat. no. MT 30-009-CI. Tumor Cell Lines

[0099] Two human tumor cell types, BXPC3 human pancreatic cancer cells and H460 non small lung carcinoma cells, were used. The BXPC3 and H460 cells were originally obtained from American Type Cell Culture (ATCC). All tumor cells were maintained at 37 °C in a humidified 5% C0₂ atmosphere in T75 tissue culture flasks containing 20 mL of Roswell Park Memorial Institute (RPMI) 1640 containing 2 mM L-glutamine, 10% FBS and 1% penicillin and streptomycin (100 IU/mL penicillin and 100 μg/mL streptomycin). The tumor cells were split at a ratio of 1:5 every 4-5 days by trypsinization and resuspended in fresh medium in a new flask. Cells were harvested for experiments at 70-90% confluency.

Test Articles

[0100] Stock solutions of BBOAG-ester and BBOAG-amide were prepared at a

concentration of 200 mM and 100 mM in DMSO. Five μL· of this solution was diluted in 10.0 mL of 0.5% serum containing RPMI media to give the desired 100 μΜ and 50 μΜ solutions in 0.05% DMSO.

Study Procedures

Design

[0101] The cancer cells were seeded at 4000 cells/well for H460 cells and 6000 cells/well for BXPC3 cells and incubated 24 hours. The cell killing activity of BBOAG-ester and BBOAG-amide were each assayed at 50 μΜ and 100 μΜ concentrations. The tumor cells were treated for 24 hours with the test article, and after 24 hours of treatment the number of viable tumor cells was determined using the CTG assay.

Cell Seeding for Experiments

[0102] Cells were grown to 70-90% confluency, medium was removed, and the cell monolayers were washed briefly by adding 5 mL of phosphate buffer saline (PBS) followed by aspiration. Trypsin-ethylenediaminetetraacetic acid (EDTA) (4 mL) was added to each flask, and the flask was placed in the tissue culture incubator for 5 minutes. Serum-containing medium (10 mL) was added to halt the enzymatic reactions, and cells were disaggregated by repeated resuspension with serological pipette. The cell-containing medium (20 μί) was added to 20 μΐ_^ of 0.4% Trypan Blue solution, mixed, and 10 μΐ_^ of this cell-containing mixture was placed in a chamber of the hemocytometer. The number of viable cells was determined by counting the number of viable cells (cells that excluded Trypan Blue) in the four corner squares of the hemocytometer chamber at lOOx magnification, to get the average number of cells present. The volume of cells needed was determined by the following formula:

where # of cells counted (mL) = average # of cells on hemocytometer x 2 (dilution factor) x 10⁴.

[0103] The number of cells targeted for the study was 4xl0³ per well for H460 cells and 6x10 per well for BXPC3 cells in 100 μΐ_^ of medium. The actual number of cells were counted and seeded in the wells of a 96 well-plate. The cells were incubated for -24 hours before addition of test article.

Treatment with Test Article

[0104] Media in the plate was removed by aspiration, and 100 μΐ_^ of BBOAG-ester or BBOAG-amide at a final concentration of 50 μΜ or 100 μΜ was added to the cells. After exposure to the test articles for 24 hours, the number of viable cells in each well was determined and the percent of viable cells relative to control (in the absence of test article) were calculated. Additionally, a set of wells was treated with cell culture medium in the absence of cells to obtain a value for background luminescence. A separate set of cells was seeded at the same time in a clear 96-well plate and observed under the microscope at 24 hours, following addition of BBOAG-ester or BBOAG-amide to estimate the amount of cells present after treatment.

Determination of the Number of Viable Cells by the CellTiter Glo® Assay

[0105] The number of viable cells was determined by using a CTG assay. Specifically, reagents were mixed and allowed to come to room temperature according to instructions from Promega, Inc. (Madison, WI). Cell plates were removed from the cell culture incubator and left on the bench for 30 minutes until they reached room temperature. 100 μΐ_^ per well of CTG reagent was added with the 12-channel Eppendorf pipettor. The cells were lysed by shaking the plate for two minutes in a shaker. The cells were kept in room temperature for 10 minutes to stabilize the luminescent signal. The luminescence was measured using the FLUOstar OPTIMA plate reader (BMG Labtech, Inc., Durham, NC).

Calculation of Cell Killing Activity

[0106] Data from luminescence readings was copied onto EXCEL spreadsheets, and cell growth relative to untreated cells was calculated, using the following equation:

Results and Conclusion

[0107] The cell killing effects of 6,8-bzs-benzylthio-octanoic acid (BBOA), the ester conjugate compound BBOAG-ester, and the amide conjugate compound BBOAG-amide are provided in Table 1. At the 50 μΜ concentration, BBOAG-ester exhibited higher cell kill effects in both BXPC3 cells and H460 cells than 6,8-bzs-benzylthio-octanoic acid (BBOA).

The gemcitabine amide conjugate compound BBOAG-amide at 50 μΜ concentration exhibited approximately the same amount of cell kill as 6,8-bzs-benzylthio-octanoic acid (BBOA).

However, at the 100 μΜ concentration, the gemcitabine amide conjugate BBOAG-amide showed significantly higher cell kill effects than both 6,8-bzs-benzylthio-octanoic acid (BBOA) and the gemcitabine ester conjugate BBOAG-ester.

EXAMPLE 4- ZV VIVO ANT/CANCER EEEECTS OF 6,8-BIS-BENZYLTHIO- OCTANOIC ACID-GEMCITABINE AMIDE CONJUGATE

[0108] 6,8-5zs-benzylthio-octanoic acid-gemcitabine amide conjugate (BBOAG-amide) was used to treat mice implanted with a human pancreatic xenograft (BXPC-3). Experimental procedures and results are described below

Part I - Experimental Procedures

Animals

[0109] CDl-Nu/Nu female mice, 28 days old obtained from Charles River Laboratories, were used. Mice were housed five to a cage in a micro-isolator room. Light-dark cycles were 12 h each daily, with light from 7 a.m. to 7 p.m. Food (Purina Rodent Chow) and water (distilled sterile-filtered water, pH 7) were provided ad libitum.

[0110] An acclimation period of 7 days was allowed between the arrival of the animals at the study site and tumor inoculation, before the animals were used in experimentation. Mice were then inoculated subcutaneously (SC) in the right flank with 2xl0⁶ tumor cells (human pancreatic BxPC-3 tumor cells). The tumor cells were suspended in 0.1 mL of Dulbeco's Phosphate Buffered Salt (PBS) solution, using a 1 cc syringe with a 27 5/8 gauge needle.

Tumor dimensions (length and width) were measured up to 3 times weekly (using Vernier calipers) and the tumor volume was calculated using the prolate ellipsoid formula: (length x width")/2. The mice were monitored daily for physical conditions and mortality. Body weight was determined immediately prior to administration of test or control articles. Test agents were administered, via intraperitoneal (IP) injection using a 3 cc syringe with a 25 5/8 gauge needle, three times per week for the duration of the study. Treatment began when the tumor was approximately 120-130 mm .

Test Article

[0111] The test articles used in this study were 6,8-bis-benzylthio-octanoic acid (BBOA), and 6,8-bzs-benzylthio-octanoic acid-gemcitabine amide conjugate (BBOAG-amide). BBOA was dissolved in 1M triethanolamine (i.e., 1M triethanolamine in water) at a concentration of 50 mg/mL. Immediately prior to dosing, the 50 mg/mL BBOA solution was diluted to an appropriate concentration with 5% dextrose in water (D5W) such that the dose volume was -80 mL/kg. BBOAG-amide was dissolved in neat DMSO at a concentration of 50 mg/mL.

Immediately prior to dosing, the 50 mg/mL BBOAG-amide solution was diluted to an appropriate concentration with 0.9% NaCl, such that the dose volume was -80 mL/kg.

Tumor Cell Line Origins and Tumor Cell Culture Prior to Inoculation into Mice

[0112] BxPC-3 cells, a human pancreatic tumor cell line was used. Tumor cells were originally obtained from American Type Cell Culture (ATCC) (Manassas, VA). These tumor cells had been tested negative for viral contamination using the Mouse Antibody Production (MAP) test, performed by Charles River Labs Molecular Division, upon the receipt of the tumor cells from ATCC.

[0113] The tumor cells were maintained at 37°C in a humidified 5% C0₂ atmosphere in T225 tissue culture flasks containing 50 mL of Roswell Park Memorial Institute (RPMI)-1640 solution with 10% Fetal Bovine Serum (FBS) and 2 mM L-glutamine. Cells were split at a ratio of 1:3 every 4-5 days by trypsinization and resuspended in fresh medium in a new flask. Cells were harvested for experiments in the same way at 70-90% confluency.

Study Design and Procedures

[0114] Six treatment groups of mice (n=6/group) with BxPC-3 xenograft were subjected to test agents injected intraperitoneally (IP) via a 3 cc syringe with a 25 5/8 gauge needle, three times per week for the duration of the study. There was one group of six tumor bearing mice (n= 6 ) that was used as the non-treated control group. Parameters are provided in Table 1.

Variables Assessed During the Study

[0115] Mice were monitored daily for physical conditions and mortality. Per IACUC rules, mice were euthanized if the tumor size exceed 2-cm in diameter; display symptoms of pain or distress; evident by sudden rapid weight loss (>15% of original weight); lethargic behavior; difficulty in moving; vocalization; changes in respiratory rate, overall appearance and activity; signs of erythema, ulceration, infection, necrosis of the tumor; unable to reach food and water; and symptoms of pain or distress. [0116] Mice that survived were those that did not die, or were not euthanized per IACUC rules.

Tumor Size Measurements

[0117] Tumor dimensions (length and width) were measured using Vernier calipers and the tumor volume was calculated using the prolate ellipsoid formula: (length x width )/2. Tumor volume was assessed twice weekly and immediately prior to each administration of test or control articles.

Part II - Results

[0118] Results are provided in Tables 2-4 below. Table 2 provides the average tumor volume in mice treated with BBOAG-amide. Results in Table 2 show that administration of BBOAG-amide to mice with human pancreatic xenograft provides an anti-cancer effect. Table 3 provides the average tumor volume in mice treated with BBOA. Table 4 provides the number of animals that died in each treatment group of six animals receiving BBOA, BBOAG- amide, or no treatment (i.e., a control group).

INCORPORATION BY REFERENCE

[0119] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[0120] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0121] WHAT IS CLAIMED IS:

Claims

1. A compound represented by Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

R 1 and R 2 each represent independently hydrogen or Z;

A 1 and A 2 each represent independently Q-Cealkyl, aryl or aralkyl;

n is 1, 2, 3, or 4; and

provided that there is only one occurrence of Z.

2. The compound of claim 1, wherein A 1 and A 2 are aralkyl.

3. The compound of claim 1, wherein A 1 and A 2 are benzyl.

4. The compound of any one of claims 1-3, wherein n is 3.

5. The compound of any one of claims 1-4, wherein R 1 is Z, and R 2 is hydrogen.

6. The compound of any one of claims 1-4, wherein R 2 is Z, and R 1 is hydrogen.

7. The compound of claim 1, wherein the compound is

a pharmaceutically acceptable salt thereof.

8. The compound of claim 1, wherein the compound is

or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition comprising a compound of any one of claims 1-8 and a pharmaceutically acceptable carrier.

10. A method for treating cancer in a patient, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of claims 1-8.

11. The method of claim 10, wherein the cancer is a solid tumor or a hematological

malignancy.

12. The method of claim 10, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, ovarian cancer, a primary melanoma, a metastatic melanoma, liver cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer, or prostate cancer.

13. The method of claim 10, wherein the cancer is lung cancer or breast cancer.

14. The method of claim 10, wherein the cancer is pancreatic cancer.

15. The method of any one of claims 10-14, wherein the conjugate compound is administered by intravenous administration.

16. The method of any one of claims 10-15, wherein the patient is a human.