WO2009032172A2

WO2009032172A2 - Platinum compositions as treatment for oct-related cancers

Info

Publication number: WO2009032172A2
Application number: PCT/US2008/010213
Authority: WO
Inventors: Stephen J. Lippard; Katherine S. Lovejoy
Original assignee: Massachusetts Institute of Technology
Current assignee: Massachusetts Institute of Technology
Priority date: 2007-08-31
Filing date: 2008-08-28
Publication date: 2009-03-12
Anticipated expiration: 2010-02-28
Also published as: WO2009032172A3

Abstract

The present invention provides compositions, preparations, formulations, kits, and methods useful for treating subjects having cancer or at risk of developing cancer, in some cases, cancers which express an organic cation transporter (OCT). Some embodiments of the invention may comprise a compound including platinum (e.g., platinum(II) or platinum(IV)) and at least one organic ligand. In some embodiments, the compound may comprise an organic ligand which enhances interaction between the compound and an OCT.

Description

PLATINUM COMPOSITIONS AS TREATMENT FOR OCT-RELATED CANCERS

Statement Regarding Federally Sponsored Research or Development This invention was made with the support under the following government contract: 5-R37-CA034992-25 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field of the Invention

The present invention relates to compositions, kits, and methods for treatment of cancers expressing organic cation transporters (OCTs).

Background of the Invention

Platinum-based drugs are among the most active and widely-used anticancer agents and cisplatin represents one of three FDA-approved, platinum-based cancer chemotherapeutics. Although cisplatin is effective against a number of solid tumors, especially testicular and ovarian cancer, its clinical use has been limited because of its toxic effects as well as the intrinsic and acquired resistance of some tumors to this drug. To overcome these limitations, platinum analogs with lower toxicity and greater activity in cisplatin-resistant tumors have been developed and tested, resulting in the approval of carboplatin and oxaliplatin in the United States (see FIG. IA). Carboplatin has the advantage of being less nephrotoxic, but its cross-resistance with cisplatin has limited its application in otherwise cisplatin-treatable diseases. Oxaliplatin, however, exhibits a different anticancer spectrum from that of cisplatin. It has been approved as the first or second line therapy in combination with 5-fluoruracil/leucovorin for advanced colorectal cancer, for which cisplatin and carboplatin are essentially inactive. In spite of their distinct antitumor specificities, cisplatin and oxaliplatin, as well as other platinum compounds, share similar mechanisms of action. In particular, their cytotoxicity arises primarily from covalent binding to DNA after aquation to form mono- and diaqua complexes, initiating a series of biochemical cascades and eventually leading to cell death.

Because cisplatin and oxaliplatin target similar DNA sites for binding and form similar types of DNA adducts, mainly 1,2- and 1,3-intrastrand cross-links involving purine nucleotides, the mechanisms responsible for their distinct tumor specificities may involve events other than their interaction with and binding to DNA. Studies aiming to identify such mechanisms have focused largely on the cellular processing of cisplatin- and oxaliplatin-DNA adducts. However, as described herein, differences in the mechanism(s) controlling the cellular uptake and efflux of these platinum compounds, although rarely investigated, may also be important, since reduced intracellular accumulation is a common observation in cisplatin-resistant cells.

Recent studies suggest a direct involvement of the human copper influx transporter, Ctrl, in the cellular uptake of cisplatin, carboplatin and oxaliplatin to a varying extent (Song et al., 2004). Studies in tumor cell lines suggest, however, that Ctrl may not affect the formation and corresponding cytotoxicity of cisplatin-DN A adducts (Holzer et al., 2004). The human copper efflux transporters, ATP7B and

ATP7A, also recognize these platinum compounds (Komatsu et al., 2000; Samimi et al., 2004a; Samimi et al., 2004b) and their elevated expression has been associated with cisplatin resistance (Aida et al., 2005; Miyashita et al., 2003; Nakayama et al., 2004; Samimi et al., 2003). The importance of these interactions in modulating the differential activity and tumor specificity of the platinum compounds is currently unknown.

The interaction between an agent and an organic cation transporter (OCT) may be important for the development of treatment for cancers which express OCTs. The organic cation transporters (OCTs), OCTl, OCT2, and OCT3 are in the class of plasma membrane transporters belonging to the solute carrier (SLC) 22A family. The OCTs mediate intracellular uptake of a broad range of structurally diverse organic cations. In some cases, the organic cations have molecular weights of 400 Da or less. Substrates of OCTs include endogenous compounds, such as choline, creatinine and monoamine neurotransmitters, and a variety of xenobiotics such as tetraethylammonium (TEA, a prototypic organic cation), l-methy-4-phenylpyridinium (MPP+, a neurotoxin) and clinically used drugs such as metformin, cimetidine and amantadine. In humans, OCTl is primarily expressed in the liver and less so in the intestine, whereas OCT2 is predominantly expressed in the kidney. OCT3 is expressed in many tissues including placenta, heart, liver and skeletal muscle. The expression of the OCTs has also been detected in a number of human cancer cell lines. OCTl and OCT2 have been identified as critical mediators of oxaliplatin transport and toxicity in human tissue. mRNA from OCTl was detected in 20 of 20 tumor samples from colon cancer patients and mRNA from OCT2 was detected in 11 of 20 samples. Oxaliplatin is a neutral compound and is typically transported after loss of the oxalate group to form mono- or dicationic species. Although oxaliplatin and cisplatin form similar adducts on DNA, cisplatin is a poor substrate for OCTl and OCT2 and is less active against colorectal cancer than oxiplatin.

European Patent 0 199 524 describes the use of some platinum(II) complexes, other than cisplatin, carboplatin, and oxaliplatin, in treating cancer in general. However, the use of platinum(II) complexes for specifically treating cancers which express OCT has not been described.

Accordingly, improved compositions and methods are needed.

Summary of the Invention

The present invention provides methods for treating a subject having a cancer which expresses an organic cation transporter (OCT), comprising administering a therapeutically-effective amount of a compound having the formula,

wherein R¹, R², and R³ can be the same or different and each is a group comprising at least one of ammonia, an amine, or a leaving group, any being optionally substituted, or, any two or three of R¹, R², and R³ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted; R⁴ is a group comprising a heterocycle including at least one nitrogen, or a group comprising an aryl group, any being optionally substituted; R⁵, R⁶, R⁷, and R⁸ can be the same or different and each is a group comprising at least one of ammonia, an amine, a heterocycle including at least one nitrogen, an aryl group, or a leaving group, any being optionally substituted, or, any two or three of R⁵, R⁶, R⁷ and R⁸ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted; R⁹ and R¹⁰ can be the same or different and are groups comprising hydroxyl, alkoxy, aryloxy, or acyloxy, any being optionally substituted; X is a counterion; and n and m are 1 or n and m are 2; and wherein the compound has a molecular weight of 700 g/mol or less, to a subject having a cancer which expresses an OCT. In some embodiments, the subject is otherwise free of indications for treatment with said compound.

The present invention also provides methods comprising promoting the inhibition or treatment of a cancer which expresses OCT in a subject susceptible to or exhibiting symptoms of a cancer which expresses OCT via administration to the patient of a composition comprising a compound having any of the formulas described above.

The present invention also provides kits for treatment of a cancer which expresses an OCT, comprising a composition comprising a compound having any of the formulas described above, and instructions for use of the composition for treatment of a cancer which expresses an OCT.

The present invention also relates to a composition of matter comprising a compound having the formula,

wherein R , R , and R can be the same or different and each is a group comprising at least one of ammonia, an amine, or a leaving group, any being optionally substituted, or, any two or three of R¹, R², and R³ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted; R⁴ is a heterocycle including at least one nitrogen, or a group comprising an aryl group, any being optionally substituted; R⁵, R⁶, R⁷, and R⁸ can be the same or different and each is a group comprising at least one of ammonia, an amine, a heterocycle including at least one nitrogen, an aryl group, or a leaving group, any being optionally substituted, or, any two or three of R⁵, R⁶, R⁷ and R⁸ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted; R⁹ and R¹⁰ can be the same or different and are groups comprising hydroxyl, alkoxy, aryloxy, or acyloxy, any being optionally substituted; X is a counterion; and n and m are 1 or n and m are 2; wherein the compound has a molecular weight of 700 g/mol or less.

The present invention also relates to a composition of matter comprising a compound having the formula, R² R¹-Pt-R³

wherein R¹, R², and R³ can be the same or different and each is a group comprising at least one of ammonia, an amine, or a leaving group, any being optionally substituted, or, any two or three of R¹, R², and R³ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted, wherein at least two of R¹, R², and R³ are leaving groups; R⁴ is a heterocycle including at least one nitrogen, or a group comprising an aryl group, any being optionally substituted; X is a counterion; and wherein the compound has a molecular weight of 700 g/mol or less.

The present invention also relates to pharmaceutical compositions comprising any of the compositions and/or compounds described above or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, additives, and/or diluents. The present invention also relates to compositions for treating a subject having a cancer which expresses an OCT, wherein the composition comprises any of the compositions and/or compounds described above. The present invention also relates to the use of any of the compositions and/or compounds described above in the preparation of a medicament for treating a subject having a cancer which expresses an OCT.

Brief Description of the Drawings FIGS. IA-B show examples of platinum compounds. FIG. 2 A shows a plot of cytotoxicity of cells expressing OCTl (white circles) and of corresponding MOCK cells (black circles).

FIG. 2B shows a plot of cytotoxicity of cells expressing OCT2 (white circles) and of corresponding MOCK cells (black circles).

FIG. 2C shows a plot of cytotoxicity of cells expressing OCT3 (white circles) and of corresponding MOCK cells (black circles).

FIG. 2D shows the cytotoxicity of oxaliplatin in cells expressing OCTl (white symbols) and in the corresponding empty vector-transfected cells (MOCK cells) (black symbols) in the presence (squares) or absence (circles) of disopyramide, an OCT inhibitor. FIG. 2E shows the cytotoxicity of oxaliplatin in cells expressing OCT2 (white symbols) and in the corresponding empty vector-transfected cells (MOCK cells) (black symbols) in the presence (squares) or absence (circles) of cimetidine, an OCT inhibitor.

FIG. 3 A shows the cellular accumulation rates of platinum in cells expressing OCTl and in the corresponding MOCK cells after a 2-hour exposure to cisplatin, carboplatin and oxaliplatin, in the presence (white bars) and absence (black bars) of disopyramide, an OCT inhibitor.

FIG. 3B shows the cellular accumulation rates of platinum in cells expressing OCT2 and in the corresponding MOCK cells after a 2-hour exposure to cisplatin, carboplatin and oxaliplatin, in the presence (white bars) and absence (black bars) of cimetidine, an OCT inhibitor.

FIG. 3 C shows the cellular accumulation rates of platinum in cells expressing OCT3 and in the corresponding MOCK cells after 2-hour exposure to cisplatin, carboplatin and oxaliplatin, in the presence (white bars) and absence (black bars) of cimetidine, an OCT inhibitor.

FIG. 4 A shows a graph of the content of platinum bound to DNA for OCTl- transfected MDCK cells after a 2-hr exposure to oxaliplatin in the presence (white bars) or absence (black bars) of disopyramide, an OCT inhibitor.

FIG. 4B shows a graph of the content of platinum bound to DNA for OCT- transfected MDCK cells after 2-hr exposure to oxaliplatin in the presence (white bars) or absence (black bars) of cimetidine, an OCT inhibitor.

FIG. 5 shows platinum-DNA adduct formation after incubation with oxaliplatin or [Pt(J?, i?-DACH)(H₂O)₂]²⁺ in phosphate buffer with either sodium chloride (PB-Cl) or sodium sulfate (PB-SO₄) buffer. FIG. 6 shows the expression of OCTl and OCT2 in colon cancer cell lines and colon tissue samples.

FIG. 7A shows the expression of human OCTl, OCT2 and OCT3 in stably transfected cell lines, as seen in lanes 1-6: MDCK-MOCK, MDCK-hOCTl, HEK- MOCK, HEK-hOCT2, HEK-MOCK and HEK-hOCT3. FIG. 7B shows the cellular uptake of model substrates (TEA for human OCTl and OCT2, and MPP⁺ for human OCT3) in OCT-transfected cells (black bars) and in the corresponding MOCK cells (white bars). Disopyramide (120 μM) was used as an inhibitor for human OCTl and cimetidine (1.5 mM) was used as an inhibitor for human

0CT2 and 0CT3. Data are expressed as mean ± SD of six measurements.

FIG. 8 shows a table listing primers used for RT-PCR.

FIG. 9 shows data for growth inhibition properties and ability to act as OCT substrates of several platinum compounds of the invention.

FIG. 1OA shows a graph of the comparison of growth inhibition for the ten of the platinum compounds studied, expressed as IC₅₀ in uM. The smaller bars indicate that the compound exhibits more effective growth inhibition.

FIG. 1OB shows a graph of compounds tested as hOCTl and hOCT2 substrates. Values on the ordinate are expressed as the "fold difference" in IC_5O between cells with the transporter and cells without. Larger bars indicate an enhanced ability of the compound to serve as hOCTl or hOCT2 substrates.

FIG. 11 shows the structures of cisplatin, oxaliplatin and cDPCP.

FIG. 12A shows a graph of the cell growth inhibition assay for cDPCP in MDCK cells (i) with hOCTl and (ii) without hOCTl .

FIG. 12B shows a graph of the cell growth inhibition assay for oxaliplatin in MDCK cells (i) with hOCTl and (ii) without hOCTl.

FIG. 12C shows a table of IC_5O values for cDPCP and oxaliplatin in various cells.

FIG. 13 shows (a) a schematic diagram of the X-ray crystal structure of cDPCP- modified DNA showing the DNA sequence and location of the platinum adduct for the complex which was studied by X-ray crystallography; (b) the structure of the cDPCP- damaged DNA duplex; (c) a close-up view of the monofunctional Pt-dG adduct of the structure in FIG. 13B; and (d) a platinated base pair overlaid with an ideal B-form DNA.

FIG. 14A shows stereoscopic views of the cDPCP-dG adduct on duplex DNA. FIG. 14B shows an electron density map of the adduct shown in FIG. 14 A.

FIG. 15A shows a graph of the percentage of repair of cisplatin and cDPCP as a function of time.

FIG. 15B shows a portion of a gel electrophoresis study comparing the repair of cisplatin and [Pt(dien)Cl]Cl adducts. FIG. 15C shows a plot comparing the successful transcription bypass and repair of various Pt-DNA adducts.

FIG. 16A shows a schematic representation of the active site of RNA polymerase II, with a cDPCP-dG adduct modeled into templated DNA at the +1 site. FIG. 16B shows a schematic representation of the space filling views of the Pt adduct given in FIG. 16 A

FIG. 17 shows DNA unwinding by agarose gel electrophoresis for (a) cisplatin and (b) cDPCP. FIG. 18 shows a graph of the results of r_f vs. r_b determination for platination with cDPCP and cisplatin on pBR322 plasmid DNA.

FIG. 19 shows a graph of the results of r_f vs. r_b determination for platination with cDPCP and cisplatin on pSV-β-galactosidase plasmid DNA.

FIG. 20 shows a graph of the bypass of various platinum adducts by the transcribing complex as assayed in live cells using a platinated pS V-β-galactosidase reporter plasmid.

FIG. 21 shows a diagram of site-specifically platinated probe assembly and in vitro assay for assessing repair by the excision repair pathway.

FIG. 22 gives the sequences of DNA oligomer components of the 156mer NER probe.

FIGS. 23 A and 23B show representative results of gel electrophoresis studies of nucleotide excision repair products.

Other aspects, embodiments and features of the invention will become apparent from the following detailed description when considered in conjunction with the accompanying drawings. The accompanying figures are schematic and are not intended to be drawn to scale. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. All patent applications and patents incorporated herein by reference are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Detailed Description

The invention provides compositions, preparations, formulations, kits, and methods useful for treating subjects having cancer or at risk of developing cancer. In some embodiments, methods and compositions of the invention are useful for treating cancers which express an organic cation transporter (OCT). In some aspects, the invention provides compounds and related compositions for use in treating subjects known to have (e.g., diagnosed with) cancer or subjects at risk of developing cancer. In some embodiments, methods of the invention include administering to a subject a therapeutically effective amount of a compound, or a therapeutic preparation, composition, or formulation of the compound as described herein, to a subject having a cancer which expresses an OCT, who is otherwise free of indications for treatment with said compound. In a particular embodiment, the subject is a human.

The present invention relates to the discovery that compounds comprising platinum(II) or platinum(IV) and at least one organic ligand may be particularly effective in the treatment of cancers which express an OCT. The compound may be, for example, a Pt(II) complex or a Pt(IV) complex. The organic ligand may facilitate interaction between the compound and cells, or portions thereof, associated with the OCT-related cancer. For example, the compound may comprise an organic ligand which enhances interaction between the compound and an OCT, which may facilitate cellular uptake of the compound. In some cases, the compound may comprise one or more organic ligands.

In some embodiments, the organic ligand is a non-leaving group, that is, the organic ligand does not dissociate from the compound or is not replaced by another ligand during, for example, cellular uptake, activation in the cell, interaction with a DNA molecule, and/or other processes involved with the treatment of the OCT-related cancer. The compound may also comprise one or more organic or inorganic leaving groups. Additional ligands may coordinate to the metal center, including neutral ligands and/or charged ligands. Neutral ligands include ligands which may coordinate the metal center but do not alter the oxidation state of the metal center. For example, solvent molecules such as water, ammonia, pyridine, and acetonitrile may be neutral ligands. Charged ligands include ligands which may coordinate the metal center and may alter the oxidation state of the metal center. Examples of charged ligands include halides, carboxylates, and the like.

Some embodiments of the invention may comprise a ligand comprising at least one cationic (e.g., positively-charged) group. For example, the compound may be a salt comprising a cation and anion (e.g., counterion). In some cases, the compound may be a neutral compound and a ligand bound to the compound may comprise a cationic group. The compound may also comprise more than one cationic group. In some embodiments, the invention provides compounds having any of the following structures as effective agents against cancers which express OCT:

R² R⁹ R²

. I R¹-Pt— R³ R¹-Pt— R³

R ^R6K'k I .':^RR⁸⁷

R¹⁰

wherein:

R¹, R , and R³ can be the same or different and each is a group comprising at least one of ammonia, an amine, or a leaving group, any being optionally substituted, or, any two or three of R¹, R², and R³ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted;

R⁴ is a group comprising a heterocycle including at least one nitrogen, or a group comprising an aryl group, any being optionally substituted;

R⁵, R⁶, R⁷, and R⁸ can be the same or different and each is a group comprising at least one of ammonia, an amine, a heterocycle including at least one nitrogen, an aryl group, or a leaving group, any being optionally substituted, or, any two or three of R⁵, R⁶, R⁷ and R⁸ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted;

R⁹ and R¹⁰ can be the same or different and are groups comprising hydroxyl, alkoxy, aryloxy, or acyloxy, any being optionally substituted;

X is a counterion; and n and m are 1 or n and m are 2.

In some embodiments, at least one of R¹, R², and R³ is a leaving group. In some embodiments, at least two of R¹, R², and R³ is a leaving group.

In some embodiments, at least one of R⁵, R⁶, R⁷, and R⁸ is a leaving group. In some embodiments, at least two of R⁵, R⁶, R⁷, and R⁸ is a leaving group.

In some embodiments, R⁴ is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine-l,9-diamine, benzylamine, or substituted derivatives thereof. In some embodiments, R⁴ is pyridine. In some embodiments, R⁴ is benzylamine.

In some embodiments, at least one of R⁵, R⁶, R⁷, and R⁸ is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine-l,9-diamine, benzylamine, or substituted derivatives thereof.

In some embodiments, at least one of R⁵, R⁶, R⁷, and R⁸ comprises a cationic group. For example, the compound may comprise a ligand including a cationic group such as N-methylpyridinium. In some embodiments, compounds of the invention may comprise a bidentate ligand which, when bound to a metal center, forms a metallacycle structure with the metal center. Bidentate ligands suitable for use in the present invention include species which have at least two sites capable of binding to a metal center. For example, the bidentate ligand may comprise at least two heteroatoms that coordinate the metal center, or a heteroatom and an anionic carbon atom that coordinate the metal center. Examples of bidentate ligands suitable for use in the invention include, but are not limited to, alkyl and aryl derivatives of moieties such as amines, phosphines, phosphites, phosphates, imines, oximes, ethers, hybrids thereof, substituted derivatives there of, aryl groups (e.g,. bis-aryl, heteroaryl-substituted aryl), heteroaryl groups, and the like. Specific examples of bidentate ligands include ethylene diamine, 2,2'-bipyridine, acetylacetonate, oxalate, and the like.

In some embodiments, compounds of the invention may comprise a tridentate ligand, which includes species which have at least three sites capable of binding to a metal center. For example, the bidentate ligand may comprise at least three heteroatoms that coordinate the metal center, or a combination of heteroatom(s) and anionic carbon atom(s) that coordinate the metal center.

In some cases, the compound may comprise a "tethering group" to facilitate the transport of the compound into cells, i.e., via an OCT. In some embodiments, the compound comprises an organic ligand (e.g., tethering group) that may interact with an OCT to enhance transport across the cellular membrane and may then dissociate from the compound upon reduction of the compound within the cell. For example, the tethering group may be positioned in an axial position of a Pt(IV) compound, such that the compound is reduced inside the cell to form a Pt(II) compound via loss of the axial payload. In some cases, the tethering group may comprise a group having a positive charge, such as N-methylpyridinium. In some cases, the tethering group may comprise an aromatic group. Examples of such compounds include the following,

Some embodiments of the invention comprise one or more leaving groups. As used herein, a "leaving group" is given its ordinary meaning in the art and refers to an atom or a group capable of being displaced by a nucleophile. Examples of suitable leaving groups include, but are not limited to, halides (such as chloride, bromide, and iodide), alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy, carboxylate), arylcarbonyloxy, mesyloxy, tosyloxy, trifluoromethane-sulfonyloxy, aryloxy, methoxy, N,0-dimethylhydroxylamino, pixyl, oxalato, malonato, and the like. A leaving group may also be a bidentate, tridentate, or other multidentate ligand. In some embodiments, the leaving group is a halide or carboxylate. In some embodiments, the leaving group is chloride.

Some embodiments of the invention comprise compounds having two leaving groups positioned in a cis configuration, i.e., the compound may be a cis isomer. However, it should be understood that compounds of the invention may also have two leaving groups positioned in a trans configuration, i.e., the compound may be a trans isomer. Those of ordinary skill in the art would understand the meaning of these terms.

In some embodiments, R⁹ and R¹⁰ can be the same or different and each is hydroxyl, phenoxide, or 2-[(2-carboxyacetoamido)methyl]-l-methylpyridinium.

In some embodiments, the compound has a molecular weight of 700 g/mol or less (e.g., 700 Da or less). Some embodiments of the invention provide the compound as a salt comprising a positively-charged platinum complex and a counterion (e.g., "X"). The counterion X may be a weak or non-nucleophilic stabilizing ion. In some cases, the counterion is a negatively-charged and/or non-coordinating ion. Examples of counterions include halides, such as chloride.

In one set of embodiments, the compound has the structure,

wherein R¹ and R² can be the same or different and each is a leaving group; R⁴ is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine- 1 ,9-diamine, benzylamine, or substituted derivatives thereof. In some embodiments, the leaving group is a halide. In some embodiments, the leaving group is chloride. In one embodiment, the compound has the structure,

wherein X is a counterion such as chloride (e.g., c/s-diammine(pyridine)chloro- platinum(II) or " cDPCP")- In other embodiments, the compound may be trans- diammine(pyridine)chloroplatinum(II).

In another embodiment, the compound has the structure,

In one embodiment, the compound has the structure,

In one set of embodiments, the compound has the structure,

wherein R⁷ is a leaving group; R⁸ is a group comprising a heterocycle including at least one nitrogen, an aryl group, or a leaving group, any being optionally substituted, or, any two or three of R⁵, R⁶, R⁷ and R⁸ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted; and R⁹ and R¹⁰ can be the same or different and are groups comprising hydroxyl, alkoxy, aryloxy, or acyloxy, any being optionally substituted. In some embodiments, the leaving group is a halide. In some embodiments, the leaving group is chloride.

Some examples of compounds of the invention are shown in FIG. IB, wherein X is a counterion. Other non-limiting examples of compounds of the invention include [Pt(dien)Cl]X, where X is a counterion and "dien" is diethylenetriamine, and [PtIA₂L], where A₂ is 2,9-dimethyl-l,10-phenanthroline and L is an N-donor heterocyclic ligand (e.g., 1-methylcytosine).

Pt(II) and Pt(IV) complexes of the invention may be synthesized according to methods known in the art, including various methods described herein. For example, the method may comprise reaction of cisplatin with one or more ligand sources. In some cases, a Pt(IV) complex can be obtained by reaction of the parent Pt(II) species with, for example, hydrogen peroxide at temperatures ranging between 25-60°C in an appropriate solvent, such as water or N,N-dimethylformamide.

One aspect of the invention is directed to a method for treating a subject having a cancer which expresses an organic cation transporter (OCT), wherein the method comprises administering a therapeutically-effective amount of a compound, as described herein, to a subject having a cancer which expresses an OCT, who is otherwise free of indications for treatment with said compound. In some cases, the cancer expresses hOCTl . In some cases, the cancer expresses hOCT2. OCT-related cancers may typically be found in the colon, liver, and kidney, though it should be understood that OCT-related cancers may be found in other locations as well. Examples of cancers which express OCT include colorectal, rectal, and intestinal cancer. As used herein, a "cancer which expresses an organic cation transporter (OCT)" or an "OCT-related cancer" refers to a cancer comprising cells which have an N-[methyl-3H]-4- phenylpyridinium ([3H]MPP⁺) uptake that is reduced by at least 50% in the presence of an OCT inhibitor, such as disopyramide (hOCTl) inhibitor or cimetidine (hOCT2 inhibitor), relative to their [3H]MPP⁺ uptake in the absence of an OCT inhibitor. In some cases, the cancer which expresses OCT comprises cells which have a [3H]MPP⁺ uptake that is reduced by at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or, in some cases, greater, relative to their [3H]MPP⁺ uptake in the absence of an OCT inhibitor.

Those of ordinary skill in the art would be able to determine which cancers express OCT using screening tests. For example, samples (e.g., cells) of a particular cancer may be incubated with [3H]MPP⁺, wherein one sample is incubated in the presence of a specified OCT inhibitor and one sample is incubated in the absence of an OCT inhibitor. For each sample, aliquots of the cell Iy sates may be used for scintillation counting and bicinchoninic acid (BCA) protein assay to determine the [3H]MPP⁺ uptake for both samples. Comparison of the [3H]MPP⁺ uptake for the samples may be used to determine if the uptake is sufficiently reduced by the presence of an OCT inhibitor, thereby determining whether or not the cancer expresses an OCT.

In some cases, the invention provides methods for treating a subject having a cancer which expresses a high level of OCT. As used herein, a cancer which expresses a "high level of OCT" refers to a cancer that comprises mRNA of genes that encode OCT in an amount that is at least 50% of the amount found in HCT-116 colorectal adenocarcinoma cells, as measured by real-time RT-PCR. For example, the cancer may comprise mRNA of genes that express OCT in an amount that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, of the amount found in HCT-116 colorectal adenocarcinoma cells, as measured by real-time RT-PCR. In one set of embodiments, the cancer may comprise mRNA of genes that express hOCTl or hOCT2 in an amount that is at least 75% of the amount found in HCT-116 colorectal adenocarcinoma cells, as measured by real-time RT-PCR. Examples of cancers that express a high level of OCT (e.g., hOCTl or hOCT2) include colorectal cancers, as well as other cancers that may be found in the colon, liver, and kidney.

In some embodiments, the subject is otherwise free of indications for treatment for sarcomas, lymphoid leukemias, lymphosarcoma myelocytic leukemia, malignant lymphoma, squamous cell carcinoma, adenocarcinoma, scirrhous carcinoma, malignant melanoma, seminoma, teratoma, choriocarcinoma, embryonalcarcinoma, cystadenocarcinoma, endometriocarcinoma, or neuroblastoma.

In some embodiments, the method involves providing a subject that has been identified (e.g., diagnosed) as having, or being at risk for having, an OCT-related cancer. That is, a diagnostic method has been applied to the subject to determine, for example, the presence and/or amount of an organic cation transporter and/or mRNA of genes that encode OCT within the subject. The diagnostic method may involve evaluating indication of an OCT-related cancer or the potential for an OCT-related cancer based upon the determination of an organic cation transporter and/or mRNA of genes that encode OCT within the subject, thereby determining that the subject is known to be at risk for an OCT-related cancer or has an OCT-related cancer. For example, a subject may be diagnosed as having an OCT-related cancer by identification of a particular type of cancer within the subject, wherein the cancer has been identified as an OCT-related cancer, as described herein. In some embodiments, methods of the invention may comprise promoting the inhibition or treatment of a cancer which expresses OCT in a subject susceptible to or exhibiting symptoms of a cancer which expresses OCT via administration to the patient of a composition comprising a compound as described herein.

The invention also comprises homologs, analogs, derivatives, enantiomers, diastereomers, tautomers, cis- and trø/«-isomers, and functionally equivalent compositions of compounds described herein. "Functionally equivalent" generally refers to a composition capable of treatment of patients having OCT-related cancer, or of patients susceptible to OCT-related cancers. It will be understood that the skilled artisan will be able to manipulate the conditions in a manner to prepare such homologs, analogs, derivatives, enantiomers, diastereomers, tautomers, cis- and trans-isomers, and functionally equivalent compositions. Homologs, analogs, derivatives, enantiomers, diastereomers, tautomers, cis- and trans-isomers, and functionally equivalent compositions which are about as effective or more effective than the parent compound are also intended for use in the method of the invention. Such compositions may also be screened by the assays described herein for increased potency and specificity towards a cancer which expresses an OCT, preferably with limited side effects. Synthesis of such compositions may be accomplished through typical chemical modification methods such as those routinely practiced in the art. Another aspect of the present invention provides any of the above-mentioned compounds as being useful for the treatment of cancer and particularly OCT-related cancers.

The invention further comprises compositions, preparations, formulations, kits, and the like, comprising any of the compounds as described herein. In some cases, treatment of an OCT-related cancer may involve the use of compositions or "agents" as described herein. That is, one aspect of the invention involves a series of compositions

(e.g., pharmaceutical compositions) or agents useful for treatment of cancer or tumor characterized by the expression of an OCT. These compositions may also be packaged in kits, optionally including instructions for use of the composition for the treatment of such conditions. These and other embodiments of the invention may also involve promotion of the treatment of cancer or tumor according to any of the techniques and compositions and combinations of compositions described herein.

Aspects of the invention may be used to prevent the growth of a tumor or cancer, and/or to prevent the metastasis of a tumor or cancer. In some embodiments, compositions of the invention may be used to shrink or destroy a cancer. It should be appreciated that compositions of the invention may be used alone or in combination with one or more additional anti-cancer agents or treatments (e.g., chemotherapeutic agents, targeted therapeutic agents, pseudo-targeted therapeutic agents, hormones, radiation, surgery, etc., or any combination of two or more thereof). In some embodiments, a composition of the invention may be administered to a patient who has undergone a treatment involving surgery, radiation, and/or chemotherapy. In certain embodiments, a composition of the invention may be administered chronically to prevent, or reduce the risk of, a cancer recurrence (particularly recurrence of an OCT-related cancer).

In another aspect, the present invention provides "pharmaceutical compositions" or "pharmaceutically acceptable" compositions, which comprise a therapeutically effective amount of one or more of the compounds described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled- release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream or foam; sublingually; ocularly; transdermally; or nasally, pulmonary and to other mucosal surfaces. 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.

The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or poly anhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

As set out herein, certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term "pharmaceutically-acceptable salts" in this respect refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al., "Pharmaceutical Salts," J. Pharm. ScI 1977, 66,1-19) The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra).

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.

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

The compound may be orally administered, parenterally administered, subcutaneously administered, and/or intravenously administered. In certain embodiments, a compound or pharmaceutical preparation is administered orally. In other embodiments, the compound or pharmaceutical preparation is administered intravenously. Alternative routes of administration include sublingual, intramuscular, and transdermal administrations.

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, and the particular mode of administration. The amount of active ingredient that 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, this amount will range from about 1% to about 99% of active ingredient, from about 5% to about 70%, or from about 10% to about 30%.

In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound of the present invention.

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.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; absorbents, such as kaolin and bentonite clay; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made in a suitable machine in which a mixture of the powdered compound is moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-described excipients. Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Dissolving or dispersing the compound in the proper medium can make such dosage forms. Absorption enhancers can also be used to increase the flux of the compound across the skin. Either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel can control the rate of such flux.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

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.

Examples of suitable aqueous and nonaqueous carriers, which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Delivery systems suitable for use with the present invention include time-release, delayed release, sustained release, or controlled release delivery systems, as described herein. Such systems may avoid repeated administrations of the active compounds of the invention in many cases, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, for example, polymer based systems such as polylactic and/or polyglycolic acid, polyanhydrides, and polycaprolactone; nonpolymer systems that are lipid-based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. Specific examples include, but are not limited to, erosional systems in which the composition is contained in a form within a matrix, or diffusional systems in which an active component controls the release rate. The formulation may be as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems. In some embodiments, the system may allow sustained or controlled release of the active compound to occur, for example, through control of the diffusion or erosion/degradation rate of the formulation. In addition, a pump-based hardware delivery system may be used in some embodiment of the invention.

Use of a long-term release implant may be particularly suitable in some cases. "Long-term release," as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the composition for at least about 30 or about 45 days, for at least about 60 or about 90 days, or even longer in some cases. Long-term release implants are well known to those of ordinary skill in the art, and include some of the release systems described above. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.

When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, about 0.1% to about 99.5%, about 0.5% to about 90%, or the like, of active ingredient in combination with a pharmaceutically acceptable carrier. The administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition to be treated. For example, the composition may be administered through parental injection, implantation, orally, vaginally, rectally, buccally, pulmonary, topically, nasally, transdermally, surgical administration, or any other method of administration where access to the target by the composition is achieved. Examples of parental modalities that can be used with the invention include intravenous, intradermal, subcutaneous, intracavity, intramuscular, intraperitoneal, epidural, or intrathecal. Examples of implantation modalities include any implantable or injectable drug delivery system. Oral administration may be useful for some treatments because of the convenience to the patient as well as the dosing schedule.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

The compositions of the present invention may be given in dosages, generally, at the maximum amount while avoiding or minimizing any potentially detrimental side effects. The compositions can be administered in effective amounts, alone or in a cocktail with other compounds, for example, other compounds that can be used to treat cancer. An effective amount is generally an amount sufficient to inhibit OCT-related cancer within the subject.

One of skill in the art can determine what an effective amount of the composition is by screening the ability of the composition using any of the assays described herein. The effective amounts will depend, of course, on factors such as the severity of the condition being treated; individual patient parameters including age, physical condition, size and weight; concurrent treatments; the frequency of treatment; or the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. In some cases, a maximum dose be used, that is, the highest safe dose according to sound medical judgment. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and then gradually increasing the dosage until the desired effect is achieved. In some embodiments, a compound or pharmaceutical composition of the invention is provided to a subject chronically. Chronic treatments include any form of repeated administration for an extended period of time, such as repeated administrations for one or more months, between a month and a year, one or more years, or longer. In many embodiments, a chronic treatment involves administering a compound or pharmaceutical composition of the invention repeatedly over the life of the subject. For example, chronic treatments may involve regular administrations, for example one or more times a day, one or more times a week, or one or more times a month. In general, a suitable dose such as a daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally doses of the compounds of this invention for a patient, when used for the indicated effects, will range from about 0.0001 to about 100 mg per kg of body weight per day. The daily dosage may range from 0.001 to 50 mg of compound per kg of body weight, or from 0.01 to about 10 mg of compound per kg of body weight. However, lower or higher doses can be used. In some embodiments, the dose administered to a subject may be modified as the physiology of the subject changes due to age, disease progression, weight, or other factors.

If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

While it is possible for a compound of the present invention to be administered alone, it may be administered as a pharmaceutical formulation (composition) as described above.

The present invention also provides any of the above-mentioned compositions useful for treatment of cancer characterized by expression of an OCT packaged in kits, optionally including instructions for use of the composition for the treatment of cancer. That is, the kit can include a description of use of the composition for participation in any biological or chemical mechanism disclosed herein associated with cancer or tumor. The kits can further include a description of activity of cancer characterized by expression of an OCT in treating the pathology, as opposed to the symptoms of the cancer. That is, the kit can include a description of use of the compositions as discussed herein. The kit also can include instructions for use of a combination of two or more compositions of the invention. Instructions also may be provided for administering the drug by any suitable technique, such as orally, intravenously, or via another known route of drug delivery. The invention also involves promotion of the treatment of cancer characterized by expression of an OCT according to any of the techniques and compositions and composition combinations described herein. The compositions of the invention, in some embodiments, may be promoted for treatment of abnormal cell proliferation, cancers, or tumors, particularly OCT-related cancers or includes instructions for treatment of accompany cell proliferation, cancers, or tumors, particularly OCT-related cancers as mentioned above. In another aspect, the invention provides a method involving promoting the prevention or treatment of cancer via administration of any one of the compositions of the present invention, and homologs, analogs, derivatives, enantiomers and functionally equivalent compositions thereof in which the composition is able to treat OCT-related cancers. As used herein, "promoted" includes all methods of doing business including methods of education, hospital and other clinical instruction, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with compositions of the invention in connection with treatment of cell proliferation, cancers or tumors. "Instructions" can define a component of promotion, and typically involve written instructions on or associated with packaging of compositions of the invention.

Instructions also can include any oral or electronic instructions provided in any manner. The "kit" typically defines a package including any one or a combination of the compositions of the invention and the instructions, or homologs, analogs, derivatives, enantiomers and functionally equivalent compositions thereof, but can also include the composition of the invention and instructions of any form that are provided in connection with the composition in a manner such that a clinical professional will clearly recognize that the instructions are to be associated with the specific composition.

The kits described herein may also contain one or more containers, which can contain compounds such as the species, signaling entities, biomolecules and/or particles as described. The kits also may contain instructions for mixing, diluting, and/or administrating the compounds. The kits also can include other containers with one or more solvents, surfactants, preservatives, and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose) as well as containers for mixing, diluting or administering the components to the sample or to the patient in need of such treatment.

The compositions of the kit may be provided as any suitable form, for example, as liquid solutions or as dried powders. When the composition provided is a dry powder, the powder may be reconstituted by the addition of a suitable solvent, which may also be provided. In embodiments where liquid forms of the composition are sued, the liquid form may be concentrated or ready to use. The solvent will depend on the compound and the mode of use or administration. Suitable solvents for drug compositions are well known and are available in the literature. The solvent will depend on the compound and the mode of use or administration.

The kit, in one set of embodiments, may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a positive control in the assay. Additionally, the kit may include containers for other components, for example, buffers useful in the assay. As used herein, a "subject" or a "patient" refers to any mammal (e.g., a human), such as a mammal that may be susceptible to tumorigenesis or cancer associated with the expression of an OCT. Examples include a human, a non-human primate, a cow, a horse, a pig, a sheep, a goat, a dog, a cat or a rodent such as a mouse, a rat, a hamster, or a guinea pig. Generally, or course, the invention is directed toward use with humans. A subject may be a subject diagnosed with cancer or otherwise known to have cancer. In some embodiments, a subject may be diagnosed as, or known to be, at risk of developing cancer. In some embodiments, a subject may be diagnosed with, or otherwise known to have, an OCT-related cancer. In certain embodiments, a subject may be selected for treatment on the basis of a known OCT-related cancer in the subject. In some embodiments, a subject may be selected for treatment on the basis of a suspected OCT- related cancer in the subject. In some embodiments, a OCT-related cancer may be diagnosed by detecting a mutation associate in a biological sample (e.g., urine, sputum, whole blood, serum, stool, etc., or any combination thereof. Accordingly, a compound or composition of the invention may be administered to a subject based, at least in part, on the fact that a mutation is detected in at least one sample (e.g., biopsy sample or any other biological sample) obtained from the subject. In some embodiments, a cancer may not have been detected or located in the subject, but the presence of a mutation associated with an OCT-related cancer in at least one biological sample may be sufficient to prescribe or administer one or more compositions of the invention to the subject. In some embodiments, the composition may be administered to prevent the development of an OCT-related cancer. However, in some embodiments, the presence of an existing OCT-related cancer may be suspected, but not yet identified, and a composition of the invention may be administered to prevent further growth or development of the cancer.

It should be appreciated that any suitable technique may be used to identify or detect mutation and/or over-expression associated with an OCT-related cancer. For example, nucleic acid detection techniques (e.g., sequencing, hybridization, etc.) or peptide detection techniques (e.g., sequencing, antibody-based detection, etc.) may be used. In some embodiments, other techniques may be used to detect or infer the presence of an OCT-related cancer (e.g., histology, etc.).

The presence of an OCT-related cancer can be detected or inferred by detecting a mutation, over-expression, amplification, or any combination thereof at one or more other loci associated with a signaling pathway of an OCT-related cancer. A "sample," as used herein, is any cell, body tissue, or body fluid sample obtained from a subject. Non-limiting examples of body fluids include, for example, lymph, saliva, blood, urine, and the like. Samples of tissue and/or cells for use in the various methods described herein can be obtained through standard methods including, but not limited to, tissue biopsy, including punch biopsy and cell scraping, needle biopsy; or collection of blood or other bodily fluids by aspiration or other suitable methods.

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 a subject at a reasonable benefit/risk ratio applicable to any medical treatment. Accordingly, a therapeutically effective amount prevents, minimizes, or reverses disease progression associated with an OCT-related cancer. Disease progression can be monitored by clinical observations, laboratory and imaging investigations apparent to a person skilled in the art. A therapeutically effective amount can be an amount that is effective in a single dose or an amount that is effective as part of a multi-dose therapy, for example an amount that is administered in two or more doses or an amount that is administered chronically. The effective amount of any one or more compounds may be from about 10 ng/kg of body weight to about 1000 mg/kg of body weight, and the frequency of administration may range from once a day to once a month. However, other dosage amounts and frequencies also may be used as the invention is not limited in this respect. A subject may be administered one or more compounds described herein in an amount effective to treat one or more cancers described herein.

In the compounds and compositions of the invention, the term "alkyl" refers to the radical of 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 some embodiments, a straight chain or branched chain alkyl may have 30 or fewer carbon atoms in its backbone, and, in some cases, 20 or fewer. In some embodiments, a straight chain or branched chain alkyl may have 12 or fewer carbon atoms in its backbone (e.g., C1-C12 for straight chain, C₃-Ci₂ for branched chain), 6 or fewer, or 4 or fewer. Likewise, cycloalkyls may have from 3-10 carbon atoms in their ring structure, or 5, 6 or 7 carbons in the ring structure. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, hexyl, cyclochexyl, and the like.

The term "heteroalkyl" refers to an alkyl group as described herein in which one or more carbon atoms is replaced by a heteroatom. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of heteroalkyl groups include, but are not limited to, alkoxy, amino, thioester, and the like.

The terms "alkenyl" and "alkynyl" 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.

The terms "heteroalkenyl" and "heteroalkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the heteroalkyls described above, but that contain at least one double or triple bond respectively.

As used herein, the term "halogen" or "halide" designates -F, -Cl, -Br, or -I. The terms "carboxyl group," "carbonyl group," and "acyl group" are recognized in the art and can include such moieties as can be represented by the general formula:

W wherein W is H, OH, O-alkyl, O-alkenyl, or a salt thereof. Where W is O-alkyl, the formula represents an "ester." Where W is OH, the formula represents a "carboxylic acid." The term "carboxylate" refers to an anionic carboxyl group. In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiolcarbonyl" group. Where W is a S-alkyl, the formula represents a "thiolester." Where W is SH, the formula represents a "thiolcarboxylic acid." On the other hand, where W is alkyl, the above formula represents a "ketone" group. Where W is hydrogen, the above formula represents an "aldehyde" group.

The term "aryl" refers to aromatic carbocyclic groups, optionally substituted, having a single ring (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fused rings in which at least one is aromatic (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl). That is, at least one ring may have a conjugated pi electron system, while other, adjoining rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls. The aryl group may be optionally substituted, as described herein. "Carbocyclic aryl groups" refer to aryl groups wherein the ring atoms on the aromatic ring are carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and polycyclic or fused compounds (e.g., two or more adjacent ring atoms are common to two adjoining rings) such as naphthyl groups. In some cases, the The term "alkoxy" refers to the group, -O-alkyl. The term "aryloxy" refers to the group, -O-aryl. The term "acyloxy" refers to the group, -O-acyl.

The term "aralkyl" or "arylalkyl", as used herein, refers to an alkyl group substituted with an aryl group.

The terms "heteroaryl" refers to aryl groups comprising at least one heteroatom as a ring atom.

The term "heterocycle" refers to refer to cyclic groups containing at least one heteroatom as a ring atom, in some cases, 1 to 3 heteroatoms as ring atoms, with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and the like. In some cases, the heterocycle may be 3- to 10-membered ring structures or 3- to 7-membered rings, whose ring structures include one to four heteroatoms. The term "heterocycle" may include heteroaryl groups, saturated heterocycles (e.g., cycloheteroalkyl) groups, or combinations thereof. The heterocycle may be a saturated molecule, or may comprise one or more double bonds. In some case, the heterocycle is a nitrogen heterocycle, wherein at least one ring comprises at least one nitrogen ring atom. The heterocycles may be fused to other rings to form a polycylic heterocycle. The heterocycle may also be fused to a spirocyclic group. In some cases, the heterocycle may be attached to a compound via a nitrogen or a carbon atom in the ring.

Heterocycles include, for example, thiophene, benzothiophene, thianthrene, furan, tetrahydrofuran, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, dihydropyrrole, pyrrolidine, imidazole, pyrazole, pyrazine, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, triazole, tetrazole, oxazole, isoxazole, thiazole, isothiazole, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, oxazine, piperidine, homopiperidine (hexamnethyleneimine), piperazine (e.g., N-methyl piperazine), morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, other saturated and/or unsaturated derivatives thereof, and the like. The heterocyclic ring can be optionally substituted at one or more positions with such substituents as described herein. In some cases, the heterocycle may be bonded to a compound via a heteroatom ring atom (e.g., nitrogen). In some cases, the heterocycle may be bonded to a compound via a carbon ring atom. In some cases, the heterocycle is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine-l,9-diamine, or the like.

The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: N(R')(R")(R'") wherein R', R", and R'" each independently represent a group permitted by the rules of valence. An example of a substituted amine is benzylamine.

Any of the above groups may be optionally substituted. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds, "permissible" being in the context of the chemical rules of valence known to those of ordinary skill in the art. It will be understood that "substituted" also includes that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. In some cases, "substituted" may generally refer to replacement of a hydrogen with a substituent as described herein. However, "substituted," as used herein, does not encompass replacement and/or alteration of a key functional group by which a molecule is identified, e.g., such that the "substituted" functional group becomes, through substitution, a different functional group. For example, a "substituted phenyl group" must still comprise the phenyl moiety and can not be modified by substitution, in this definition, to become, e.g., a pyridine ring. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.

Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃, -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, amino, halide, alkylthio, oxo, acylalkyl, carboxy esters, -carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, -carboxamidoalkylaryl, -carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy-, aminocarboxamidoalkyl-, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like.

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. In the claims, as well as in the specification above, all transitional phrases such as

"comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

EXAMPLES AND EMBODIMENTS Although the platinum-based anticancer drugs, cisplatin, carboplatin and oxaliplatin, have similar DN A-binding properties, only oxaliplatin has been shown to be active against colorectal tumors. This study demonstrates that the human organic cation transporters, OCTl and OCT2, can markedly increase oxaliplatin, but not cisplatin or carboplatin, accumulation and cytotoxicity in OCT-transfected cells. The cytotoxicity of oxaliplatin was greater than that of cisplatin in six colon cancer cell lines, but decreased by the OCT inhibitor, cimetidine, to a level similar to that of cisplatin. In some cases, structure-activity studies identified that organic functionalities on non-leaving groups coordinated to platinum may enhance selective uptake by OCTs. These results indicate that OCTs may be useful as determinants of oxaliplatin cytotoxicity and may contribute to its antitumor specificity for colorectal cancer. The results of the present study indicate that the influx transporters, OCTl and OCT2, may be useful as determinants of the anticancer activity of oxaliplatin and may contribute to differences in the tumor specificities of platinum-based compounds. Furthermore, expression of OCTs in tumors may be useful as markers for selecting specific platinum based therapies in individual patients. The development of new anticancer drugs, specifically targeted to OCTs, represents a novel strategy for targeted drug therapy. The results of the present structure- reactivity studies indicate specific tactics for realizing this goal.

The goals of the present study were to characterize the interaction of cisplatin, carboplatin and oxaliplatin with human OCTl, OCT2 and OCT3; to determine whether the OCTs play a role in the cytotoxicity of these and related platinum compounds; to determine whether interactions with OCTs contribute to the differential antitumor specificity of oxaliplatin versus cisplatin; and to understand in a broader context the underlying chemical principles that determine these differences. The data indicate that OCTl and OCT2 play a role in mediating the uptake and consequent cytotoxicity of oxaliplatin, but not cisplatin or carboplatin. Structure-activity relationship studies suggest that the 1 ,2-diaminocyclohexane (DACH) moiety of oxaliplatin is an important pharmacophore for its interaction with the OCTs and that an organic component on the non-leaving portion of the platinum complexes may be useful in enhancing cellular uptake via OCTs. Finally, the experiments described below suggest that interactions with OCTl and OCT2 may be important contributors to the sensitivity of colorectal cancer to oxaliplatin.

Example 1 The following example describes various experimental procedures for the study of platinum compounds in the treatment of cancers expressing the OCT .

Drugs and Reagents: Cisplatin, carboplatin, oxaliplatin, cimetidine, disopyramide, N-methyl-4-phenylpyridinium (MPP⁺) and thiazolyl blue tetrazolium bromide were purchased from Sigma (St. Louis, MO). Solutions of carboplatin (10 mM) and oxaliplatin (5 mM) were freshly prepared in water. A solution of cisplatin (2 mM) was made in Ix phosphate buffered saline (PBS). These stock solutions were immediately aliquoted and stored frozen at -20⁰C and discarded one month after preparation, [methy 1-³H] -MPP⁺ was from Perkin Elmer (Boston, MA) and tetraethylammonium (TEA) bromide [ethyl- 1-¹⁴C] was from American Radiolabeled Chemicals (St. Louis, MO). Hygromycin B and G418 were from Invitrogen (Carlsbad, CA). The cell culture medium Dubecco's Modified Eagle Medium (DMEM), RPMI and fetal bovine serum (FBS) were from the Cell Culture Facility of University of California, San Francisco (San Francisco, CA).

Cell Lines and Transfection: Madin-Darby canine kidney (MDCK) cells stably transfected with the full length human OCTl cDNA (MDCK-hOCTl) and with the empty vector (MDCK-MOCK) were established previously in our laboratory (Shu et al.₅ 2003),Human embryonic kidney (HEK) 293 cells transfected with pcDNA5/FRT vector (Invitrogen) containing the full length human OCT2 cDNA (HEK-hOCT2) and with the empty vector (HEK-MOCK) were established using Lipofectamine™ 2000 (Invitrogen) per manufacturer's instructions. The stable clones were selected with 75 μg/mL of hygromycin B. HEK 293 cells transfected with pcDNA3 vector containing the full length human OCT3 cDNA (HEK-hOCT3) and with the empty vector (HEK-MOCK) were also established using Lipofectamine™ 2000. The stable clones were selected with 600 μg/mL G418. The pcDNA3 vector containing the full length human OCT3 cDNA was provided by the Institute of Pharmacology and Toxicology, University of Bonn, Germany. All the colon cancer cell lines (LS 180, SW620, DLD, HCTl 16, HT20 and RKO) used in the present study were from American Type Culture Collection (Manassas, VA).

Cell Culture: The culture medium for stably transfected MDCK and HEK 293 cells was DMEM supplemented with 10% FBS and 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen). To maintain the transgene expression, G418 (Invitrogen, 600 μg / mL) was added to the culture medium for MDCK transfected cells, human OCT3 transfected HEK 293 cells (HEK-hOCT3) and the corresponding HEK- MOCK cells; Hygromycin B (Invitrogen, 75 μg / mL) was added to the culture medium for human OCT2 transfected HEK 293 cells (HEK-hOCT2) and the corresponding HEK- MOCK cells. The culture medium for all the colon cancer cell lines (RKO, DLD, HT29, HCTl 16, LS 180 and SW620) is RPMI containing 10% FBS, 100 units / mL penicillin and 100 μg / mL streptomycin. All the cells were grown at 37⁰C in a humidified atmosphere with 5 % CO₂/ 95 % air.

Drug Sensitivity Assay: Cytotoxicity of the platinum compounds was measured by MTT (thiazolyl blue tetrazolium bromide) assay. Cells were seeded in 100 μL of the culture medium without any antibiotics in 96-well plates at a predetermined cell density. For HEK 293 cells, poly-D-lysine coated plates were used. After overnight incubation, the platinum compounds were then added to the culture medium to give the indicated final concentrations. For the OCT inhibitor studies, the inhibitors (disopyramide or cimetidine) were added to the medium at a specified concentration immediately before the addition of the platinum compounds. After incubation for a specified time period, the drug-containing medium was replaced with fresh, drug-free medium and the incubation was continued for a total of 72 hours (start from the addition of platinum compounds). Then the MTT assay was performed similarly as previously described (Alley et al., 1988). The IC₅₀ (the drug concentration which inhibits 50% of cell growth) values were obtained by fitting the percent of the maximal cell growth at different drug concentrations (F) with the equation, F=I 0Ox(I -CV(IC_5O ^v+C^γ)), using WinNonlin

(Pharsight, Mountain View, CA). The maximal cell growth was the cell growth in the medium without any platinum compounds; C is the concentration of the platinum compound and γ is the slope factor. Cellular Uptake of TEA or MPP⁺: MDCK or HEK 293 Cells were grown in 24- well plates to > 90% confluence in the culture medium without any antibiotics. The poly-D-lysine coated plates were used for HEK 293 cells. The cells were washed with 1 x PBS first and then incubated in the uptake buffer (1 x PBS) containing 10 μM ¹⁴C- TEA or 2 μM ³H-MPP⁺ as specified. For the OCT inhibitor studies, the indicated OCT inhibitor (disopyramide or cimetidine) was added to the uptake buffer at specified concentration together with the radioactive substrate. The uptake was performed at room temperature for 2 min (¹⁴C-TEA uptake) or 5 min (³H-MPP⁺ ) and then the cells were washed with ice-cold PBS for three times. The cells were then lysed with the lysis buffer (0.1 N NaOH, 0.1% SDS) for scintillation counting and BCA protein assay (Pierce, Rockford, IL) to determine the uptake.

Cellular Accumulation of Platinum: The cellular accumulation of platinum was determined as previous described (Holzer et al., 2004) with some modifications. Briefly, the cells were grown in 100 mm x 20 mm dishes in the culture medium without any antibiotics to over 90% confluence. For HEK 293 cells, poly-D-lysine coated dishes were used. For platinum accumulation, the cells were incubated in the culture medium containing the indicated concentrations of the platinum compounds at 37°C in 5% CO₂ for 2 hr unless specified. After incubation, the dishes were immediately placed on ice and the cells were washed with 6 mL of ice-cold PBS for three times and collected with a rubber policeman. The cell pellets were obtained by centrifugation at 400 x g and at 4⁰C for 15 min. For the OCT inhibitor studies, the incubation medium also contained the indicated inhibitor (disopyramide or cimetidine) in addition to the platinum compounds. The resulting cell pellets were then dissolved into 200 μl of 70% nitric acid at 65°C for at least 2.5 hours, and then distilled water containing 10 ppb of iridium (Sigma) and 0.1% Triton X-IOO was added to the samples to dilute nitric acid to 7%. The platinum content was measured by inductively plasma coupled mass spectrometry (ICP-MS) in the Analytical Facility at University of California at Santa Cruz (Santa Cruz, CA). Cell lysates from a set of identical cultures were used for BCA protein assay.

Platinum-DNA Adduct Formation: The platinum content associated with genomic DNA was determined as previously described (Samimi et al., 2004, Clin Cancer Res 10, 4661-4669) with some modifications. Briefly, the cells were grown in 100 mm x 20 mm dishes in the culture medium without any antibiotics to over 90% confluence. For HEK 293 cells, poly-D-lysine coated dishes were used. Then, the cells were incubated in the culture medium containing the specified concentrations of the platinum compounds at 37⁰C in 5% CO₂ for 2 hours (or 25 min as specified). In some experiments, phosphate buffer (PB: 1.06 mM KH₂PO₄, 2.97 mM Na₂HPO₄, pH 7.4) containing 155 mM NaCl (PB-Cl buffer) or 103 mM Na₂SO₄ (PB-SO₄ buffer) was used instead of the culture medium as specified. For the OCT inhibitor study, the incubation medium (buffer) also contained disopyramide or cimetidine). If PB-Cl or PB-SO₄ buffer was used, the cells were washed with the same buffer once before drug incubation. After incubation, the cells were washed with ice-cold PBS, scraped and pelleted. Genomic DNA was isolated from the cell pellets using Wizard^® Genomic DNA Purification Kit (Promega, Madison, WI) following the manufacturer's instruction. Briefly, the cells were lysed with Nuclei Lysis Solution. After RNA digestion and protein precipitation, the lysates were centrifuged and the resulting supernatant was aliquoted. The genomic DNA prepared from two different aliquots of the supernatant was used for platinum and DNA content determination, respectively. For the determination of platinum, the DNA samples were treated with 70% nitric acid at 65°C and diluted in the same way as described above. The platinum content was analyzed using ICP-MS and the DNA content was measured by absorption spectroscopy.

RNA Isolation: Cultured cells were grown in 100 mm x 20 mm dishes to 70- 80% confluence. Total RNA was isolated using RNeasy^® Mini Kit (Qiagen, Valencia, CA) following manufacturer's instruction, quantified by spectroscopy and stored at - 80°C until use. Samples of tumor and normal colon mucosa were collected from colon cancer resection from Department of Surgery, Queen Mary Hospital, University of Hong Kong. Tissues were frozen in liquid nitrogen within half an hour after they were resected. Nonneoplastic mucosa from colon was dissected free of muscle and histologically confirmed to be tumor free by frozen section. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA). This study was approved by the Ethics Committee of the University of Hong Kong and the Internal Review Board of University of California, San Francisco.

RT-PCR: The first-strand cDNA was synthesized from 2 μg of total RNA using Superscript™ III First-Strand Synthesis System for RT-PCR kit (Invitrogen) in a 20 μl reaction mixture, and the random hexamers were used as the primer. FIG. 8 shows primers for RT-PCR. The sense and antisense primers for human OCTl were 5'-CTG TGT AGA CCC CCT GGC TA-3' and 5'-GTG TAG CCA GCC ATC CAG TT-3', corresponding to the nucleotide positions 408-427 and 751-770 (accession number:

NM_003057), respectively, and the size of the expected PCR product is 363 bp. The sense and antisense primers for human OCT2 were 5'-CCT GGT ATG TGC CAA CTC CT-3' and 5'-CAC CAG GAG CCC AAC TGT AT-3\ corresponding to the nucleotide positions 590-609 and 904-923 (accession number: NM_003058), respectively, and the size of the expected PCR product is 334 bp. The sense and antisense primers for human OCT3 were 5'-ATC GTC AGC GAG TTT GAC CT-3' and 5'-TTG AAT CAC GAT TCC CAC AA-3', corresponding to the nucleotide positions 445-464 and 749-768 (accession number: NM_021977), respectively, and the size of the expected PCR product is 324 bp. The sense and antisense primers for human GAPDH were 5'-AAT CCC ATC ACC ATC TTC CA-3' and 5'-TGT GGT CAT GAG TCC TTC CA-3', corresponding to the nucleotide positions 289-308 and 587-606 (accession number: NM_002046), respectively, and the size of the expected PCR product is 318 bp. The sense and antisense primers for dog GAPDH were 5'-GGT GAT GCT GGT GAG TA-3' and 5'- GTG GAA GCA GGG ATG ATG TT-3 ' , corresponding to the nucleotide positions 256- 275 and 607-626 (accession number: AB038240), respectively, and the size of the expected PCR product is 371 bp. All sets of primers were designed to anneal with sequences in different exons of the genes. An annealing temperature of 58°C was used for PCR amplification. A cycle number of 40 was used for the detection of human OCTl and OCT2 in the colon cancer cell lines and colon tissue samples. A cycle of 30 was used to detect human OCTl, OCT2 or OCT3 in the corresponding OCT-transfected cells and the MOCK cells. For the detection of human or dog GAPDH, a PCR cycle number of 30 was used in all the conditions.

Statistical Analysis: The differences between the mean values were analyzed for significance using Student's t test. P values < 0.05 were considered statistically significant.

Example 2

The following examples describe the synthesis of various platinum analogs. Potassium tetrachloroplatinate(II) was a gift from Engelhard Corp. and the starting materials cisplatin and potassium aminetrichloroplatinate(II) were synthesized as reported (Dhara, 1970, Indian Journal of Chemistry 8, 193-194; Giandomenico et al., 1995, Inorganic Chemistry 34, 1015-1021). ¹H NMR spectra were acquired on a Varian 300 MHz spectrometer. FT-IR spectra were measured on an Avatar 380 FT-IR (Thermo

Nicolet, Waltham, MA). ESI-MS spectra were obtained on an Agilent Technologies 1100 Series LCMS instrument. Previously reported procedures were used to prepare [Pt(Cl₂)(en)] (Dhara, 1970, Indian Journal of Chemistry 8, 193-194), cis- [Pt(NH₃)(Cy)Cl₂] (Giandomenico et al., 1995, Inorganic Chemistry 34, 1015-1021), and [Pt(J^-DACH)Cl₂] (Hoeschele et al., 1988, Inorganic Chemistry 27, 4106-4113). The [Pt(SS-DACH)Cl₂] and [Pt(S, S-DACH)oxalato] were synthesized as described (Kidani, 1978, Journal of Medicinal Chemistry 21, 1315-1318) FTIR and ¹H NMR spectra of all compounds matched literature spectra. Example 2.1

Preparation of ^' [Pt(NHs) ₂(trans-l ,2-(OCO)₂CeHiQ)J: The compound was prepared as described for the Pt-DACH derivative (Al-Allaf et al., 2003, Transition Metal Chemistry 28, 717-721). Insufficient solubility, similar to that reported for the DACH compound, prevented analysis by NMR spectroscopy. IR (KBr, cm^"1) 3266 (sh), 2920 (s), 2850 (s), 1618 (s), 1556 (sh), 1384 (vs), 1279 (w), 1222 (m), 1111 (w), 1030 (w), 772 (w), 719 (w), 588 (b). ESI-MS: [M+H]⁺ = 400.2 amu (observed), 400.3 amu (calculated). Example 2.2

Preparation Of[Pt(R₁R-DACH)(H₂O)₂J²⁺: [?t(R,R-O ACH)Cl₂] was dissolved in distilled water (200 μM) and incubated with silver nitrate (400 μM) in dark for 10 hours. [Pt(i?,i?- DACH)(H₂O)₂]^2"1" was obtained by filtering the reaction mixture to remove the silver chloride precipitate.

Example 3

The following example describes OCT expression and function in stably transfected cell lines.

The expression of human OCTl, OCT2 or OCT3 was determined in the cell lines stably transfected with human OCTs (MDCK-hOCTl, HEK-hOCT2 and HEK-hOCT3) or the corresponding empty vectors (MOCK cells) by RT-PCR as described in the Experimental Procedures. The dog and human GAPDH were used as the expression control for the transfected MDCK and HEK 293 cells, respectively. (See Table A). FIG. 7 A shows the expression of human OCTl, OCT2 and OCT3 in stably transfected cell lines, as seen in lanes 1-6: MDCK-MOCK, MDCK-hOCTl, HEK-MOCK, HEK- hOCT2, HEK-MOCK and HEK-hOCT3. FIG. 7B shows the cellular uptake of model substrates (TEA for human OCTl and OCT2, and MPP⁺ for human OCT3) in OCT-transfected cells (black bars) and in the corresponding MOCK cells (white bars). Disopyramide (120 μM) was used as an inhibitor for human OCTl and cimetidine (1.5 mM) was used as an inhibitor for human OCT2 and OCT3. Data are expressed as mean ± SD of six measurements.

The expression and function of human OCTs in the stably transfected cells was confirmed by RT-PCR and by examining the uptake of the model OCT substrates (TEA for OCTl and OCT2, MPP⁺ for OCT3). The expression of the mRNA transcripts of OCTl, OCT2 and OCT3 and uptake of model compounds were higher in OCT- transfected cells (MDCK-hOCTl , HEK-hOCT2 or HEK-hOCT3) in comparison to empty vector-transfected control counterparts (MOCK cells), as shown in FIGS. 7A-B. OCT inhibitors (disopyramide (120 μM) for OCTl, cimetidine (1.5 mM) for OCT2 and OCT3) substantially decreased the uptake of the model compounds in the OCT- transfected cells (p < 0.001). (FIG. 7B)

Table A. Expression of OCTl and OCT 2 in colon cancer cell lines and colon tissue samples determined by real time RT-PCR.

Samples OCTl Expression OCT2 Expression

DLD 0.966 ± 0.222 0.00144 ±0.00153

LS 180 1.78 ±0.260 0.00827 ± 0.00686

SW620 1.61 ±0.256 0.00205 ± 0.00109

HCTl 16 0.947 ± 0.248 0.00650 ± 0.00435

HT29 2.06 ± 0.739 0.0121 ±0.00578

RKO 2.34 ± 0.526 0.000226 ±0.000170

Nl 0.355 ±0.110 0.00405 ± 0.0036

N2 1.96 ±0.906 0.00565 ± 0.00347

N3 6.83 ±0.661 0.0181 ±0.0227

N4 3.72 ±0.552 0.0293 ±0.0173

Tl 5.92 ± 0.362 0.759 ±0.150

T2 0.406 ±0.147 0.0140 ±0.00714

T3 1.99 ±0.304 0.291 ±0.124

T4 4.75 ±0.616 0.379 ± 0.0633

T5 4.96 ± 0.843 0.127 ±0.0324

16 10.4±l.ll 1.03 ±0.0853

11 1.68 ±0.132 0.0630 ± 0.0266

T8 0.198 ±0.0282 0.00684 ± 0.00607

T9 1.42 ±0.364 0.0390 ±0.0158

TlO 1.00 ± 0.238 1.00 ±0.189

TH 1.04 ±0.0777 0.240 ± 0.0526

T12 1.72 ±0.327 1.31 ±0.0827

T13 3.00 ±0.265 2.97 ±0.196

T14 6.36 ±2.32 0.638 ±0.152

T15 0.299 ± 0.0524 0.840 ± 0.0504

T16 0.753 ±0.132 0.178 ±0.0441

T17 2.73 ±0.721 0.114 ±0.0556

T18 2.37 ±0.384 0.0271 ±0.0111

T19 0.957 ± 0.223 2.64 ±0.312

T20 1.50 ±0.264 5.29 ± 0.468

Example 4

The following example describes the effect of OCTs on the cytotoxicity of cisplatin, carboplatin and oxaliplatin. The cytotoxicity of oxaliplatin in OCTl-, OCT2- and OCT3-transfected cells and in the corresponding MOCK cells was determined as described in the Experimental Procedures above. Cells were seeded in 96-well plates at a density of 5,000 cells/well for the transfected MDCK cells and 12,000 cells/well for the transfected HEK 293 cells and exposed to the test compounds for 7 hours on the following day. After a total of 72 hours, cell growth was determined by an MTT assay. FIG.2A shows a plot of cytotoxicity of OCTl -transfected cells (white circles) and of corresponding MOCK cells (black circles). FIG.2B shows a plot of cytotoxicity of OCT2-transfected cells (white circles) and of corresponding MOCK cells (black circles).

FIG. 2C shows a plot of cytotoxicity of OCT3-transfected cells (white circles) and of corresponding MOCK cells (black circles). In addition, the cytotoxicity of oxaliplatin in OCT-transfected cells and in the corresponding empty vector-transfected cells in the presence of an OCT inhibitor was simultaneously determined in a similar fashion. When the OCT inhibitors were used, disopyramide (150 μM) or cimetidine (1.5 mM) was added to the incubation medium immediately before the addition of oxaliplatin.

The IC₅₀ values (μM) of cisplatin, carboplatin and oxaliplatin in (A) human OCTl-, (B) OCT2- and (C) OCT3-transfected cell lines were determined in parallel with those in the corresponding MOCK cells using MTT assay as described in the

Experimental Procedures. Briefly, the cells were seeded in 96-well plates at a density of 5,000 cells/well for the transfected MDCK cells or 12,000 cells/well for the transfected HEK 293 cells. The platinum drugs were added on the following day. After the specified time periods of drug exposure, the drug-containing medium was replaced with fresh, drug-free medium, and the incubation was continued for a total of 72 hours

(starting from the time when the drug was added). After incubation, the cell growth was determined by an MTT assay. Data are expressed as mean ± SD from 3 to 6 independent experiments with each performed in quadruplicate. The resistance factor (RF) was defined as the ratio of the mean IC₅₀ value in the MOCK cells to that in the OCT- transfected cells.

FIG. 2D shows the cytotoxicity of oxaliplatin in OCTl -transfected cells (white symbols) and in the corresponding empty vector-transfected cells (MOCK cells) (black symbols) in the presence (squares) or absence (circles) of disopyramide, an OCT inhibitor. FIG. 2E shows the cytotoxicity of oxaliplatin in OCT2 -transfected cells (white symbols) and in the corresponding empty vector-transfected cells (MOCK cells) (black symbols) in the presence (squares) or absence (circles) of cimetidine, an OCT inhibitor. The lines represent the predicted data obtained by fitting the observed data using WinNonlin as described in the Experimental Procedures. Presented are the data from a typical experiment. Three to six independent experiments were performed and similar results were obtained. For clarity, the standard deviation bars in panel FIG. 2D and FIG. 2E were eliminated.

The IC₅₀ values of oxaliplatin, determined in MTT assays, in MDCK-MOCK cells after different time periods (7, 24 and 72 hr) of drug exposure were all significantly higher than those in MDCK-hOCTl cells. Resistance factors (RF), defined as the ratio of the IC₅₀ value in MOCK cells to that in the corresponding OCT-transfected cells, ranged from 5.73 to 8.48 (p < 0.01 or p < 0.001; Table IA and FIG. 2A). In contrast, the IC₅₀ values of both cisplatin and carboplatin were similar in the OCTl-transfected and in the MDCK-MOCK cells with RF values close to unity (p > 0.05) (Table IA). Co- incubation with a known OCTl inhibitor, disopyramide (150 μM), substantially increased the IC₅₀ value of oxaliplatin in MDCK-hOCTl (control vs. disopyramide- treated: 3.79 ± 1.57 μM vs. 22.8 ± 10.5 μM) by 6.01 -fold (p < 0.05) with little effect in MDCK-MOCK (control vs. disopyramide-treated: 30.4 ± 9.28 vs. 32.2 ± 13.0 μM, p > 0.05) tested in parallel (FIG. 2D). Disopyramide itself did not manifest any cytotoxicity up to a concentration of 400 μM under the same test conditions (data not shown). These results indicate that OCTl can enhance the cytotoxicity of oxaliplatin, but not that of cisplatin or carboplatin. A similar pattern of observations was obtained in human OCT2- transfected cells, but the increase in oxaliplatin cytotoxicity was more pronounced (FIG. 2B). The IC₅₀ values of oxaliplatin after different time periods (7, 24 and 72 hr) of exposure were greater in HEK-MOCK cells than in HEK-OCT2 cells with RF values ranging from 48.4 to 76.7 (p < 0.05 to p < 0.001) (Table IB and FIG. 2B). However, the IC₅₀ values of cisplatin and carboplatin were only slightly greater in HEK-MOCK cells than in HEK-OCT2 cells with RF values around 2 after 7-hour drug exposure (Table IB).

Co-incubation with an OCT inhibitor, cimetidine (1.5 mM), increased the oxaliplatin IC₅₀ (control vs. cimetidine-treated: 0.039 ± 0.025 μM vs. 2.81 ± 1.63 μM) by 72-fold (p < 0.05) in HEK-hOCT2 cells, with only a 3.18-fold increase in HEK-MOCK cells (control vs. cimetidine-treated: 2.99 ± 1.51 vs. 9.50 ± 2.95 μM, p < 0.05) (FIG. 2E). Cimetidine itself did not exhibit cytotoxicity up to a concentration of 5 mM under the same test conditions (data not shown). These results indicate that OCT2 can enhance the cytotoxicity of oxaliplatin with only slight effects on the cytotoxicities of cisplatin and carboplatin. In contrast to OCTl and OCT2, overexpression of human OCT3 did not affect the cytotoxicity of any of the platinum drugs (Table 1C and FIG. 2C). Table 1. Drug sensitivity of cisplatin, carboplatin and oxaliplatin in the OCT-transfected cells.

A. Cytotoxicity, expressed as ICs₀, of the platinum drugs in MDCK-MOCK and MDCK-hOCTl cells.

Drug Exposure Time MDCK-MOCK MDCK hOCTl

Platinum Drugs (hour) (μM) (μM) RF cisplatin 7 19.6 ± 7.56 15.4 ± 2.84 1.27 carboplatin 7 258 ± 86.3 227 ± 85.8 1.13 oxaliplatin 7 33.0 ± 9.12 3.89 ± 1.30 8.48*** oxaliplatin 24 14.3 ± 5.55 1.79 ± 0.58 7.95** oxaliplatin 72 9.64 ± 1.85 1.68 ± 0.27 5.73***

B. Cytotoxicity, expressed as IC₅O, of the platinum drugs in HEK-MOCK and HEK- hOCT2 cells.

Drug Exposure Time HEK-MOCK HEK-hOCT2

Platinum Drugs (hour) (μM) (μM) RF cisplatin 7 2.95 ± 0.23 1.32 ± 0.18 2.23*** carboplatin 7 110 ± 46.3 61.6 ± 46.3 1.78 oxaliplatin 7 2.99 ± 1.51 0.039 ± 0.025 76.7** oxaliplatin 24 1.50 ± 0.69 0.02 ± 0.001 73.8* oxaliplatin 72 0.93 ± 0.056 0.019 ± 0.004 48.4***

C. Cytotoxicity, expressed as IC₅₀, of the platinum drugs in HEK-MOCK and HEK- hOCT3 cells.

Drug Exposure Time HEK-MOCK HEK-hOCT3

Platinum Drugs (hour) (μM) (μM) RF cisplatin 7 2.83 ± 0.90 2.44 ± 0.71 1.16 carboplatin 7 84.8 ± 9.71 48.1 ± 23.4 1.76 oxaliplatin 7 1.47 ± 0.28 2.22 ± 0.41 0.66 oxaliplatin 24 0.47 ± 0.05 0.62 ± 0.19 0.75 oxaliplatin 72 0.47 ± 0.12 0.69 ± 0.19 0.68

*: p < 0.05; **: p < 0.01 and ***: p < 0.001

Example 5

The following example described platinum accumulation rates in cells after exposure to cisplatin, carboplatin and oxaliplatin. The cellular accumulation rates of platinum in OCTl-, OCT2- and OCT3-transfected cells and in the corresponding MOCK cells after incubation with cisplatin, carboplatin and oxaliplatin in the presence and absence of an OCT inhibitor (disopyramide for OCTl, cimetidine for OCT2 and OCT3) were determined as described in the Experimental Procedures. Briefly, (A) MDCK cells were incubated in the antibiotic-free medium containing cisplatin (3 μM), carboplatin (15 μM) or oxaliplatin (3 μM) at 37°C and 5% CO₂ for 2 hours. For the inhibitor studies, the incubation medium also contained disopyramide (150 μM). (B) HEK 293 cells were incubated in the antibiotic-free medium containing cisplatin (0.3 μM), carboplatin (10 μM) or oxaliplatin (0.3 μM) at 37°C and 5% CO₂ for 2 hours. For the inhibitor studies, the incubation medium also contained cimetidine (1.5 mM). (C) The study was performed similarly as (B) except that the concentrations of cisplatin, carboplatin and oxaliplatin in the incubation medium were 2 μM, 10 μM and 2 μM, respectively. After drug exposure, the cells were washed with ice-cold PBS three times and harvested by scraping and centrifugation. The cell-associated platinum was determined by ICP-MS and normalized for protein content. Data are expressed as mean ± SD from a single experiment performed in triplicate. Experiments were replicated for OCTl and OCT2, and similar results were obtained.

FIG. 3 A shows the cellular accumulation rates of platinum in OCTl-transfected cells in the corresponding MOCK cells after 2-hour exposure to cisplatin, carboplatin and oxaliplatin, in the presence (white bars) and absence (black bars) of disopyramide, an OCT inhibitor. FIG. 3B shows the cellular accumulation rates of platinum in OCT2- transfected cells in the corresponding MOCK cells after 2-hour exposure to cisplatin, carboplatin and oxaliplatin, in the presence (white bars) and absence (black bars) of cimetidine, an OCT inhibitor. FIG. 3 C shows the cellular accumulation rates of platinum in OCT3-transfected cells in the corresponding MOCK cells after 2-hour exposure to cisplatin, carboplatin and oxaliplatin, in the presence (white bars) and absence (black bars) of cimetidine, an OCT inhibitor.

The cellular platinum accumulation rate after two hours of exposure to oxaliplatin (3 μM) was 2.90-fold higher (p < 0.001) in MDCK-hOCTl cells (8.53 ± 0.52 pmol / (mg protein-hr)) than that in MDCK-MOCK cells (2.94 ± 0.11 pmol / (mg protein-hr)) (FIG. 3A). Co-incubation with disopyramide (150 μM) resulted in a two-fold decrease in the rate of platinum accumulation in MDCK-hOCTl cells (control vs. disopyramide-treated; 8.53 ± 0.52 vs. 4.04 ± 0.04 pmol / (mg protein-hr), p < 0.001) with little effect in MDCK-MOCK cells (control vs. disopyramide-treated; 2.94 ± 0.11 vs. 3.23 ± 0.31 pmol / (mg protein-hour), p > 0.05) (FIG. 3A). However, the cellular accumulation rates of platinum after 2-hr exposure to cisplatin (3 μM) or carboplatin (15 μM) in MDCK- hOCTl cells (cisplatin: 3.88 ± 0.15 pmol / (mg protein-hr)); carboplatin: 2.77 ± 0.36 pmol / (mg protein-hr)) were not significantly different from those in MDCK-MOCK cells (cisplatin: 3.70 ± 0.45 pmol / (mg protein-hr)); carboplatin: 2.22 ± 0.07 pmol / (mg protein-hr)) and were not inhibited by disopyramide (FIG. 3A). These results indicate that human OCTl contributes substantially to the uptake of oxaliplatin, but not cisplatin or carboplatin in OCTl-transfected cells. The platinum accumulation rate in HEK- hOCT2 (20.2 ± 1.54 pmol / (mg protein-hr)) was markedly higher (21.7-fold, p < 0.001) than that in HEK-MOCK cells and was substantially reduced in the presence of cimetidine (control vs. cimetidine; 20.2 ± 1.54 vs. 1.80 + 0.13 pmol / (mg protein-hr)). However, the cellular accumulation rate of platinum in HEK-hOCT2 cells after 2-hr exposure to cisplatin (0.3 μM) or carboplatin (10 μM) (cisplatin: 0.738 ± 0.055 pmol / (mg protein-hr); carboplatin: 4.17 ± 0.18 pmol / (mg protein-hr)) was only modestly higher (1.38-fold for carboplatin, p < 0.001; 2.08-fold for cisplatin, p < 0.01) than that in HEK-MOCK cells.

Co-incubation with cimetidine (1.5 mM) produced only a small decrease (less than 1.5-fold, p < 0.01) in platinum accumulation rate after exposure of HEK-hOCT2 cells to either cisplatin or carboplatin with little effect in HEK-MOCK cells. These results indicate that OCT2 plays a critical role in the uptake of oxaliplatin in the transfected cells with a much lower effect on the uptake of cisplatin or carboplatin. In contrast to OCTl and OCT2, OCT3 overexpression did not substantially affect the uptake of any of these platinum drugs (FIG. 3C).

Example 6

The following example describes platinum-DNA adduct formation after 2-hr exposure to oxaliplatin to determine whether the oxaliplatin taken up by cells via the human OCTl and OCT2 transporters was available for DNA binding.

The content of platinum bound to DNA after 2-hr exposure to oxaliplatin in the presence (open bars) or absence (solid bars) of an OCT inhibitor (disopyramide for OCTl, cimetidine for OCT2) was determined as described in the Experimental Procedures. Briefly, (A) Transfected MDCK cells were incubated in the antibiotic-free medium containing oxaliplatin (10 μM) with or without disopyramide (150 μM). (B) Transfected HEK 293 cells were incubated in the antibiotic-free medium containing oxaliplatin (0.6 μM) with or without cimetidine (1.5 mM). After incubation at 37°C and 5% CO₂ for 2 hours, the cells were washed with ice-cold PBS three times and harvested. The genomic DNA was isolated from the cells and the platinum content associated with

DNA was determined by ICP-MS and normalized for DNA content. Data are expressed as mean ± SD from a typical experiment performed in triplicate. (Columns represent the mean, while the bars indicate SD.) Two independent experiments were conducted and similar results were obtained.

FIG. 4 A shows a graph of the content of platinum bound to DNA for OCT- transfected MDCK cells after 2-hr exposure to oxaliplatin in the presence (white bars) or absence (black bars) of disopyramide, an OCT inhibitor. FIG. 4B shows a graph of the content of platinum bound to DNA for OCT-transfected MDCK cells after 2-hr exposure to oxaliplatin in the presence (white bars) or absence (black bars) of cimetidine, an OCT inhibitor. The platinum-DNA adduct level in MDCK-hOCTl cells (0.0457 ± 0.001 lpmol / μg DNA, r_b (ratio of bound platinum atoms per nucleotide) = 1.51 ± 0.04 x 10^"5) was 4.15-fold greater (p < 0.001) than that in MDCK-MOCK cells (0.0110 ± 0.0010 pmol / μg DNA, r_b = 3.63 ± 0.33 x 10^"6) after exposure to oxaliplatin (FIG. 4A). Co-incubation with disopyramide (150 μM) significantly decreased (2.11 -fold, p < 0.001) platinum-DNA adduct formation in MDCK-hOCTl cells (control vs. disopyramide-treated; 0.0457 ± 0.0011 pmol / μg DNA, r_b= 1.51 ± 0.04 x 10^"5 vs. 0.0217 ± 0.0019 pmol / μg DNA, r_b= 7.16 ± 0.63 x lO^'6) with no effect in MDCK- MOCK cells. The platinum-DNA adduct level in HEK-hOCT2 cells (0.0284 ± 0.0020 pmol / μg DNA, r_b = 9.37 ± 0.66 x 10^"6) was 28.8-fold higher (p < 0.001) than that in HEK-MOCK after exposure to oxaliplatin (FIG. 4B) and was markedly reduced by cimetidine (0.00216 ± 0.00031 pmol / μg DNA, r_b= 9.37 ± 0.66 x 10^"6 vs. 7.13 ± 1.02 x 10^"7). Cimetidine produced only a small decrease (1.70-fold, p < 0.05) in HEK-MOCK cells.

Example 7

The following example describes the investigation of Structure- Activity Relationships (SAR) for Platinum-OCTl Interaction. To investigate the SAR for platinum-OCTl interactions, the drug sensitivities (IC₅o) and RF values of 9 platinum complexes (FIG. 1) in both MDCK-MOCK and MDCK-hOCTl cells were determined (Table 2A): the higher the RF value, the higher the interaction. Also, the drug sensitivities and RF values of the platinum complexes (FIG. 1) in both HEK-MOCK and HEK-hOCT2 cells were determined (Table 2B). Nature of the non-leaving group(s): RF values less than two were obtained for platinum complexes with diamine non-leaving groups including cisplatin, carboplatin and [Pt(NH₃)₂(trans-l, 2-(OCO)₂C₆Hio], indicating that platinum compounds with this purely inorganic non-leaving unit are poorly recognized by OCTl (Table 2A). However, when the non-leaving group(s) contained an organic component as in [PtCl₂(Cn)], which has two methylene groups between the amine functionalities, the RF value increased to 3.26. Moreover, with increasing size of the organic component of the non-leaving group(s), the interaction of a platinum compound with OCTl increased. For example, the platinum compounds CiS-[Pt(NH₃)(Cy)Cl₂], the R₅R- and S,S-isomers of oxaliplatin and [Pt(DACH)Cl₂], which all have a 6-C cyclohexyl moiety as part of their non-leaving group, had high RF values (9.02-28.4) (Table 2A). Therefore, it appeared that the structure of the non-leaving group(s) of a platinum compound is an important determinant of its interaction with OCTl. Lastly, different isomers of the 1,2- diaminocyclohexane-substituted platinum complexes appeared to interact similarly with OCTl . The R₅R- and S,S-isomers of oxaliplatin (R,R vs. S₅S: 22.4 vs. 20.7) and [Pt(DACH)Cl₂] (R₅R vs. S₅S: 22.9 vs. 28.4) have similar RF values (Table 2A).

Nature of the leaving group(s): Changes in the leaving group induce small or no changes in the RF values of platinum complexes. For example, all the DACH compounds (R₅R- and S,S-isomers of oxaliplatin and [Pt(DACH)Cl₂]) had similar RF values (20.7-28.4, Table 2A), although the leaving group of oxaliplatin (oxalate) is very different from that of [Pt(DACH)Cl₂] (chloride) (Table 2A). In addition, cisplatin, carboplatin and [Pt(NH₃)₂(trans-l ,2-(OCO)₂C₆H₁₀], all of which have different leaving groups but identical non-leaving groups, had similar RF values (1.13-1.97, Table 2A). Moreover, a cyclohexane ring, when present in the non-leaving group(s) of a platinum complex, such as in those DACH compounds, markedly increases OCTl interaction (RF: 20.7-28.4) in comparison to diamine ligands (RF: 1.13-1.97). However, when the cyclohexane ring was incorporated into the leaving group, as in [Pt(NH₃)₂(trans-l,2- (OCO)₂C₆Hio], it had minimal effect on the OCTl interaction, the RF value of [Pt(NH₃)₂(trans-l,2-(OCO)2C₆H₁₀] being 1.97 (Table 2A).

Example 8

The following example describes the study and identification of the chemical form of oxaliplatin that is the substrate(s) of OCTl . Multiple chemical species exist in equilibrium when platinum complexes are dissolved in an aqueous solution containing high concentrations of chloride ion. Therefore, identification of the chemical species that are taken up by OCTl would contribute to the understanding of the SAR of platinum- OCTl interactions. In chloride containing media, such as plasma ([Cl-] - 103 mM; Howe-Grant et al., 1980, Metal Ions in Biological Systems (Sigel, H., ed., 11, 63-125) and our cell culture medium, the oxalate leaving group of oxaliplatin can be replaced by chloride, resulting in [Pt(i?,i?-D ACH)Cl₂]. The latter can be further aquated to form the mono-, [Pt(i?,i?-D ACH)(H₂O)Cl]⁺, and dicationic, [Pt(J?, i?-DACH)(H2O)₂]²⁺, species. The monoaqua and diaqua cations are the active forms of oxaliplatin, which bind to DNA. Considering the general properties of OCT substrates, which are positively charged small organic compounds, the mono- and / or diaqua chemical species, having one or two positive charges, may be the chemical forms taken up by OCTl .

To investigate experimentally the oxaliplatin-derived species taken up by OCTl, the platinum-DNA adduct formation in both MDCK-hOCTl and MDCK-MOCK cells after incubation with oxaliplatin (20 μM) in chloride free buffer (PB-SO4) was measured. In this buffer, oxaliplatin was predicted to remain predominantly intact because the affinity of sulfate for platinum(II) is much lower than that of chloride. Displacement of the oxalate group by water was expect to be a relatively slow process. In addition, short incubation times (25 min) to minimize conversion of oxaliplatin to intermediate aquated species were used.

FIG. 5 shows platinum-DNA adduct formation after incubation with oxaliplatin or [Pt(J?,i?-DACH)(H₂O)₂]²⁺ in PB-Cl or PB-SO₄ buffer. Transfected MDCK cells were incubated with oxaliplatin (20 μM) or [Pt(J?, i?-DACH)(H₂O)₂]²⁺ (1 μM) in PB-Cl or PB- SO₄ buffer at 37°C and 5% CO₂ for 25 min. Oxaliplatin was freshly prepared and was added to PB-SO₄ buffer immediately, and to PB-Cl buffer half an hour before cell incubation. After incubation, the cells were washed with ice-cold PBS three times and harvested. Genomic DNA was isolated from the harvested cells and the DNA-associated platinum content was determined by ICP-MS and normalized for the DNA content. Data are expressed as mean ± SD of three measurements. Under these conditions, the Pt-DNA adduct level in MDCK-hOCTl cells

(0.00398 ± 0.00089 pmol / μg DNA, r_b = 1.31 ± 0.29 x 10^"6) was similar to (p > 0.05) that in MDCK-MOCK cells (0.00320 ± 0.00042 pmol / μg DNA, η, = 1.05 ± 0.14 * 10^"6) (FIG. 5), suggesting that unmodified oxaliplatin is not an OCTl substrate. Secondly, to determine whether an aquated form of oxaliplatin was taken up by OCTl, the platinum-

DNA adduct formation was measured after incubation with oxaliplatin (20 μM) in the chloride-containing buffer, PB-Cl, for 25 min. Under these conditions, it is likely that conversion to the monochloro/monoaqua cation will occur, with displacement of the oxalate ligand. The DNA-associated platinum level was substantially higher (2.74-fold, p < 0.01) in MDCK-hOCTl cells (0.00933 ± 0.00124 pmol / μg DNA, r_b = 3.08 ± 0.41 x 10^"6) than that in MDCK-MOCK cells (0.00340 ± 0.00087 pmol / μg DNA, r_b = 1.12 ± 0.29 x 10^"6) (FIG. 5) consistent with this expectation. Platinum-DNA adduct formation after direct incubation with the diaqua compound, [Pt(Z?, .K-D ACH)(H₂O)₂J²⁺ (1 μM), in the PB-SO₄ buffer for 25 min, was also determined. Under these conditions, the platinum complex was expected to be a mixture of diaqua (82.8 %) and aqua/hydroxo (17.1%) species. Here the percentage was calculated based on the pKa values of 6.14 and 7.56 for the diaqua and aqua/hydroxo forms of oxaliplatin, respectively, and the pH value of 7.4 for the incubation buffer). The DNA-associated platinum level in MDCK- hOCTl cells (0.0139 ± 0.0020 pmol / μg DNA, r_b = 4.59 ± 0.66 x 10-6) was similar to (p > 0.05) that in MDCK-MOCK cells (0.0142 ± 0.0028 pmol / μg DNA, r_b = 4.69 ± 0.92 x 10-6) (FIG. 5), suggesting that the diaqua form is not an OCTl substrate. Whether or not the aqua/hydroxo form, which carries one positive charge, can be taken up by OCTl remained unclear. Taken together, these studies suggest that a monoaquated form of oxaliplatin, either the chloro or hydroxo species, both of which carry one positive charge, is the actual substrate of OCTl .

Example 9 The following example describes the expression of OCTl and OCT2 in colon cancer cell lines and tissue samples. Total RNA was isolated from colon cancer cells and normal or cancerous colon tissues. The expression of OCTl and OCT2 in these samples was detected by RT-PCR as described in the Experimental Procedures. A PCR cycle number of 40 was used in all the samples. Human GAPDH expression was used as a loading control and a PCR cycle number of 30 was used for its amplification. As shown in FIG. 6, expression of OCTl mRNA was detected in the six colon cancer cell lines tested in this study (LS 180, DLD, SW620, HCTl 16, HT29 and RKO) with the highest expression level in HT29 cells. Four normal colon tissue samples and twenty colon tumor samples exhibited variable OCTl expression levels. OCT2 was not detected in any of the cell lines or in the normal colon tissue samples; however, 11 of the

20 tumor samples demonstrated significant OCT2 expression (FIG. 6).

Example 10 The effect of an OCT inhibitor, cimetidine, on drug sensitivity of cisplatin and oxaliplatin in colon cancer cell lines was studied. To evaluate the potential role of OCTl in the cytotoxicity of oxaliplatin and to determine whether OCTl contributes to the differences in activities of cisplatin and oxaliplatin, the sensitivities (IC₅₀) of both oxaliplatin and cisplatin in the colon cancer cells in the presence and absence of an OCT inhibitor, cimetidine (1.5 mM), were determined. The resistance factor (RF) due to the presence of cimetidine was defined as the ratio of the IC_5O value in the presence of cimetidine to that in the absence of cimetidine. As shown in Table 2B₅ the sensitivity of oxaliplatin was higher (lower IC₅₀) than that of cisplatin in each of the tested colon cancer cell lines in the absence of cimetidine (control, the mean ± SE Of IC₅₀ in the six cell lines: 3.88 ± 1.42 μM (oxaliplatin) vs. 10.5 ± 2.02 μM (cisplatin)). However, in the presence of cimetidine, oxaliplatin sensitivity was substantially decreased in each of the cell lines (RF values ranged from 5.04 to 11.4 (p < 0.001)), resulting in IC₅₀ values comparable to, or even higher than those of cisplatin (mean ± SE of IC₅₀ in the six cell lines: 29.1 ± 10.7 μM (oxaliplatin) vs. 19.4 ± 4.32 μM (cisplatin)). The effect of cimetidine on cisplatin sensitivity was small (range of RF values: 1.44-2.47, Table 2B).

Example 11

The following example describes the investigation of the drug sensitivity of the nine platinum complexes shown in FIG. IA. The IC₅₀ values (μM) of all the 9 platinum complexes shown in FIG. IA, except for carboplatin, in MDCK-MOCK and MDCK-hOCTl after 7 hours of drug exposure were determined in parallel using an MTT assay as described in the Experimental Procedures. Briefly, MDCK cells were seeded at a density of 5,000 cells/well in 96-well plates and exposed to the test compounds for 7 hours on the following day. After incubation for a total of 72 hours, the cell growth was determined by an MTT assay. The resulting data is shown in Table 2 A. The data for carboplatin was taken from Table IA and was not determined simultaneously with the other compounds. The resistance factor (RF) was defined as the ratio of the mean IC₅₀ value in MDCK-MOCK cells to that in MDCK-hOCTl cells.

Also, the IC₅₀ values (μM) of oxaliplatin and cisplatin in the colon cancer cell lines were determined in the presence or absence (control) of cimetidine (1.5 mM) in a similar fashion. The cell seeding density was 6,000-, 8,000-, 6,000-, 15,000- 12,000- and 4,000 cells/well for HCTl 16, HT29, RKO, SW620, LS 180 and DLD cells, respectively. When cimetidine (1.5 mM) was used, it was added to the wells immediately before the addition of the platinum drugs. The resistance factor (RF) was defined as the ratio of the mean IC_5O value in the presence to that in the absence of cimetidine. The resulting data is shown in Table 2B.

Data are expressed as mean ± SD from six measurements for Tables 2A-C and each measurement was performed in quadruplicate.

Table 2. Drug sensitivity of platinum compounds.

A. Drug sensitivity of structurally diverse platinum complexes in OCT1-transfected cells

Platinum complexes MDCK-MOCK (μmol/L) MDCK-hOCT1 (μmol/L) Resistance factor

CfcpUUn 6.3 + (17-1 3Λ ± tt.30 1.7*

Cnrboplatin 2W) ± 86 23(1 ± Sh 1.1

|Pl (NI hU'rans - 1 ,2-(CXX) ),C,,H ,„)] 21 ± 2.9 11 ± 2.7 2.0 '

Ce-IPl(Nl 1.-,XCy)Cl₂] 1.4 ± 0.15 0.16 ± (1030 •).() '

Oxulψlatui 11 ± 3.7 0.48 ± O.W 22 '

|Pl(£.f-DΛCH)υxalah>| XO ± 11 1.4 1 1.2 21 '

IPI(AA-DACH)CI.,] 15 ± 3.2 0.65 i 0.26 23 '

|Pt (.W⁾ACH)CU 16 ± 3.7 O37 ± 0.18 28 '

B. Drug sensitivity of structurally diverse platinum complexes in OCT2-transfected cells

Platinum complexes HEK-MOCK (μmol/L) HEK+IOCT2 (μmol/L) Resistance factor

Cisplatin 2J6 ± O52 1.2 i α.v» 2.1*

Cartxiptuliu 11(1 ± -K> 62 ± -16 lϋ

[PmIMImIU-IMOCO)₂C_tH₁₀)] 19 ± 5.7 9.9 i 2.8 l.q*

[PU-nJCW 6/. ± 1.5 1.1 ± 0.42 ύtϊ ' c-HPtfNHjXCylCl.,] OXi ± <1(M3 0.020 1 0.0065 iι ^!

O.xullplalin 4.1 ± 1.69 o.ii ± iurio 37 '

|Pl(£.f-DACH)uuilato] 9.0 + 1.7 0.27 ± UOfti »'

[Pl(AA-DACH)CU 2.1 ± a <» 0.071 ± 0.026 2« '

[Pl(SS-DACHtCI₂I <ts ± n.71 0.M ± α041 Xi ' C. The sensitivity of the colon cancer cell lines to oxaliplatin and cisplatin in the presence or absence ol cimetidine

Cell Ones Oxaliplatin Cisplatin

Control Cimetidine treated Resistance Control Cimetidine Resistance laclor treated (actor

HCTl 16 > 2.4 i 1.4 11 ± 6.2 7.9 ' 5.4 ± 1.3 10 i 3.2 1.9*

Hrø 1 4.6 ± 1.4 52 ± 19 JH 12. ± 3.9 Sl ± U 2.5*

KKO » 1.6 ± 0.56 9.7 i 2.7 5.9 ' &fι ± 2.4 13 ± AA 1.5

SW620* 2.8 + 1.0 14 ± 2.8 5.0 ' 13 ± 2.0 ?> ± 4.9 1Λ*

I-Si ai ' 13 ± 0.41 8.4 ± 2.8 6.4 ' 5.7 ± US 8.3 + 3.4 1.4

DID JJ ± 6,0 71 ± 13 6.7 ' 18 ± 7.6 32 ± 10 1.7!

NOTE: The IC₅₀ values (μmol/1.) of αli the platinum complexes, except for camoplatin. after 7 hours of drug exposure were determined in parallel using a M'lT assay as described in Λlaterktls and Methods (A and B). The data for camopkitin in (A) and (B) were taken from Table IA and B. respectively, and were not determined simultaneously with the other compounds. The resistance tactπr was defined as the ratio of the mean IC^ value in the MOCK cells to that in the OCT-transfected cells. The [C₃, values (μmol/1.) of oxuliplatin and cisplalin in the colon cancer cell Unas (7 hours ol'drug exposure) were determined in the presence or absence (control) of cimelidine ( 1.3 innuil/M in parallel (C). The cell seeding density was 6.(100, 8/MX).6,000. 15,(JM. 12WK). and 4.000 cells per well for HCT! 16, HT29, RKO. SW620, I.SISO. mid DlJJ cells, respectively. VVhen cimelidine ( 1.5 rninolA.) was used, it was added t<j the immediately before the addition of the platinum drugs. The resistance factor was defined as the ratio of the mean IC_M value in the presence to that in the absence of cimetidine. All the datii are expressed as mean ± SU of six measurements, and each measurement was done in quadruplicate. V> < 0.01. IP >: 0.001. tTtic IC₃₁ value of oxaliplatin is significantly lower than Unit of cisplatin in the absence of cimetidine. V < 0.05.

Example 12

The striking activity of cisplatin in an otherwise fatal disease, testicular cancer, has been established by thirty years of clinical experience. However, acquired and intrinsic resistance limits its application to a relatively narrow range of tumor types. To broaden the anticancer spectrum of this platinum agent, thousands of structural analogs have been tested. Cisplatin analogs with two ammine ligands, such as carboplatin and nedaplatin (approved in Japan), are cross-resistant with cisplatin. Analogues with different ligands display more diverse activity profiles. Notably, oxaliplatin, with DACH in place of the two ammine ligands, in combination with 5-fluoruracil/leucovorin produced response rates twice that of 5-fluoruracil/leucovorin regimens alone in the treatment of colorectal cancer, against which cisplatin is inactive. Efforts to understand the differences in oxaliplatin versus cisplatin antitumor activity have focused mainly on the cellular processing of cisplatin- and oxaliplatin-DNA adducts. Defects in MMR cause modest to moderate resistance to cisplatin but not to oxaliplatin. Differences in the mechanism(s) controlling cellular uptake and efflux of these platinum compounds, although rarely studied, can also contribute to their disparate activities considering the nature of their chemical structures.

In the present study, it was observed that the influx transporters, OCTl and OCT2, can play a critical role in the cellular uptake and consequent cytotoxicity of oxaliplatin (Table 1 and FIG. 2). In contrast, the two transporters were relatively unimportant in mediating the uptake and cytotoxicity of cisplatin and carboplatin (Table 1). Overexpression of OCTl and, more strikingly, OCT2 in transfected cells not only increased the rate of cellular platinum accumulation but also elevated the level of platinum-DNA adducts after oxaliplatin exposure (FIG. 3 and FIG. 4). These effects were blocked by known OCT inhibitors. The data strongly suggest that oxaliplatin can be an excellent substrate of human OCTl and OCT2, and the cellular uptake of platinum mediated by these transporters has ready access to the key pharmacological target (DNA). These results are in contrast to platinum uptake mediated by human Ctrl , which appears to sequester the drug in some intracellular compartment, rendering it substantially inaccessible to the pharmacological target. It should be noted that a modest increase in cisplatin uptake (FIG. 3B) and sensitivity (2.23-fold, p < 0.001, Table IB) was observed in HEK-hOCT2 cells in comparison to HEK-MOCK cells, suggesting that cisplatin may be a weak substrate of OCT2.

It is noteworthy that expression of OCTl or OCT2, even at low levels, may play a significant role in the cytotoxicity of oxaliplatin. More than a three-fold increase (3.18-fold) was consistently observed in the IC₅₀ value of oxaliplatin in HEK-MOCK cells in the presence of the OCT inhibitor, cimetidine (FIG. 2E), but not for cisplatin or carboplatin (data not shown). The decrease in oxaliplatin sensitivity in HEK-MOCK cells by the OCT inhibitor may be due to inhibition of intrinsic OCTl and/or 0CT2 activity in HEK 293 cells. Both transporters were detected in HEK-MOCK cells in PCR studies using a cycle number of 40 (data not shown). Furthermore, cimetidine consistently produced a significant decrease in the cellular uptake of oxaliplatin, but not of cisplatin or carboplatin in HEK-MOCK cells (FIG. 3B, 3C). Taken together, the data suggest that low levels of expression of OCTl and OCT2 play a role in sensitizing cells to oxaliplatin. Structure-activity relationship studies revealed that the nature of the amine ligand bound to platinum can be important for interaction with OCTl, with an organic component being required for effective interaction, while the structure of the leaving ligand seems to be less important. A monoaqua derivative of oxaliplatin, either the chloro or hydroxo species, not a divalent diaqua complex, was shown to likely be a likely substrate of OCTl (FIG. 5). These results are consistent with previous work showing that OCTs interact with small molecular weight monovalent organic cations (Jonker and Schinkel, 2004). Although the structure-activity relationships were established for platinum-OCTl interactions, it is likely that the conclusions would apply to platinum- OCT2 interactions because the two transporters have largely overlapping substrate specificities. These studies establish the basis for the design of additional platinum complexes to facilitate the development of a detailed structure-activity relationship, which could be used to predict their interaction with OCTs. This may provide the ability to target platinum complexes for therapy against tumors that express OCTl and OCT2. The structure-activity relationship studies further suggest that OCTs might not play a major role in determining the cytotoxicity of platinum compounds with two ammine ligands, such as cisplatin, carboplatin and nedaplatin. In contrast, OCTs may be important for mediating cytotoxicity of platinum compounds with organic amine ligands (Table 2C). Cell lines that are resistant to cisplatin are cross-resistant to the diammine complexes, carboplatin and nedaplatin, but not to the DACH compounds, oxaliplatin and tetraplatin, which share a similar activity profile. Differences in the activity profiles of these compounds parallel the differences in their interaction with OCTs, suggesting that interactions with OCTl and OCT2 may explain, at least in part, differences in the activities and tumor specificities of platinum complexes.

It is likely that the activity of oxaliplatin in colorectal cancer can be explained, at least in part, by the selective uptake via OCTs. In this study, OCTl expression was detected in all twenty human colon cancer tissue samples and OCT2 expression in 11 out of 20 tissue samples (FIG. 6). Similar levels of OCTl were also detected in the six tested human colon cancer cell lines although OCT2 was not detectable (FIG. 6).

However, both OCTl and OCT2 expression have been detected in another human colon cancer cell line, Caco-2. Drug sensitivity to oxaliplatin was greater than that of cisplatin in each of the six colon cancer cell lines (Table 2C). The higher activity of oxaliplatin in comparison to that of cisplatin in these colon cancer cells may be attributed to the selective uptake of oxaliplatin mediated by the intrinsic OCTl in these cells, since similar activities of oxaliplatin and cisplatin were observed in these cells when OCTl was blocked by cimetidine (Table 2C).

Based on the expression of OCTl and OCT2 in the colon cancer tissue samples and the OCT-dependent activity of oxaliplatin in the cell lines, these transporters may be considered important determinants of oxaliplatin activity in colorectal cancer. Also, it is possible that variable expression of OCTs, especially OCT2, may account for the variability in response to oxaliplatin treatment. In some cases, expression levels of OCTl and OCT2 may be used as markers for the rational selection of oxaliplatin-based versus irrinotecan-based combination therapies for treatment of individuals with colorectal cancer. Such selection is currently primarily based on side-effect profiles or clinical experience. In some cases, oxaliplatin-based therapy may be selected for patients with high levels of OCTl and OCT2 in their tumor samples. In addition, genotyping for non-functional and reduced function polymorphisms of OCTl and OCT2 may be incorporated in the decision-making process.

Currently, platinum based therapies are used in the treatment of a variety of tumors including testicular cancer, ovarian cancer, small cell lung cancer and head and neck cancers. In these therapies, cisplatin is often the drug of choice. However, the studies described herein suggest that when OCTl or OCT2 is expressed in the tumor, oxaliplatin can give improved results, in some cases. Recently, OCTl and OCT2 expression has been observed in a number of human cancer cell lines, suggesting that these transporters may be expressed in the corresponding tumors.

Example 13

The following example describes the synthesis of compounds 1-6, shown below, as well as the investigation of their growth inhibition properties and ability to act as OCT substrates.

The potassium tetrachloroplatinum(II) used was from Engelhard Corp. and cisplatin was synthesized as reported previously. All other chemicals and solvents used were commercially available. ¹H NMR and ¹⁹⁵Pt NMR spectra were obtained on Varian 300 and 500 MHz spectrometers, respectively. Electrospray ionization-MS (ESI-MS) spectra were obtained on an Agilent Technologies 1100 Series liquid chromatography/MS instrument. Fourier transform-IR (FT-IR) spectra were measured on an Avatar 380 FT-IR. Example 13.1 : Synthesis of trfl«s-rPt(TSΗ^(piperazine^*)Cl₂l Cl (I).

The compound was synthesized according to a previously published method. Briefly, cisplatin (300 mg, 1.0 mmol) was dissolved in 30 mL DMF and stirred at room temperature for five min. Solutions of t-butyl-1-piperazine carboxylate (372.5 mg, 2.0 mmol) and silver nitrate (339.7 mg, 2.0 mmol), each in 1 mL DMF, were then added and the solution was stirred for 24 h in the dark at room temperature. A dark yellow precipitate formed and was collected on a fine glass frit filter and lyophilized overnight to remove remaining DMF. The dried solid was taken up in 30 mL ddH₂O and 2 mL concentrated HCl and stirred at room temperature in the dark for 24 h. The solution was filtered twice to remove the white precipitate, heated to 90°C for 60 min and cooled to room temperature. A pale yellow precipitate was removed by filtration and the filtrate was concentrated to ~10 mL by rotary evaporation and kept at 4°C for 18 h. A fine yellow precipitate was observed and collected by filtration. After a second crop was collected from the mother liquor, a total of 73.4 mg was obtained (18%, 0.18 mmol). ¹H- NMR (D₂O, 300 MHz) δ=2.689 (s, IH) 3.207-3.522 (m, 8H) 3.656 (s, IH) 5.440 (s, IH); ¹⁹⁵Pt-NMR (D₂O, 500 MHz) δ=-2180; ESI-MS m/z calculated (M+HC1) 405.61 amu, found 405.7 amu. Example 13.2: Synthesis of \?t(R. i?-DACH¥acac)l Cl (2).

A portion of [?t(R,R-O ACH)Cl₂] (0.200 g, 0.52 mmol) was dissolved in deionized water (10 mL) and, after addition of a solution of silver nitrate (0.171 g, 1.00 mmol) in 5 mL water, the reaction was stirred for 4 h at room temperature. Precipitated AgCl was removed by centrifugation and sodium acetylacetonate monohydrate (0.074 g, 0.526 mmol) was added. The pH was adjusted to 7 with an aqueous solution of 10% sodium hydroxide and the reaction was stirred for 3 h at room temperature. After concentration to ~2 mL by rotary evaporation, the resulting precipitate was collected by vacuum filtration. The white solid was washed with ice cold water, redissolved in minimal hot water (~60°C) and allowed to crystallize to yield colorless crystals (13.2 mg, 0.032 mmol, 6.2%). ¹H-NMR (CD₃OD, 300 MHz) δ=5.519 (s, IH) 2.325 (t, 2H, J=5.4Hz) 1.963 (d, 2H, J=13.8Hz) 1.775 (s, 7H) 1.565 (d, 2H, J=4.2Hz) 1.261 (t, 3H, J=1.8Hz) 1.137 (t, 2H, J=9.6Hz); ¹⁹⁵Pt NMR (CD₃OD, 500 MHz) δ=-1787.66; ESI-MS m/z calculated 408.13 amu, found 408.1 amu; FT-IR (KBr) v (cm^'1) 3434.38, 3105.24, 2941.71, 2859.12, 1635.16, 1569.60, 1537.76, 1384.09, 1341.00, 1167.95, 1126.49, 1063.08. Example 13.3: Synthesis of rPt(7?.i?-DACHΪF6-acac)lCl (3 ).

The compound was synthesized as described for compound 2. ¹⁹⁵Pt NMR (DMF, 500 MHz) δ = -2651; ESI-MS m/z calculated (M+H) : 516.07, found: compound did not fly. FT-IR (KBr) v (cm^'1) 3453.00, 2930.49, 2846.60, 1688.39, 1640.89, 1534.96, 1384.08, 1207.39, 1141.40.

To a solution of [Et₄N][PtNH₃Cl₃] (1.11 mmol) in 4 mL H₂O was added a solution of benzylamine in 0.5 mL H₂O. The orange mixture was stirred for 9 h in the dark at room temperature, although an orange precipitate was observed after only 30 min. The orange precipitate was collected by filtration and washed with water (4x), ethanol (3x) and ethyl ether (2x) to yield an orange solid (73 mg, 17%). ¹H-NMR (d- DMF, 300 MHz) δ=4.034 (t, 2H) 4.292 (s, 2H) 5.315 (s, IH) 7.376 (m, 3H) 7.473 (dd, 2H, J=I.5 Hz, 6.6 Hz); ¹⁹⁵Pt NMR (CD₃OD, 500 MHz) δ=-2163.4. ESI-MS m/z calculated (M+Na): 412.9901, found: 412.9905. Example 13.5: Synthesis of rPt(dien)Cl]Cl (5).

Compound was synthesized from cw-[Pt(dmso)₂Cl₂] as described previously and characterization matched previously published data. ¹H NMR (D₂O, 300 MHz) δ: 2.984 (8H, t); ESI-MS m/z calculated (M+H): 334.04, found: 334.0. FT-IR (KBr) v (cm^"1) 3443 (vs), 3193 (vs), 31 18 (vs), 3056 (vs), 2955 (vw), 2922 (s), 1606 (vs), 1598 (vs), 1462 (m), 1448 (s), 1439 (s).

Synthesis was carried out as previously described. Briefly, a portion of cDDP (300 mg, 1 mmol) was dissolved in 50 mL ddH₂O, to which pyridine (79.9 mg, 1.01 mmol) was added. The stirred solution was heated to 60⁰C for 2 h, then was cooled to room temperature and stirred for 72 h. The volume was reduced to 5 mL by rotary evaporation and unreacted cisplatin was removed by filtration. The filtrate was rotovapped to dryness and the solid was recrystallized from 0.1 N HCl, 0.5 N HCl, and methanol to yield 29.1 mg of white needles (8.4%) ESI-MS m/z calculated (M+H): 344.69, found: 344.0. ¹⁹⁵Pt NMR (CD₃OD, 500 MHz) δ=-2312. A unit cell was collected by x-ray crystallography that matched published data.

Compounds 1 -6 were then evaluated for their growth inhibition properties and their ability to act as OCT substrates, according to the procedures described above, and the resulting data is shown in FIG. 9. FIG. 1OA shows a graph of the comparison of growth inhibition for the ten of the platinum compounds studied, expressed as IC₅₀ in uM (left bars indicate OCTl (+); right bars indicate OCT2(+)). The smaller bars indicate more effective growth inhibition. FIG. 1 OB shows a graph of compounds tested as hOCTl and hOCT2 substrates (left bars indicate OCTl (+) vs. OCTl (-); right bars indicate OCT2(+) vs. OCT2(-)). Values on the ordinate are expressed as the "fold difference" in IC50 between cells with the transporter and cells without. Larger bars indicate better hOCTl or hOCT2 substrates.

Compound 1 was tested and shows MDCK-MOCK cytotoxicity of 44.5 μM and a MDCK-hOCTl cytotoxicity of 5.87 μM, corresponding to a 5.1 1 -fold increase in cytotoxicity. Without wishing to be bound by theory, a possible explanation for the increase in cytotoxicity may be that the outward-facing charged N-heteroatom on compound 1 may adversely affect affinity for the transporter.

Compound 4 was similarly tested, and was observed to be a highly cytotoxic OCTl substrate having a cytotoxicity four times that of oxiplatin in the OCT(+) cell line. Compound 6 was similarly tested, and was shown to have a MDCK-MOCK cytotoxicity of 704 μM and a MDCK-hOCTl cytotoxicity of 8.09 μM, corresponding to a 87-fold increase in cytotoxicity. Without wishing to be bound by theory, factors which may contribute to the improved rate of transport of compound 6 may be the aromaticity and/or planarity of the pyridine ligand, and/or the fact that compound 6 is positively- charged even prior to aquation.

Example 14

The following examples (e.g., Examples 14-22) describe the identification of unique chemical and biological properties of a cationic, monofunctional platinum(II) complex, cis-diammine(pyridine)chloroplatinum(II), cis-[Pt(NH₃)₂(py)Cl]⁺ or cDPCP, a coordination compound. In the following examples, this compound is shown to be an excellent substrate for organic cation transporters 1 and 2 (SLC22A1 and SLC22A2). These transporters are abundantly expressed in human colorectal cancers where they mediate uptake of oxaliplatin, cis- [Pt(D ACH)(oxalate)] (DACH = trans-i?,i?-l,2- diamminocyclohexane), an FDA-approved first-line therapy for colorectal cancer.

Unlike oxaliplatin, however, cDPCP can bind DNA monofunctionally, as revealed by an X-ray crystal structure of cis- {Pt(NH₃)₂(py)}²⁺ bound to the N7 atom of a single guanosine residue in a DNA dodecamer duplex, as described more fully below. Although the quaternary structure resembles that of B-form DNA, there is a base pair step to the 5' side of the Pt adduct with large shift and slide values, features that are characteristic of cisplatin intrastrand cross-links. cDPCP was shown to effectively block transcription from DNA templates carrying adducts of the complex, unlike DNA lesions of other monofunctional platinum(II) compounds like {Pt(dien)}²⁺. cDPCP-DNA adducts were removed by the nucleotide excision repair apparatus. Characteristics such as these indicate that cDPCP and related complexes may be considered as therapeutic options for treating colorectal and other OCT-related cancers bearing appropriate cation transporters. The following examples describe the results of structural and mechanistic investigations of cis-diammine(pyridine)chloroplatinum(II) (cDPCP, FIG. 11). The experiments reveal (i) the uptake of cDPCP mediated by organic cation transporters (OCT) 1 and 2; (ii) the X-ray crystal structure of a DNA dodecamer duplex containing a monofunctional adduct of the complex bound to a central guanosine residue; (iii) the ability of cDPCP-DNA adducts to inhibit transcription; and (iv) reduced repair of cDPCP-DNA adducts by the mammalian excinuclease relative to those of cisplatin.

Cellular accumulation of platinum anticancer agents represents the first step in their mechanism of action. Differences in relative influx and efflux contribute to drug resistance and are factors in the differential response of various tumor types to the drugs. Recently, OCTl and OCT2 were identified as mediators of oxaliplatin transport and toxicity in human tissue. In previous studies, mRNA from OCTl was detected in 20 of 20 tumor samples from colon cancer patients and mRNA from OCT2 was detected in 11 of 20 samples. There are three members of the human OCT family. hOCTl is expressed primarily in the liver, hOCT2 in the kidney, and hOCT3 in the liver, heart, brain, and placenta. All are present in the intestinal/colorectal area to varying degrees. OCTs generally interact with organic substrates having M_r < 400 Da and overall positive charge. Oxaliplatin, a neutral compound, is typically transported only after loss of the oxalate group to form mono- or dicationic species. Although oxaliplatin and cisplatin form adducts on DNA, cisplatin is often a poor substrate for OCTl and 2 as compared to oxaliplatin, and is not active against colorectal cancer.

As described more fully below, structure-function studies of Pt-based OCT substrates indentified the monofunctional, cationic complex cDPCP as having outstanding antitumor properties for OCT-related cancers. The antitumor properties of cDPCP were initially established in a sarcoma 180 ascites murine model study. The compound was subsequently shown to block DNA replication at single dG sites by replication mapping experiments, and a series of biochemical experiments established that cDPCP forms a fundamentally different adduct profile than the bifunctional cross- links of cisplatin or oxaliplatin. Concern over the potential lability of heterocyclic N- donor ligands, such as pyridine, in Pt(II) complexes have led to extensive investigations of the stability of their DNA adducts. Although some complexes containing three N- donor ligands, such as N-methyl-2,7-diazapyrenium, do form bifunctional adducts after loss of the heterocycle, neither pyridine or ammonia were observed to dissociate from cDPCP upon DNA binding.

In the following examples, the molecular mechanism of action of cDPCP was studied. The four early phases by which platinum compounds exert their anticancer activity are (1) cellular accumulation, (2) activation (typically by aquation), (3) DNA binding, and (4) the initial cellular responses to the DNA damage. The following examples, in part, study whether or not cDPCP exhibits specificity for entry into cells expressing OCTs while also exhibiting potency. The cellular accumulation of cDPCP due to organic cation transporters was also examined, as well as the effect of transporter expression on the sensitivity of mammalian kidney cells to the compound. To determine the influence of cDPCP on DNA geometry, a site-specifically platinated dodecamer duplex containing a centrally located cis-{Pt(NH₃)₂(py)}²⁺-dG adduct was constructed and its structure was determined by X-ray crystallography. The consequences of cDPCP binding to a plasmid DNA were also studied. To investigate processing of cDPCP and cisplatin adducts by DNA damage-recognition proteins, an in vitro system was utilized to study repair of monofunctional platinum lesions by nucleotide excision repair (hereafter referred to as excision repair) and a cell-based β-galactosidase reporter assay was utilized to study the ability of the adducts to inhibition transcription by RNA polymerase II.

Example 15

The following example describes various experimental procedures for the study of platinum compounds in the treatment of cancers expressing OCT.

Materials: Potassium tetrachloroplatinate(II) was obtained as a gift from Engelhard Corporation (now BASF, Iselin, NJ). Cisplatin and m-[Pt(NH₃)₂(py)Cl]Cl were synthesized as described. Phosphoramidites and other reagents for DNA synthesis were purchased from Glen Research. Crystallization reagents were obtained from Hampton Research and Sigma. Enzymes were purchased from New England Biolabs. Plasmids pBR322 and pSV-β-galactosidase were purchased from New England Biolabs and Promega, respectively, and were amplified in 100 mL LB cultures of E. coli XLl- Blue cells containing ampicillin as a selecting agent and purified on Maxi-prep columns (Qiagen). [γ-³²P]ATP was obtained from Perkin Elmer. All other chemicals and solvents were purchased from commercial suppliers.

Cellular Accumulation and Compound Cytotoxicity and Cell Lines and Transfection: Madin-Darby canine kidney (MDCK) cells were stably transfected with full-length human OCT 1 cDN A (MDCK-hOCT 1 ) and the empty vector (MDCK-

MOCK), as previously established. Human embryonic kidney (HEK) 293 cells stably transfected with the full-length OCT2 cDNA (HEK-hOCT2) and with the empty vector (HEK-MOCK) were also previously described (Zhang et al., 2006, Cancer Research, 66, 8847-8857). Cell Culture: The stably transfected MDCK and HEK 293 cells were cultured in

DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin (Invitrogen) and the respective selection antibiotics and grown at 37 °C in a humidified atmosphere with 5% CO₂.

Compound Cytotoxicity: Cytotoxicities of the compounds were determined by plating cells in 96-well plates at a predetermined density. Cells were then incubated overnight and platinum complexes were added to the culture medium. After 7 h, the medium was replaced with fresh, Pt-free medium and the incubation was continued for a total of 72 h after the initial addition. MTT assays were performed as previously described (Alley et al., 1988, Cancer Research, 48, 589-601.). Cellular Accumulation of Platinum: Studies of cellular accumulation of platinum were performed as described (Zhang et al., 2006, Cancer Research, 66, 8847-8857).

X-ray Crystal Structure Determination of Platinated DNA Duplex: Two deoxyoligonucleotides (5'-CCTCTCGTCTCC-3' and its complementary strand) were synthesized and purified by standard methods (e.g., see Caruthers, 1991, Ace. Chem. Res. 24, 278-284.). The site-specifically platinated duplex was prepared and purified as previously described (Silverman et al., 2002, J. Biol. Chem., 277, 49743-49749; Spingler et al., 2001, Inorg. Chem., 40, 5596-5602.). Details of crystallization experiments, X-ray diffraction data collection, structure determination, and refinement are found in Examples 17 and 22. Final coordinates for the refined model are deposited into the

Protein Data Bank with accession code 3CO3.

DNA Unwinding Assays: Details of plasmid platination and agarose gel analysis are found in Examples 18 and 22. Transcription in Living Human Cells. Transcription Probe Preparation: The pSV-β-galactosidase vector (Promega), containing a lacZ gene under the control of an

SV40 promoter and enhancer, was amplified in XLl -Blue, purified on a Maxi-prep column (Qiagen) and globally platinated with either c/s-[Pt(NH₃)₂(py)Cl]⁺ [Pt(dien)Cl]⁺ to yield r_b values between 0 and 0.13. Excess platinum was removed by spin dialysis (Nanosep columns, Pall Biosciences, 3K MWCO), and DNA and Pt concentrations were quantified by UV-vis and atomic absorption spectroscopy, respectively.

Transcription Assay: Experimental details are provided in Examples 19 and 22. Nucleotide Excision Repair Assay: 156mer probes were prepared as described in

Examples 20 and 22. Assays were performed as described previously (Reardon, JT, Sancar, 2006, Methods Enzymol, 408, 189-213; Zamble et al., Biochemistry, 35, 10004-

10013) with 10 frnol of the platinated repair probe and 75 μg cell-free HeLa extract.

Reactions were allowed to proceed for 60 min at 30 °C and were stopped by the addition of SDS and proteinase K to final concentrations of 0.34% and 20 μg/mL, respectively.

After extraction with 25:24:1 phenol: chloroform :isoamyl alcohol and careful ethanol precipitation with 10 μg linear polyacrylamide as co-precipitant, reaction products were analyzed by 10% urea-PAGE.

Example 16 The following example describes the cellular accumulation and Pt-induced loss of viability in OCT+/- Cells. Mammalian cells stably transfected with a full-length human OCTl or OCT2 cDNA, or transfected with an empty vector as a control, were used to study the cellular accumulation and cytotoxicity of cw-diammine(pyridine)- chloroplatinum(II) (e.g., c/s-[Pt(NH₃)₂(py)Cl]⁺ or cDPCP) and oxaliplatin. In this example, cDPCP was 87-fold more cytotoxic in OCTl (+) than OCTl (-) cells, whereas oxaliplatin was only 12-fold more effective. The cytotoxicity of cDPCP in OCT2(+) cells increased by a factor of 137 over OCT2(-) cells, compared to a 53-fold increase with oxaliplatin and is shown in FIG. 12, as described herein. Examination of treated cells for platinum content revealed that accumulation of cDPCP is about 68-fold higher in hOCT2-containing cells than in cells not expressing the transporter. In hOCTl- containing cells, a 23-fold increase in platinum accumulation was measured.

Table 3 gives a comparison of the accumulation of cDPCP and oxaliplatin by hOCTl and hOCT2 cells, as measured by ICP-MS and expressed as mean + SD from six measurements. Cells were incubated with either 10 uM (OCTl experiment) or 2 uM platinum (OCT2) for 2 h. The corresponding numbers for oxaliplatin were 23 -fold for hOCT2 and 4.7-fold for hOCT2. Measurements of platinum levels on DNA after cDPCP treatment were not obtained, but DNA platination by oxaliplatin closely tracked cellular accumulation of platinum in cells expressing hOCTl and hOCT2 and can be reversed with the OCTl and OCT2 inhibitors disopyramide and cimetidine, respectively (Zhang et al., 2006).

FIG. 12A and FIG. 12B show the cell growth inhibition assays for cDPCP and oxaliplatin, respectively, in MDCK cells with and without hOCTl. FIG. 12C gives the IC₅₀ values for both compounds in MDCK-OCTl vs. -MOCK and HEK-OCT2 vs. - MOCK cells, expressed as mean ± SD from three experiments, with quadruplicate measurements obtained in each experiment.

Table 3. Accumulation of cDPCP and oxaliplatin by hOCTl and hOCT2 cells. pmol Pt/mg protein

MDCK-MOCK MDCK-hOCTl Fold change cDPCP 33.1 ± 0.5 779 ± 67 23

Oxaliplatin 13.9 ± 0.9 65.6 ± 4.1 5

HEK-MOCK HEK-hOCT2 Fold change cDPCP 18.7 ± 1.6 1278 ± 69 68

Oxaliplatin 5.65 ± 0.72 130 ± 11 23

Example 17

The following example describes DNA binding characteristics of cDPCP and the X-ray structure.

A DNA dodecamer duplex containing a site-specific c/5-{Pt(NH₃)₂(py)}²⁺-dG adduct was synthesized, crystallized, and the X-ray diffraction data was collected to 2.17 A resolution. Diffraction-quality crystals were grown by using the hanging-drop vapor diffusion method at 4 ⁰C. Crystallization solutions contained 120 mM Mg(OAc)₂, 50 mM sodium cacodylate pH 6.5, 1 mM spermine, and 28% w/v polyethylene glycol (PEG) 4000. Hanging drops contained 2 μL of 0.2 mM DNA and 2 μL crystallization solution. All solutions were prepared and sterile filtered immediately prior to use. Crystals were transferred to a cyroprotectant solution (120 mM Mg(OAc)₂, 50 mM sodium cacodylate pH 6.5, 1 mM spermine, 30% w/v polyethylene glycol (PEG) 4000, and 15% v/v glycerol), mounted on loops, and flash frozen in liquid nitrogen. Data sets . for single-wavelength anomalous diffraction (SAD) studies were collected at 100 K on beam line 24-ID-C at the Advanced Photon Source (APS) at Argonne National

Laboratory and processed in HKL2000. Higher resolution data to 2.17 A were later collected on beam line 9-2 of the Stanford Synchrotron Radiation Laboratory (SSRL) and processed with HKL2000. Collection statistics for both data sets are summarized in Table 4. SAD phases were calculated using the program SHARP and the initial model was constructed manually with the program Coot. After several cycles of rigid-body refinement in Refmac5 (Murshudov et al., 1997) and subsequent model building in Coot, the structure was subjected to restrained TLS refinement against the high-resolution data set. Initial TLS parameters were obtained from the TLS Motion Determination server. Sixteen water molecules were added to locations with appropriate hydrogen bonding distances (<3.5 Angstroms) to the DNA and electron density greater than 1.5σ on a 2F₀- F_c map. A simulated-annealing composite omit map was calculated using CNS. Final refinement statistics are given in Table 4. Geometric parameters were calculated with the program 3DNA (Lu et al., 2003). All crystallographic images were created using PyMOL. The structure was solved using phases obtained from single-wavelength anomalous diffraction data from the Pt atom. There is one molecule in the asymmetric unit and a solvent content of 56%. The structure, depicted in FIG. 13, contains linear, B- form DNA with platinum coordinated to N7 of guanine in the major groove. FIG.

13 shows the X-ray crystal structure of cDPCP-modified DNA. FIG. 13 A shows a schematic diagram of the DNA sequence and location of the platinum adduct for the complex studied by X-ray crystallography. FIG. 13B shows the structure of the cDPCP- damaged DNA duplex, which can maintain a linear B-form conformation upon binding of the Pt complex. FIG. 13C shows a close-up view of the monofunctional Pt-dG adduct, with 2F₀-F₀ maps contoured at lσ (3) and 15σ (inside circle 2), which show significant electron density around the platinum atom. FIG. 13D depicts the platinated base pair (4) overlaid with ideal B-form DNA (6). Platinum binding forces the DNA bases out into the major groove, causing significant increases in the shift and slide values of the base pair step to the 5' side of the adduct. Table 5 lists the base pair step parameters for the DNA duplex, with entries 6 and

7 showing steps containing the platinum adduct. Table 6 lists base pair parameters for the DNA duplex, with entry 7 showing the bp containing the platinum adduct. Watson- Crick hydrogen bonding between the base pairs is maintained throughout the dodecamer duplex. The double helix is unwound by approximately 8° in the vicinity of the platination site, a value in agreement with previous NMR spectroscopic results of a heptamer duplex containing the analogous cw-{Pt(NH₃)₂(4-Me-py)}²⁺-dG adduct.

The pyridine ligand of the cw-{Pt(NHs)₂(py)}²⁺ moiety is directed toward the 5' end of the platinated strand. FIG. 14A shows various stereoscopic views of the cDPCP- dG adduct on duplex DNA and FIG. 14B shows the 2F₀-F _c electron density map contoured at lσ. This orientation can facilitate formation of a hydrogen bond between the NH₃ ligand trans to pyridine and 06 of the guanosine residue (N-O distance, 2.8 A). Analysis of the structure revealed that the platinated G-C base pair is displaced toward the major groove in order to accommodate the platinum adduct, resulting in a base pair step to the 5' side of the platinum adduct with abnormally large shift and slide values of 1.20 A and 0.82 A, respectively (FIG. 13).

Table 4. X-ray data collection and refinement statistics.

I Data collection statistics* I

Data set SAD High res.

Beamline APS 24-ID-C^f SSRL 9-2*

Wavelength (A) 1.072 0.984

Space group C222_! C222,

Unit cell dimensions (A) a 45.8 46.4 b 66.4 66.0

C 56.6 56.1

Resolution range (A) 50 -2.72 50 - 2.17

Obs. reflections 14263 32977

Unique reflections 2264 4765

Completeness 94.2 (73.6) 96.0 (76.0)

I/σ 23.2 (6.7) 18.1 (2.7)

**merze 7.3 (17.6) 10.7 (55.6)

Refinement statistics I

22.5

Kfree 25.4

RMSD bond lengths (A) 0.006

RMSD bond angles (°) 1.435

Average B-factors (A²) 42.4

Platinum ligand 44.4

Solvent 42.9

^♦Values in parentheses are for the highest resolution shell.

¹ADSC Q315 detector, 360 frames, ΔΦ = 1°, exposure time = 2 s

*MAR 325 detector , 180 frames, ΔΦ = 1°, exposure time = 5 s

*R_merge = Σ|I-D 0 0 0 U .

V = Σ||F_O| - |F_C||/Σ|F_O|. tⁱRf_ree ⁼ R obtained for a test set of reflections (5% of diffraction data).

Table 5. Base pair step parameters for the DNA duplex, where the parameters are defined as follows: Dx, shift; Dy, slide; Dz, rise; /, tilt (°); a, roll (°); Ω, twist (°). All values are in A unless otherwise noted.

Step Dx Dy Dx I Ω

Q

1 C1-G24/C2-G23 -0.73 -0.28 3.43 -3.79 0.64 35.04

2 C2-G23/T3-A22 -0.19 0.20 3.27 0.62 0.89 32.64

3 T3-A22/C4-G21 0.56 0.91 3.13 5.56 -1.76 39.47

4 C4-G21/T5-A20 -1.29 0.23 3.49 -2.99 4.57 30.10

5 T5-A20/C6-G19 0.47 0.06 3.50 2.81 3.42 40.20

6 C6-G19/G7-C18 1.20 0.82 3.50 6.37 2.79 31.29

7 G7-C18/T8-A17 -1.18 -0.37 3.01 -4.64 1.19 30.84

8 T8-A17/C9-G16 0.87 0.30 3.43 2.17 2.52 41.84

9 C9-G16/T10-A15 -0.47 0.20 3.19 0.31 3.68 28.69

10 T10-A15/C11-G14 1.12 0.67 3.43 2.33 5.03 39.78

11 C11 -G14/C12-G13 0.01 0.06 3.51 -2.48 -1.22 37.79 Table 6. Base pair step parameters for the DNA duplex, where the parameters are defined as follows: Sx, shear; Sy, stretch; Sz, stagger; K, buckle; ω, propeller twist (°); σ, opening. All values are in A unless otherwise noted.

Base pair Sx Sy Sz K ω σ

1 C1-G24 0.18 0.01 0.47 -4.35 -10.04 1.95

2 C2-G23 0.30 0.04 0.67 -9.65 -14.55 -1.07

3 T3-A22 0.41 0.07 0.47 -4.23 -15.84 2.09

4 C4-G21 0.60 0.03 -0.14 1.40 -17.81 1.80

5 T5-A20 0.07 -0.19 -0.09 -0.18 -18.77 -5.95

6 C6-G19 0.45 -0.26 -0.09 -6.76 -12.71 -3.37

7 G7-C18 -0.07 0.02 -0.35 -8.24 -16.97 -0.24

8 T8-A17 -0.22 0.04 -0.15 3.64 -13.99 -5.82

9 C9-G16 0.13 -0.08 -0.04 -2.52 -16.54 3.83

10 T10-A15 -0.10 0.05 -0.05 5.28 -18.20 0.89

11 C11-G14 0.20 0.08 0.22 -1.48 -7.70 3.76

12 C12-G13 0.18 0.28 0.52 -7.85 -6.11 3.87

Example 18

The following example describes investigation of solution structural properties of various platinum complexes with DNA. A supercoiled pBR322 DNA was employed and global platination with cDPCP was studied in solution as a function of bound platinum per nucleotide (r_t,), as determined by atomic absorption spectroscopy, in order to assess whether the adduct unwinds the duplex in solution. A plasmid pBR322 was allowed to react with cisplatin or cw-[Pt(NH₃)₂(py)Cl]Cl in 24 mM HEPES, pH 7.4, and 10 mM NaCl over 24 h at 37 ⁰C in the dark. The DNA concentration was 19.6 μM overall (in base pairs) and the platinum concentration ranged from 1.5 to 100 μM. Unbound Pt was removed after 24 h by spin microdialysis. Spin cartridges were washed until no further platinum was detected in the wash solution (5 x 100 μL ddH₂O) and DNA-bound Pt was quantified by atomic absorption spectroscopy (Analyst 300, Perkin Elmer). DNA concentrations were measured by UV-vis absorption spectroscopy (Cary 50) at 260 nm. Agarose gel electrophoresis was used to determine the extent of DNA unwinding induced by the Pt-DNA adducts. Samples were concentrated by ethanol precipitation and loaded onto an ethidium-free 1% agarose gel in a glycerol-based loading buffer. Following electrophoresis at 75 V, gels were stained with 1 μg/mL ethidium bromide and imaged in a Fluor-S gel scanner using QuantityOne software (BioRad).

The above method is based on the principle that negatively supercoiled circular DNA typically becomes positively wound when the duplex is locally unwound, a phenomenon encountered upon the formation of intrastrand cross-links by cisplatin. Cisplatin unwinds the duplex by 13° per bound platinum atom, visibly altering the electrophoretic mobility of the supercoiled DNA on agarose gels. This result is largely a consequence of the formation of intrastrand cross-links, since monofunctional platinum complexes like [Pt(dien)Cl]⁺, which can only bind to a single base, unwind duplex DNA by only 6° per Pt atom. Essentially no unwinding of the superhelix by cDPCP was observed at r_b values up to 0.034.

FIG. 17 shows images of agarose gel electrophoresis tests that were performed to study DNA unwinding with (a) cisplatin and (b) cDPCP, with values being reported as r_f, then r_b (rf/r_b). In FIG. 17, the "lanes" correspond to: (a) cisplatin, (1) 0.072/0; (2) 0.13/0; (3) 0.39/0.005; (4) 0.65/0.006; (5) 1.04/0.019; (6) 1.57/0.038; cDPCP: (7) 0.072/0.001; (8) 0.13/0.002; (9) 0.39/0.007; (10) 0.65/0.014; (11) 1.04/0.020; (12) 1.57/0.019; and (b) cDPCP, (1) 0.039/0; (2) 0.052/0; (3) 0.065/0; (4) 0.078/0.001 ; (5) 0.13/0.002; (6) 0.26/0.003; (7) 0.39/0.007; (8) 0.52/0.011 ; (9) 0.65/0.014; (10) 0.78/0.012; (11) 0.91/0.017; (12) 1.04/0.020; (13) 1.17/0.023; (14) 1.3/0.024; (15) 1.43/0.026; (16) 1.57/0.019; (17) 1.83/0.026; (18) 2.08/0.030; (19) 2.34/0.033; (20) 2.6/0.034. These results are consistent with the formation of monofunctional Pt-DNA adducts in solution. The amount of DNA-bound cDPCP per amount added was, within experimental error, substantially the same as that of cisplatin, as revealed by plots of r_b versus r_f, the formal ratio of platinum added per nucleotide. FIG. 18 shows the results of r_f vs. r_b determination for platination with cDPCP and cisplatin on pBR322 plasmid DNA. The error bars show one standard deviation and data points were measured in triplicate. FIG. 19 shows the results of r_f vs. r_b determination for platination with (i) cDPCP, (ii) cisplatin and (iii) [Pt(dien)Cl]Cl on pSV-β-Galactosidase plasmid DNA. The error bars show one standard deviation and data points were measured in triplicate.

Example 19

The following example describes the inhibition of transcription by cDPCP in HeLa cells. Among the proteins and protein complexes that encounter cisplatin-DNA adducts is the transcription apparatus. Unlike DNA polymerases, which briefly pause at, and then bypass, cisplatin cross-links, presumably without major downstream effects, RNA polymerases are typically affected by the presence of these adducts. The progression of human RNA polymerase II (Pol II) along the DNA strand is substantially blocked by cisplatin-DNA adducts, and the arrest and subsequent ubiquitylation of Pol II initiate transcription-coupled repair, a subpathway of nucleotide excision repair, as well as programmed cell death, or apoptosis.

In this example, plasmids containing the lacL gene downstream of an S V40 promoter were modified with cisplatin, cDPCP, or [Pt(dien)Cl]⁺ at r_b levels from 0 to 0.13 (FIG. 19) and transfected into HeLa cells. HeLa cells (ATCC) were cultured in DMEM with 10% fetal bovine serum at 5% CO₂ in a humidified chamber and restarted upon reaching passage number 20. Cells were transfected with platinated or unplatinated control plasmids according to the manufacturer's protocol (Lipofectamine 2000, Invitrogen Corp.). After 24 h incubation, cells were washed twice with PBS, incubated for 15 min in lysis buffer (25mM bicine, pH 7.8, 0.05% Tween 20, 0.05% Tween 80) and scraped from the plate.

The products of β-galactosidase activity were assayed colorimetrically after 24 h by addition of ort/jø-nitrophenyl-β-galactoside (ONPG). After 15 s vigorous vortexing and centrifugation (18,000 x g), the supernatant was treated with 2.2 raM ONPG and 50 mM β -mercaptoethanol in 100 mM sodium phosphate, pH 7.3, and 1 mM MgCl₂.

Following incubation at 37 °C for 4 h and addition of sodium carbonate to 0.75 M, the absorbance of the solution was measured at 420 run and values from the cells transfected with platinated plasmid were normalized to those from cells transfected with nonplatinated plasmid. The results shown in FIGS. 15A-C illustrate the repair of, and inhibition of transcription by, cDPCP-DNA adducts. FIG. 15A shows a graph of the percentage of repair of cisplatin and cDPCP as a function of time. FIG. 15B shows a portion of a gel, comparing the repair of cDPCP with repair of cisplatin and [Pt(dien)Cl]Cl adducts, with gel lanes corresponding to: (1) ladder w/ band at 25 nt, (2) cisplatin 0 min, (3) cisplatin 30 min, (4) cisplatin 60 min, (5-7) cDPCP 0/30/60 min, (8- 10) [Pt(dien)Cl]Cl 0/30/60 min. (The entire gel is shown in FIG. 23A.) Error bars represent the standard deviation of three separate experiments.

FIG. 15C shows a plot comparing successful transcription bypass and repair of various Pt-DNA adducts. Adducts of cDPCP, much like those of cisplatin, allowed minimal bypass by RNA polymerase but exhibited a relatively low repair value, similar to adducts of [Pt(dien)Cl]Cl. Repair values report % excision products detected after 60 min and transcription bypass values are given at η,= 0.0039 for the β-gal live-cell assay. Repair error bars are the standard deviation of five, five, and two separate experiments for cisplatin, cDPCP, and [Pt(dien)Cl]Cl, respectively. Transcription error bars are the standard deviation of samples prepared in triplicate. The entire experiment was performed twice.

FIG. 20 shows the bypass of various platinum adducts by the transcribing complex as assayed in live cells using a platinated pSV-β-galactosidase reporter plasmid. The platination level (r_b value) is plotted vs. % transcription bypass relative to cells treated with unplatinated plasmid. A bypass % of 100% indicates that there was no decrease in transcription of the reporter protein due to platination. Error bars represent the standard deviation of three samples from the same cell preparation and the experiment was repeated twice. As shown by the studies described above, bypass of platinum adducts by the Pol

II complex led to increased transcription of the lacZ gene and absorbance at 420 run arising from ONPG cleavage by b-galactosidase. A difference between Pol II bypass of cisplatin vs. [Pt(dien)Cl]⁺ adducts was observed, relative to that for the unplatinated control plasmids (FIG. 15), with [Pt(dien)Cl]⁺ requiring more than 5 times the platination level as cisplatin to block progression of RNA Pol II completely. In contrast, transcription inhibition by the monofunctional cDPCP adducts very nearly matched that of cisplatin and was more effective than inhibition by [Pt(dien)Cl]⁺, in this study. Transcription of the cisplatin-modified plasmid was effectively inhibited at an i^ value of 2.5x10^"3. Inhibition by [Pt(dien)Cl]⁺-modified plasmids was the essentially same as that of the unplatinated control at η, = 7.8xlO^'3, whereas transcription from the cDPCP- modified plasmid was reduced to 16% that of the control at an T_b value of 3.9x10^"3 (FIG. 15).

Example 20 The following example describes the removal of lesions by excision repair. The major product of cisplatin- induced DNA damage, the intrastrand d(GpG) cross-link, is repaired by the excision repair pathway. The integrity of this pathway in human cells can be an indicator of the sensitivity of a tumor to platinum-based therapy. Human cells from disorders in which excision repair deficiency is a phenotype, such as xeroderma pigmentosum and Cockayne syndrome, are exquisitely sensitive to cisplatin damage. A test for the presence of a key protein in the excision repair pathway, ERCCl, is in FDA Phase III trials for use as a predictive factor in tailoring chemotherapy to patients with non-small cell lung cancer. Conversely, increased efficiency of excision repair leads to rapid removal of cisplatin adducts and is associated with cisplatin resistance in human tumor cells.

Repair probes were made by synthesizing, annealing, and ligating five short oligomers to form dsDNA strands of 156 base pairs in length (FIGS. 21 and 22) and were purified and radiolabeled with ³²P as described previously (Zamble et al., 1996, Biochemistry, 55, 10004-10013.; Reardon et al., 2006, Methods Enzymol, 408, 189- 213). The platination step was performed using either cisplatin, CW-[Pt(NHs)₂(Py)Cl]⁺ or [Pt(dien)Cl]⁺.

A 156mer DNA probes site-specifically platinated with adducts of cisplatin, cDPCP, or [Pt(dien)Cl]⁺, and radioactively labeled with an internal g-³²P modification, were used in combination with repair-active mammalian cell-free extracts, to analyze excision repair. FIG. 21 shows a diagram of site-specifically platinated probe assembly and in vitro assay for assessing repair by the excision repair pathway. FIG. 22 shows the sequences of DNA oligomer components of the 156mer NER probe. The samples were resolved on denaturing polyacrylamide electrophoresis gels and the radioactive intensity was quantified with a phosphorimager. FIGS. 23 A-B show representative polyacrylamide electrophoresis gels showing nucleotide excision repair products. FIG. 23 A shows an electrophoresis gel of the following samples: (1)100 bp ladder, (2) cisplatin 0 min, (3) cisplatin 30 min, (4) cisplatin 60 min, 5-7) cDPCP 0/30/60 min, 8-10) [Pt(dien)Cl]Cl 0/30/60 min. FIG. 23B shows an electrophoresis gel of the following samples: (1) cisplatin 0 min, (2) cisplatin 60 min, 3-(8) cDPCP 0/15/30/60/90/120 min, illustrating the kinetics of repair for cDPCP. Percent repair was determined by comparing the intensity of unrepaired 156mer vs. that of the primary excision products at 25-29 bp. Repair efficiency of the three different site-specifically platinated 156mer probes in CHO (Chinese hamster ovary) nuclear extracts was quantitated as 3.5% for cisplatin-modified DNA, 1.0% for the cDPCP adduct, and 0.3% for [Pt(dien)Cl]Cl adducted DNA after 60 min reaction times (FIGS. 15 and 23).

The rate and amount of repair were comparable to values previously observed for cisplatin and oxaliplatin 1 ,2-intrastrand d(GpG) cross-link-containing probes and less than the 10% repair observed for cisplatin 1,3-intrastrand d(GpTpG) cross-linked probes. Kinetic experiments revealed that repair of the cDPCP adduct leveled off after 60 min (FIGS. 15, 23), a result consistent with previous measurements. Example 21

As noted above, cDPCP can be a substrate for OCTl and OCT2 as shown by the increased accumulation by, and sensitivity of, cells that express these critical transporters compared to those lacking them. The dramatic increases in cellular accumulation and sensitivity suggest that cDPCP has greater tumor targeting potential than oxaliplatin. Compared with oxaliplatin, cDPCP is much less toxic to cells that do not express OCTl or 2, suggesting that cDPCP may be able to target colorectal or liver cancer, but with an advantageous reduction in the severity of side effects for tissues that do not express OCTl or OCT2. The presence of these transporters in certain organs, such as kidney and liver, may involve the use of co-treatments to mitigate any toxic side effects. Nephrotoxicity, the dose-limiting side effect for cisplatin therapy, may be less problematic in oxaliplatin treatment. Liver toxicity is a non-dose-limiting side effect of cisplatin and oxaliplatin.

Compared to cisplatin, cDPCP generally causes only minor distortions to double helical DNA upon binding to a guanine N7 atom in the major groove. Characteristics of the cisplatin 1 ,2-intrastrand d(GpG) cross-link include a roll angle of 26° between the bound guanines and 40° bend towards the major groove. It is generally known in the art that these structural distortions can inhibit transcription, leading either to nucleotide excision repair or to apoptosis. A recent X-ray crystal structure of an RNA polymerase II elongation complex containing a cisplatin 1 ,2-d(GpG) intrastrand cross-link in the DNA template strand suggested that the platinum adduct inhibits transcription by prohibiting translocation of the cross-link from the +2/+3 site to the +2/+1 site. This barrier may stem from the inability of the covalently linked dinucleotide to twist by -90° for crossing the bridge helix in the +2/+1 site. By contrast, monofunctional adducts of cDPCP offer minimal or essentially no translocation barrier because cDPCP binds covalently only to a single DNA base. Modeling of the c/5-{Pt(NH₃)₂(py)}²⁺-dG adduct into the template strand of the elongation complex suggested the absence of a significant barrier to translocation between the +2 and +1 sites. However, when the adduct was modeled in the +1 site, where the incoming NTP is matched to the template strand, of an elongation complex solved crystallographically, it was observed that the pyridine ligand would likely sterically clash with the bridge helix, in effect twisting the base out of its native conformation (FIG. 16). This distortion could lead to NTP misincorporation, which in turn might stall the polymerase. Without wishing to be bound by theory, this hypothesis can explain why cDPCP lesions inhibit transcription, whereas adducts of the trans isomer and other monofunctional platinum compounds like [Pt(dien)Cl]Cl, which would not cause such steric hindrance, are much less potent inhibitors. Like cisplatin, transcription may be strongly inhibited by cDPCP both in cell extracts and in live cells, but a significantly higher level of platination with [Pt(dien)Cl]⁺ is required to block transcription to the same extent.

FIG. 16A shows a schematic representation of the active site of RNA polymerase II, with a cDPCP-dG adduct (16) modeled into template DNA (10) at the +1 site, where incoming NTPs are matched and added to synthesized RNA (12). Complementary, non- template DNA is shown by 14. This model demonstrates how cDPCP adducts may shift the template base out of its native conformation (outlined by square 20) by steric interactions between the pyridine ligand and the Pol II bridge helix (18). FIG. 16B shows a schematic representation of various space-filling views of the Pt adduct and bridge helix where the Pol II coordinates are taken from PDB code 2NVQ and cDPCP- dG coordinates are from this work.

As described herein, because cDPCP can largely escape repair and yet inhibit transcription very effectively, its adducts should persist longer than those of cisplatin yet produce a similar number of downstream consequences that might raise the therapeutic potential of cDPCP relative to cisplatin. The design of anticancer agents specifically as transcription inhibitors has been proposed, based on the premise that an extended delay in the restoration of transcription would induce apoptosis by p53-dependent and - independent pathways. If true, persistence of transcription blocks would promote cell death and enhance the potency of cDPCP. Combined with the high selectivity of cDPCP for cells expressing hOCTl and hOCT2, which are broadly expressed in human colorectal cancer, these findings support the candidacy of this unique monofunctional cationic complex as an anticancer drug.

What is claimed:

Claims

1. A method for treating a subject having a cancer which expresses an organic cation transporter (OCT), comprising: administering a therapeutically-effective amount of a compound having the formula,

wherein:

R¹, R², and R³ can be the same or different and each is a group comprising at least one of ammonia, an amine, or a leaving group, any being optionally substituted, or, any two or three of R¹, R², and R³ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted;

R⁵, R⁶, R⁷, and R⁸ can be the same or different and each is a group comprising at least one of ammonia, an amine, a heterocycle including at least one nitrogen, an aryl group, or a leaving group, any being optionally substituted, or, any two or three of R , R⁶, R⁷ and R⁸ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted;

X is a counterion; and n and m are 1 or n and m are 2; wherein the compound has a molecular weight of 700 g/mol or less, to a subject having a cancer which expresses an OCT.

2. A method, comprising: promoting the inhibition or treatment of a cancer which expresses OCT in a subject susceptible to or exhibiting symptoms of a cancer which expresses OCT via administration to the patient of a composition comprising a compound having the formula,

or wherein:

R⁵, R⁶, R⁷, and R⁸ can be the same or different and each is a group comprising at least one of ammonia, an amine, a heterocycle including at least one nitrogen, an aryl group, or a leaving group, any being optionally substituted, or, any two or three of R⁵, R , R and R can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted;

X is a counterion; and n and m are 1 or n and m are 2, wherein the compound has a molecular weight of 700 g/mol or less.

3. A method as in any preceding claim, wherein the cancer expresses hOCTl .

4. A method as in any preceding claim, wherein the cancer expresses hOCT2.

5. A method as in any preceding claim, wherein the subject is otherwise free of indications for treatment for sarcomas, lymphoid leukemias, lymphosarcoma myelocytic leukemia, malignant lymphoma, squamous cell carcinoma, adenocarcinoma, scirrhous carcinoma, malignant melanoma, seminoma, teratoma, choriocarcinoma, embryonalcarcinoma, cystadenocarcinoma, endometriocarcinoma, or neuroblastoma.

6. A method as in any preceding claim, wherein at least one of R¹, R², and R³ is a leaving group.

7. A method as in any preceding claim, wherein at least two of R¹, R², and R³ is a leaving group.

8. A method as in any preceding claim, wherein at least one of R , R , R , and R is a leaving group.

9. A method as in any preceding claim, wherein at least two of R , R , R , and R is a leaving group.

10. A method as in any preceding claim, wherein the leaving group is a halide or carboxylate.

11. A method as in any preceding claim, wherein the leaving group is chloride.

12. A method as in any preceding claim, wherein R⁴ is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine-l,9-diamine, benzylamine, or substituted derivatives thereof.

13. A method as in any preceding claim, wherein at least one of R , R , R , and R is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine- 1,9-diamine, benzylamine, or substituted derivatives thereof.

14. A method as in any preceding claim, wherein at least one of R , R , R , and R comprises a cationic group.

15. A method as in any preceding claim, wherein R⁹ and R¹⁰ can be the same or different and each is hydroxyl, phenoxide, or 2-[(2-carboxyacetoamido)methyl]-l- methylpyridinium.

16. A method as in any preceding claim, wherein the compound has the structure,

wherein R¹ and R² can be the same or different and each is a leaving group; R⁴ is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine- 1 ,9-diamine, benzylamine, or substituted derivatives thereof.

17. A method as in claim 16, wherein the leaving group is a halide.

18. A method as in claim 16, wherein the leaving group is chloride.

19. A method as in any preceding claim, wherein the compound has the structure,

wherein R⁷ is a leaving group; R⁸ is a group comprising a heterocycle including at least one nitrogen, an aryl group, or a leaving group, any being optionally substituted, or, any two or three of R⁵, R⁶, R⁷ and R⁸ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted; and R⁹ and R¹⁰ can be the same or different and are groups comprising hydroxyl, alkoxy, aryloxy, or acyloxy, any being optionally substituted.

20. A method as in claim 19, wherein the leaving group is a halide.

21. A method as in claim 19, wherein the leaving group is chloride.

22. A method as in any preceding claim, wherein the compound has the structure,

23. A method as in any preceding claim, wherein the compound has the structure,

24. A method as in any preceding claim, wherein the compound has the structure,

25. A kit for treatment of a cancer which expresses an OCT, comprising: a composition comprising a compound having the formula,

or wherein:

R¹, R², and R³ can be the same or different and each is a group comprising at least one of ammonia, an amine, or a leaving group, any being optionally substituted, or, any two or three of R¹, R², and R³ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted; R⁴ is a group comprising a heterocycle including at least one nitrogen, or a group comprising an aryl group, any being optionally substituted;

R⁹ and R¹⁰ can be the same or different and are groups comprising hydroxyl, alkoxy, aryloxy, or acyloxy, any being optionally substituted; X is a counterion; and n and m are 1 or n and m are 2, wherein the compound has a molecular weight of 700 g/mol or less; and instructions for use of the composition for treatment of a cancer which expresses an OCT.

26. A kit as in claim 25, wherein the cancer expresses hOCTl .

27. A kit as in claim 25, wherein the cancer expresses hOCT2.

28. A kit as in claim 25, wherein the subject is otherwise free of indications for treatment for sarcomas, lymphoid leukemias, lymphosarcoma myelocytic leukemia, malignant lymphoma, squamous cell carcinoma, adenocarcinoma, scirrhous carcinoma, malignant melanoma, seminoma, teratoma, choriocarcinoma, embryonalcarcinoma, cystadenocarcinoma, endometriocarcinoma, or neuroblastoma.

29. A kit as in claim 25, wherein at least one of R¹, R², and R³ is a leaving group.

30. A kit as in claim 25, wherein at least two of R¹, R², and R³ is a leaving group.

31. A kit as in claim 25, wherein at least one of R⁵, R⁶, R⁷, and R⁸ is a leaving group.

32. A kit as in claim 25, wherein at least two of R⁵, R⁶, R⁷, and R⁸ is a leaving group

33. A kit as in claim 25, wherein the leaving group is a halide or carboxylate.

34. A kit as in claim 25, wherein the leaving group is chloride.

35. A kit as in claim 25, wherein R⁴ is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine-l,9-diamine, benzylamine, or substituted derivatives thereof.

36. A kit as in claim 25, wherein at least one of R⁵, R⁶, R⁷, and R⁸ is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine-1,9- diamine, benzylamine, or substituted derivatives thereof.

37. A kit as in claim 25, wherein at least one of R⁵, R⁶, R⁷, and R⁸ comprises a cationic group.

38. A kit as in claim 25, wherein R⁹ and R¹⁰ can be the same or different and each is hydroxyl, phenoxide, or 2-[(2-carboxyacetoamido)methyl]-l-methylpyridinium.

39. A kit as in claim 25, wherein the compound has the structure,

wherein R¹ and R² can be the same or different and each is a leaving group; R is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine- 1 ,9-diamine, benzylamine, or substituted derivatives thereof.

40. A kit as in claim 39, wherein the leaving group is a halide.

41. A kit as in claim 39, wherein the leaving group is chloride.

42. A kit as in claim 25, wherein the compound has the structure,

wherein R is a leaving group; R is a group comprising a heterocycle including at least one nitrogen, an aryl group, or a leaving group, any being optionally substituted, or, any two or three of R⁵, R⁶, R⁷ and R⁸ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted; and R⁹ and R¹⁰ can be the same or different and are groups comprising hydroxyl, alkoxy, aryloxy, or acyloxy, any being optionally substituted.

43. A kit as in claim 42, wherein the leaving group is a halide.

44. A kit as in claim 42, wherein the leaving group is chloride.

45. A kit as in claim 25, wherein the compound has the structure,

46. A kit as in claim 25, wherein the compound has the structure,

H₃ CI

47. A kit as in claim 25, wherein the compound has the structure,

.

48. A pharmaceutical composition, comprising: a compound having the formula,

wherein:

X is a counterion; and n and m are 1 or n and m are 2; wherein the compound has a molecular weight of 700 g/mol or less; or, a pharmaceutically acceptable salt thereof; and one or more pharmaceutically acceptable carriers, additives, and/or diluents.

49. A pharmaceutical composition, comprising: a compound having the formula,

wherein:

R¹, R², and R³ can be the same or different and each is a group comprising at least one of ammonia, an amine, or a leaving group, any being optionally substituted, or, any two or three of R¹, R², and R³ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted, wherein at least two of R¹, R , and R are leaving groups;

R⁴ is a heterocycle including at least one nitrogen, or a group comprising an aryl group, any being optionally substituted; X is a counterion; and wherein the compound has a molecular weight of 700 g/mol or less; or, a pharmaceutically acceptable salt thereof; and one or more pharmaceutically acceptable carriers, additives, and/or diluents.

50. A pharmaceutical composition as in any preceding claim, wherein at least one of R¹, R², and R³ is a leaving group.

51. A pharmaceutical composition as in any preceding claim, wherein at least two of R , R , and R is a leaving group.

52. A pharmaceutical composition as in any preceding claim, wherein at least one of R⁵, R⁶, R⁷, and R⁸ is a leaving group.

53. A pharmaceutical composition as in any preceding claim, wherein at least two of R⁵, R⁶, R⁷, and R⁸ is a leaving group.

54. A pharmaceutical composition as in any preceding claim, wherein the leaving group is a halide or carboxylate.

55. A pharmaceutical composition as in any preceding claim, wherein the leaving group is chloride.

56. A pharmaceutical composition as in any preceding claim, wherein R⁴ is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine-1,9- diamine, benzylamine, or substituted derivatives thereof.

57. A pharmaceutical composition as in any preceding claim, wherein at least one of R⁵, R⁶, R⁷, and R⁸ is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine-l,9-diamine, benzylamine, or substituted derivatives thereof.

58. A pharmaceutical composition as in any preceding claim, wherein at least one of R , R , R , and R comprises a cationic group.

59. A pharmaceutical composition as in any preceding claim, wherein R⁹ and R¹⁰ can be the same or different and each is hydroxyl, phenoxide, or 2-[(2- carboxyacetoamido)methyl] - 1 -methylpyridinium.

60. A pharmaceutical composition as in any preceding claim, wherein the compound has the structure,

61. A pharmaceutical composition as in claim 60, wherein the leaving group is a halide.

62. A pharmaceutical composition as in claim 60, wherein the leaving group is chloride.

63. A pharmaceutical composition as in any preceding claim, wherein the compound has the structure,

64. A pharmaceutical composition as in claim 63, wherein the leaving group is a halide.

65. A pharmaceutical composition as claim 63, wherein the leaving group is chloride.

66. A pharmaceutical composition as in any preceding claim, wherein the compound has the structure,

H₃ CI

67. A pharmaceutical composition as in any preceding claim, wherein the compound has the structure,

H₃Nl' ^VCI.

68. A composition of matter, comprising: a compound having the formula,

wherein:

R¹, R², and R³ can be the same or different and each is a group comprising at least one of ammonia, an amine, or a leaving group, any being optionally substituted, or, any two or three of R¹, R², and R³ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted; R⁴ is a heterocycle including at least one nitrogen, or a group comprising an aryl group, any being optionally substituted;

^ ft 7 fi

R , R , R , and R can be the same or different and each is a group comprising at least one of ammonia, an amine, a heterocycle including at least one nitrogen, an aryl group, or a leaving group, any being optionally substituted, or, any two or three of R⁵, R⁶, R⁷ and R⁸ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted;

R⁹ and R¹⁰ can be the same or different and are groups comprising hydroxyl, alkoxy, aryloxy, or acyloxy, any being optionally substituted; X is a counterion; and n and m are 1 or n and m are 2; wherein the compound has a molecular weight of 700 g/mol or less.

69. A composition of matter, comprising: a compound having the formula,

R² R¹-Pt— R³ X

wherein:

R¹, R², and R³ can be the same or different and each is a group comprising at least one of ammonia, an amine, or a leaving group, any being optionally substituted, or, any two or three of R¹, R², and R³ can be joined together to form a bidentate ligand or tridentate ligand, any being optionally substituted, wherein at least two of R¹, R², and R are leaving groups;

R⁴ is a heterocycle including at least one nitrogen, or a group comprising an aryl group, any being optionally substituted; X is a counterion; and wherein the compound has a molecular weight of 700 g/mol or less.

70. A composition as in any preceding claim, wherein at least one of R¹, R², and R³ is a leaving group.

71. A composition as in any preceding claim, wherein at least two of R¹, R², and R³ is a leaving group.

72. A composition as in any preceding claim, wherein at least one of R⁵, R⁶, R⁷, and R is a leaving group.

73. A composition as in any preceding claim, wherein at least two of R⁵, R⁶, R⁷, and R⁸ is a leaving group.

74. A composition as in any preceding claim, wherein the leaving group is a halide or carboxylate.

75. A composition as in any preceding claim, wherein the leaving group is chloride.

76. A composition as in any preceding claim, wherein R⁴ is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine-l,9-diamine, benzylamine, or substituted derivatives thereof.

77. A composition as in any preceding claim, wherein at least one of R , R , R , and R is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine- 1 ,9-diamine, benzylamine, or substituted derivatives thereof.

78. A composition as in any preceding claim, wherein at least one of R⁵, R⁶, R⁷, and

R comprises a cationic group.

79. A composition as in any preceding claim, wherein R⁹ and R¹⁰ can be the same or different and each is hydroxyl, phenoxide, or 2-[(2-carboxyacetoamido)methyl]-l- methylpyridinium.

80. A composition as in any preceding claim, wherein the compound has the structure,

81. A composition as in claim 80, wherein the leaving group is a halide.

82. A composition as in claim 80, wherein the leaving group is chloride.

83. A composition as in any preceding claim, wherein the compound has the structure,

84. A composition as in claim 84, wherein the leaving group is a halide.

85. A composition as in claim 84, wherein the leaving group is chloride.

86. A composition as in any preceding claim, wherein the compound has the structure,

87. A composition as in any preceding claim, wherein the compound has the structure,

88. A method as in any preceding claim, wherein the cancer comprises mRNA of genes that express OCT in an amount that is at least 75% of the amount found in HCT- 116 colorectal adenocarcinoma cells, as measured by real-time RT-PCR.

89. A kit as in any preceding claim, wherein the cancer comprises mRNA of genes that express OCT in an amount that is at least 75% of the amount found in HCT-116 colorectal adenocarcinoma cells, as measured by real-time RT-PCR.

90. A method as in any preceding claim, wherein the subject is otherwise free of indications for treatment with said compound.

91. A method as in any preceding claim, further comprising the act of providing a subject to whom a diagnostic method has been applied, the diagnostic method comprising: determining the presence of an amount of an organic cation transporter and/or mRNA of genes that encode OCT within a subject; evaluating indication of an OCT-related cancer or the potential for an OCT- related cancer based upon the determining step; and determining that the subject is known to be at risk for an OCT-related cancer or has an OCT-related cancer.

92. A composition for treating a subject having a cancer which expresses an organic cation transporter (OCT), wherein the composition comprises a compound having the formula,

R² R²

R *⁵. ^R I ⁹ R 7⁷

R¹-Pt-R³ _R>< . R¹-Pt— R³

R⁶ _n R⁸ R< _R I ₁10

wherein:

93. A composition as in claim 92, wherein the compound has the structure,

H₃ CI

94. Use of a composition comprising a compound having the formula,

R S⁵ _V ^R I R 7⁷

X

R R⁶^ ^P I ^t _1n-R 8⁸ m R¹⁰ or wherein:

X is a counterion; and n and m are 1 or n and m are 2; wherein the compound has a molecular weight of 700 g/mol or less, in the preparation of a medicament for treating a subject having a cancer which expresses an organic cation transporter (OCT).