WO2010062381A1

WO2010062381A1 - Organelle-specific drug delivery

Info

Publication number: WO2010062381A1
Application number: PCT/US2009/006218
Authority: WO
Inventors: Robert Shorr; Robert Rodriguez; Paul Bingham; Zuzana Zachar; Lakmal W. Boteju; Patrick P. Zaretski
Original assignee: Robert Shorr; Robert Rodriguez; Paul Bingham; Zuzana Zachar; Boteju Lakmal W; Zaretski Patrick P
Priority date: 2008-11-28
Filing date: 2009-11-20
Publication date: 2010-06-03

Abstract

A pharmaceutical composition constructed from a compound comprising a fatty acid, or analogue thereof, conjugated to at least one polymer, non-polymer, or lipid-based particle and/or at least one therapeutic, imaging, or diagnostic agent, modulates the binding affinity of the compound to a carrier molecule in the blood of warm-blooded animals in such a way as to modulate the circulation time of the pharmaceutical composition, and consequently the penetration and distribution of the compound into a tumor mass, as well as to demonstrate selective uptake and transport into a specific organelle in a diseased cell. More specifically, increased depth of tumor cell layer penetration and more even distribution throughout a tumor mass, with a resulting active cellular uptake and transport into diseased-cell mitochondria, leading to enhanced diagnostic, imaging, or therapeutic benefit, is contemplated.

Description

Organelle-Specific Drug Delivery Field of the Invention

This invention relates to therapeutic, diagnostic, and imaging compositions, and more particularly to pharmaceutical compositions comprised of fatty acids and conjugates, or analogues thereof, which demonstrate active uptake, transport, and concentration into specific organelles of diseased cells, such as mitochondria, with limited or non-detectable uptake and concentration in non-diseased cells.

Background of the Invention The American Cancer Society projects that, in 2008, over 1,400,000 Americans will be diagnosed with some form of cancer, excluding skin cancer. More than 560,000 Americans will die from the disease and related causes in the same year. Indeed, in any given year, cancer may be the number one or two disease-related cause of death in the United States. Over ten million Americans are currently living with cancer, having survived surgery, radiation, and/or chemotherapy, and these survivors are routinely monitored for local relapse or metastatic disease. Surgical intervention provides the highest probability of cure. However, the morbidity associated with surgery is high, dramatically affecting the patient's quality of life, and local relapse or disease progression may still occur despite successful surgery. Systemic chemotherapy has a theoretical advantage over surgery in its ability to reach inoperable tumors, circulating cancer-causing stem or metastatic cells, and non-detectable metastatic nodules. Stem cells which are present in limited numbers in a tumor mass are especially important to be treated given their proposed role in tumor regeneration and local relapse. On the other hand, beneficial concentration of drug either throughout a tumor mass or within tumor cells is difficult to achieve. In current cancer chemotherapy, for drugs relying on elevated plasma concentration to drive a diffusion gradient, higher drug doses are often given to push diffusion-based tumor penetration and intracellular concentration. However, current evidence indicates that the distribution of many anticancer drugs in tumor tissue is incomplete, with only a fraction of administered drug ever reaching the appropriate site. Also, such higher dosing is often accompanied by undesirable side effects and increased morbidity. Furthermore, even those drug delivery technologies which enhance accumulation within a tumor mass often remain ultimately unable to deliver sufficient drug to the intended molecular target, which may be further sequestered within a subcellular organelle.

Other barriers to safer, more effective cancer chemotherapy are a drug's lack of selectivity for a tumor type versus healthy cells and tissue; the presence, absence, or variability of a molecular target among cancer cells within a tumor mass or metastatic site (even within the same patient or patient group); drug cycle stage dependency; changes to metabolic and hypoxic gradients, microenvironmental pH, cellular matrix enzymes, and stroma (both permissive and non-permissive) due to poorly organized and leaky vascularization and limited lymphatic drainage; systemic or cellular resistance that may be inducible; and tight cellular packing and back pressure that limits drug ability to penetrate to where it needs to go. This latter challenge is especially critical for tumor sites not amenable to surgery or radiation. (See Minchinton A. and Tannock I. 2006. Drug penetration in solid tumours. Nature Rev Cancer 6:583-592, passim, herein incorporated by reference.) To be most effective, then, anticancer drugs must selectively penetrate tumor tissue in such a manner as to reach all of the cancer cells and expose them to drug at a concentration sufficient to exert a therapeutic or diagnostic benefit. It would therefore be beneficial to provide technology that enhances the ability to deliver drugs to the intended molecular target in a diseased-cell-specific manner that increases tumor penetration and intracellular uptake at lower dosage levels, thereby minimizing the amount of deleterious side effects. The physicochemical properties of drugs (e.g., molecular weight, shape, charge, and solubility) help determine the rate of tissue diffusion. The penetration of a drug is also dependent on its consumption (e.g., metabolism, binding to tissue elements, or uptake), which functions to remove free drug from the circulation. Retention in tumor cells can be due to binding at the site of lethal activity, usually DNA, although basic drugs can be sequestered in acidic organelles such as perinuclear endosomes. Although these parameters are important for determining overall drug disposition, which might relate to toxicity for certain body organs, and for devising a logical schedule of administration, they give limited information about access to target tumor cells. For example, mitoxantrone has a high distribution volume because of sequestration within cells caused by DNA binding and entrapment in acidic vesicles but has poor penetration into tissue. An anticancer drug might even show an average concentration that is higher in the tumor than in normal tissues, but if tissue penetration is poor, only cells close to blood vessels will be exposed to an effective concentration.

On the other hand, regulation of both tissue and blood vessel growth breaks down in solid cancers, and it is observed that tumor cells proliferate more rapidly than blood capillaries, thereby reducing vascular density. Irregular blood flow, compression of blood and lymphatic vessels by cancer cells, increased interstitial fluid pressure, and the composition and structure of the extracellular matrix are all factors which can both limit the delivery of anticancer drugs to cells distant from functioning blood vessels as well as potentially interfere with a drug's mechanism of action. As a consequence of the poorly- organized vasculature in solid tumors, there is limited delivery of oxygen and other nutrients to cancer cells that are distant from functional blood vessels, leading to the build-up of metabolic products, such as lactic and carbonic acid, which lower the extracellular pH. While tumor cell proliferation decreases with increasing distance from tumor blood vessels, parallel with decreasing nutrient and oxygen concentration, hypoxia becomes prevalent in some tumor regions. There is in fact a large body of evidence indicating that hypoxic cells are relatively resistant to radiation treatment and can repopulate the tumor after radiotherapy.

As anticancer drugs distribute within tumors, they form gradients from tumor blood vessels that change with time as the drug is cleared from the body. The permeability of vessel walls influences drug penetration but is thought to be insignificant in many tumors where blood-vessel fenestrations have been observed. Drugs penetrate normal tissues by both diffusion and convection, with a net flow of fluid from blood vessels balanced by resorption into lymphatics. However, tumors often lack functional lymphatics, which can lead to increased levels of interstitial fluid pressure in tumors, which in turn is likely to reduce convection and thereby inhibit the distribution of macromolecules. Additionally, the intervessel distances in tumors can often be large, and can result in some cells receiving subtherapeutic drug exposure. The tacit assumption that drugs distribute efficiently throughout the tissues of the body thus does not hold for anticancer drugs. There are consequently at least three potential reasons why cells that are distant from blood vessels might be resistant to conventional chemotherapy: most anticancer drugs exert selective toxicity on cycling cells, so that non-proliferating or slowly proliferating cells are resistant; some drugs might be less active in hypoxic, acidic, or nutrient-deprived microenvironments; and cells distant from blood vessels might be exposed to low concentrations of drug because of limited drug access.

Macromolecules like albumin or transferrin (MW > 45 kDa) and many biocompatible water-soluble polymers and nanoparticles take advantage of the vascular permeability, or "leakiness," seen in the interstitium of tumors, and its concomitant ability to accumulate plasma components. These compounds are not cleared rapidly from the sites of a lesion and thus remain there for a prolonged time, usually more than a few days. This enhanced permeability and retention (EPR) effect has become a common theme for anticancer drug delivery systems seeking more cancer-selective targeting using macromolecular drugs, the intention being the reduction of side effects due to the passive nature of the EPR effect and its failure to affect cellular uptake or distribution.

However, as most anticancer agents are low-weight molecular compounds, anticancer agents usually require changes in drug formulation to modify their ability to penetrate tumor tissue. Potential examples include formulations of doxorubicin encapsulated in liposomes, which effectively alter the pharmacokinetics of the free drug and take advantage of the permeability of tumor vessels to liposomal particles, and abraxane, a nanoparticle formulation of paclitaxel bound to albumin that was developed to circumvent the need for cremophor as a solubilizing agent. Additionally, nanoparticles, emulsions, liposomes, and polymers (e.g., polyethylene glycol (PEG), polyvinyl pyridolone, polyethylene imine (PEI), polyvinyl pyridolone, and dextrans) have been widely used with diverse compositions. Overall, any changes in formulation should also have effects on pharmacokinetics that lead to improved tissue penetration.

There are alternatives to drug reformulation which also affect a drug's circulation lifetime tumor penetration ability, and/or efficacy. Drugs may be covalently or non- covalently bound to carriers, with these bonds sensitive to conditions such as pH and cleavage by specific enzymes. Such bonds may therefore be reversible, resulting in prodrug formulations. Surface coatings may also be used. In addition to providing the benefits of the EPR effect through increasing blood circulation lifetime, inhibition of metabolism or excretion can also demonstrate sustained release, thereby decreasing the need for higher and/or more frequent drug dosing. Antibodies or receptor ligands to tumor-associated targets have also been explored as drug carriers. Finally, some cationic dyes and drugs with selectivity for other cell compartments depending on their lipophilic/hydrophilic character accrue within tumor mitochondria due to the enhanced mitochondrial membrane potential associated with many cancer cells. (See Kandela K, Bartlett JA, and Indig GL. 2002. Effect of molecular structure on the selective phototoxicity of triarylmethane dyes towards tumor cells. Photochem Photobiol Sci 1 :309-14, herein incorporated by reference.)

Both in vitro and tumor-bearing animal studies have shown that serum albumin is taken up in malignancies, which is pharmaceutically relevant because a variety of cancer drugs can be bound to albumin. In a representative study, a research team evaluated radiolabeled human serum albumin (HSA) in 23 patients using SPET imaging. Increased tumor uptake was found in 35% of the examined patients. While small cell carcinoma did not accumulate the labeled HSA, squamous cell carcinoma and adenocarcinoma were most responsive. In all cases, the HSA uptake did not follow the vascularity. {See Clorius JH, Sinn H, Manke H, Schrenk H, Blatter J, Werling C, Friedrich EA, Voges J, Banner M, Sturm V, Drings P, and van Kaick G. 1995. Serum albumin (SA) accumulation by bronchogenic tumours: a tracer technique may help with patient selection for SA-delivered chemotherapy. Eur JNucl Med 22:989-996, herein incorporated by reference.) Additional studies of tumor- targeting approaches have shown that passive tumor mass uptake can be hindered by "gridlock" within the tumor mass; inhibition of cell uptake; less than desired penetration; and the "missing" of an intracellular target, which is especially evident with drugs intended to reach a cellular organelle, such as the nucleus or mitochondria.

The mitochondria of healthy cells produce ATP for cellular processes using enzyme pathways that are regulated by upstream events such as oxygen levels, nutrition, and exercise. Enzyme cofactors and molecules that participate in the modulation of oxidative levels of reactive oxygen and nitrogen species (RONS) are taken up into cell and transported to the mitochondria. The machinery for cell absorption and delivery to mitochondria, as well as select mitochondrial enzymes, also contribute both to self-regulation and regulation of downstream events such as growth and differentiation or cell death. Regulation of nutritional uptake and organelle delivery, enzyme complex composition and stoichiometry, enzymatic activities, concomitant expression levels, and post-translational events such as phosphorylation and dephosphorylation are tissue-specific. Changes in the upstream and downstream regulation of mitochondrial enzymes, signal transduction cascades, and mitochondria structure and number have been associated with metabolic disease states, including cancer. Metabolic changes in cancer cell mitochondria may be reflected in calcium fluxes, oxidation-reduction (redox) potential, kinetics of change in response to stimuli, and the production of toxic by-products that require catabolic detoxification. Nevertheless, many tumor cells cannot efficiently adapt to metabolic stress and can be induced to die by metabolic catastrophe (i.e., where high energy demand is contrasted by insufficient energy production). (See Jin S, DiPaola RS, Mathew R, and White E. 2007. Metabolic catastrophe as a means to cancer cell death. J Cell Sci 120, 379-383, herein incorporated by reference.) It would be advantageous, then, to formulate a pharmaceutical agent which would promote such metabolic catastrophe within tumor cells upon delivery to the appropriate targets therein. A cofactor in the pyruvate dehydrogenase (PDH), alpha-ketoglutarate (αKDH), and branched chain alpha-keto acid dehydrogenase (BCAKDH) complexes, lipoic acid (1,2- dithione-3-pentanoic acid) is a sulfur-containing antioxidant with metal-chelating and anti- glycation capabilities. The anti-glycation capacity of lipoic acid combined with its capacity for hydrophobic binding enables lipoic acid to prevent glycosylation of albumin in the bloodstream. Lipoic acid exists as two enantiomers, with the R-enantiomer being more biologically active than the S-enantiomer. Naturally-occurring lipoic acid is the R-form, but synthetic lipoic acid (known as alpha lipoic acid) is a racemic mixture of R-form and S-form.

Lipoic acid is the oxidized part of a redox pair, capable of being reduced to dihydrolipoic acid (DHLA). Both lipoic acid and DHLA can chelate heavy metals that could generate free radicals, having been found both to inhibit copper- and iron-mediated oxidative damage in vitro and to inhibit excess iron and copper accumulation in vivo. In general, DHLA has superior antioxidant activity to lipoic acid; by donating two hydrogens, DHLA can neutralize free radicals without itself becoming a free radical. Thus, lipoic acid is active against OH^', HClO, and O₂, but not against O₂ ^'' or H₂O₂. DHLA is active against OH^' and HClO, but not against H₂O₂ or O₂. Both lipoic acid and DHLA can directly scavenge physiologically-relevant reactive oxygen and nitrogen species (RONS) in vitro, but it is unclear whether lipoic acid acts directly to scavenge RONS in vivo. Given the important role of lipoic acid in the regulation of RONS metabolism, then, it may be inferred that conjugates, derivatives, or analogues of lipoic acid would have a similar effect on RONS metabolism. It is unclear if cancer cells synthesize lipoate or increase its uptake from plasma.

However, it has been reported that reduction of exogenous lipoic acid to dihydrolipoate occurs in most mammalian tissues, including red blood cells. Normal human erythrocytes were found to reduce lipoate to dihydrolipoate only in the presence of glucose but not deoxyglucose, suggesting that reduction of lipoate requires glucose metabolism and is NADPH-dependent. {See Constantinescu A, Pick U, Handelman GJ, Haramaki N, Han D, Podda M, Tritschler HJ, and Packer L. 1995. Reduction and transport of lipoic acid by human erythrocytes. Biochem Pharmacol 50:253-261, herein incorporated by reference.) Additionally, it has been shown through the use of red blood cell ghosts and liposomes that a transport system exists for lipoic acid and dihydrolipoate. A Russian research team demonstrated that ³⁵S-lipoic acid uptake by red blood cells taken from breast cancer patients was increased compared to that from healthy donors, this difference being less significant in cases of acute mastitis or benign tumors. Furthermore, the rate of ³⁵S lipoic acid uptake by red blood cells from cancer patients was much higher than that found for healthy donor cells. {See Savvov VI and Karpov LM. 1982. Characteristics of ³⁵S-lipoic acid absorption by the blood cells in breast cancer (article in Russian). Vopr Onkol 28:11-13, herein incorporated by reference.) It is possible, then, that an active uptake mechanism (e.g., a channel or pump) exists for lipoates which may be modified or upregulated in cancer cells. It is also possible that, given the need for lipoates in the mitochondria, transport proteins or chaperones for delivery of lipoates to mitochondria may also be present. Additional activities may be associated with such proteins (e.g., transport from the cellular membrane to and across the outer or inner mitochondrial membrane), or multiple proteins may be present.

US Patents 6,090,842, 6,235,772, and 6,387,945 all to Packer et al, all herein incorporated by reference, teach lipoic acid analogues of various specific structures, and/or methods of production thereof, which may be used to treat conditions involving reactive oxygen species or redox mechanisms, including cancer. However, there is no indication in either patent that these derivatives are useful for improving therapeutic or diagnostic agent targeting for and penetration into a molecular target, such as may be found within a tumor mass or tumor cell. Furthermore, there is no suggestion in these patents of conjugation to a second useful molecular entity which enhances the properties of penetration, distribution, uptake, and transport.

US Patents 6,331,559 and 6,951,887 to Bingham et al, as well as US Patent Application No. 12/105,096 by Bingham et al., all herein incorporated by reference, disclose a novel class of lipoic acid derivative therapeutic agents which selectively target and kill both tumor cells and certain other types of diseased cells. These teachings further disclose pharmaceutical compositions, and methods of use thereof, comprising an effective amount of such lipoic acid derivatives along with a pharmaceutically acceptable carrier. However, while these patents describe the structures of and general use for these lipoic acid derivatives, there is no indication in either patent that these derivatives are useful for improving therapeutic or diagnostic agent targeting for and penetration into a molecular target, such as may be found within a tumor mass or tumor cell. Furthermore, there is no suggestion in these patents of conjugation to a second useful molecular entity which enhances the properties of penetration, distribution, uptake, and transport.

As it has been demonstrated that naturally-occurring fatty acids and analogues thereof conjugated to a polymer, nonpolymer, or lipid-based particle and/or a therapeutic, imaging, and/or diagnostic agent can be of diagnostic, imaging, or treatment benefit in the management of cancer; that such compounds can become bound to serum albumin (e.g., HSA), enhancing diagnostic, imaging, and therapeutic benefit by the promotion of passive tumor mass accumulation; that such compounds are not only actively taken up into cancer cells and concentrated in tumor mitochondria but also show limited if any cellular or mitochondrial uptake in healthy cells; and that such compounds can be constructed so as to contain and deliver diagnostic, imaging, or therapeutic molecules, such as but not limited to drugs, radioisotopes, or nanoparticles, to diseased cells, it would as a result be desirable to increase the circulation time of these compounds in the bloodstream via linkage to a carrier molecule, as well as to provide additional diagnostic, imaging, or therapeutic capability by conjugation of the compound to a drug or other non-drug atom or molecule.

Objects of the Invention and Industrial Applicability

Consequently, it is an object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of a disease, condition, or syndrome characterized by cellular hyperproliferation, such as cancer, which exhibits selective distribution and cell layer penetration into a tumor mass, as well as selective uptake and transport into specific organelles in tumor cells.

It is a further object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of such an aforementioned disease, condition, or syndrome which causes minimal side effects upon administration. It is a still further object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of such an aforementioned disease, condition, or syndrome which is easily manufactured at the least possible cost and is capable of being stored for the longest possible period. It is a still further object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of such an aforementioned disease, condition, or syndrome which modulates mitochondrial energy metabolism, especially via the structure, activity, and/or expression level of the PDH, αKDH, and/or BCAKDH complexes in tumor cell mitochondria, in combination with an additional diagnostic, imaging, or therapeutic capability.

It is a still further object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of such an aforementioned disease, condition, or syndrome which exhibits increased ability to penetrate into tumor mass cell layers and deliver its diagnostic, imaging, or therapeutic agent. It is a still further object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of such an aforementioned disease, condition, or syndrome which modulates the structure, activity, and/or expression level of at least one enzyme or enzyme complex, such as the PDH, αKDH, and/or BCAKDH complexes in tumor cell mitochondria, in combination with additional diagnostic, imaging, or therapeutic capability.

Summary of the Invention

To achieve the aforementioned aims, the present invention broadly provides herein the description of molecular structures intended to improve tumor cell layer penetration and distribution of a pharmaceutical composition into a tumor mass of a warm-blooded animal, including humans, by increased cellular uptake and transport into a specific organelle, and more specifically by targeted transport and uptake of a diagnostic, imaging, or therapeutic agent to the mitochondria of a cell characterized by hyperproliferation, such as a cancer cell. This pharmaceutical composition comprises an effective amount of at least one compound, this compound being at least one naturally-occurring fatty acid, or an analogue thereof , including but not limited to conjugates and analogues of naturally-occurring fatty acids, such as but not limited to those of octanoic acid or lipoate, including those described in US Patents 6,331,559 and 6,951,887 and US Patent Application No. 12/105,096, all herein incorporated by reference; this compound conjugated to at least one molecule which increases the solubility or blood circulation life of this fatty acid or analogue thereof and/or at least one diagnostic, therapeutic, and/or therapeutic agent; and at least one pharmaceutically- acceptable carrier or excipient of such compounds. The fatty acid portion of such compositions, in addition to promoting cellular uptake and transport to specific organelles, may also separately provide simultaneous or synergistic treatment benefit. Alternatively, the fatty acid portion of a conjugate or analogue may serve only to deliver a detectable moiety, drug, pro-drug, radioisotope, metal, chelator, or DNA- or RNA-interacting agent to a specific organelle or molecular target contained therein for diagnosis, imaging or treatment benefit.

As molecules which are not only a derivative of one which is found normally within mitochondria but also one which is instrumental to the increased glycolytic activity of tumor cells as suggested by the Warburg effect, the compounds of the present invention demonstrates selective cell layer penetration and distribution into a tumor mass, as well as selective targeting for and uptake and transport into specific organelles within tumor cells, such as but not limited to the mitochondria. Increased depth of tumor cell layer penetration and more even distribution throughout a tumor mass assisted by active cell transport is contemplated for cancer. Penetration and distribution to desired organelles are anticipated to exceed that observed with non-cell-selective delivery vehicles that depend on diffusion gradients and that are limited by distance from blood vessels, thereby facilitating diagnosis, imaging, or treatment and providing both detection and therapeutic benefit. Furthermore, such selective tumor cell uptake minimizes the side effects the administration of this pharmaceutical composition would have on healthy non-transformed cells and tissue. The compounds of the present invention have the general formulae:

or

to show that unsaturation or polyunsaturation may occur, wherein x = 1 to 18; R₁, R₂, and/or R₆ is an aromatic component, -COOH, -OH, or -NH₃; R₃, R₄, and/or R₅ is an aromatic component, an alkyl, an aryl, S, O, and/or N; Ri, R₂, R₃, R₄, R₅, and/or R₆ may be phosphorylated; and Ri, R₂, R₃, R₄, R₅, and/or R₆ may be so modified as to modulate the binding affinity of the compound to carrier molecules in vivo so as to regulate the amount of circulating time the compound spends in the blood, which in turn leads to a modification in the dosage of the pharmaceutical composition administered to a patient.

It is specifically contemplated that the carrier molecule is serum albumin (e.g., HSA), although any protein or molecule in the blood or plasma is appropriate. Ri, R₂, R₃, R₄, R₅, and/or R₆ may be modified either to decrease the binding affinity between the compound and the carrier molecule, thereby increasing the rate of release of the compound off of the carrier molecule, or vice versa.

Additionally, at least one diagnostic, imaging, and/or therapeutic agent is linked to the active compound by a chemical bond at R₁, R₂, R₃, R₄, R₅, and/or R₆, and such agents may either be a drug, prodrug, or useful non-drug atom or molecule (e.g., boron). This bond may be a non-covalent bond such as an ionic bond or hydrophobic interaction; a non-reversible covalent bond; or a reversible covalent bond, such as but not limited to an ester linkage. The reversible covalent bond is susceptible to enzymatic cleavage by such an enzyme as, but not limited to, a peptidase, lipase, or glycosidase, and may be modulated by kinase or phosphatase activity. Furthermore, each of these enzymes may be particular to a specific disease, and in turn be tissue-specific, cell-specific, or organelle-specific.

In an additional embodiment of the present invention, Ri, R₂, R₃, R₄, R₅, and/or R₆ may be a polymer, such as but not limited to PEG; polyethyleneimine (PEI); polyglutamic acid; dextrans; or other polymers known in the art. The bond linking the polymer to the active compound may be a non-covalent bond such as an ionic bond or hydrophobic interaction; a non-reversible covalent bond; or a reversible covalent bond, such as but not limited to an ester linkage. As with non-polymeric additions, the reversible covalent bond is susceptible to enzymatic cleavage by such an enzyme as, but not limited to, a peptidase, lipase, or glycosidase, and may be modulated by kinase or phosphatase activity. Furthermore, each of these enzymes may again be particular to a specific disease, and in turn be tissue-specific, cell-specific, or organelle-specific.

In a further embodiment of the present invention, the active compound is linked at Ri, R₂, R₃, R₄, R₅, and/or R₆ to a lipid-based particle, such as but not limited to a nanolipid particle such as a gold or carbon nanoparticle; a liposome; a polymeric lipid particle; a solid lipid; or other lipid-based particle known in the art. As with the polymeric or non-polymeric additions, the reversible covalent bond is susceptible to enzymatic cleavage by such an enzyme as, but not limited to, a peptidase, lipase, or glycosidase, and may be modulated by kinase or phosphatase activity. Furthermore, each of these enzymes may again be particular to a specific disease, and in turn be tissue-specific, cell-specific, or organelle-specific. However, in an additional embodiment of the present invention, the active compound may be surface-coated onto the lipid-based particle, and that lipid-based particle then impregnated onto a biomedical or surgical device such as a stent.

The carrier molecule linked to the active compound may be associated with diseased cells, such as those characterized by hyperproliferation, including cancer cells. Indeed, the carrier molecule may be specific to cancer cells or to the erythrocytes of cancer patients. This carrier molecule may be detectable by an in vivo or ex vivo diagnostic or prognostic assay for disease status, such as through antibody detection of the carrier molecule or use of an assay includes polymerase chain reaction amplification. The carrier molecule may thus also be the molecular target for the diagnosis, imaging, or treatment of such a disease, the means of which may include small-molecule inhibition, the use of antibodies or small- interfering RNA, and so on. Additionally, the carrier molecule may be acted upon by a translocase or channel, such as one found on a cellular or organellar membrane, such that transport can occur by facilitated diffusion or active transport. It is specifically contemplated that such a translocase or channel be found on the surface of a mitochondrion. This carrier molecule may first interact with a translocase or channel mediator, such as a molecular chaperone. Finally, the carrier molecule may be found in different isoforms, with such isoforms depending on the type of disease presented. However, it is also possible that the carrier molecule, translocase, channel, and/or transport mediator is absent in the diseased state, in which case such absence is useful in the diagnosis or treatment of the disease, such as by gene therapy. As the general structure may be metabolized within the cell or mitochondrion, it is expressly intended that metabolites of the above-referenced structure are within the scope of the present invention. Also, the R-isomer of a particular active compound may possess greater physiological activity than does the S-isomer. Consequently, the active compound should be present either solely in its R-isomer form or in a mixture of the R- and S-isomers.

Brief Description of the Figures

The following drawings are illustrative of embodiments of the invention and are not intended to limit the scope of the application as encompassed by the entire specification and claims.

FIGURE 1 illustrates an overlay of both a fluorescently-labelled compound of the present invention with a specific mitochondrial tracker showing mitochondrial accumulation of that compound in C6 glioma cells.

FIGURE 2 depicts the exclusive mitochondrial uptake of a fluorescently-labelled compound of the present invention in ras-transformed NIH 3T3 cells versus the complete lack of mitochondrial uptake seen in normal, non-transformed NIH 3T3 cells.

Similarly, FIGURE 3 shows the exclusive mitochondrial uptake of a fluorescently- labelled compound of the present invention in H460 lung cancer cells versus the complete lack of mitochondrial uptake seen in normal, non-transformed lung epithelial cells. Also similarly, FIGURE 4 demonstrates the exclusive mitochondrial uptake of a fluorescently-labelled compound of the present invention in breast cancer cells versus the complete lack of mitochondrial uptake seen in normal, non-transformed breast epithelial cells.

FIGURES 5 through 19 illustrate chromatography results of a study analyzing protein binding with various compounds of the present invention. Detailed Description of the Invention

The present invention is generally directed to molecular structures intended to improve penetration and distribution of a pharmaceutical composition into a tumor mass as well uptake and transport of the pharmaceutical composition to specific organelles within tumor cells, and more specifically by targeted delivery of a diagnostic, imaging, or therapeutic agent to the mitochondria of a cell characterized by hyperproliferation, such as a cancer cell, in warm-blooded animals. Such animals include those of the mammalian class, such as humans, domestic animals including dogs and cats, horses, cattle, etc., subject to disease and other pathological conditions and syndromes characterized by cellular hyperproliferation, including cancer. Nevertheless, other diseases characterized by cellular hyperproliferation may be amenable to diagnosis, imaging, or treatment by the pharmaceutical composition of the present invention. Non-limiting examples of other diseases characterized by cellular hyperproliferation include age-related macular degeneration; Crohn's disease; cirrhosis; chronic inflammatory-related disorders; diabetic retinopathy; granulomatosis; immune hyperproliferation associated with organ or tissue transplantation; an immunoproliferative disease or disorder (e.g., inflammatory bowel disease, psoriasis, rheumatoid arthritis, or systemic lupus erythematosus); vascular hyperproliferation secondary to retinal hypoxia; or vasculitis. The pharmaceutical composition of the present invention comprises an effective amount of at least one compound, this compound being at least one naturally-occurring fatty acid, or an analogue thereof, conjugated to at least one molecule which increases the solubility or blood circulation life of this active compound and/or at least one diagnostic, therapeutic, and/or therapeutic agent, and a pharmaceutically-acceptable carrier or excipient therefor. The compounds of the present invention have the general formulae:

or

R₁ R,

\

R, R,

(CH=CH)_x R₆

to show that unsaturation or polyunsaturation may occur, wherein x = 1 to 18; R₁, R₂, and/or

R₆ is an aromatic component, -COOH, -OH, or -NH₃; R₃, R₄, and/or R₅ is an aromatic component, an alkyl, an aryl, S, O, and/or N; R₁, R₂, R₃, R₄, R₅, and/or R₆ may be phosphorylated; and Ri, R₂, R₃, R₄, R₅, and/or R₆ may be so modified as to modulate the binding affinity of the compound to carrier molecules in vivo so as to regulate the amount of circulating time the compound spend in the blood, which in turn leads to a modification in the dosage of the pharmaceutical composition administered to a patient.

It is specifically contemplated that the carrier molecule is serum albumin (e.g., HSA), although any protein or molecule in the blood or plasma is appropriate. R], R₂, R₃, R₄, R₅, and/or R₆ may be modified either to decrease the binding affinity between the compound and the carrier molecule, thereby increasing the rate of release of the compound off of the carrier molecule, or vice versa.

Additionally, a diagnostic, imaging, and/or therapeutic or agent is linked to the compound by a chemical bond at Ri, R₂, R₃, R₄, R₅, and/or R₆, and the therapeutic agent may either be a drug or a useful non-drug atom or molecule (e.g., boron). This bond may be a non-covalent bond such as an ionic bond or hydrophobic interaction; a non-reversible covalent bond; or a reversible covalent bond, such as but not limited to an ester linkage. The reversible covalent bond is susceptible to enzymatic cleavage by such an enzyme as, but not limited to, a peptidase, lipase, or glycosidase, and may be modulated by kinase or phosphatase activity. Furthermore, each of these enzymes may be particular to a specific disease, and in turn be tissue-specific, cell-specific, or organelle-specific.

In an additional embodiment of the present invention, Rj, R₂, R₃, R₄, R₅, and/or R₆ may be a polymer, such as but not limited to PEG; PEI; polyglutamic acid; dextrans; or other polymers known in the art. The bond linking the polymer to the compound may be a non- covalent bond such as an ionic bond or hydrophobic interaction; a non-reversible covalent bond; or a reversible covalent bond, such as but not limited to an ester linkage. As with non- polymeric additions, the reversible covalent bond is susceptible to enzymatic cleavage by such an enzyme as, but not limited to, a peptidase, lipase, or glycosidase, and may be modulated by kinase or phosphatase activity. Furthermore, each of these enzymes may again be particular to a specific disease, and in turn be tissue-specific, cell-specific, or organelle- specific.

In a further embodiment of the present invention, the compound is linked at Ri, R₂,

R₃, R4, R₅, and/or R₆ to a lipid-based particle, such as but not limited to a nanolipid particle such as a gold or carbon nanoparticle; a liposome; a polymeric lipid particle; a solid lipid; or other lipid-based particle known in the art. As with the polymeric or non-polymeric additions, the reversible covalent bond is susceptible to enzymatic cleavage by such an enzyme as, but not limited to, a peptidase, lipase, or glycosidase, and may be modulated by kinase or phosphatase activity. Furthermore, each of these enzymes may again be particular to a specific disease, and in turn be tissue-specific, cell-specific, or organelle-specific. However, in an additional embodiment of the present invention, the compound may be surface-coated onto the lipid-based particle, and that lipid-based particle then impregnated onto a biomedical or surgical device such as a stent.

As molecules which are not only a derivative of one which is found normally within mitochondria but also one which is instrumental to the increased glycolytic activity of tumor cells as suggested by the Warburg effect, the compounds of the present invention are particularly well-suited for the selective delivery into and effective concentration within the mitochondria of cells and tissues characterized by hyperproliferation, such as tumorous ones, thereby sparing normal cells and tissue from the effects of the composition. Furthermore, such specific tumor organelle uptake minimizes the side effects the administration of this pharmaceutical composition would have on healthy non-transformed cells and tissue.

By inhibiting mitochondrial energy metabolism through the activation of PDKs and/or inhibition of PDPs or by inhibiting the conversion of pyruvate to the less-toxic molecule acetoin through inhibition of the activity of the El subunit of the PDH, αKDH, and/or BCAKDH complexes, thereby increasing RONS production within the mitochondrion, the compounds of the present invention cause both the loss of mitochondrial membrane potential and other mitochondrial consequences in the diseased cell, such as reversible redox signalling or irreversible oxidative stress, resulting in the irreversible initiation of cell death through either apoptosis or necrosis.

The carrier molecule linked to the compound may be associated with diseased cells such as those characterized by hyperproliferation, including cancer cells. Indeed, the carrier molecule may be specific to cancer cells or to the erythrocytes of cancer patients. This carrier molecule may be detectable by an in vivo or ex vivo diagnostic or prognostic assay for disease status, such as through antibody detection of the carrier protein or use of an assay includes polymerase chain reaction amplification. The carrier molecule may thus also be the molecular target for the treatment or diagnosis of such a disease, the means of which may include small-molecule inhibition, the use of antibodies or small-interfering RNA, and so on. Additionally, the carrier molecule may be acted upon by a translocase or channel, such as one found on a cellular or organellar membrane, such that transport can occur by facilitated diffusion or active transport. It is specifically contemplated that such a translocase or channel be found on the surface of a mitochondrion. This carrier molecule may first interact with a translocase or channel mediator, such as a molecular chaperone. Finally, the carrier molecule may be found in different isoforms, with such isoforms depending on the type of disease presented. However, it is also possible that the carrier molecule, translocase, channel, and/or transport mediator is absent in the diseased state, in which case such absence is useful in the diagnosis or treatment of the disease, such as through gene therapy.

As the general structure may be metabolized within the cell or mitochondrion, it is expressly intended that metabolites of the above-referenced structure are within the scope of the present invention. Also, the R-isomer of a particular compound may possess greater physiological activity than does the S-isomer. Consequently, the compound should be present either solely in its R-isomer form or in a mixture of the R- and S-isomers.

The compositions of the present invention may further include a pharmaceutically- acceptable carrier or excipients. Examples of pharmaceutically-acceptable carriers are well known in the art and include those conventionally used in pharmaceutical compositions, such as, but not limited to, antioxidants, buffers, chelating agents, flavorants, colorants, preservatives, absorption promoters to enhance bioavailability, antimicrobial agents, and combinations thereof. The amount of such additives depends on the properties desired, which can readily be determined by one skilled in the art.

The pharmaceutical compositions of the present invention may routinely contain salts, buffering agents, preservatives, and compatible carriers, optionally in combination with other therapeutic ingredients. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically-acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically- and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, palicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

The present invention additionally provides methods for treating or diagnosing a patient, or facilitating the imaging of a tumor mass within a patient, with diagnostic, imaging, or therapeutic agents by delivering to diseased cells an effective amount of at least one diagnostic, imaging, or therapeutic agent, linked to the compound, for implementing the prevention, diagnosis, imaging, or treatment of a disease, condition, or syndrome, or symptoms thereof, including those characterized by cellular hyperproliferation, such as but not limited to cancer. Modulating the PDH, αKDH, and/or BCAKDH complexes as an improved treatment of cancer is especially contemplated, including treatment of primary tumors by the control of tumor cell proliferation, angiogenesis, metastatic growth, apoptosis, and treatment of the development of micrometastasis after or concurrent with surgical removal; and radiological or other chemotherapeutic treatment of a primary tumor. The pharmaceutical composition of the present invention is useful in such cancer types as primary or metastatic melanoma, lymphoma, sarcoma, lung cancer, liver cancer, Hodgkin's and non- Hodgkin's lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer. Appropriate diagnostic, imaging, or therapeutic agents may be, but are not limited to, a drug, a pro-drug, a radioisotope, a chelator, a glycolytic inhibitor, a ligand-binding moiety such as but not limited to an antibody or aptamer, a receptor-binding moiety, a microtubule- interacting agent, a cytostatic agent, a folic acid inhibitor, an alkylating agent, a topoisomerase inhibitor, a tyrosine kinase inhibitor, podophyllotoxin or derivatives thereof, an antitumor antibiotic, a chemotherapeutic agent, an apoptosis-inducing agent, an anti- angiogenic agent, nitrogen mustards, nucleic acid intercalating agents, a detectable moiety, other DNA- or RNA-interacting agents, and combinations thereof. Such therapeutic agents may further include other metabolic inhibition reagents. Many such therapeutic agents are known in the art. The variety in linked agents provides for simultaneous, sequential, or separate use in treating such conditions as needed to amplify or ensure patient response to the treatment method.

The methods of the present invention may be practiced using any mode of administration that is medically acceptable, and produces effective levels of the compounds without causing clinically unacceptable adverse effects. Although formulations specifically suited for parenteral administration are preferred, the compositions of the present invention can also be formulated for inhalational, oral, topical, transdermal, nasal, ocular, pulmonary, rectal, transmucosal, intravenous, intramuscular, subcutaneous, intraperitoneal, intrathoracic, intrapleural, intrauterine, intratumoral, or infusion methodologies or administration, in the form of aerosols, sprays, powders, gels, lotions, creams, suppositories, ointments, and the like. If such a formulation is desired, other additives well-known in the art may be included to impart the desired consistency and other properties to the formulation.

Those skilled in the art will recognize that the particular mode of administering the therapeutic or diagnostic agent depends on the particular agent selected; whether the administration is for treatment, diagnosis, or prevention of a disease, condition, syndrome, or symptoms thereof; the severity of the medical disorder being treated or diagnosed; and the dosage required for therapeutic efficacy. For example, a preferred mode of administering an anticancer agent for treatment of leukemia would involve intravenous administration, whereas preferred methods for treating skin cancer could involve topical or intradermal administration. As used herein, "effective amount" refers to the dosage or multiple dosages of the therapeutic or diagnostic agent at which the desired therapeutic or diagnostic effect is achieved. Generally, an effective amount of the therapeutic or diagnostic agent may vary with the activity of the specific agent employed; the metabolic stability and length of action of that agent; the species, age, body weight, general health, dietary status, sex and diet of the subject; the mode and time of administration; rate of excretion; drug combination, if any; and extent of presentation and/or severity of the particular condition being treated. The precise dosage can be determined by an artisan of ordinary skill in the art without undue experimentation, in one or several administrations per day, to yield the desired results, and the dosage may be adjusted by the individual practitioner to achieve a desired therapeutic effect or in the event of any complication. Importantly, when used to treat cancer, the dosage amount of the therapeutic agent used should be sufficient to inhibit or kill tumor cells while leaving normal cells substantially unharmed.

The therapeutic or diagnostic agent included in the pharmaceutical compositions of the present invention can be prepared in any amount desired up to the maximum amount that can be administered safely to a patient. The amount of the diagnostic agent or therapeutic agent may range from less than 0.01 mg/mL to greater than 1000 mg/mL, preferably about 50 mg/mL.

Generally, the pharmaceutical composition of the present invention will be delivered in a manner sufficient to administer to the patient an amount effective to modulate the structure and/or activity of the PDH, αKDH, and/or BCAKDH complexes. The dosage amount may thus range from about 0.3 mg/m² to 2000 mg/m², preferably about 60 mg/m². The dosage amount may be administered in a single dose or in the form of individual divided doses, such as from one to four or more times per day. In the event that the response in a subject is insufficient at a certain dose, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent of patient tolerance. Multiple doses per day are contemplated to achieve appropriate systemic or targeted levels of the therapeutic or diagnostic agent.

The following non-limiting examples are provided to facilitate understanding of the pharmaceutical compositions of the present invention. EXAMPLE 1

Uptake of 6,8-6/.S-BODIPY Mercaptooctanoic Acid by Different Tumor and Normal

Cells

Materials and equipment:

Cell lines: rat C6 (ATCC); mouse fibroblast, NIH 3T3, ras-transformed NIH 3T3 (NIH 3T3 T24) (gift from Dafna Bar-Sagi); breast tumor MDA-MB-435 cells; normal human mammary epithelial cells (HMEC); non-small lung cell carcinoma tumor cells (H460 and A549); and human small airway epithelial cells (SAEC). (See Table 1.)

Cell growth conditions: Sterile, 18mm² cover slips were placed inside 35mm dishes. Dishes were seeded with 2 x 10⁵ cells and grown overnight in media appropriate for each cell line. (See Table 1.)

Labelling protocol: Growth medium was replaced with labeling medium containing

2.5μM 6,8-6/.S-BODIPY mercaptooctanoic acid (bBMOA) and incubated for 160 minutes at

37⁰C. At this time, 25nM Mitotracker 580 red (Invitrogen) was added to the medium and cells were incubated for an additional 20 minutes. (Total labelling time: 3hrs bBMOA, 20 minutes Mitotracker.) At the end of the 20 minutes incubation, labelling medium was removed, and cells were washed twice with PBS and fixed in 10% formalin for 10 minutes. Cover slips were removed from the dish, washed twice with PBS and once with H₂O and mounted with Prolong Gold (Invitrogen) anti-fade mounting medium. Images were collected using an Axiovert 200M (Zeiss) deconvolution microscope at a fixed exposure time. FITC filter was used to detect bBMOA, and rhodamine filter was used to detect Mitotracker 580 red.

Table 1. Source, Growth and Labelling media for Cell lines.

Results: FIGURE 1 illustrates an overlay of both a BODIP Y-labelled compound of the present invention with the Mitotracker Red, showing mitochondrial accumulation of that compound in C6 glioma cells.

FIGURE 2 depicts the exclusive mitochondrial uptake of a BODIPY-labelled compound of the present invention in røs-transformed NIH 3T3 cells versus the complete lack of mitochondrial uptake seen in normal, non-transformed NIH 3T3 cells.

Similarly, FIGURE 3 shows the exclusive mitochondrial uptake of a BODIPY- labelled compound of the present invention in H460 lung cancer cells versus the complete lack of mitochondrial uptake seen in normal, non-transformed SAEC cells.

Also similarly, FIGURE 4 demonstrates the exclusive mitochondrial uptake of a BODIPY-labelled compound of the present invention in MDA-MB-435 breast cancer cells versus the complete lack of mitochondrial uptake seen in normal, non-transformed HMEC breast epithelial cells. (N.B. The MDA-MB-435 cell line was reclassified in September 2006 as M 14 human melanoma. (Christgen M. and Lehmann U. 2007. MDA-MB-435: the questionable use of a melanoma cell line as a model for human breast cancer is ongoing. Cancer Biol Ther 6(9): 1355-1357, herein incorporated by reference)

EXAMPLE 2

Thioctan/Human Serum Albumin Binding Study

Method:

A method described by Cheng et al. (See Cheng Y, Ho E, Subramanyam B and Tseng J. 2004. Measurements of drug-protein binding by using immobilized human serum albumin liquid chromatography-mass spectrometry. J Chromatogr B, 809:67-73, herein incorporated by reference) was modified. Procedure: Column:

A Chiral HSA column (Chromtech Ltd., UK) 50 x 4.0mm was used. Run Conditions:

A mobile phase of 20% acetonitrile in 5OmM ammonium acetate, pH 7.4, isocratic, at a flow rate of 0.8mL/min was used. The wavelength used for detection was 220nm, with a sensitivity setting of 0.05 AUFS. 20μL of a O.lmg/mL solution was injected in each assay.

Results: Chromatograms (KB. time scales (x-axes) may be different on figures):

Lipoic Acid (Racemic): Retention time = 4.43 min. (See FIGURE 5)

(R)-Lipoic Acid: Retention time = 4.02 min. (See FIGURE 6)

(S)-Lipoic Acid: Retention time - 4.07 min. (See FIGURE 7)

CPI-613 (Racemic): Retention Times = 38.39, 43.81 min. (See FIGURE 8)

(R)-CPI-613: Retention time = 36.38 min. (See FIGURE 9)

(S)-CPI-613: Retention time = 38.80 min. (See FIGURE 10)

CPI-613 Amine: Retention times = 3.41, 3.72 min. (See FIGURE 11)

CPI-613 Alcohol: Retention Time = 9.76, 10.60 min. (See FIGURE 12)

CPI-613 Amide: Retention Time = 4.47, 5.72 min. (See FIGURE 13)

CPI-613 Bolton-Hunter Analog: Retention times 13.6, 16.93 min. (See FIGURE 14)

CPI-613 Methyl Ester: (Has low solubility in the mobile phase). Gives a broad peak around 55 min. (See FIGURE 15)

CPI-613 Diethylamide: Retention times = 7.89, 12.66 min. (See FIGURE 16)

CPI-613 Monohydroxyethyl Amide: Retention times = 3.67, 4.88 min. (See FIGURE

17)

CPI-613 Dihydroxyethyl Amide: Retention times = 3.09, 3.96 min. (See FIGURE

18)

CPI-613 K⁺-SaIt: Apparently, the potassium salt converts to CPI-613 under pH = 7.4. Retention times 44.0 and 48.9 min. (See FIGURE 19)

Table 2. Summary of results from protein binding study.

Analyses:

This study was conducted at a buffer pH of 7.4, similar to that of human blood. Racemic lipoic acid did not resolve into two enantiomers under these run conditions. In separate runs, the retention times of lipoic acid and its two enantiomers were almost similar.

EXAMPLE 3 PEGylated Analogues

The various non-limiting examples of PEGylated compounds of the present invention presented below have been manufactured and are herein disclosed.

MW - 1194.6

The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims. Furthermore, while exemplary embodiments have been expressed herein, others practiced in the art may be aware of other designs or uses of the present invention. Thus, while the present invention has been described in connection with exemplary embodiments thereof, it will be understood that many modifications in both design and use will be apparent to those of ordinary skill in the art, and this application is intended to cover any adaptations or variations thereof. It is therefore manifestly intended that this invention be limited only by the claims and the equivalents thereof.

Claims

The invention to be claimed is:

1. A compound having the general structure:

Ri R₂

\ /

R₃ R₄

and salts thereof, wherein x = 1 to 18; Ri, R₂, and/or R₆ is an aromatic component, -COOH, -OH, or - NH₃; and Ri, R₂, R₃, R₄, R₅, and/or R₆ may be so modified as to modulate the binding affinity of the compound to carrier molecules in vivo.

2. A compound having the general structure:

and salts thereof, wherein x = 1 to 18; Ri, R₂, and/or R₆ is an aromatic component, -COOH, -OH, or -

NH₃; the (CH=CH)_x constituent may be polyunsaturated; and Ri, R₂, R₃, R₄, R₅, and/or R₆ may be so modified as to modulate the binding affinity of the compound to carrier molecules in vivo.

3. The compound of claim 1 or 2, wherein R₃, R₄, and/or R₅ is an aromatic component, an alkyl, an aryl, S, O, and/or N.

4. The compound of claim 3, wherein the alkyl is saturated.

5. The compound of claim 4, wherein the alkyl is unsaturated.

6. The compound of claim 1 or 2, wherein Ri, R₂, R₃, R», R₅, and/or R₆ may be phosphorylated.

7. The compound of claim 1 or 2, wherein Ri, R₂, R₅, and/or R₆ is a therapeutic agent.

8. The compound of claim 7, wherein the therapeutic agent is a drug, a prodrug, a ligand-binding moiety, a receptor-binding moiety, a radioisotope, a metal, a chelating agent, an alkylating agent, a DNA-interacting agent, or a RNA-interacting agent.

9. The compound of claim 8, wherein the ligand-binding moiety is an antibody.

10. The compound of claim 9, wherein the antibody is a single-chain antibody.

11. The compound of claim 8, wherein the ligand-binding moiety is an aptamer.

12. The compound of claim 1 or 2, wherein Ri, R₂, R₅, and/or R₆ is a diagnostic agent.

13. The compound of claim 12, wherein the diagnostic agent is useful in vivo.

14. The compound of claim 12, wherein the diagnostic agent is useful ex vivo.

15. The compound of claim 12, wherein the diagnostic agent is selected from the group consisting of a drug, a prodrug, a ligand-binding moiety, a receptor-binding moiety, a radioisotope, a fluorescent label, a fluorescent label moiety, a contrast agent, a metal, a chelating agent, an alkylating agent, a DNA-interacting agent, or a RNA-interacting agent.

16. The compound of claim 15, wherein the ligand-binding moiety is an antibody.

17. The compound of claim 16, wherein the antibody is a single-chain antibody.

18. The compound of claim 15, wherein the ligand-binding moiety is an aptamer.

19. The compound of claim 1 or 2, wherein R₁, R₂, R₅, and/or R₆ is an imaging agent.

20. The compound of claim 19, wherein the imaging agent is a ligand-binding moiety, a receptor-binding moiety, a radioisotope, a fluorescent label, a fluorescent label moiety, a contrast agent, a metal, and a chelating agent.

21. The compound of claim 20, wherein the ligand-binding moiety is an antibody.

22. The compound of claim 21, wherein the antibody is a single-chain antibody.

23. The compound of claim 20, wherein the ligand-binding moiety is an aptamer.

24. The compound of claim 1 or 2, wherein the carrier molecule interacts with a translocase or channel.

25. The compound of claim 24, wherein the carrier molecule interacts first with a translocase or channel mediator.

26. The compound of claim 25, wherein the mediator is a molecular chaperone.

27. The compound of claim 24, wherein the translocase works via facilitated diffusion.

28. The compound of claim 24, wherein the translocase works via active transport.

29. The compound of claim 1 or 2, wherein the carrier molecule is a protein.

30. The compound of claim 29, wherein the carrier protein is serum albumin.

31. The compound of claim 1 or 2, wherein a diagnostic, imaging, or therapeutic agent is linked to the compound by a chemical bond at Ri, R₂, R₃, R₄, R₅, and/or R₆.

32. The compound of claim 31, wherein the chemical bond is selected from the group comprising a non-reversible covalent bond, a reversible covalent bond, and a non- covalent bond.

33. The compound of claim 32, wherein the reversible covalent bond is an ester linkage.

34. The compound of claim 33, wherein the reversible covalent bond is susceptible to enzymatic cleavage.

35. The compound of claim 34, wherein the enzyme responsible for the enzymatic cleavage includes, but is not limited to, a peptidase, a lipase, or a glycosidase.

36. The compound of claim 34, wherein the enzyme is disease-specific.

37. The compound of claim 36, wherein the enzyme is tissue-specific.

38. The compound of claim 36, wherein the enzyme is cell-specific.

39. The compound of claim 36, wherein the enzyme is organelle-specific.

40. The compound of claim 32, wherein the non-covalent bond is an ionic bond.

41. The compound of claim 32, wherein the non-covalent bond is a hydrophobic interaction.

42. The compound of claim 6, wherein the linkage between the diagnostic, imaging, and/or therapeutic agent may be modulated by kinase activity.

43. The compound of claim 6, wherein the linkage between the diagnostic, imaging, and/or therapeutic agent may be modulated by phosphatase activity.

44. The compound of claim 42 or 43, wherein the alteration in binding affinity allows for reversible covalent binding.

45. The compound of claim 42 or 43, wherein the alteration in binding affinity allows for noncovalent binding.

46. The compound of claim 42 or 43, wherein the alteration in binding affinity allows for irreversible binding.

47. The compound of claim 1 or 2, wherein Ri, R₂, R₃, R₄, R₅, and/or R_O may be modified to decrease the binding affinity between the compound and the carrier molecule.

48. The compound of claim 47, wherein the decreased binding affinity permits an increased rate of release of the compound off the carrier molecule.

49. The compound of claim 1 or 2, wherein Ri, R₂, R₃, R₄, R₅, and/or R₆ may be modified to increase the binding affinity between the compound and the carrier molecule.

50. The compound of claim 49, wherein the increased binding affinity permits a decreased rate of release of the compound off the carrier molecule.

51. The compound of claim 1 or 2, wherein Ri, R₂, R₅, and/or R₆ may be a polymer.

52. The compound of claim 51, wherein the polymer is polyethylene glycol, polyethyleneimine, polyglutamic acid, dextrans, or other polymers known in the art.

53. The compound of claim 52, wherein the polymer is linked to the compound by a chemical bond.

54. The compound of claim 51, wherein the chemical bond is selected from the group comprising a non-reversible covalent bond, a reversible covalent bond, and a non- covalent bond.

55. The compound of claim 54, wherein the reversible covalent bond is an ester linkage.

56. The compound of claim 55, wherein the reversible covalent bond is susceptible to enzymatic cleavage.

57. The compound of claim 56, wherein the enzyme is disease-specific.

58. The compound of claim 57, wherein the enzyme is tissue-specific.

59. The compound of claim 57, wherein the enzyme is cell-specific.

60. The compound of claim 57, wherein the enzyme is organelle-specific.

61. The compound of claim 54, wherein the non-covalent bond is an ionic bond.

62. The compound of claim 54, wherein the non-covalent bond is a hydrophobic interaction.

63. The compound of claim 1 or 2, wherein the compound is associated with a lipid-based particle.

64. The compound of claim 63, wherein the lipid-based particle is a nanolipid particle, a liposome, a polymeric lipid particle, a solid lipid, or other lipid-based particle known in the art.

65. The compound of claim 64, wherein the nanolipid particle is a gold-containing nanolipid.

66. The compound of claim 64, wherein the nanolipid particle is a carbon- containing nanolipid.

67. The compound of claim 63, wherein the lipid-based particle is linked to the compound by a chemical bond.

68. The compound of claim 67, wherein the chemical bond is selected from the group comprising a non-reversible covalent bond, a reversible covalent bond, and a non- covalent bond.

69. The compound of claim 68, wherein the reversible covalent bond is an ester linkage.

70. The compound of claim 69, wherein the reversible covalent bond is susceptible to enzymatic cleavage.

71. The compound of claim 70, wherein the enzyme is disease-specific.

72. The compound of claim 71, wherein the enzyme is tissue-specific.

73. The compound of claim 71 , wherein the enzyme is cell-specific.

74. The compound of claim 71 , wherein the enzyme is organelle-specific.

75. The compound of claim 68, wherein the non-covalent bond is an ionic bond.

76. The compound of claim 68, wherein the non-covalent bond is a hydrophobic interaction.

77. The compound of claim 63, wherein the compound may be surface-coated onto the lipid-based particle.

78. The compound of claim 77, wherein the lipid-based particle is impregnated onto a biomedical or surgical device.

79. The compound of claim 78, wherein the biomedical or surgical device is a stent.

80. The compound of claim 1 or 2, wherein the carrier molecule is associated with diseased cells.

81. The compound of claim 80, wherein the disease is characterized by cellular hyperproliferation.

82. The compound of claim 81, wherein the disease is cancer.

83. The compound of claim 80, wherein the carrier molecule interacts with a translocase or channel specific to diseased cells.

84. The compound of claim 82, wherein the carrier molecule is specific to the erythrocytes of cancer patients.

85. The compound of claim 80, wherein the carrier molecule is detectable by a diagnostic or prognostic assay for disease status.

86. The compound of claim 85, wherein the assay is antibody detection of the carrier molecule.

87. The compound of claim 85, wherein the assay includes polymerase chain reaction amplification.

88. The compound of claim 1 or 2, wherein the carrier molecule is the molecular target for the diagnosis, imaging, or treatment of a disease.

89. The compound of claim 88, wherein the disease is characterized by cellular hyperproliferation.

90. The compound of claim 89, wherein the disease is cancer.

91. The compound of claim 88, wherein the means of diagnosis, imaging, or treatment is small-molecule inhibition.

92. The compound of claim 88, wherein the means of diagnosis, imaging, or treatment uses ligand-binding moieties.

93. The compound of claim 92, wherein the moieties are antibodies.

94. The compound of claim 92, wherein the moieties are aptamers.

95. The compound of claim 88, wherein the means of diagnosis, imaging, or treatment uses small-interfering RNA.

96. The compound of claim 83, wherein the translocase or channel is found on a cell membrane.

97. The compound of claim 83, wherein the translocase or channel is found on an organelle membrane.

98. The compound of claim 97, wherein the translocase or channel is found on the cytoplasmic face of the outer mitochondrial membrane.

99. The compound of claim 29, wherein the carrier protein is found in different isoforms.

100. The compound of claim 99, wherein the isoform found is based on the type of disease presented.

101. The compound of claim 29, wherein the carrier protein and/or translocase, channel, and/or transport mediator therefor is absent in the diseased state.

102. The compound of claim 101, wherein such absence is useful in the diagnosis, imaging, or treatment of the disease.

103. The compound of claim 102, wherein such a disease may be treated by gene therapy.