WO2012167255A1 - Anticancer prodrug therapies - Google Patents

Abstract

The invention provides prodrug compounds with superior efficacy and reduced incidence of nonspecific adverse effects when used for the treatment of cancers expressing carboxylesterase 2. Also provided are pharmaceutical compositions containing these prodrug compounds of the invention, together with at least one pharmaceutically acceptable carrier. Also provided are methods of treating or preventing cancers comprising administering to a mammal in need thereof, therapeutically effective amounts of at least one of the prodrug compounds of the invention.

Description

ANTICANCER PRODRUG THERAPIES

RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S. Provisional Patent Application No. 61/492,895, filed June 3, 2011, which is hereby incorporated by reference in its entirety.

GOVERNMENT INTEREST

This invention was made with the government support under grant number R21 CA141101 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.

TECHNICAL FIELD

The invention relates to prodrug compounds with superior efficacy and reduced incidence of nonspecific adverse effects when used for the treatment of cancers expressing carboxylesterase 2. Also provided herein are pharmaceutical compositions comprising at least one prodrug compound of the invention, together with at least one pharmaceutically acceptable carrier. Also provided herein are methods for the treatment or prophylaxis of cancers comprising administering to a mammal in need thereof, therapeutically effective amounts of pharmaceutical composition(s) comprising at least one prodrug compound of the invention and one or more pharmaceutically acceptable carriers.

BACKGROUND OF INVENTION

Doxorubicin is a broad-spectrum anthracycline, anti-tumor drug used for the treatment of leukemias, lymphomas and solid tumors and is a main-line drug for the treatment of breast cancer. Unfortunately, Doxorubicin exhibits frequent and dose- limiting or even drug-limiting cardiotoxicity. Additionally, most multidrug-resistant tumors and cancer cells display resistance to Doxorubicin. While these undesirable characteristics have limited the clinical usefulness of Doxorubicin, the drug remains one of the oldest and most used anthracycline anti-tumor compounds due to its substantial efficacy against sensitive cancer cells. For this reason, there has been an intensive search for similar anthracycline compounds or derivatives of Doxorubicin having the same or similar anti-tumor activity with greater specificity for tumor tissue.

Prodrug targeting strategies have been used to direct Doxorubicin to tumor cells to increase the specificity of this drug, thereby reducing the non-specific toxicity and related side effects. Antibody Directed Enzyme Prodrug Therapy (ADEPT) and Gene Directed Enzyme Prodrug Therapy (GDEPT) were promising methods for tumor localization of a prodrug-activating enzyme that have been studied for anthracycline-based drugs, and particularly Doxorubicin, as Doxorubicin is a widely-used anti-tumor agent that is relatively easy to derivatize. The most effective Doxorubicin prodrugs to utilize this enzyme-activated approach incorporated a peptide or sugar recognized and cleaved by endogenous or non-native-enzymes near the tumor, or its supporting vasculature, with the goal of redirecting the activity and reducing the dose-limiting cardiotoxic side effects of Doxorubicin.

Similarly, Scheeren and coworkers (Mol. Cancer Therap., 1 : 901-911, 2002; J. Org. Chem., 2001 : 8815-8830, 2001) developed a plasmin-activated tripartate

Doxorubicin prodrug, ST-9802, with reduced cardio and systemic toxicity. Plasmin is a protease over-expressed by numerous cancer types and, although it is found in the bloodstream, its activity is inhibited by a2-antiplasmin and a2-macroglobulin. ST-9802 showed no release of Doxorubicin after incubation in bovine serum for 3 days, indicating good plasmin specificity. While the toxicity of ST-9802 was reduced, efficacy was lost as well and it failed to match the tumor growth inhibition of Doxorubicin in mice bearing human MCF-7 breast tumors even at an equitoxic dose. A variant of ST-9802 with an elongated Katzenellenbogen-type spacer ST-9905 fared better in mouse efficacy experiments, but could only match the activity of Doxorubicin (FASEB J., 18: 565-567, 2004).

Although groundbreaking in their day, these prodrug designs ultimately failed as their anti-tumor activity was substantially decreased along with their cardiotoxicity and other adverse effects. Indeed, none of the Doxorubicin-containing prodrugs described above outperformed Doxorubicin in mouse xenograft assays.

A related enzyme-based prodrug strategy that has demonstrated clinical usefulness includes the prodrugs irinotecan and capecitabine that are activated by carboxylesterase enzymes, most prominently carboxylesterase 1 and 2 (CES1 and CES2) (Xu, G., et al. Clin. Cancer Res., 8:2605-2611, 2002; Bencharit, S., et al, Nature Struct. Biol. 9:337-42, 2002; Shimma, N., et al, Bioorg. Med. Chem., 8: 1697-1706, 2000; Walko, C. M., et al, Clin. Therap., 27:23-44, 2005; Guang, X., et al, Clin. Cancer Res., 8:2605-11, 2002). Both of these prodrugs require hydrolysis of a carbamate functional group, minimizing spurious activation by ubiquitous esterases. CES2 is an endoplasmic reticulum-localized esterase normally involved in xenobiotic detoxification and is commonly expressed in tumor tissue including liver, colon, kidney, thyroid and pancreatic tumors. A recent phase II study of first-line therapy against metastatic pancreatic cancer with irinotecan showed significant efficacy (Ueno, H., et al., Cancer Chemother. Pharmacol., 59:447-54, 2007). Unfortunately, CES2 is also expressed by several normal tissues including stomach, colon, liver, and kidney, resulting in the serious non-tumor tissue toxicities seen with this prodrug approach.

Another prodrug delivery strategy that has shown at least preliminary success, is the design of prodrugs that are passively selective for tumors and their microenvironment. A prodrug designed to use this strategy is currently in phase II clinical trials and involves covalent bonding of a doxorubicin acylhydrazone tethered to a maleimide, DOXO-EMCH, which is in turn tethered to the thiol of cysteine-34 of albumin, which resides in a hydrophobic crevice of the albumin protein (Lebrecht, D., et al., Int. J. Cancer, 120:927- 34, 2006; Kratz, F., et al, Drug Delivery 5:281-99, 1998; Kratz, F., et al, J. Med. Chem. 45(25):5523-33, 2002; Kratz, F., et al, Expert Opin. Investig. Drugs, 16: 1037-1058, 2007). Using this approach, targeted delivery to tumors occurs by exploiting the enhanced permeability and retention effect (EPR effect), wherein the size of albumin, 60 kDa, enables it to selectively exit the leaky vasculature of tumors, where it is retained due to poor lymphatic drainage. Release of doxorubicin is then accomplished by hydrolysis of the acyl hydrazone at the low pH associated with the extracellular environment of many tumors. Further, albumin is endocytosed by many tumor cells, and release of doxorubicin occurs in the low pH environment of the late endosomes or lysosomes. Because the half- life of albumin is about 19 days, and because albumin is not taken up by normal cells, a significant percentage of the doxorubicin slowly accumulates at the tumor.

Unlike free doxorubicin, DOXO-EMCH caused complete remission in a murine renal cancer and in two breast cancer xenograft models (Lebrecht, D., et al., Int. J. Cancer, 120:927-34, 2006; Kratz, F., et al, J. Med. Chem. 45(25):5523-33, 2002). Further, DOXO-EMCH showed significantly less toxicity than doxorubicin in mouse, rat and dog models, including cardiotoxicity even at four-fold higher doses.

Therefore, despite these recent successes, there is a need for better prodrug strategies for maintaining or increasing the anti-cancer efficacy of Doxorubicin while significantly decreasing the cardiotoxicity and other non-tumor specific toxicity related adverse effects of Doxorubicin. Preferably new prodrugs would take advantage of these known targeting systems (enzyme activated prodrugs and the enhanced permeability and retention strategy).

SUMMARY OF INVENTION

The present inventors have designed and tested a prodrug that specifically targets a highly active doxorubicin derivative to extravascular tumor tissue using a two-step approach that greatly increases the tumor specificity of the drug and thereby significantly decreases non-tumor tissue toxicities, including cardiotoxicity associated with both anthracycline and particularly doxorubicin, anti-cancer drugs.

The inventors previously developed a derivative of doxorubicin, doxazolidine, which functions by introducing repair-resistant DNA cross-links at 5 '-GC-3 ' sequences (Fenick, D. J., et al, J. Med. Chem., 40:2452-61 , 1997) (U.S. Patent Application Serial No. 12/091 ,321 , which is incorporated herein by reference, in its entirety). Doxazolidine bears an oxazolidine ring from reaction of formaldehyde with the 3 ',4 '-amino alcohol functionality of doxorubicin, and the cross-linking of DNA occurs upon nucleophilic ring opening of the oxazolidine by the 2-amino group of a G-nucleic acid base.

Doxazolidine has several significant advantages over doxorubicin:

1) Doxazolidine is several orders of magnitude more toxic to a wide variety of cancer cells than doxorubicin (Post, et al, J. Med. Chem. 48:7648- 57, 2005; Spenser, D.M.S., et al, Mutation Res., 638: 1 10-21 , 2008). A comparison of log IC50 values for doxazolidine and doxorubicin appears in Table 1.

Table 1. Log IC50 values for 3 h treatment of cancer cells and rat cardiomyocytes with doxorubicin or doxazolidine. Growth was measured at 80% confluence.

*This cell line has recently been reclassified from breast to ovarian. Doxazolidine is toxic to resistant cancer cells that display the multidrug resistance phenotype (MDR) and to cancer cells that are an order of magnitude resistant to doxorubicin because of low expression of topoisomerase 2 (Kalet, B.T., et al, J. Med. Chem. 50:4493-4500, 2007). Doxazolidine is thought to overcome MDR in part because it is not cationic at physiological pH and because it rapidly forms covalent bonds to DNA (Lampidis, T.J., et al, Biochemistry 36:2679-85, 1997). Referring to Table 1, MCF-7/ADR and SHP-77 cells exhibit an MDR phenotype but are highly sensitive to doxazolidine.

Doxazolidine exhibits greater specificity for tumor cells and therefore less cardiotoxicity. Whereas doxorubicin is more toxic to rat cardiomyocytes than to cancer cells, doxazolidine is more toxic to cancer cells than cardiomyocytes (Kalet, B.T., et al, J. Med. Chem. 50:4493-4500, 2007).

Similar to irinotecan and capecitabine, doxazolidine may be formed as a prodrug (pentyl PABC-Doxaz (hereinafter "PPD")) (see Figure 1 for chemical structures) that is primarily activated by carboxylesterase 2 (CES2) (the mechanism for the activation of PPD is shown in Figure 2) (Barthel, B.L., et al, J. Med. Chem. 51 :298-304, 2008).

Delivery of doxazolidine as a CES2-activated prodrug achieves further specificity for tumor cells beyond the increase in specificity seen with doxazolidine over doxorubicin (Barthel, B.L., et al, J. Med. Chem. 51 :298-304, 2008; Burkhart, D.J., et al, J. Med.

Chem. 49:7002-12, 2006).

Doxazolidine 's selectivity for tumors can be even further increased by using the enhanced permeability and retention effect (the EPR effect) in conjunction with PPD. To do so, PPD is derivatized with 6-(epsilon)-maleimidocaproylhydrazine (EMCH) to form the acylhydrazone that covalently binds to albumin in vivo. With respect to the albumin drug delivery strategy, PPD possesses many positive characteristics, including profound stability in human plasma over 24 h and stability at pH 5 over 24 h, thereby ensuring its survival at conditions required for hydrolysis of the acyl hydrazone attachment. Further, PPD is uncharged at pH 5 and is more membrane permeable than doxorubicin, which is a cation at pH 5, allowing PPD to more easily pass through the bilayer of a late

endosome/lysosome when released from albumin.

With this prodrug of doxazolidine, a double tumor-specificity enhancement is achieved as PPD is first released from albumin only at low pH in the extracellular environment of tumors and at the low pH within tumor cells in late endosomes/lysosomes. Upon release from albumin, doxazolidine is then released from PPD in cancer cells by the endogenous action of CES2. This strategy provides a double filter: the EPR effect and the requirement of CES2, to greatly enhance the tumor specificity of doxazolidine and thereby minimize toxic effects in non-tumor tissues.

This double filter doxazolidine prodrug (hereinafter "PPD-EMCH") is superior to DOXO-EMCH for cancers that express CES2 because it releases doxazolidine, which is more active than doxorubicin and overcomes many resistance mechanisms. Cancer cells that express CES2 respond to PPD and tumor growth is significantly inhibited by PPD in mouse xenograft models of liver cancer and non-small cell lung cancer created with CES2-expressing cells (Barthel, B., et al, J. Med. Chem., 52(23):7678-88, 2009).

While drug delivery systems that rely on multiple targeting systems have suffered from insufficient activity when too little drug reaches the tumor site(s), the prodrugs of the invention overcome this problem because PPD can be given at a higher dose than doxorubicin due to its inherent tissue specificity, these prodrugs circulate in the blood stream for a long period following administration, presumably due to the rapid in vivo association with albumin, and doxazolidine is at least an order of magnitude more active than doxorubicin. The prodrugs of the invention may be used as a cytotoxic component, working cooperatively with a cell signaling effector as part of a multidrug protocol with CES2 expression as a biomarker for efficacy.

Provided herein are anti-cancer prodrug compounds or medicaments comprising at least one prodrug of the invention, for the treatment or prophylaxis of cancer and related neoplastic diseases.

Also provided herein is a pharmaceutical composition comprising at least one anti- cancer prodrug of the invention, with at least one pharmaceutically acceptable carrier.

Also provided herein are pharmaceutical packages containing a pharmaceutical composition comprising therapeutically-effective amounts of at least one anti-cancer prodrug of the invention, optionally together with at least one pharmaceutically-acceptable carrier. The pharmaceutical compositions can be administered separately, simultaneously or sequentially, with other anti-cancer compounds or anti-cancer therapies.

Also provided herein are pharmaceutical kits containing a pharmaceutical composition of at least one prodrug of the invention, optionally together with at least one pharmaceutically-acceptable carrier; prescribing information and a container. The prescribing information may describe the administration, and/or use of these

pharmaceutical compositions alone or in combination with other anti-cancer therapies.

Also provided herein are methods for the treatment or prophylaxis of tumors in a mammal comprising administering to a mammal in need thereof therapeutically effective amounts of any of the above-described pharmaceutical compositions, including, for example, the pharmaceutical compositions comprising at least one prodrug doxorubicin derivative of the invention.

Other aspects of the invention will be set forth in the accompanying description of embodiments, which follows and will be apparent from the description or may be learnt by the practice of the invention. However, it should be understood that the following description of embodiments is given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art and are encompassed within the scope of this invention.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1, shows chemical structures of the drug and prodrug compounds.

Figure 2, shows the structure of carboxylesterase 2-activated doxazolidine, pentyl PABC-Doxaz (PPD), and the mechanism for its activation. Once the carboxylesterase has cleaved, the prodrug at the terminal carbamate, decarboxylation, 1 ,6-elimination and subsequent decarboxylation occur rapidly and spontaneously under physiological conditions to release active doxazolidine. Selective cleavage at the terminal carbamate is shown because of the increased activity of PPD versus the simple carbamate, pentyl Doxaz.

Figure 3 shows two synthetic schemes for the preparation of prodrugs of the invention. Abbreviations: PABA p- aminobenzyl alcohol, THF tetrahydrofuran, DIEA diisopropylethylamine, Doxaz doxazolidine, EMCH 6- maleimidocaproylhydrazine, pentyl PABC-PNP pentyloxycarbonyl-p-aminobenzyl p-nitrophenyl carbonate, PPD pentyl PABC-doxazolidine, PPD-EMCH 6-maleimidocaproylhydrazone of pentyl- PABC- Doxaz.

Figure 4 shows the scheme for the conjugation of PPD-EMCH to the thiol (HS) of Cysteine-34 of albumin and acid catalyzed hydrolysis of the acyl hydrazone to release PPD. Figure 5 shows the 1H-NMR of PPD-EMCH taken at 55 °C in deuterochloroform. Mass spectroscopy produces an exact mass of 1048.3751 (error = 4.4 ppm) for the species [M+Na+].

Figure 6-9 provide microscopic images of the heart tissue of control and treated mice receiving administration of doxorubicin, PPD and PPD-EMCH.

Figure 10A provides data showing tumor growth and survival rates after tumor implantation for control and treated mice receiving administration of PPD and PPD- EMCH.

Figure 10B provides data showing tumor growth and survival rates after tumor implantation for control and treated mice receiving administration of PPD-EMCH formulated in DMSO and PPD-EMCH formulated in Pluronic F127.

Figure 11 provides data showing the rate of decline of PPD in mouse serum for DMSO and Pluronic F127 formulations.

Figure 12 provides data showing the rate of decline of PPD-EMCH in mouse serum for DMSO and Pluronic F127 formulations as well as the corresponding formation of DOX.

Figure 13 provides data showing the rate of binding of PPD-EMCH to mouse serum albumin.

DESCRIPTION OF EMBODIMENTS

The present invention is drawn to prodrugs of the anti-cancer compound doxazolidine that exhibit greatly enhanced tumor tissue specificity and greatly reduced non-tumor tissue toxicity, including greatly reduced cardiotoxicity. These drugs also display greater tumor cell toxicity than doxorubicin.

The anti-cancer prodrug compounds of the present invention include compounds having the chemical structure of Formula I:

Formula I

wherein:

R¹ is substituted or unsubstituted C_2-10 alkyl, alkyne, alkene, aryl, heteroalkyl, heteroaryl;

R² is Ci_io alkyl, aryl, cycloalkyl, arylalkyl, heteroalkyl, heterocycyl.

Preferably, Rl is pentyl.

Preferably R² is ethyl, butyl or pentyl. Most preferably, R² is pentyl (forming the prodrug PPD-EMCH).

Methods of Making Prodrugs of the Invention

Although numerous doxorubicin-prodrug designs incorporate a carbamate functional group at the Dox 3 '- amine, the synthesis of an analogous doxazolidine derivative is not similarly straightforward because the oxazolidine ring of doxazolidine is very hydrolytically sensitive, especially at low pH. The synthesis of the initial PPD and subsequent synthesis of PPD-EMCH is described in detail in Example 1 of this disclosure.

Five doxazolidine-carbamates were synthesized and evaluated for cancer cell growth inhibition. The first three carbamates were the simple ethyl, butyl, and pentyl carbamates. Of these, the butyl carbamate is the most active. This success prompted the synthesis of butyl and pentyl carbamates each with a Katzenellenbogen self-eliminating spacer (p- aminobenzyl alcohol, PABA) to separate the CES2 enzyme active site from the bulk of the prodrug. The structure and cleavage of the most promising carbamate, N- (pentyloxycarbonyl-p-aminobenzyloxycarbonyl)doxazolidine (pentyl PABC-Doxaz, "PPD"), is shown in Figure 2.

PPD is stable with respect to acid-catalyzed hydrolysis even at pH 2 at 37 °C for 24 h. It shows good growth inhibition of Hep G2 and N-Hep G2 human liver cancer cells and poor growth inhibition of rat cardiomyocytes H9c2(2-1), a measure of cardiotoxicity. Poor growth inhibition of green monkey kidney cells, Vera cells, is another indicator of low toxicity toward normal cells. PPD shows less growth inhibition of Hep G2 liver cancer cells than doxazolidine, characteristic of enzyme-activated prodrugs. The data suggest that only 10 to 20% of the dose is converted to Doxazolidine over 24 h. Slow conversion of the clinical drugs, capecitabine and irinotecan, is also observed and is thought to be a desirable property for a carboxylesterase-activated prodrug to minimize side effects. CES2 activates PPD and no activity is observed with recombinant CES1. The cancer cell lines and rat cardiomyocytes were also compared for expression of CES 1 and CES2 by Western blot analysis using antibodies from Dr. Potter. Of particular interest is the high level expression of CES2 by the two liver cancer cell lines, Hep G2 and N-Hep G2, and the lower level expression by the SK-HEP-1 liver cancer cell line. Hence, tumor response of liver cancer cells (Table 2) parallels CES2 expression.

Table 2. Cell growth inhibition values (log IC50 (M)) with drug treatment time 3 or 24 h as indicated. Hep G2 cells are liver cancer cells and N-Hep G2 cells are a variant that are tumorigenic in NOD SCID mice. SK-HEP- 1 cells are liver cancer cells that do not express high levels of CES2. Mia PaCa2 cells are pancreatic cancer cells, and MCF-7/Adr cells are resistant ovarian cancer cells that express MDR1 (formerly classified as breast cancer cells).

The prodrug compounds of this invention may have one or more asymmetric centers or planes and it will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in, and be isolated in, optically active and racemic forms. Addionally, some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any chiral (enantiomeric and diastereomeric) racemic, optically-active, polymorphic, or stereoisomeric forms, or mixtures thereof, of the prodrug compounds of the invention. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in these compounds, and all such stable isomers are also contemplated in the present invention. It is well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine anti-cancer and anti-tumor activity using the in vitro and in vivo tests described herein, or using other similar tests which are well known in the art. In each instance, all chiral, (enantiomeric and diastereomeric) and racemic forms and all geometric isomeric forms of a structure are intended to be disclosed, unless the specific

stereochemistry or isomeric form is indicated in this disclosure. Additionally, the invention includes solvates, and pharmaceutically-acceptable salts of the compounds of Formula I.

The term "solvate" refers to an aggregate of a molecule with one or more solvent molecules.

A "pharmaceutically acceptable salt" as used herein, includes salts that retain the biological effectiveness of the free acids and bases of the specified compound and that are not biologically or otherwise undesirable. Compounds of the invention may possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a mineral or organic acid or an inorganic base, such salts including sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates,

dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-l,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates,

methylbenzoates, dinitromenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, pheylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycollates, tartrates, methanesulfonates, propanesulfonates, naphthalene- 1 -sulfonates, naphthalene-2-sulfonates, and mandelates. Because a single compound of the present invention may include more than one acidic or basic moieties, the compounds of the present invention may include mono, di or tri-salts in a single compound.

In the embodiments of the present invention in which the anti-cancer compound is formed as a base, the desired pharmaceutically-acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an acidic compound, particularly an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

In the embodiments of the present invention in which the anti-cancer compound is formed as an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base. Preferred inorganic salts are those formed with alkali and alkaline earth metals such as lithium, sodium, potassium, barium and calcium. Preferred organic base salts include, for example, ammonium, dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2-hydroxyethyl) ammonium, phenylethylbenzylamine, dibenzylethylenediamine, and the like. Other salts of acidic moieties may include, for example, those salts formed with procaine, quinine and N-methylglusoamine, plus salts formed with basic amino acids such as glycine, ornithine, histidine, phenylglycine, lysine and arginine.

The therapeutically-active compounds of the present invention are effective over a wide dosage range and are generally administered in a therapeutically-effective amount. The dosage and manner of administration will be defined by the application of the anticancer agent and can be determined by routine methods of clinical testing to find the optimum dose. The maximum tolerated dose (MTD) for weekly i.v. injection of PPD- EMCH for three weeks is established with a dose escalation experiment starting at 8 mg/kg, escalating to 16 and 24 mg/kg. The MTD for three weekly injections of DOXO- EMCH is 24 mg/kg. It will be understood, however, that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

When employed as pharmaceuticals, the prodrug compounds of Formula I are administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. Preferably, the compounds of the present invention are administered via intravenous administration. Such pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active anti-cancer compound of Formula I.

The pharmaceutical compositions of the present invention contain, as the active ingredient, one or more of the compounds of Formula I above, associated with pharmaceutically-acceptable formulations and carriers. In making the compositions of this invention, the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container. An excipient is usually an inert substance that forms a vehicle for a drug. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 30% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

Some examples of suitable excipients for use in the pharmaceutical compositions of the invention include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, gum Arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

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 non-aqueous 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 antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

PPD is only modestly soluble in water and is currently formulated for in vivo experiments by dissolution in 5% DMSO/95% D5W (an isotonic solution of dextrose). PPD-EMCH should be more water soluble than PPD because of the additional hydrogen bond donor and acceptor groups associated with the maleimido-hydrazone functionality. If saline or D5W prove insufficient for solublization, various excipients including cyclodextrins, Cremophor ELP, and pharmaceutical grade Tween 80 together with D5W may be used to enhance solubility and prepare a useful pharmaceutical composition for parenteral administration.

A back-up methodology for achieving the EPR effect includes a liposomal formulation for PPD. A relevant methodology is provided by the successful liposomal formulation of Gemcitabine that is also an uncharged drug at physiological pH, which proved successful in an orthotopic model of pancreatic cancer.

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 wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in 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 monosterate and gelatin.

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 is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the drug 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. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter. A preferred formulation of the invention is a mono-phasic pharmaceutical composition suitable for intravenous administration for the treatment or prophylaxis of cancer consisting essentially of a therapeutically-effective amount of a prodrug compound of Formula I, and a pharmaceutically acceptable carrier.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules or as a solution or a suspension in an aqueous or non-aqueous liquid, or an oil-in- water or water-in-oil liquid emulsions, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and the like, each containing a predetermined amount of a compound or compounds of the present invention as an active ingredient. A compound or compounds of the present invention may also be administered as 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: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example,

carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monosterate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) 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 be employed as fillers in soft and hard- filled 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 by molding in a suitable machine a mixture of the powdered compound 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 sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in

microencapsulated form.

The tablets or pills of the present invention may be coated or otherwise

compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate. 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 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 compounds of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to an active ingredient, 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 an active ingredient, 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 compounds of the invention to the body. Such dosage forms can be made by dissolving, dispersing or otherwise incorporating one or more compounds of the invention in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

Pharmaceutical formulations include those suitable for administration by inhalation or insufflation or for nasal or intraocular administration. For administration to the upper (nasal) or lower respiratory tract by inhalation, the compounds of the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane,

dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of one or more compounds of the invention and a suitable powder base, such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered-dose inhaler.

For intranasal administration, compounds of the invention may be administered by means of nose drops or a liquid spray, such as by means of a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and Medihaler (Riker). Drops, such as eye drops or nose drops, may be formulated with an aqueous or nonaqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered by means of a simple eye dropper-capped bottle or by means of a plastic bottle adapted to deliver liquid contents dropwise by means of a specially shaped closure.

The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

The dosage formulations provided by this invention may contain the compounds of Formula I, either alone or in combination with other therapeutically active ingredients, and pharmaceutically acceptable inert excipients. The term 'pharmaceutically acceptable inert excipients' includes at least one of diluents, binders, lubricants/glidants, coloring agents and release modifying polymers.

Suitable antioxidants may be selected from amongst one or more pharmaceutically acceptable antioxidants known in the art. Examples of pharmaceutically acceptable antioxidants include butylated hydroxyanisole (BHA), sodium ascorbate, butylated hydroxytoluene (BHT), sodium sulfite, citric acid, malic acid and ascorbic acid. The antioxidants may be present in the dosage formulations of the present invention at a concentration between about 0.001% to about 5%, by weight, of the dosage formulation.

Suitable chelating agents may be selected from amongst one or more chelating agents known in the art. Examples of suitable chelating agents include disodium edetate (EDTA), edetic acid, citric acid and combinations thereof. The chelating agents may be present in a concentration between about 0.001% and about 5%, by weight, of the dosage formulation.

The dosage form may include one or more diluents such as lactose, sugar, cornstarch, modified cornstarch, mannitol, sorbitol, and/or cellulose derivatives such as wood cellulose and microcrystalline cellulose, typically in an amount within the range of from about 20%> to about 80%>, by weight.

The dosage form may include one or more binders in an amount of up to about 60%) w/w. Examples of suitable binders include methyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl pyrrolidone, eudragits, ethyl cellulose, gelatin, gum arabic, polyvinyl alcohol, pullulan, carbomer, pregelatinized starch, agar, tragacanth, sodium alginate, microcrystalline cellulose and the like.

Examples of suitable disintegrants include sodium starch glycolate, croscarmellose sodium, crospovidone, low substituted hydroxypropyl cellulose, and the like. The concentration may vary from 0.1% to 15%, by weight, of the dosage form.

Examples of lubricants/glidants include colloidal silicon dioxide, stearic acid, magnesium stearate, calcium stearate, talc, hydrogenated castor oil, sucrose esters of fatty acid, microcrystalline wax, yellow beeswax, white beeswax, and the like. The

concentration may vary from 0.1% to 15%, by weight, of the dosage form.

Release modifying polymers may be used to form extended release formulations containing the compounds of Formula I. The release modifying polymers may be either water-soluble polymers, or water insoluble polymers. Examples of water-soluble polymers include polyvinylpyrrolidone, hydroxy propylcellulose, hydroxypropyl methylcellulose, vinyl acetate copolymers, polyethylene oxide, polysaccharides (such as alginate, xanthan gum, etc.), methylcellulose and mixtures thereof. Examples of water-insoluble polymers include acrylates such as methacrylates, acrylic acid copolymers; cellulose derivatives such as ethylcellulose or cellulose acetate; polyethylene, and high molecular weight polyvinyl alcohols.

Another embodiment of the invention relates to the use of any of the prodrug compounds or compositions described herein in the preparation of a medicament for the treatment of cancer.

Methods of Using Prodrug Compounds of the Invention

The invention includes methods of treating a cancer in a mammal by administering to a mammal in need thereof, therapeutically effective amounts of at least one compound of Formula I. The compounds of the invention are particularly effective in the treatment of a cancer expressing CES2, including non-small cell lung cancers (NSCLC), liver, colon, kidney, thyroid, pancreatic, head and neck, prostate, ovarian, breast cancers and sarcoma.

Therefore one embodiment of the invention is a method of treating a mammal in need of anti-cancer therapy by first selecting a cancer patient who is predicted to benefit or not benefit from therapeutic administration of a prodrug of Formula I by first detecting in a sample of tumor cells from the mammal a level of CES2 expression or enzymatic activity, comparing the level of the CES2 in the tumor cell sample to a control level of the CES2 selected from the group consisting of: i)a control level of CES2 that has been correlated with sensitivity to the prodrug; and ii) a control level of the biomarker that has been correlated with resistance to the prodrug; and selecting the patient as being predicted to benefit from therapeutic administration of the prodrug, if the level CES2 in the mammal's tumor cells is statistically similar to or greater than the control level of CES2 that has been correlated with sensitivity to the prodrug, or if the level of CES2 in the mammal's tumor cells is statistically greater than the level of CES2 that has been correlated with resistance to the prodrug; or selecting the mammal as being predicted to not benefit from therapeutic administration of the prodrug, if the level of CES2 in the mammal's tumor cells is statistically less than the control level of CES2 that has been correlated with sensitivity to the prodrug, or if the level of CES2 in the mammal's tumor cells is statistically similar to or less than the level of CES2 that has been correlated with resistance to the prodrug.

In these methods, the prodrug compounds of the invention may be administered in conjunction with a cell signaling effector as part of a multidrug protocol.

No significant cardiotoxicity from PPD-EMCH is anticipated for the following reasons:

1) Mouse xenograft experiments show little evidence of cardiotoxicity from i.v. injection of PPD.

2) The IC50 for doxorubicin inhibition of the growth of rat cardiomyocytes occurs at 30-fold lower concentration than for PPD. This is consistent with no detectable CES2 in cardiomyocytes by Western blot analysis.

3) Doxazolidine inhibits half the growth of cardiomyocytes at the same

concentration as doxorubicin but inhibits the growth of cancer cells at over two orders of magnitude lower concentration. This likely stems from doxazolidine induction of cancer cell death by a mechanism different than that used by doxorubicin.

4) DOXO-EMCH showed no evidence of cardiotoxicity in a rat model and in a phase I clinical study.

Doxorubicin cardiotoxicity is most often associated with doxorubicin induction of oxidative stress coupled with disruption of iron homeostasis. A clinical cardioprotective drug, ICRF-187 (dexrazoxane), functions as a cell membrane permeable derivative of the metal chelator EDTA that complexes iron released through doxorubicin disruption of iron homeostasis. The effect of ICRF-187 on doxorubicin and doxazolidine-treated cancer cells and cardiomyocytes has been measured. The results (Table 3) show that ICRF-187 protects cardiomyocytes but unfortunately, also cancer cells from doxorubicin. However, ICRF-187 shows almost no effect on inhibition of cancer cell growth by doxazolidine while providing some protection of cardiomyocytes. Protection of cardiomyocytes from treatment with doxazolidine likely stems from protection from the doxorubicin released upon hydrolysis of the doxazolidine.

Therefore, one embodiment of the invention is a method of treating cancer in a mammal by the administration of an anti-cancer compound of Formula I in coadministration with ICRF-187 for the amelioration of cardiotoxicity without affecting the response of the tumor cells.

Table 3. Log IC₅₀ data for growth inhibition of cancer cells and cardiomyocytes by doxorubicin and doxazolidine in the presence and absence of ICRF-187. Cells were pretreated with 90 μΜ ICRF-187, then co-treated with 90 μΜ ICRF-187 and doxorubicin, doxazolidine (doxaz) or vehicle for 3 h and growth was measured when controls reached 80% confluence.

The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. EXAMPLES

Example 1. Studies with DOXO-EMCH

Studies of DOXO-EMCH were carried out to mirror the results obtained by Kratz and co- workers (Kratz, F., et al, J. Med. Chem., 5523-33, 2002). These studies provided an analytical foundation to approach the synthesis and experiments associated with PPD- EMCH. Although DOXO-EMCH is a useful model, it does not assure similar success with the synthesis and function of PPD-EMCH because DOXO-EMCH is a water-soluble cation at physiological pH, whereas PPD-EMCH is an unprotonated, hydrophobic compound. Further, acid catalyzed hydrolysis of DOXO-EMCH releases an active anti- tumor drug, doxorubicin; whereas, acid catalyzed hydrolysis of PPD-EMCH releases a prodrug PPD of an active anti-tumor drug doxazolidine. So, PPD-EMCH provides a double filter for selection of tumors over normal tissues to minimize side effects.

1.1 Synthesis of DOXO-EMCH

The synthesis of DOXO-EMCH was done initially in accordance with that previously published by Willner and co-workers (Bioconjugate Chem., 4:521-527, 1993). Problems arose in the initial addition of the 6-maleimidocaproylhydrazine to the C-13 ketone of doxorubicin. HPLC results did not give a good yield of product, only 50-60%. Upon further analysis, we determined TFA was not needed to catalyze the reaction, and instead used pyridine. With pyridine, chromatograms from the HPLC showed 90% DOXO-EMCH relative to 10% DOX. The pyridine may have improved the yield by serving as a base to facilitate formation of the hydrazone. Another problem we encountered in the synthesis was purification of the final product. According to Willner' s method, 5 volumes of acetonitrile (ACN) were to be added to a concentrated methanolic solution of crude DOXO-EMCH to achieve crystallization after 48 h at 4 °C. By this method, only 10-20%) of the desired product precipitated. To obtain a better yield, the crystallization step was done 4 times with 6 volumes of ACN used in each step. A lesser amount of methanol was needed each time, as less product remained in solution. Even with the multiple crystallizations, a final yield of only 59% was obtained. Various other methods for crystallization were explored, including using different solvents and increasing the initial solubility in methanol by heat, but none gave better results. 1.2 Rate of Hydrolysis of DOXO-EMCH at Varying pH

Subsequent pH studies to determine the rate of hydrolysis of the hydrazone were carried out as a benchmark for later hydrolysis experiments with PPD-EMCH. The results of the hydrolysis experiments showed that at lower pH, the hydrolysis reaction proceeded very quickly in the formation of DOX. Moreover, at higher pH the hydrazone proved to be very robust in that its degradation is very slow.

1.3 Conjugation of DOXO-EMCH to HSA

A pilot study was done to determine the binding of DOXO-EMCH to HSA. Two reactions were run at varying concentrations of DOXO-EMCH to prove conjugation by HPLC. Conjugation was confirmed by chromatography, and the peaks for bound drug corresponded to those observed with PPD-EMCH and HSA conjugation, thus supporting the results we obtained in the conjugation of PPD-EMCH to human serum albumin (HAS), which is discussed further below. No further analysis was done on DOXO-EMCH. 2. Studies with PPD-EMCH

The primary goals of experiments performed with PPD-EMCH were to establish a synthesis, create a formulation for later studies done in vivo, show binding to HSA, and determine the rate of hydrolysis of the PPD-EMCH-HSA conjugate. A high probability for success was not obvious because DOXO-EMCH is a cation at physiological pH and PPD- EMCH is unprotonated. Further, the structure of PPD-EMCH is significantly larger and more complex than the structure of DOXO-EMCH. The results of the following experiments show that PPD-EMCH displays similar qualities to DOXO-EMCH, which provide a foundation for advancement into in vivo mouse studies.

A primary strategy and backup strategy for the synthesis of PPD-EMCH from readily available starting materials are shown in Figure 3. The primary strategy has been accomplished by reaction of PPD with the trifluoroacetic acid salt of 6- maleimidocaproylhydrazine.

A backup strategy, also shown in Figure 3, proposes synthesis of doxazolidine- EMCH from DOXO-EMCH followed by reaction with pentyloxycarbonyl-p-aminobenzyl p-nitrophenyl carbonate (pentyl PABC-PNP). The goal is a synthetic methodology for production of gram quantities of PPD, PPD-EMCH, and DOXO-EMCH. DOXO-EMCH was also prepared for control experiments to be described below. Structures and purity of new compounds were established from HPLC multidimensional NMR experiments and mass spectral measurements. 2.1 Synthesis of PPD-EMCH.

The initial synthesis of PPD from DOX'HCl was carried out as a precursor to the synthesis of PPD-EMCH. The synthesis of PPD was reported in the literature by Burkhart and co-workers (J. Med. Chem., 49:7002-12, 2006) but several changes were made to the procedure to improve the yield of pure material. The initial synthesis of Doxaz from DOX'HCl was done as described in the literature with comparable results, as verified by HPLC and NMR (Barthel, B.L., J. Med. Chem., 52(23):7678-88, 2009). However, the final synthesis of PPD required changes to the reaction and the workup. In the coupling of Doxaz to the PABA carbamate (step 3), the literature reports incubation of its p- nitrophenyl carbonate (1 equiv relative to doxazolidine) with doxazolidine in anhydrous DMSO with no reagents present to help facilitate the reaction. We found that the use of 1 equiv of hydroxybenzotriazole (HOBT) to activate the PNP carbonate for nucleophilic attack by the 3 ' nitrogen of doxazolidine increased the yield as indicated by HPLC analysis.

In the workup, two radial chromatography methods were used instead of one. The first, as documented in the literature, eluted with 30: 1 chloroform:methanol which removed non-red impurities. The second elution was done with 40:60:7.5 toluene:ethyl acetate: acetic acid which removed red impurities from the PPD product. These impurities were most likely unreacted doxazolidine and doxoform. The addition of the second radial chromatography method gave very good separation of the desired product from the impurities. Subsequent analysis by HPLC showed increased purity of the collected peaks compared to what was documented in the literature. The NMR data for PPD were the same as previously reported (Burkhart, D.J., et al, J. Med. Chem. 49:7002-12, 2006).

The synthesis of PPD-EMCH initially followed that reported by Willner et al. for the synthesis of DOXO-EMCH, which provided a foundation for an idealized method for PPD-EMCH. The initial conditions consisted of a methanolic solution of doxorubicin and EMCH with TFA to help activate the C- 13 carbonyl for nucleophilic attack by the hydrazide of EMCH. The same method was used on PPD with minimal success. Final yields were dismal, on the order of 50% by HPLC, so several steps were taken to produce better results. The most significant change to Willner's method was the choice of solvent. By switching from methanol to ethanol, measurements by HPLC showed the reaction going to over 90% completion within several hours. Though the kinetics yielding the improved results are not clear, the success is notable nonetheless. Perhaps solubility or transition state kinetics is most likely the reason for the observed increase.

The final procedure for the synthesis of PPD-EMCH was to add 0.75 equiv of TFA and 3 equiv EMCH-TFA salt to an anhydrous ethanol solution of PPD, and run overnight at room temperature while stirring. At 90% product by HPLC, the reaction was stopped and the product isolated by precipitation of PPD/PPD- EMCH through addition of at least 3 volumes of PBS (pH 7.4). The pH of all solutions was kept above 7 because the hydrazone is acid sensitive. Further purification was accomplished by radial

chromatography. First, PPD was eluted with 90: 1, chloroform:methanol. Second, PPD- EMCH was eluted with 20: 1, chloroform:methanol. The structure of the final PPD-EMCH product was established by NMR and mass spectrometry. 400 MHz NMR spectrum of PPD-EMCH in deuteriochloroform at 56°C; scheme: δ 0.90 ppm (8-CH3), 1.33 (γ'), 1.34 (Y-CH2 + 5-CH2), 1.36 (5 '-Me), 1.63 (β', δ'), 1.66 (P-CH2), 1.71 (α'), 1.72 (2'), 2.14 (8), 2.27 (20, 2.46 (8), 2.96 (14-ΟΗ), 3.00 (10), 3.24 (10), 3.5 (ε'), 4.06 (40, 4.07 (4-OMe), 4.09 (50, 4.13 (-CH2), 4.16 (30, 4.67 (9-ΟΗ), 4.74 (14), 4.94 (0-CH2-N), 5.03 (0-CH2- Ν), 5.06 (Βη), 5.16 (Βη), 5.26 (7), 5.40 ( ), 6.65 (Μ), 6.89 (ΝΗ), 7.30 (PABC-3"), 7.39 (3), 7.41 (PABC-2'O, 7.76 (2), 8.02 (1), 9.9 (ΝΗ), 13.15 (11-ΟΗ), 13.78 (6-ΟΗ).

3.1 Preclinical in vitro studies of PPD-EMCH.

The extensive studies of Kratz and co-workers with DOXO-EMCH serve as a valuable framework for the evaluation of PPD-EMCH. Conjugation of PPD-EMCH to HSA was established by HPLC using absorption at 480 nm by the dox chromophore for detection. The molecular mass was established by mass spectrometry of material separated from HSA by HPLC. The rate and extent of conjugation of PPD-EMCH to HSA in human plasma will also be established relative to the rate and extent of conjugation of DOXO- EMCH, again using HPLC and mass spectrometric techniques. Conjugation of PPD- EMCH to HSA and hydro lytic release of PPD from the conjugate are shown with structures in Figure 4.

3.2 Formulation of PPD and PPD-EMCH

Formulation studies were done with PPD and PPD-EMCH to establish a viable means of injection of the drugs intravenously in future mouse studies. One way to approach formulation is with the octanol/water partition coefficient of the molecule, also known as the logP value. The partition coefficient is a measurement of the differential in solubility between an aqueous phase and an oil phase. In particular, dH₂0 and octanol were used. The logP corresponds to the log of the fraction of drug in the octanol layer over the aqueous layer. The determined values for PPD-EMCH and PPD were 2.3 and 2.1, respectively. Based on the log scale, this means there is over 2000 times more of each drug in the oil than in the aqueous layer. PPD-EMCH most likely shows less water solubility than PPD due to the addition of the 6-carbon hydrophobic linker. The low solubility in the aqueous layer was helpful to know for making the ideal formulation.

The desired formulations for mouse experiments required a concentration of 8 mg of PPD-EMCH (DOX equivalent weight) per kg mouse body weight and 10 mg PPD (DOX equivalent weight)/kg body weight in a 100 injection. Low aqueous solubility is not a problem with DOX because the aminol nitrogen is positively charged at

physiological pH (7.4), thus giving the molecule high water solubility. Derivatives of Doxaz are lacking this feature because the amine is not charged, and with PPD derivatives, there is less chance for a charge because the nitrogen is in a carbamate functional group. However, under basic condition, >pH9, one of the two phenolic protons can be removed to give a new negative charge to the molecule. The charge increases the solubility of the drug in water, and thus is an important feature to exploit in the formulation. A pH 9 solution was used for the final formulation because it represents the maximal pH without causing significant amounts of inflammation, and is a standard cutoff point for basic formulations. The first pKa of the phenolic protons of these molecules is estimated to be 9 because of resonance stabilization of the deprotonated oxygen by the quinone functionality. Although not all of the molecules will be deprotonated at pH 9, approximately half of the molecules will have a -1 charge and be more soluble in the aqueous formulation.

In addition to formulation in base, a cosolvent system was used to help maximize the solubility of the drug. A flow chart created by Lee, Y., et al. (Lee, Y.; Int. J. Pharm. 253: 111-19, 2003) outlines a method for finding suitable formulations. In particular, for low aqueous solubility, and a high logP value, the recommendation is to use a cosolvent formulation. A co-solvent system of 60% polyethylene glycol 400 (PEG400), and 3% dimethylacetamide (DMA) was used. The remaining 37% was dH₂0 at pH 9. With this formulation, PPD-EMCH at a concentration of 8 mg/kg DOX equiv was mostly in solution with a minor amount in suspension, and PPD at a concentration of 10 mg/kg DOX equiv went fully into solution. These results are consistent with the higher logP value for PPD-EMCH, which would cause a lower concentration to be in solution with 37%) of the volume aqueous. 3.3 Conjugation of PPD-EMCH with HSA

Experiments to conjugate PPD to HSA showed remarkable success with nearly 95% of drug bound to HSA. HSA (600 micromolar) was reacted with 100 micromolar PPD-EMCH at ambient temperature for 15 min. Similar results were obtained with 1 : 1 and 2: 1 HSA:PPD-EMCH and with bovine serum albumin (BSA) replacing HSA. HPLC chromatograms of the reaction of PPD-EMCH with human serum albumin (HSA) showed the formation of the PPD-EMCH-HSA conjugate. HSA bearing the DOX chromophore absorbs at 480 nm. Without the DOX chromophore HSA absorption only occurs at 280 nm.

3.4 MS/MALDI Characterization

Further characterization of the PPD-EMCH-HSA conjugate was done by

MS/MALDI analysis. A reaction of PPD-EMCH-HSA was run, and the bound peak was collected by HPLC. The collected material was put into dialysis tubing and allowed to equilibrate for 8 hr in deionized water to remove any salts or other impurities. The resulting solution was lyophilized and analyzed by MS/MALDI. The spectrum of pure

HSA appears symmetrical with a maximum peak at 66.5 kDa. The spectrum of lyophilized PPD-EMCH-HSA conjugate shows a maximum peak at 67 kDa. Since a sample of pure PPD-EMCH-HSA would yield a peak with a maximum at 67.5 kDa, the results indicate the presence of both bound and unbound HSA because the molecular mass of PPD-EMCH is 1.02 kDa. Though these results are not conclusive, coupled with the HPLC data, they provide strong evidence that one molecule of PPD-EMCH is attached to HSA, presumably at Cys34. Another interpretation of the observed results is the presence of the EMCH-HSA conjugate that results from hydrolysis of the hydrazone. If the PPD-EMCH-HSA conjugate were unstable under the unbuffered conditions required for MALDI analysis, the EMCH-HSA conjugate would be present in the solution. The corresponding mass for the EMCH-HSA conjugate would be 66.72 kDa. The most likely scenario for the observed results is a mixture of PPD-EMCH-HSA, EMCH-HSA and HSA.

3.5 Rate of Hydrolysis of the PPD-EMCH-HSA Conjugate

A study on the rate of hydrolysis of the PPD-EMCH-HSA conjugate was carried out to determine the stability at a range of pH values that the conjugate may encounter in vivo. Hydrolysis occurs at the C-13 hydrazone to release PPD and EMCH-HSA (Figure 4). The rate of hydrolysis of the hydrazone was monitored at pH 4, 5, 6, and 7. The lower pH values correspond to conditions that may be seen by the conjugate in the area surrounding tumor cells (pH 5), and within lysosomes of tumors (pH 4-5). At lower pH values, our results confirmed appreciable hydrolysis over time to release PPD. A high rate of hydrolysis is important because it confirms that the hydrazone of PPD-EMCH is hydroxytically sensitive at low pH. Conversely, at a higher pH the hydrazone was found to be very robust with a minimal amount of hydrolysis over time. The lack of hydrolysis at high pH is important because at physiological pH (7.4) the drug conjugate needs to be stable while the albumin circulates throughout the body until it reaches the site of the tumor.

The results demonstrate that PPD-EMCH will be a useful prodrug for the treatment of cancers that express CES2 using the double filter methodology to protect normal cells and tissues. The first filter is by the EPR effect, which selectively delivers the PPD- EMCH-HSA conjugate to the leaky vasculature of the tumor. The low pH of the tumor and/or the low pH of the endosome/lysosome that delivers PPD-EMCH-HSA to the cancer cell should release PPD-EMCH from the albumin. Intracellular CES2 then activates the prodrug to doxazolidine, which cross-links DNA of the tumor cells to induce apoptosis. Because PPD is hydrophobic, PPD released from PPD-EMCH-HSA in a cancer cell that doesn't express significant amounts of CES2 might logically migrate to another cancer cell that does express CES2. Similarly, doxazolidine, which is also hydrophobic, released in one cell might migrate to another cell. This phenomenon, commonly called the bystander effect, increases prodrug effectiveness independent of activating enzyme expression.

Experimental General Remarks

Abbreviations: DOX doxorubicin, DOX'HCl doxorubicin hydrochloride, HPLC high performance liquid chromatography, NMR nuclear magnetic resonance, DMSO dimethylsulfoxide, dH₂0 deionized water, HSA human serum albumin, BSA, bovine serum albumin, PABA p-aminobenzyl alcohol, THF tetrahydrofuran, DIEA

diisopropylethylamme, Doxaz doxazolidine, EMCH 6-maleimidocaproylhydrazine, pentyl PABC-PNP pentyloxycarbonyl-p-aminobenzyl p-nitrophenyl carbonate, PPD pentyl PABC-doxazolidine, PPD- EMCH 6-maleimidocaproylhydrazone of pentyl-PABC-Doxaz, TFA trifluoroacetic acid, PBS phosphate buffered saline, DMA dimethylacetamide.

General HPLC instruments and methods

Analytical HPLC methods were performed using a Hewlett-Packard/ Aligent 1050/1100 chromatograph with an auto injector, diode array UV-vis absorption detector. Method 1.1 : Analytical HPLC injections were onto an Aligent Zorbax Eclipse XDB-C18 reversed phase column, 4.6 mm x 150 mm, eluting at 1.0 mL/min. A gradient of acetonitrile/20 mM sodium phosphate buffer (pH 6.9), 80% buffer to 55% at 10 min, 55% to 40% at 12 min, 40% to 80% at 13 min. Retention times: at 480 nm, DOX (9.4 min), DOXO-EMCH (1 1.2 min).

Method 1.2: Analytical HPLC injections were onto an Aligent Zorbax 3.5 μιη Eclipse 300 SB-C8 reversed phase column, 4.6 mm x 150 mm, eluting at 1.0 mL/min. A gradient of acetonitrile/20 mM sodium phosphate buffer (pH 7.6), 60% buffer isocratic until 2 min, 60% to 45% at 5 min, 45% to 40 % at 10 min, 40% to 30% at 13 min, 30% to 60% at 15 min. Retention times: at 480 nm DOXO-EMCH (2.7 min), DOXO-EMCH-HSA (7.8 min); at 280 nm HSA (6.8 min), DOXO-EMCH-HSA (7.8). Method 1.3 : Analytical HPLC injections were onto an Aligent Zorbax 5 μιη Eclipse XDB-C 18 reversed phase column, 4.6 mm x 150 mm, eluting at 1.0 mL/min. A gradient of acetonitrile/20 mM sodium phosphate buffer (pH 7), 80% buffer to 60% at 5 min, 60% to 30% at 10 min, 30% to 25% at 13 min, 25% to 20% at 16 min, isocratic until 19 min, 20% to 80% at 20 min. Retention times: at 480 nm PPD- EMCH (15 min), PPD (16 min). Method 1.4: Analytical HPLC injections were onto an Aligent Zorbax 5 μιη Eclipse XDB-C 18 reversed phase column, 4.6 mm x 150 mm, eluting at 1.0 mL/min. A gradient of acetonitrile/20 mM sodium phosphate buffer (pH 7.6), 100% buffer isocratic until 25 min, 100% to 30%> at 40 min, 30% isocratic until 50 min, 30% to 100% until 60 min. Retention times: at 480 nm PPD- EMCH-HSA (36 min), PPD- EMCH (43.5 min); at 280 nm HSA (35 min), PPD-EMCH- HSA (36 min) Method 1.5 : Analytical HPLC injections were onto an Aligent Zorbax 3.5 μιη Eclipse 300 SB-C8 reversed phase column, 4.6 mm x 150 mm, eluting at 1.0 mL/min. A gradient of acetonitrile/20 mM sodium phosphate buffer (pH 7.6), 60% buffer isocratic until 2 min, 60% to 45% at 5 min, 45% to 40 % at 10 min, 40% to 30% at 13 min, 30% to 60% at 15 min. Retention times: at 480 nm PPD-EMCH-HSA (7.6 min), PPD-EMCH (10.2 min);at 280 nm HSA (6.75 min), PPD-EMCH-HSA 7.5 min).

NMR spectra were obtained using a Varian Unity INOVA 400 MHz spectrometer. Concentrations of materials bearing a doxorubicin chromophore were measured using a Hewlett-Packard 8452A Diode Array spectrophotometer. The molar extinction coefficient of DOX-based molecules was assumed to be ε=1 1 ,500 M^_1cm^_1 at 480 nm. When calculating the molar concentrations of HSA and BSA, a molecular weight of 66 kDa was used. Radial chromatography was accomplished using a 7924T Chromatotron (Harrison Research) with a radius of 1 1 cm and a thickness of 2 mm for the silica plate. MS/MALDI analysis was performed on an Applied Biosystems Voyager-DE STR System 4004 mass spectrometer. The intensity of the laser was set within 1800-2100 on the instrument, with 50-100 laser shots/spectrum depending on what yielded the best results. The matrix solvent was alpha-cyano-4-hydroxycinnamic acid. A linear flight method was used.

1.1 Synthesis of DOXO-EMCH

The synthesis of DOXO-EMCH was accomplished using the procedure reported by Willner et al, with several changes to improve the yield (Willner, D., et al.,

Bioconjugate Chem., 4:521-27, 1993). DOX'HCl (20 mg, 34 μιηοΐ) was dissolved in 6 mL of methanol. Pyridine (12.53 μί) was added to the solution, followed by 35.4 mg

EMCH'TFA. The reaction was stirred at room temperature overnight. By HPLC, the reaction was 90% complete. The solvent was evaporated to dryness by rotary evaporation. A minimal amount of methanol was used to dissolve the solid, and six volumes of acetonitrile at 4 °C were added to the solution. The resulting solution was allowed to sit undisturbed at 4 °C for 48 h for crystallization. The precipitate was collected, and the crystallization method was repeated 4 times. The resulting solids were combined and washed three times with 1 : 10 methanokacetonitrile. The final yield of DOXO-EMCH was 11.59 mg, 58%. HPLC Method 1.1 was used. NMR spectra corresponded to those previously given by Willner (Bioconjugate Chem. 4:521-27. 1993).

1.2 Rate of Hydrolysis of DOXO-EMCH at Varying pH

Four 500 solutions of 0.2x PBS were prepared at pH 4, 5, 6, 7. The proper pH was achieved upon addition of phosphoric acid. DOXO-EMCH was added to each solution at a final concentration of 50 μΜ. Aliquots (50 μί) were removed from each solution at t (min) = 2, 18, 35, 50. Other time points are distributed unevenly based on the extent of the reaction. The relative amount of DOXO-EMCH to DOX was determined by the integral values of the respective peaks on the HPLC chromatograms, monitored at 480 nm. HPLC Method 1.1 was used. 1.3 Conjugation of DOXO-EMCH to HSA.Two reactions were run at varying concentrations of DOXO-EMCH. Two solutions of 100 μΜ and 200 μΜ DOXO-EMCH and a solution of 600 μΜ HSA were incubated in PBS at pH 7.4 for 20 min at room temperature (26 °C). The reaction was then analyzed by HPLC. Albumin absorption was monitored at 280 nm and DOXO-EMCH was monitored at 480 nm. HPLC method 1.2 was used.

2. Experimental for Studies with PPD-EMCH

2.1 Synthesis of PPD-EMCH a) PPD. The synthesis of PPD corresponds to that already established by Burkhart et al (J. Med. Chem. 49:7002-12, 2006). Several modifications were made to the synthesis which gave advantageous results over those reported. In step 3, 1 equiv of

hydroxybenzotriazole (HOBT) relative to doxazolidine was used in the synthesis of the doxazolidine carbamate. Two radial chromatography methods were used in the workup. The first eluted with 30: 1 chloroform:methanol which removed non-red impurities. The second required elution with 40:60:7.5 toluene:ethyl acetate: acetic acid to remove red impurities from the PPD product. NMR data were the same as previously reported (Burkhart et al, J. Med. Chem. 49:7002-12, 2006).

b) PPD-EMCH. EMCH-TFA (3 equiv) and 0.75 equiv TFA were added to an anhydrous ethanol (dried by 3 A molecular sieves, superactivated by heating to 150 °C under a vacuum of 0.1 Torr) solution (8.57 mL) of PPD (15 mg, 18.3 μιηοΐ, 1 equiv) and stirred under a nitrogen atmosphere overnight at room temperature. The extent of the reaction was monitored by HPLC (Method 1.3). Once 90% complete by HPLC, the solution was evaporated to dryness and the residue redissolved in anhydrous DMSO. PPD/PPD-EMCH was precipitated by adding at least 3 volumes of PBS (pH 7.4). The precipitate was washed with dH₂0 (pH > 6.5) and redissolved in DMSO. The precipitation was repeated three times, followed by further purification by radial chromatography, eluting PPD and PPD-EMCH with 90: 1 and 20: 1 chloroform:methanol, repectively. NMR spectra were obtained at 56°C in deuteriochloroform (Figure 5).

2.2 Formulation of PPD and PPD-EMCH

a) Partition Coefficient. A concentrated solution of PPD-EMCH or PPD in DMSO was evaporated under high vacuum (10^~2 Torr) until a dry solid remained. Octanol was added in 20 aliquots until most of the solid was in solution. The resulting saturated solution, with a small suspension of drug, was centrifuged at 16,000 xg in a tabletop centrifuge for 5 min. The concentration of drug in the supernatant was determined by absorbance at 480 nm, assuming an extinction coefficient of 11,500 M^_1cm^_1. The spectrophotometer was blanked with a 1000 solution of 25% DMSO, 75%> dH₂0, and 50 μΐ, octanol. An equal volume of dH₂0 was then added to the octanol-drug solution. The solution was shaken continuously for 5 min. A large emulsion layer appears at the interface of the two solvents and the solution was allowed to sit undisturbed for 24 hours, or until the emulsion layer was gone. A seperatory funnel was then used to separate the two layers. Care was taken not to re-emulsify the layers by vigorous movement. Once separated, the concentration of drug was measured in each layer by absorbance at 480 nm. Measurements were done in triplicate and the log of the ratio ([drug]_octanoi:[drug]water) of the average values corresponded to the logP. The final concentrations of drug in octanol and water were calculated to account for the initial amount of drug within the saturated octanol solution. Literature supports the idea that concentration of solute can affect the final partition coefficient, so several concentrations were used to establish a standard value. Our results verified a constant logP value over a range of concentrations.

b) Formulation. A cosolvent system of DMA, PEG-400, and dH₂0 at pH 9 was used. A solution of PPD-EMCH (8 mg/kg DOX equiv) or PPD (10 mg/kg DOX equiv) in DMSO, assuming 0.02 kg/mouse and a 100 injection size, was evaporated to dryness. The red solid was redissolved in DMA (3 μί) and vortexed until the drug was dissolved. PEG400 (60 μί) was then added and the mixture vortexed (10 s). Deionized water at pH 9 (37 μί) was then added and the mixture vortexed a final time. For PPD-EMCH, most of the drug was in solution, but some remained in suspension. For PPD, all of the drug was in solution. DMA and DMSO were found to be interchangeable in the formulation.

2.3 Conjugation of PPD-EMCH to HSA

a) Conjugation. Recombinant HSA (20 μΐ,, 10%) was incubated with 27.5 μΐ, PBS at pH 7.4 for 5 min at room temperature. PPD-EMCH in DMSO (2.5 μΐ,, 0.005 μιηοΐ) was added to a final concentration of 100 μΜ and allowed to react for 15 min. Care was taken to mix quickly, but gently, because some of the protein crashes out upon addition of

DMSO, but will redissolve upon mixing. The reaction was monitored by HPLC. Varying concentrations were used: 1 : 1 and 2: 1, HSA:PPD-EMCH, all with similar success. The same results were obtained when using BSA under the same conditions. HPLC Method

1.4 was used for the initial conjugation, and HPLC Method 1.5 was used for the final successful conjugation under the same conditions as the initial.

b) MS/MALDI. The reaction for MALDI analysis was run by the same conjugation procedure as previously stated in 2.3a, except using 1 : 1 molar concentrations of

HSA:PPD-EMCH. The PPD-EMCH-HSA adduct was collected from the HPLC and further purified by dialysis (12 kDa molecular weight cutoff) for at least 8 h into 4 L dH₂0 to remove any salts and other low mass impurities. The resulting liquid was lyophilized by speed-vac to dryness. The resulting red and white solid was dissolved in dH₂0 (pH > 7) to a final protein concentration of protein of 2 mg/mL. The MALDI was run as described in General Remarks and data showed a peak shift and broadening to 67 kDa from 66.5 kDa for pure recombinant HSA, likely indicating bound drug.

2.4 pH Stability of the PPD-EMCH-HSA Conjugate

Incubation of PPD-EMCH and HSA was done at pH 4, 5, 6, 7. The buffer for pH 4 was made from a 0.3 M sodium acetate buffer at pH 3.7. A high buffer capacity was required because the protein itself appears to have a significant effect on pH. Upon addition of the reaction solution, the pH increased to 4, and was checked periodically throughout the reaction. If the pH increased above 4, a 0.1 M solution of glacial acetic acid was added in 3 μΐ, aliquots until the pH went back to 4. The buffers for pH 5 and 6 were 10 mM phosphate buffer adjusted to the proper pH with 0.1 M phosphoric acid. The buffer at pH 7 was a 2x PBS solution. Recombinant HSA (102 μΐ,, 0.3 μιηοΐ) was added to 102 μΐ_^ lx PBS buffer (pH 7.4) at ambient temperature. The solution was allowed to stand for 5 min. PPD-EMCH in DMSO (38 L, 0.1 μιηοΐ) was then added to the HSA buffer solution, quickly mixed, and allowed to react for 15 min. Then 275 μΐ_^ of buffer at the tested pH was added to the solution. Degradation of the adduct was monitored by HPLC over a time span that correlated to the predicted rate of hydrolysis, starting with early timepoints in all samples. In all mixtures, care was taken not to introduce air bubbles into the solution. For pH 4, 5, and 6, the HPLC Method 1.5 was used. At pH 7, Method 1.5 was used with a Zorbax 300SB-C18, 5 μιη, 4.6 x 250 mm column.

Example 2. Cardiotoxicity of DOX, PPD, and PPD-EMCH in an Orthotopic Liver Cancer Model

Human hepatocellular carcinoma (HCC) N-Hep G2 cells were genetically modified to express the protein luciferase (N-Hep G2/luc), which allows for visualization of cells in a living animal. Immunocompromised (nu/nu) mice were injected

subcutaneously with N-Hep G2/luc and tumors allowed to grow. The tumors were then collected, diced into small cubes, and implanted into the liver of a new nude mouse recipient. The mice were screened for successful tumor implant and growth by positive luciferin signal and positive mice were divided into 4 cohorts: control, 6 mg/kg

doxorubicin, 6 mg/kg PP-Doxaz (DMSO formulated), and 5 mg/kg PPD-EMCH (DMSO formulated). Each cohort received 3 intravenous treatments, spaced 10 days apart. Hearts taken from the treated mice were stained with hematoxylin and eosin to visualize cellular morphology and assess the cardiotoxicity of the drugs. Hearts from untreated mice (Figure 6) exhibited normal, undamaged morphology. With DOX treatment (3 x 6 mg/kg, Figure 7), the muscle fibers show a characteristic degeneration by vacuoles, indicated by colorless, circular holes in the tissue (asterisks indicate a few examples). Vacuolar degeneration was less severe when mice were treated with 3 x 6 mg/kg PPD (Figure 8) and nearly absent with 3 x 5 mg/kg PPD-EMCH (Figure 9). Formulation difficulties prevented higher doses of PPD or PPD-EMCH. Therefore, at approximately equal dosing, PPD-EMCH is less cardiotoxic than the parent drug PPD, and far less cardiotoxic than doxorubicin. Example 3. Pancreatic Cancer in a Subcutaneous Xenograft

Nude mice were implanted subcutaneously with L3.5 human pancreatic cancer cells. Resulting tumors were treated with either PPD at 5 mg/kg in a DMSO formulation or PPD-EMCH at two doses (5 or 6 mg/kg) in a formulation of DMSO and Tween-80 (polysorbate-80) Tumor growth results are shown on the left panel of Figure 10A. While there was little difference in antitumor efficacy, there was a large difference in survival

(shown on the right of Figure 10A) and measured either by animal death or removal of the animal due to tumor burden. Treatment with PPD-EMCH at 6 mg/kg resulted in a significant improvement in survival, with 60% of the animals remaining in the study by day 33, while PPD and PPD-EMCH at 5 mg/kg groups had only 20% of mice remaining in the study. Therefore, PPD-EMCH has the capability for significant benefit, given its improved toxicity profile, if the dose can be increased, for example through a change in prodrug formulation.

Tumors were grown as before, but treatments were performed with PPD-EMCH at 6 mg/kg in the DMSO/Tween-80 formulation as in Figure 10A, and PPD-EMCH at 10 mg/kg formulated with a 6:1 mass ratio of Pluronic F127:prodrug. Tumor growth results are shown on the left panel of Figure 10B. Antitumor efficacy was significantly greater in the 10 mg/kg PPD-EMCH group than in either of the other groups. Additionally, survival (measured either by death or by removal of animals due to tumor burden), shown on the right of Figure 10B, was improved in 10 mg/kg relative to 6 mg/kg.

These experiments indicate that PPD-EMCH is better than PPD, primarily with respect to survival and, perhaps, efficacy at higher doses. Improvements in the formulation (for example, by inclusion of Pluronic F127) make it possible to achieve higher doses of PPD-EMCH as well as PPD. Example 4. Contribution of Pluronic F127 Towards Stability of PPD and PPD-EMCH in Mouse Serum

PPD and other carboxylesterase-activated prodrugs are unstable in mouse serum due to the presence of circulating esterase activity. This phenomenon is unique to rodents and does not exist in humans, but presents problems when examining pharmacokinetics and toxicology, as this circulating esterase activity results in early cleavage of the prodrug and circulation of the active compound. This experiment tested whether the inclusion of Pluronic F 127 in the formulation had an effect on the rate at which the prodrugs are cleaved by mouse serum esterase activity.

As shown in Figure 11, PPD was incubated at 37 °C for varying lengths of time and the amount of prodrug loss was measured by HPLC. The left bars for each time period indicate the old DMSO formulation, while the right bars for each time period show the stability of the new Pluronic F127 formulation, but at a 1 : 1 molar ratio, which gives better solubility than the 6: 1 mass ratio used in the animal experiments. In these tests, the presence of Pluronic F127 stabilized PPD by a factor of approximately 4 by 24 h.

Figure 12 provides results showing PPD-EMCH analyzed in a similar way. We hypothesized that the attachment of the prodrug to albumin would create steric hinderance to the esterase, hopefully resulting in far less enzymatic cleavage than with PPD. Again the leftmost of the tall bars for each time period represent the DMSO formulation, while the second of the tall bars represent the Pluronic formulation. In this analysis, the prodrug was incubated with serum, resulting in binding to serum albumin. After the specified time periods of incubation, the PPD-EMCH- Albumin was hydrolyzed with acid, resulting in the release of PPD and EMCH- Albumin. Enzymatic activity would convert to PPD-EMCH- Albumin to doxorubicin-EMCH-Albumin which, after acid incubation, would hydrolyze to DOX and EMCH-Albumin. This experiment demonstrates that there was little DOX produced (short bars, left DMSO formulation, right Pluronic formulation), even after 24 h. Whereas approximately 65% of PPD with Pluronic F127 was lost after 24 h, only about 25% of PPD-EMCH in Pluronic F127 was hydrolyzed by the serum esterase activity. As with PPD, Pluronic F127 offered some protection from hydrolysis, although not as much as in the case of PPD, in addition to allowing higher doses to be delivered.

As these experiments show, PPD-EMCH is superior to PPD with respect to serum stability, having a longer effective life in circulation. This may at least partly explain its reduced toxicity to heart tissue. A Pluronic F127 formulation, in addition to aiding in solubility and delivery, results in a measureable protection of these drugs from degradation in the serum.

Example 5. Binding Kinetics of PPD-EMCH to Mouse Serum Albumin.

PPD-EMCH was added to mouse serum, incubated for various periods of time, then the protein precipitated with ethanol, without removing the bound protein. The supernatant was then analyzed by HPLC for the presence of PPD-EMCH. Any prodrug that had bound to albumin was precipitated with the protein and was not available for analysis, allowing for measurement of the rate of binding of PPD-EMCH to mouse serum albumin, as shown in Figure 13.

The results demonstrate that PPD-EMCH binds serum albumin very quickly, and by 10 min is nearly completely bound. The half time of binding is approximately 2 min. Thus, PPD-EMCH is rapidly bound to albumin, even in a complex mixture. This fast binding prevents tissue uptake of PPD-EMCH, enzymatic hydrolysis, and problematic side effects.

The foregoing description of the present invention has been presented for purposes of illustration and description. Furthermore, the description is not intended to limit the invention to the form disclosed herein. Consequently, variations and modifications commensurate with the above teachings, and the skill or knowledge of the relevant art, are within the scope of the present invention. The embodiments described hereinabove are further intended to explain the best mode known for practicing the invention and to enable others skilled in the art to utilize the invention in such, or other, embodiments and with various modifications required by the particular applications or uses of the present invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.

Claims

What is claimed is:

1. A compound having the chemical structure:

wherein:

R¹ is substituted or unsubstituted C_2-10 alkyl, alkyne, alkene, aryl, heteroalkyl, heteroaryl; and,

R² is Ci_io alkyl, aryl, cycloalkyl, arylalkyl, heteroalkyl, heterocycyl.

2. The compound of Claim 1 , wherein R² is ethyl, butyl or pentyl.

3. The compound of Claim 1 , wherein R² is pentyl.

4. The compound of Claim 1 , wherein R¹ is pentyl.

5. A medicament comprising at least one compound of Claim 1 for the treatment or prophylaxis of cancer or related neoplastic disease.

6. A pharmaceutical composition comprising at least one compound of Claims 1-4, with at least one pharmaceutically acceptable carrier.

7. A pharmaceutical package comprising a pharmaceutical composition comprising therapeutically-effective amounts of at least one compound of Claims 1-4, optionally together with at least one pharmaceutically acceptable carrier.

8. A pharmaceutical kit containing a pharmaceutical composition of at least one compound of Claims 1-4, optionally together with at least one pharmaceutically acceptable carrier and prescribing information and a container.

9. A method for the treatment or prophylaxis of a cancer in a mammal comprising administering to a mammal in need thereof therapeutically effective amounts of at least one compound of Claims 1-4.

10. The method of Claim 9, wherein the cancer is selected from non-small cell lung cancer (NSCLC), liver, colon, kidney, thyroid, pancreatic, head and neck, prostate, ovarian, breast and sarcoma.

11. The method of Claim 9, wherein the compound is administered in conjunction with a cell signaling effector as part of a multidrug protocol with CES2 expression as a biomarker for efficacy.

12. The method of Claim 9, wherein the compound is administered in conjunction with ICRF-187.