WO2012167255A1 - Thérapies par promédicaments anticancéreux - Google Patents

Thérapies par promédicaments anticancéreux Download PDF

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WO2012167255A1
WO2012167255A1 PCT/US2012/040749 US2012040749W WO2012167255A1 WO 2012167255 A1 WO2012167255 A1 WO 2012167255A1 US 2012040749 W US2012040749 W US 2012040749W WO 2012167255 A1 WO2012167255 A1 WO 2012167255A1
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ppd
emch
compound
cancer
doxorubicin
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PCT/US2012/040749
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English (en)
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Tad H. Koch
Benjamin L. Barthel
Alexander R.H. ROWAN
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The Regents Of The University Of Colorado, A Body Corporate
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids

Definitions

  • 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.
  • pharmaceutical compositions comprising at least one prodrug compound of the invention, together with at least one pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • Doxorubicin exhibits frequent and dose- limiting or even drug-limiting cardiotoxicity.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • irinotecan 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).
  • 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.
  • 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.
  • doxorubicin 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.
  • DOXO-EMCH 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.
  • new prodrugs would take advantage of these known targeting systems (enzyme activated prodrugs and the enhanced permeability and retention strategy).
  • 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.
  • 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.
  • MDR multidrug resistance phenotype
  • 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).
  • 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).
  • PPD penentyl PABC-Doxaz
  • 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.
  • 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.
  • PPD is derivatized with 6-(epsilon)-maleimidocaproylhydrazine (EMCH) to form the acylhydrazone that covalently binds to albumin in vivo.
  • EMCH 6-(epsilon)-maleimidocaproylhydrazine
  • 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.
  • 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
  • doxazolidine 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).
  • 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.
  • 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.
  • composition comprising at least one anti- cancer prodrug of the invention, with at least one pharmaceutically acceptable carrier.
  • compositions 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.
  • 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
  • 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.
  • 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.
  • Figure 3 shows two synthetic schemes for the preparation of prodrugs of the invention.
  • PABA p- aminobenzyl alcohol THF tetrahydrofuran
  • DIEA diisopropylethylamine 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 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.
  • 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.
  • anti-cancer prodrug compounds of the present invention include compounds having the chemical structure of Formula I:
  • R 1 is substituted or unsubstituted C 2-10 alkyl, alkyne, alkene, aryl, heteroalkyl, heteroaryl;
  • R 2 is Ci_io alkyl, aryl, cycloalkyl, arylalkyl, heteroalkyl, heterocycyl.
  • Rl is pentyl
  • R 2 is ethyl, butyl or pentyl. Most preferably, R 2 is pentyl (forming the prodrug PPD-EMCH).
  • doxazolidine-carbamates 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.
  • PABA Katzenellenbogen self-eliminating spacer
  • 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.
  • 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.
  • tumor response of liver cancer cells (Table 2) parallels CES2 expression.
  • 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).
  • 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
  • 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.
  • all chiral, (enantiomeric and diastereomeric) and racemic forms and all geometric isomeric forms of a structure are intended to be disclosed, unless the specific
  • the invention includes solvates, and pharmaceutically-acceptable salts of the compounds of Formula I.
  • 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.
  • 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, ⁇ -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.
  • 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-to
  • an acidic compound particularly an inorganic acid, such as hydrochloric acid, hydro
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • aqueous and nonaqueous carriers examples 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.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • 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.
  • 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.
  • isotonic agents such as sugars, sodium chloride, and the like in the compositions.
  • 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.
  • the absorption of the drug 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.
  • 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
  • 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.
  • 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.
  • a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • 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.
  • 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,
  • disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain si
  • 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 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.
  • 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.
  • opacifying agents include polymeric substances and waxes.
  • the active ingredient can also be in
  • microencapsulated form is a microencapsulated form.
  • the tablets or pills of the present invention may be coated or otherwise
  • 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.
  • 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.
  • 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.
  • 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,
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions include those suitable for administration by inhalation or insufflation or for nasal or intraocular administration.
  • 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,
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • 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.
  • 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.
  • 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.
  • 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.
  • sterile liquid carrier for example water for injection
  • 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.
  • 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.
  • pharmaceutically acceptable antioxidants include butylated hydroxyanisole (BHA), sodium ascorbate, butylated hydroxytoluene (BHT), sodium sulfite, citric acid, malic acid and ascorbic acid.
  • BHA butylated hydroxyanisole
  • BHT butylated hydroxytoluene
  • 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.
  • suitable chelating agents include disodium edetate (EDTA), edetic acid, citric acid and combinations thereof.
  • EDTA disodium edetate
  • 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.
  • 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.
  • 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.
  • 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.
  • lubricants/glidants examples 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the prodrug compounds of the invention may be administered in conjunction with a cell signaling effector as part of a multidrug protocol.
  • 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.
  • doxorubicin 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.
  • DOXO-EMCH showed no evidence of cardiotoxicity in a rat model and in a phase I clinical study.
  • ICRF-187 (dexrazoxane)
  • EDTA metal chelator
  • 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.
  • 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.
  • 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.
  • PPD-EMCH provides a double filter for selection of tumors over normal tissues to minimize side effects.
  • 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.
  • 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 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.
  • 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
  • PPD was eluted with 90: 1, chloroform:methanol.
  • PPD- EMCH was eluted with 20: 1, chloroform:methanol.
  • the structure of the final PPD-EMCH product was established by NMR and mass spectrometry.
  • 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.
  • dH 2 0 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
  • 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.
  • 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.
  • 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 dH 2 0 at pH 9.
  • HSA 600 micromolar
  • PPD-EMCH 100 micromolar
  • BSA bovine serum albumin
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • DOX doxorubicin DOX'HCl doxorubicin hydrochloride
  • HPLC high performance liquid chromatography NMR nuclear magnetic resonance
  • DMSO dimethylsulfoxide dH 2 0 deionized water
  • HSA human serum albumin BSA
  • bovine serum albumin PABA p-aminobenzyl alcohol
  • THF tetrahydrofuran DIEA
  • 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 ⁇ 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 ⁇ 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 ⁇ 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.
  • 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.
  • DOXO-EMCH DOXO-EMCH
  • 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).
  • PPD-EMCH PPD-EMCH.
  • EMCH-TFA 3 equiv
  • 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 ⁇ , 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).
  • 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 dH 2 0 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 dH 2 0 (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.
  • 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.
  • 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 ⁇ , 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 ⁇ ,, 0.3 ⁇ ) was added to 102 ⁇ ⁇ 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 ⁇ ) was then added to the HSA buffer solution, quickly mixed, and allowed to react for 15 min. Then 275 ⁇ ⁇ 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 ⁇ , 4.6 x 250 mm column.
  • HCC Human hepatocellular carcinoma
  • 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
  • mice 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).
  • DMSO formulated 6 mg/kg PP-Doxaz
  • PPD-EMCH 5 mg/kg PPD-EMCH
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the attachment of the prodrug to albumin would create steric hinderance to the esterase, hopefully resulting in far less enzymatic cleavage than with PPD.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne des composés promédicaments présentant une efficacité supérieure et une influence réduite d'effets secondaires non spécifiques lorsqu'ils sont utilisés pour le traitement de cancers exprimant la carboxylestérase 2. L'invention concerne également des compositions pharmaceutiques contenant ces composés promédicaments selon l'invention, combinés avec au moins un support pharmaceutiquement acceptable. L'invention concerne également des procédés de traitement ou de prévention de cancers comprenant l'administration à un mammifère en ayant besoin de quantités thérapeutiquement efficaces d'au moins un des composés promédicaments selon l'invention.
PCT/US2012/040749 2011-06-03 2012-06-04 Thérapies par promédicaments anticancéreux WO2012167255A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190086418A1 (en) * 2016-02-24 2019-03-21 Cellact Pharma Gmbh Method for the diagnosis of etoposide prodrug treatable cancer
WO2019226774A1 (fr) * 2018-05-25 2019-11-28 Nantcell, Inc. Compositions de doxorubicine modifiée et méthodes associées
KR102105938B1 (ko) * 2018-10-24 2020-05-04 고려대학교 산학협력단 약물 내성 극복을 위한 항암 약물전구체

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007102888A2 (fr) * 2005-10-28 2007-09-13 The Regents Of The University Of Colorado Procédés de traitement du cancer utilisant la doxazolidine et des promédicaments de celle-ci
US20070219351A1 (en) * 2004-05-07 2007-09-20 Luigi Flume, Giuseppina Di Stefano And Marcella Lanza Process for the Preparation of Doxorubicin Conjugates with Lactosaminated Human Albumin
US20090215082A1 (en) * 2005-03-30 2009-08-27 Salamone Salvatore J Doxorubicin Immunoassay

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070219351A1 (en) * 2004-05-07 2007-09-20 Luigi Flume, Giuseppina Di Stefano And Marcella Lanza Process for the Preparation of Doxorubicin Conjugates with Lactosaminated Human Albumin
US20090215082A1 (en) * 2005-03-30 2009-08-27 Salamone Salvatore J Doxorubicin Immunoassay
WO2007102888A2 (fr) * 2005-10-28 2007-09-13 The Regents Of The University Of Colorado Procédés de traitement du cancer utilisant la doxazolidine et des promédicaments de celle-ci

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190086418A1 (en) * 2016-02-24 2019-03-21 Cellact Pharma Gmbh Method for the diagnosis of etoposide prodrug treatable cancer
JP2019507878A (ja) * 2016-02-24 2019-03-22 セルアクト・ファーマ・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツングＣｅｌｌＡｃｔ Ｐｈａｒｍａ ＧｍｂＨ エトポシドプロドラッグで処置可能な癌の診断方法
JP7143217B2 (ja) 2016-02-24 2022-09-28 セルアクト ファーマ ジーエムビーエイチ エトポシドプロドラッグで処置可能な癌の診断方法
US11892454B2 (en) 2016-02-24 2024-02-06 Cellact Pharma Gmbh Method for the diagnosis of etoposide prodrug treatable cancer
WO2019226774A1 (fr) * 2018-05-25 2019-11-28 Nantcell, Inc. Compositions de doxorubicine modifiée et méthodes associées
KR102105938B1 (ko) * 2018-10-24 2020-05-04 고려대학교 산학협력단 약물 내성 극복을 위한 항암 약물전구체
US11241501B2 (en) 2018-10-24 2022-02-08 Korea University Research And Business Foundation Anticancer prodrug for overcoming drug resistance

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