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WO2008112014A1 - Procédés et compositions permettant de traiter le cancer - Google Patents

Procédés et compositions permettant de traiter le cancer Download PDF

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Publication number
WO2008112014A1
WO2008112014A1 PCT/US2007/080588 US2007080588W WO2008112014A1 WO 2008112014 A1 WO2008112014 A1 WO 2008112014A1 US 2007080588 W US2007080588 W US 2007080588W WO 2008112014 A1 WO2008112014 A1 WO 2008112014A1
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Prior art keywords
chaetocin
cancer
cells
myeloma
pharmaceutically acceptable
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Keith C. Bible
Crescent R. Isham
Ruifang Xu
Jennifer D. Tibodeau
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Priority to US12/406,444 priority Critical patent/US20090264421A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/08—Bridged systems

Definitions

  • TECHNICAL FIELD This disclosure relates to compositions and methods for treating hematologic cancers, including myelomas, leukemias, and lymphomas, and solid cancerous tumors using thiodioxopiperazines, e.g., bridged disulfide thiodioxopiperazines, also known as epipolythiodioxopiperazines, and structurally related compounds.
  • thiodioxopiperazines e.g., bridged disulfide thiodioxopiperazines, also known as epipolythiodioxopiperazines, and structurally related compounds.
  • Chaetocin a small molecule natural product produced by Chaetomium species fungi and originally isolated in 1970 (Hauser D, Weber HP, and Sigg HP, Isolation and Configuration of Chaetocin, HeIv Chim Acta (1970) 53(5):1061-73) is a representative of a class of fungal secondary metabolites known as thiodioxopiperazines.
  • Other thiodioxopiperazines have been previously reported to have a wide range of biological activities, including antimicrobial and antifungal effects.
  • Thiodioxopiperazines may be produced by fungi to gain a competitive advantage over adjacent fungal and other saprophytic organisms through toxic and antiproliferative effects on adjacent organisms.
  • TrxRl Thioredoxin reductase-1
  • TrxRl catalyzes the NADPH-dependent reduction of thioredoxin and other substrate disulfide bonds via its selenocysteine/FAD active site.
  • Mammalian TrxRl consequently participates in diverse metabolic reactions involving oxidation-reduction cycles and is widely believed to be central to intracellular ROS mitigation.
  • TrxRl/Trx pathway may provide plausible molecular targets for cancer therapies for several reasons.
  • TrxRl and/or Trx are known to be upregulated in a variety of human cancers, including lung, colorectal, cervical, hepatic, and pancreatic, and Trx overexpression has been linked to aggressive tumor growth and poorer prognosis.
  • TrxRl enhances tumor proliferation via its regulatory effects on the Gi checkpoint during cell cycle progression.
  • TrxRl invokes a pro-survival signaling cascade. Further, cells overexpressing TrxRl are more resistant to some anticancer agents.
  • upregulated TrxRl/Trx pathway activity may in part account for how cancer cells have adapted to their generally higher basal levels of cellular oxidative stress. Therefore, despite providing a potential survival advantage to cancer cells, upregulated TrxRl/Trx pathway activity may also be required for cancer survival in light of increased ROS stress inherent in some cancer cells. In this fashion, the TrxRl/Trx pathway may contain therapeutically useful antineoplastic molecular targets.
  • TrxRl Small molecules such as lipid hydroperoxides, selenite and dehydroascorbate, as well as proteins such as protein disulfide isomerase or glutathione peroxidase along with Trx, are all known substrates of TrxRl, demonstrating its low substrate specificity.
  • inhibitors of TrxRl including auranofm, cisplatin, lipoic acid, motexafm gadolinium, myricetin, quercetin, and 1 -methyl- 1 -propyl-2-imidazo IyI disulfide (IV-2). Of these, motexafm gadolinium and IV-2 have anticancer effects putatively attributed to TrxRl inhibition and are undergoing development as candidate cancer therapeutics.
  • myeloma is an incurable cancer characterized by the clonal proliferation of B-cell lineage plasma cells, resulting in the production of monoclonal proteins in serum and/or urine and destructive boney lesions, and the deaths of about 12,000 individuals in the U.S. annually.
  • therapeutics are becoming available to treat this disease— with the potential for significant symptom palliation, induction of disease responses, and prolongation of disease-free survival- available therapeutic approaches including peripheral blood stem cell transplantation and newer therapeutics have yet to be conclusively demonstrated to appreciably impact patient overall survival in randomized trials (Barlogie et al., 2005; Fermand et al., 2005; Blade et al., 2005).
  • there is still need for improved anti-myeloma therapies are still need for improved anti-myeloma therapies.
  • chaetocin a thiodioxopiperazine natural product
  • chaetocin has potent in vitro anti-myeloma activity in IL-6-dependent and IL-6-independent myeloma cell lines. Chaetocin potently killed freshly collected sorted patient CD 138+ myeloma cells, but spared matched normal CD 138- patient bone marrow leukocytes, and displayed superior ex vivo anti-myeloma activity and selectivity in comparison to doxorubicin and dexamethasone.
  • chaetocin In vivo experiments using chaetocin confirmed the antiproliferative activity in myeloma. Furthermore, the effects of chaetocin were seen in samples obtained from patients afflicted with different types of myeloma, including smoldering myeloma and heavily pretreated myeloma patients who had previously undergone peripheral blood stem cell transplantation.
  • chaetocin is rapidly and dramatically accumulated in cancer cells via a transport system inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell, its anti-cancer activity appears mediated primarily through the imposition of oxidative stress and apoptosis induction. The ability of chaetocin to selectively kill myeloma cells appears to be mediated based upon a generally increased susceptibility of myeloma cells to oxidative stressors. This suggests that not only chaetocin, but also other agents that similarly induce cellular oxidative stress, may hold promise for further development as potential anti-myeloma therapeutics.
  • TrxRl oxidative stress mitigation enzyme thioredoxin reductase-1
  • TrxRl/Trx pathway is of central importance in limiting reactive oxygen species (ROS) accumulation in cancer cells, and as chaetocin exerts its selective anti-myeloma effects via ROS imposition, the inhibition of TrxRl by chaetocin appears to link enzyme targeting of TrxRl by chaetocin to chaetocin's selective anti-myeloma effects.
  • ROS reactive oxygen species
  • structurally related compounds e.g. , those containing intact disulfides, those containing a bridged disulfide thiodioxopiperizine ring, and/or those capable of inducing oxidative stress
  • structurally related compounds that include a bridged disulfide thiodioxopiperazine ring include chaetomin and gliotoxin.
  • the inventors have found that chaetomin and gliotoxin also inhibit the reduction of thioredoxin by thioredoxin reductase.
  • the cancer is multiple myeloma. In other embodiments, the cancer is a solid tumor.
  • Chaetocin bears chemical structural similarity to the acetylated histone lysine moiety mimicked by many histone deacetylase inhibitors (HDACIs) and has anti- myeloma activity in vitro - yet does not alter levels of acetylated histone H3 in myeloma cells.
  • HDACIs histone deacetylase inhibitors
  • FIG. 1 Chaetocin kills myeloma cells in vitro via induction of morphological apoptosis accompanied by loss of mitochondrial membrane potential, PARP cleavage and DNA ladder formation.
  • D Treatment of normal B-cells or matched negatively selected neutrophils from three normal patients with chaetocin indicates that, unlike myeloma cells, normal B-cells are not selectively killed by chaetocin.
  • B-CLL cells or matched negatively selected patient neutrophils from two B-CLL patients with chaetocin indicate that, unlike myeloma cells, B-CLL cells are not selectively killed by chaetocin. Survival indicated in A-E was assessed using a trypan blue exclusion assay, counting viable cells using a hemocytometer after 24 hour exposure to the indicated drugs and drug concentrations.
  • FIG. 4 Patient myeloma cells treated in mixed culture are selectively killed by chaetocin in comparison to other bone marrow leukocytes, and chaetocin has in vivo anti- myeloma activity.
  • A-C Unsorted bone marrow leukocytes obtained from 4 patients with multiple myeloma were treated with 100 nM Chaetocin or diluent for 24 hours and then subjected to FACS analyses to examine induced cell death in various leukocyte subpopulations. Whereas myeloma cells were readily killed by chaetocin (A, representative data shown), combined granulocytes and monocytes (B, representative data shown) were relatively spared.
  • Results from 4 unsorted patient marrow leukocyte samples are indicated in C, indicating selective killing of myeloma cells by chaetocin in mixed culture in all 4 examined patient samples.
  • A-C surviving cells were defined as those with low annexin and 7-AAD staining.
  • D. Chaetocin has in vivo anti-myeloma activity in the RRMI 8226 SCID flank xenograft mouse model. Arrows indicate times of intraperitoneal chaetocin administration, while * indicates statistically significance differences from corresponding vehicle control values (p ⁇ 0.05).
  • FIG. 6 Glutathione dramatically attenuates chaetocin-induced reductions in A549 cell colony formation, partially mediated through attenuation of intracellular accumulation of chaetocin.
  • A Glutathione or NAC, but not inhibitors of DNA (aphidicolin), RNA (DRB) or protein (cycloheximide) synthesis, attenuate chaetocin- induced reductions in colony formation in A549 cells. Results shown are representative of three independent experiments for each treatment.
  • B The ability of glutathione to attenuate chaetocin-induced inhibition of colony formation is highly time-dependent, and is maximal when glutathione is added before initiation of chaetocin exposure. Results shown are representative of two independent experiments.
  • C. and D The effects of glutathione pretreatment on intracellular (C) and extracellular (media, D) chaetocin concentration in response to treatment of A549 cells with 10 ⁇ M chaetocin for 5 min. Intracellular and extracellular chaetocin concentrations were assessed using HPLC as described in the text, with 100 ⁇ M glutathione added 5 minutes before chaetocin addition. E. "Double reciprocal" plot indicating the effects of 5 minute pretreatment with 100 mM glutathione or diluent on intracellular chaetocin concentrations resulting from exposure of A549 cells to varying concentration of chaetocin for 5 minutes. Intracellular chaetocin concentrations were assessed using HPLC as described in the text. Results shown are representative of three independent experiments.
  • Results shown are representative of three independent FACS experiments as described in the text; * p ⁇ 0.05, **p ⁇ 0.01.
  • F. Chaetocin did not appreciably alter intracellular concentrations of reduced glutathione, yet pretreatment with 1 mM glutathione led to increased levels of intracellular reduced glutathione.
  • A549 cells were exposed to the indicated concentration of chaetocin for 24 hours, with addition of glutathione or diluent 30 minutes prior to chaetocin addition. Glutathione levels were assessed by spectrophotometric assay as described below. G Relative intracellular chaetocin levels in patient normal CD 138- bone marrow cells compared to those attained in identically treated patient CD 138+ myeloma cells. Cells were treated with 10 ⁇ M chaetocin for 20 minutes prior to assay, and calculated intracellular chaetocin levels were measured by HPLC with adjusted for differences in average cell volume (calculated from measured cell radii ascertained via light microscopy).
  • CD 138+ patient myeloma cells are more sensitive to the cytotoxic effects of hydrogen peroxide than matched normal patient CD138- bone marrow leukocytes. Cells from 4 myeloma patients were exposed to 200 ⁇ M hydrogen peroxide for 24 hours prior to assay, with trypan blue exclusion used to assess surviving cells.
  • Figure 8 shows the structures of chaeotocin, chaetomin, and gliotoxin.
  • Figure 9 shows a representative synthetic scheme for preparing certain compounds according to Formula I.
  • Figure 10 shows the effects of chaetocin on colony formation in solid tumor cancer cell lines.
  • A Effects of chaetocin on colony formation in a variety of solid tumor cancer cell lines (24 h chaetocin exposures).
  • B Time-dependance of chaetocin-induced reductions in colony formation in A549 cells.
  • C Effects of chaetocin on colony formation in a variety of thyroid cancer cell lines (24 h chaetocin exposures).
  • Figure 11 demonstrates the effects of chaetocin on normal cells.
  • A. and B Effects of chaetocin on survival of MGUS plasma cells and matched patient normal leucocytes from two patients (assessed by trypan blue exclusion assay, 24 h chaetocin exposures).
  • C. and D Effects of chaetocin on survival of normal plasma cells and matched patient normal leucocytes from two patients (assessed by trypan blue exclusion assay, 24 h chaetocin exposures).
  • Figure 12 demonstrates the effects of chaetocin in a variety of thyroid cancer cell lines and in normal thyroid gland thyrocytes.
  • Presented data represent the results of colony forming assays, with 24 hour exposures to chaetocin at the indicated concentrations. Note that normal thyrocytes are distinctly less sensitive to the cytotoxic effects of chaetocin in comparison to all tested thyroid cancer cell lines.
  • Figure 13 shows results from assessment of the effects of chaetocin in dexamethasone- or doxorubicin-resistant myeloma cell lines.
  • dexamethasone-resistant MMlR cells are not cross-resistant to chaetocin (A. and B.)
  • doxorubicin-resistant RPMI 8226 D40 cells are only modestly cross-resistant to chaetocin (C. and D.). Cell numbers and viability were assessed by trypan exclusion.
  • Figure 14 demonstrates that chaetocin inhibits thioredoxin reductase activity - but not the activities of glutathione reductase or thioredoxin.
  • A Chemical structure of chaetocin.
  • B Thioredoxin reductase activity (assessed by following DTNB reduction) is inhibited by chaetocin in a dose-dependent fashion.
  • Inset time versus absorbance data showing inhibition of DTNB reduction by chaetocin at various concentrations and times.
  • Glutathione reductase activity (C, assessed by following DTNB reduction) and thioredoxin activity (D., assessed using the insulin precipitation method) are unaffected by chaetocin at concentrations that readily inhibit thioredoxin reductase.
  • Data shown represent single experiments (triplicate data points; error bars, one standard deviation) representative of a minimum of three separate experiments.
  • Figure 15 demonstrates that chaetocin and other intact thiodioxopiperazines inhibit the ability of thioredoxin reductase to reduce its native substrate thioredoxin.
  • A. Chaetocin inhibits the reduction of thioredoxin by thioredoxin reductase in a dose- dependent fashion.
  • B. Chaetocin, chetomin and gliotoxin each inhibit the reduction of thioredoxin by thioredoxin reductase; however, the related compounds S-methyl chaetocin and verruculogen lacking bridged disulfide bonds do not.
  • Figure 16 shows that chaetocin is a substrate for thioredoxin reductase.
  • A Oxidation of NADPH by thioredoxin reductase in response to various chaetocin concentrations in the absence of other thioredoxin reductase substrates, indicating that chaetocin itself has thioredoxin reductase substrate functionality. Data shown are representative of a minimum of three separate experiments.
  • B Mass spectrometry results demonstrating that chaetocin disulfide bonds are reduced in the presence of thioredoxin reductase and NADPH, but not NADPH alone.
  • Figure 17 demonstrates that transient overexpression of the downstream thioredoxin substrate and effector thioredoxin attenuates chaetocin-induced but not doxorubicin-induced cytotoxicity - thus demonstrating the potential impact of chaetocin on reduction states of critical downstream targets of thioredoxin reductase.
  • A. Transient overexpression of thioredoxin attenuates chaetocin- but not doxorubicin-induced cytotoxicity in HeLa cells. Transfected cells were treated for 24 hrs, cell viability assessed using a trypan blue inclusion assay. Inset: immunoblotting showing increased level of thioredoxin in transiently transfected HeLa cells compared to empty- vector transfected cells.
  • Figure 18 Summary of thioredoxin reductase steady-state kinetics data for thioredoxin and tested substrates.
  • Figure 19 shows that the reduction of chaetocin and thioredoxin by thioredoxin reductase displays sigmoidal kinetics.
  • Data shown represent single experiments (triplicate data points; error bars, one standard deviation) representative of a minimum of three separate experiments. Error bars not evident are hidden by data points.
  • Figure 20 shows mouse weight data after twice weekly 0.6 mg/kg IP administration of chaetocin.
  • pharmaceutically acceptable derivatives of a compound include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof.
  • Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization.
  • the compounds produced may be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs.
  • salts include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1 -para-chlorobenzyl-2-pyrrolidin- 1 '-ylmethyl- benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, nitrates, borates, me
  • esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulf ⁇ nic acids and boronic acids.
  • Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.
  • treatment means any manner in which one or more of the symptoms of a cancer, e.g., a hematologic or solid tumor cancer, are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as uses for treating diseases, disorders, or ailments in which a cancer is implicated.
  • amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.
  • IC 50 refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response.
  • EC50 refers to a dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.
  • a prodrug is a compound that, upon in vivo administration, is metabolized by one or more steps or processes or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes.
  • the prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug.
  • the compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configuration, or may be a mixture thereof. In certain cases, a particular configuration may be preferred, e.g., see FIG. 8 demonstrating a preferred stereochemistry for chaetocin. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures.
  • amino acid residues such residues may be of either the L- or D- form. The configuration for naturally occurring amino acid residues is generally L. When not specified the residue is the L form.
  • amino acid refers to ⁇ -amino acids which are racemic, or of either the D- or L-configuration.
  • the designation "d” preceding an amino acid designation refers to the D-isomer of the amino acid.
  • the designation "dl” preceding an amino acid designation refers to a mixture of the L- and D-isomers of the amino acid. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form.
  • substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
  • TLC thin layer chromatography
  • HPLC high performance liquid chromatography
  • MS mass spectrometry
  • alkyl As used herein, “alkyl,” “alkenyl” and “alkynyl” refer to carbon chains that may be straight or branched. Exemplary alkyl, alkenyl and alkynyl groups herein include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, isohexyl, allyl (propenyl) and propargyl (propynyl).
  • cycloalkyl refers to a saturated mono- or multi- cyclic ring system, in certain embodiments of 3 to 10 carbon atoms, in other embodiments of 3 to 6 carbon atoms.
  • the ring systems of the cycloalkyl groups may be composed of one ring or two or more rings which may be joined together in a fused, bridged or spiro-connected fashion. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • aryl refers to aromatic monocyclic or multicyclic groups containing from 6 to 19 carbon atoms.
  • Aryl groups include, but are not limited to groups such as unsubstituted or substituted fluorenyl, unsubstituted or substituted phenyl, and unsubstituted or substituted naphthyl.
  • heteroaryl refers to a monocyclic or multicyclic aromatic ring system, in certain embodiments, of about 5 to about 15 members, where one or more, in one embodiment 1 to 4, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
  • the heteroaryl group may be optionally fused to a benzene ring.
  • Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, quinolinyl and isoquinolinyl.
  • heterocyclyl refers to a monocyclic or multicyclic non-aromatic ring system, in one embodiment of 3 to 10 members, in another embodiment of 4 to 7 members, in a further embodiment of 5 to 6 members, where one or more, in certain embodiments, 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
  • halo refers to F, Cl, Br or I.
  • pseudohalides or pseudohalo groups are groups that behave substantially similar to halides. Such compounds can be used in the same manner and treated in the same manner as halides. Pseudohalides include, but are not limited to, cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethoxy, and azide.
  • haloalkyl refers to an alkyl group in which one or more of the hydrogen atoms are replaced by halogen.
  • carboxy refers to a divalent radical, -C(O)O-.
  • aminocarbonyl refers to -C(O)NH 2 .
  • alkylcarbonyl refers to -C(O)R, where R is alkyl.
  • arylcarbonyl refers to -C(O)R, where R is aryl.
  • aminoalkyl refers to -RNH 2 , in which R is alkyl.
  • aralkyl refers to an alkyl group that is substituted with one or more aryl groups.
  • alkaryl refers to an aryl group that is substituted with one or more alkyl groups.
  • hydroxyalkyl refers to an alkyl group in which one or more of the hydrogen atoms are replaced by hydroxyl (-OH).
  • alkoxy and RS- refer to RO- and RS-, in which R is alkyl.
  • aryloxy and arylthio refer to RO- and RS-, in which R is aryl.
  • amido refers to the divalent group -C(O)NH.
  • hydroazide refers to the divalent group -C(O)NHNH-.
  • haloalkyl may include one or more of the same or different halogens.
  • the natural product chaetocin exhibits in vitro activity against multiple myeloma, with superior ex vivo activity and selectivity in patient myeloma cells relative to the commonly utilized anti-myeloma agents dexamethasone and doxorubicin. Additionally, chaetocin was also demonstrated to inhibit growth of myeloma in vivo. Accordingly, methods employing chaetocin and other compounds or compositions described further herein for treating or ameliorating one or more symptoms associated with multiple myeloma in a mammal, e.g., a human, are provided herein. Chaetocin or other compounds described herein can be administered either alone or in combination with other known anticancer drugs.
  • chaetocin has been shown to have activity against normal plasma cells, while largely sparing granulocytes and other leukocytes. See FIG 11.
  • chaetocin and other compounds or compositions described herein for treating or ameliorating one or more symptoms associated with plasma cell B-lymphocyte lineage disorders in addition to myeloma, such as MGUS (monoclonal gammopathy of undetermined significance), amyloidosis, heavy chain diseases, cryoglobulinemia and Waldenstrom's macroglobulinemia.
  • MGUS monoclonal gammopathy of undetermined significance
  • amyloidosis amyloidosis
  • heavy chain diseases such as cryoglobulinemia and Waldenstrom's macroglobulinemia.
  • chaetocin has broad-spectrum anti-cancer activity, including activity against solid tumors, such as lung, breast, prostate, hepatoma, thyroid and colon tumors; and sarcomas. See FIG 10. Furthermore, chaetocin also has selectivity in killing solid tumor cancer cells. In particular, chaetocin has potent anticancer activity in a variety of thyroid cancer cell lines, while largely sparing normal thyrocytes (normal thyroid gland cells). See FIG 12. Accordingly, methods for treating such solid tumors employing chaetocin and other compounds or compositions described herein are also provided.
  • chaetocin may have therapeutic application to even highly chemotherapy-resistant cancers.
  • chaetocin has been shown to be a competitive and selective substrate for thioredoxin reductase-1 (TrxRl), an oxidative stress mitigation enzyme of central importance in limiting ROS accumulation in cancer cells, thereby linking chaetocin's enzymatic targeting to its anti-myeloma effects.
  • TrxRl thioredoxin reductase-1
  • chaetocin may hold promise for further development as anti-cancer therapeutics.
  • other thiodioxopiperazine-containing natural products such as gliotoxin and chaetomin
  • gliotoxin and chaetomin also are known to be cytotoxic via a mechanism of oxidative stress induction. See FIG 8 for a comparison of the structures of gliotoxin, chaetomin, and chaetocin.
  • compounds related to chaeotocin such as chaetomin and gliotoxin (e.g.
  • compounds containing a core thiodioxopiperazine structure, and particularly containing the bridged disulfide thiodioxopiperazine structure are proposed herein to have similar anti-cancer activities, and can be used in any of the methods described herein.
  • examples of such compounds include melinacidin IV, sirodesmin, hyalodendrin, sporidesmin A, leptosin, emestrin, dithiosilvatin, epicorazine, emethallicin, verticillin A and B, 19 methyl- 19'deoxy-6,6'dihydroxychaetocin, aranotin, apoaranotin, and scabrosin; see also Formulas I and II below.
  • R, R', R 2 , and R 2 ' independently, can be selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkoxy, hydroxy, aryl, heteroaryl, heteroalkyl, heterocyclyl, halo, pseudohalo, carboxy, haloalkyl, hydroxyalkyl, alkaryl, aralkyl, aminocarbonyl, aminoalkyl, alkylcarbonyl, arylcarbonyl, alkoxy, alkylthio, aryloxy, and arylthio; or wherein R and R 2 , and/or R' and R 2 ', independently, together to which the atoms to which they are attached, form a fused heterocyclic ring system which is optionally substituted with one or more alkyl, cycloalkyl, alkoxy, aryl, hydroxy, heteroaryl, heteroalkyl, heterocyclyl, halo, pseudohal
  • R and/or R' are selected from hydrogen, halo, pseudohalo, hydroxyl, alkyl having from 1 to 8 carbon atoms (e.g., 1 to 6, 1 to 5, 1 to 4, or 1 to 3 C atoms), aralkyl, where the alkyl group has from 1 to 8 carbon atoms (e.g., 1 to 6, 1 to 5, 1 to 4, or 1 to 3 C atoms), alkaryl, where the alkyl group has from 1 to 8 carbon atoms (e.g., 1 to 6, 1 to 5, 1 to 4, or 1 to 3 C atoms), hydroxyalkyl, where the alkyl group has froml to 8 carbon atoms (e.g., 1 to 6, 1 to 5, 1 to 4, or 1 to 3 C atoms), alkylcarbonyl, where the alkyl group has from 1 to 8 carbon atoms (e.g., 1 to 6, 1 to 5, 1 to 4, or 1 to 3 C atoms), and arylcarbonyl.
  • R and/or R' are selected from H, methyl, ethyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl.
  • R 2 and/or R 2 ' are selected from hydrogen, halo, pseudohalo, alkyl having from 1 to 8 carbon atoms (e.g., 1 to 6, 1 to 5, 1 to 4, or 1 to 3 C atoms), and hydroxyalkyl, where the alkyl group has from 1 to 8 carbon atoms (e.g., 1 to 6, 1 to 5, 1 to 4, or 1 to 3 C atoms).
  • the compounds for use in the compositions and methods provided herein may be obtained from commercial sources (e.g., Sigma, St. Louis, MO; Aldrich Chemical Co., Milwaukee, WI), may be isolated from fungi using known techniques, or may be prepared by methods well known to those of skill in the art or by the methods shown herein (e.g., see FIG 9).
  • One of skill in the art could modify certain of the compounds using the appropriate starting materials and standard methods in organic chemistry.
  • compositions provided herein contain therapeutically effective amounts of one or more of the compounds provided herein that are useful in the treatment or amelioration of one or more of the symptoms associated with a hematologic (e.g. , myeloma, lymphoma, or leukemia) or solid cancers, and a pharmaceutically acceptable carrier.
  • a hematologic e.g. , myeloma, lymphoma, or leukemia
  • solid cancers e.g., a hematologic (e.g. , myeloma, lymphoma, or leukemia) or solid cancers
  • Pharmaceutical carriers suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • the compositions contain one or more compounds provided herein.
  • the compounds are, in one embodiment, formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers.
  • the compounds described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).
  • compositions effective concentrations of one or more compounds or pharmaceutically acceptable derivatives thereof is (are) mixed with a suitable pharmaceutical carrier.
  • the compounds may be derivatized as the corresponding salts, esters, enol ethers or esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs prior to formulation, as described above.
  • concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats or ameliorates one or more of the symptoms of a cancer, e.g. , multiple myeloma.
  • compositions are formulated for single dosage administration.
  • the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved or one or more symptoms are ameliorated.
  • the active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems, and then extrapolated therefrom for dosages for humans.
  • the concentration of active compound in the pharmaceutical composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • Pharmaceutical dosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or 2000 mg, and in one embodiment from about 10 mg to about 500 mg of the active ingredient or a combination of essential ingredients per dosage unit form.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disorder being treated and may be determined empirically using known testing protocols or by extrapolation from in vzVo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • solubilizing compounds may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), polyethylene glycol (PEG) (e.g., PEG400), cyclodextrins or cremaphor; using surfactants, such as TWEEN®, or dissolution in aqueous sodium bicarbonate.
  • cosolvents such as dimethylsulfoxide (DMSO), polyethylene glycol (PEG) (e.g., PEG400), cyclodextrins or cremaphor
  • surfactants such as TWEEN®
  • a formulation can include from about 5% to about 85% PEG400, and/or from about 0.5% to about 30% DMSO.
  • a formulation can include 0.9N NaCl, e.g., from about 15% to about 90% 0.9N NaCl.
  • a formulation can include about 15% to about 25% PEG400, about 75% to about 85% 0.9N NaCl, and from about 0.5% to about 5% DMSO.
  • DMSO is present in an amount less than 1%, e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1%, or less.
  • the resulting mixture may be a solution, suspension, emulsion or the like.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.
  • the pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.
  • the pharmaceutically therapeutically active compounds and derivatives thereof are, in one embodiment, formulated and administered in unit- dosage forms or multiple-dosage forms.
  • Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent.
  • unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof.
  • a multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered may also contain nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, nanoparticles (e.g. gold, albumin) and other such agents.
  • auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, nanoparticles (e.g. gold, albumin) and other such agents.
  • Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared.
  • compositions may contain 0.001%-100% active ingredient, or in one embodiment 0.1-95%.
  • compositions for oral administration are either solid, gel or liquid.
  • the solid dosage forms are tablets, capsules, granules, and bulk powders.
  • Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated.
  • Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.
  • Solid compositions for oral administration In certain embodiments, the formulations are solid dosage forms, in one embodiment, capsules or tablets.
  • the tablets, pills, capsules, troches and the like can contain one or more of the following ingredients, or compounds of a similar nature: a binder; a lubricant; a diluent; a glidant; a disintegrating agent; a coloring agent; a sweetening agent; a flavoring agent; a wetting agent; an emetic coating; and a film coating.
  • binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.
  • Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid.
  • Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.
  • Glidants include, but are not limited to, colloidal silicon dioxide.
  • Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose.
  • Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate.
  • Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors.
  • Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate.
  • Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether.
  • Emetic-coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates.
  • Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.
  • the compound, or pharmaceutically acceptable derivative thereof, could be provided in a composition that protects it from the acidic environment of the stomach.
  • the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine.
  • the composition may also be formulated in combination with an antacid or other such ingredient.
  • the dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
  • dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the compounds can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the active materials can also be mixed or complexed with other active materials which do not impair the desired action, or with materials that supplement the desired action.
  • the active ingredient is a compound or pharmaceutically acceptable derivative thereof as described herein. Higher concentrations, up to about 98% by weight of the active ingredient, may be included.
  • tablets and capsules formulations may be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.
  • they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate.
  • a conventional enterically digestible coating such as phenylsalicylate, waxes and cellulose acetate phthalate.
  • Liquid compositions for oral administration include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Aqueous solutions include, for example, elixirs and syrups.
  • Emulsions are either oil-in-water or water-in- oil.
  • Elixirs are clear, sweetened, hydroalcoholic preparations.
  • Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative.
  • An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid.
  • Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives.
  • Pharmaceutically acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form include diluents, sweeteners and wetting agents.
  • Pharmaceutically acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.
  • Solvents include glycerin, sorbitol, ethyl alcohol and syrup.
  • preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol.
  • non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil.
  • emulsifying agents examples include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate.
  • Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia.
  • Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin.
  • Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.
  • Organic acids include citric and tartaric acid.
  • Sources of carbon dioxide include sodium bicarbonate and sodium carbonate.
  • Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof.
  • Flavoring agents include natural flavors extracted from plants such fruits, and synthetic blends of compounds which produce a pleasant taste sensation.
  • the solution or suspension in for example propylene carbonate, vegetable oils or triglycerides, is in one embodiment encapsulated in a gelatin capsule.
  • a gelatin capsule Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Patent Nos. 4,328,245; 4,409,239; and 4,410,545.
  • the solution e.g., for example, in a polyethylene glycol, may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for administration.
  • liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells.
  • Other useful formulations include those set forth in U.S. Patent Nos. RE28,819 and 4,358,603.
  • such formulations include, but are not limited to, those containing a compound provided herein, a dialkylated mono- or poly-alkylene glycol, including, but not limited to, 1 ,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol- 750-dimethyl ether wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.
  • BHT but
  • formulations include, but are not limited to, aqueous alcoholic solutions including a pharmaceutically acceptable acetal.
  • Alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol.
  • Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.
  • injectables, solutions, and emulsions Parenteral administration, in one or more embodiment characterized by injection, either subcutaneously, intramuscularly, intraperitoneally or intravenously is also contemplated herein.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • the injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol.
  • compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
  • a compound provided herein is dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene- vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, polyprop
  • parenteral administration of the compositions includes intravenous, subcutaneous, intraperitoneal and intramuscular administrations.
  • Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • thickening and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
  • aqueous vehicles include Sodium Chloride Injection, Ringers
  • Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil.
  • Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate.
  • Antioxidants include sodium bisulfate.
  • Local anesthetics include procaine hydrochloride.
  • Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone.
  • Emulsifying agents include Polysorbate 80 (TWEEN® 80).
  • a sequestering or chelating agent of metal ions include EDTA.
  • Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment. The concentration of the pharmaceutically active compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art.
  • the unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile, as is known and practiced in the art.
  • intravenous or intraarterial infusion of a sterile aqueous solution containing an active compound is an effective mode of administration.
  • Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.
  • Injectables are designed for local and systemic administration.
  • a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1% w/w up to about 90% w/w or more, in certain embodiments more than 1% w/w of the active compound to the treated tissue(s).
  • the compound may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product or to produce a prodrug.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined. 3. Lyophilized powders
  • lyophilized powders which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels.
  • the sterile, lyophilized powder is prepared by dissolving a compound provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder.
  • Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
  • the solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
  • a buffer such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
  • sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation.
  • the resulting solution will be apportioned into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the compound.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4 0 C to room temperature.
  • Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends upon the selected compound. Such amount can be empirically determined. 4. Topical administration
  • Topical mixtures are prepared as described for the local and systemic administration.
  • the resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.
  • the compounds or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma).
  • These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfme powder for insufflation, alone or in combination with an inert carrier such as lactose.
  • the particles of the formulation will, in one embodiment, have diameters of less than 50 microns, in one embodiment less than 10 microns.
  • the compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application.
  • Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.
  • compositions for other routes of administration may be formulated as 0.01% - 10% isotonic solutions, pH about 5-7, with appropriate salts. 5.
  • Compositions for other routes of administration may be formulated as 0.01% - 10% isotonic solutions, pH about 5-7, with appropriate salts. 5.
  • transdermal patches including iontophoretic and electrophoretic devices, and rectal administration, are also contemplated herein.
  • Transdermal patches including iotophoretic and electrophoretic devices, are well known to those of skill in the art.
  • such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.
  • rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients.
  • Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used.
  • spermaceti and wax agents to raise the melting point of suppositories include spermaceti and wax.
  • Rectal suppositories may be prepared either by the compressed method or by molding.
  • the weight of a rectal suppository in one embodiment, is about 2 to 3 gm. Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.
  • Targeted Formulations The compounds provided herein, or pharmaceutically acceptable derivatives thereof, may also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non- limiting examples of targeting methods, see, e.g., U.S. Patent Nos.
  • liposomal suspensions including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.
  • tissue-targeted liposomes such as tumor-targeted liposomes
  • liposome formulations may be prepared according to methods known to those skilled in the art.
  • liposome formulations may be prepared as described in U.S. Patent No. 4,522,811. Briefly, liposomes such as multilamellar vesicles (MLVs) may be formed by drying down egg phosphatidyl choline and brain phosphatidyl serine (7:3 molar ratio) on the inside of a flask.
  • MLVs multilamellar vesicles
  • a solution of a compound provided herein in phosphate buffered saline lacking divalent cations (PBS) is added and the flask shaken until the lipid film is dispersed.
  • PBS phosphate buffered saline lacking divalent cations
  • Nanoparticle preparations may include the use of nanoparticle preparations, optionally complexed with antibodies (e.g. anti-CD 138) or other substances, intended to enhance targeting to desired neoplastic cells or tissues.
  • antibodies e.g. anti-CD 138
  • other substances intended to enhance targeting to desired neoplastic cells or tissues.
  • the compounds or pharmaceutically acceptable derivatives may be packaged as articles of manufacture (e.g., kits) containing packaging material, a compound or pharmaceutically acceptable derivative thereof provided herein within the packaging material, and a label that indicates that the compound or composition, or pharmaceutically acceptable derivative thereof, is useful for treatment or amelioration of one or more symptoms of a cancer, including a hematologic cancer such as myeloma.
  • articles of manufacture e.g., kits
  • packaging material e.g., a compound or pharmaceutically acceptable derivative thereof provided herein within the packaging material
  • packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, e.g., U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252.
  • Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. 8. Sustained Release Formulations
  • sustained release formulations to deliver the compounds to the desired target at high circulating levels (between 10 “9 and 10 "4 M).
  • the levels are either circulating in the patient systemically, or in one embodiment, localized to a site of, e.g., paralysis. It is understood that the compound levels are maintained over a certain period of time as is desired and can be easily determined by one skilled in the art.
  • sustained and/or timed release formulations may be made by sustained release means of delivery devices that are well known to those of ordinary skill in the art, such as those described in US Patent Nos.
  • compositions can be used to provide slow or sustained release of one or more of the active compounds using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like.
  • Suitable sustained release formulations known to those skilled in the art, including those described herein, may be readily selected for use with the pharmaceutical compositions provided herein.
  • single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, caplets, powders and the like, that are adapted for sustained release are contemplated herein.
  • the sustained release formulation contains active compound such as, but not limited to, microcrystalline cellulose, maltodextrin, ethylcellulose, and magnesium stearate. As described above, all known methods for encapsulation which are compatible with properties of the disclosed compounds are contemplated herein.
  • the sustained release formulation is encapsulated by coating particles or granules of the pharmaceutical compositions provided herein with varying thickness of slowly soluble polymers or by microencapsulation.
  • the sustained release formulation is encapsulated with a coating material of varying thickness (e.g. about 1 micron to 200 microns) that allow the dissolution of the pharmaceutical composition about 48 hours to about 72 hours after administration to a mammal.
  • the coating material is a food-approved additive.
  • the sustained release formulation is a matrix dissolution device that is prepared by compressing the drug with a slowly soluble polymer carrier into a tablet.
  • the coated particles have a size range between about 0.1 to about 300 microns, as disclosed in U.S. Patent Nos. 4,710,384 and 5,354,556, which are incorporated herein by reference in their entireties.
  • Each of the particles is in the form of a micromatrix, with the active ingredient uniformly distributed throughout the polymer.
  • Sustained release formulations such as those described in U.S. Patent No. 4,710,384, which is incorporated herein by reference in its entirety, having a relatively high percentage of plasticizer in the coating in order to permit sufficient flexibility to prevent substantial breakage during compression are disclosed.
  • the specific amount of plasticizer varies depending on the nature of the coating and the particular plasticizer used. The amount may be readily determined empirically by testing the release characteristics of the tablets formed. If the medicament is released too quickly, then more plasticizer is used. Release characteristics are also a function of the thickness of the coating. When substantial amounts of plasticizer are used, the sustained release capacity of the coating diminishes. Thus, the thickness of the coating may be increased slightly to make up for an increase in the amount of plasticizer.
  • the plasticizer in such an embodiment will be present in an amount of about 15 to 30 % of the sustained release material in the coating, in one embodiment 20 to 25 %, and the amount of coating will be from 10 to 25% of the weight of the active material, and in another embodiment, 15 to 20 % of the weight of active material.
  • Any conventional pharmaceutically acceptable plasticizer may be incorporated into the coating.
  • sustained release pharmaceutical products can be formulated as a sustained and/or timed release formulation. All sustained release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non- sustained counterparts. Ideally, the use of an optimally designed sustained release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition. Advantages of sustained release formulations may include: 1) extended activity of the composition, 2) reduced dosage frequency, and 3) increased patient compliance. In addition, sustained release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the composition, and thus can affect the occurrence of side effects.
  • sustained release formulations are designed to initially release an amount of the therapeutic composition that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of compositions to maintain this level of therapeutic effect over an extended period of time.
  • the therapeutic composition In order to maintain this constant level in the body, the therapeutic composition must be released from the dosage form at a rate that will replace the composition being metabolized and excreted from the body.
  • the sustained release of an active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compounds are formulated as controlled release powders of discrete microparticles that can be readily formulated in liquid form.
  • the sustained release powder comprises particles containing an active ingredient and optionally, an excipient with at least one non-toxic polymer.
  • the powder can be dispersed or suspended in a liquid vehicle and will maintain its sustained release characteristics for a useful period of time. These dispersions or suspensions have both chemical stability and stability in terms of dissolution rate.
  • the powder may contain an excipient comprising a polymer, which may be soluble, insoluble, permeable, impermeable, or biodegradable.
  • the polymers may be polymers or copolymers.
  • the polymer may be a natural or synthetic polymer. Natural polymers include polypeptides (e.g., zein), polysaccharides (e.g., cellulose), and alginic acid. Representative synthetic polymers include those described, but not limited to, those described in column 3, lines 33-45 of U.S. Patent No.
  • the sustained release compositions provided herein may be formulated for parenteral administration, e.g., by intramuscular injections or implants for subcutaneous tissues and various body cavities and transdermal devices.
  • intramuscular injections are formulated as aqueous or oil suspensions.
  • the sustained release effect is due to, in part, a reduction in solubility of the active compound upon complexation or a decrease in dissolution rate.
  • Oils that may be used for intramuscular injection include, but are not limited to, sesame, olive, arachis, maize, almond, soybean, cottonseed and castor oil.
  • a highly developed form of drug delivery that imparts sustained release over periods of time ranging from days to years is to implant a drug-bearing polymeric device subcutaneous Iy or in various body cavities.
  • the polymer material used in an implant which must be biocompatible and nontoxic, include but are not limited to hydrogels, silicones, polyethylenes, ethylene-vinyl acetate copolymers, or biodegradable polymers.
  • the activity of the compounds provided herein for anti-cancer activity and/or cell selectivity may be measured in standard assays, e.g., in vitro cell proliferation or cell death, apoptosis, cellular uptake, and oxidative stress assays, including those described in the Examples herein.
  • the methods include administering one or more of the compounds described herein, or a pharmaceutically acceptable salt or derivative thereof, or a composition or pharmaceutical composition comprising the same, to a mammal, e.g., a human, cat, dog, horse, pig, cow, sheep, mouse, rat, or monkey.
  • a mammal e.g., a human, cat, dog, horse, pig, cow, sheep, mouse, rat, or monkey.
  • the compound is one according to Formula I, as described above.
  • the compound administered is chaetocin, or a pharmaceutically acceptable salt or derivative thereof, having the structure as set forth in FIG. 8.
  • the compound administerered is chaetomin or gliotoxin, or a pharmaceutically acceptable salt or derivative thereof, having the structures as set forth in FIG. 8.
  • the compound administered comprises a bridged disulfide thiodioxopiperazine ring.
  • the compound administered is selected from melinacidin IV, sirodesmin, hyalodendrin, sporidesmin A, leptosin, emestrin, dithiosilvatin, epicorazine, emethallicin, verticillin A and B, 19 methyl- 19'deoxy-6,6'dihydroxychaetocin, aranotin, apoaranotin, and scabrosin, or a pharmaceutically acceptable salt or derivative thereof.
  • a method for treating or ameliorating a cancer can include administering to a mammal a compound according to Formula II:
  • R, R', R 2 , and R 2 ' independently, can be selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkoxy, hydroxy, aryl, heteroaryl, heteroalkyl, heterocyclyl, halo, pseudohalo, carboxy, haloalkyl, hydroxyalkyl, alkaryl, aralkyl, aminocarbonyl, aminoalkyl, alkylcarbonyl, arylcarbonyl, alkoxy, alkylthio, aryloxy, and arylthio; or wherein R and R 2 , and/or R' and R 2 ', independently, together to which the atoms to which they are attached, form a fused heterocyclic ring system which is optionally substituted with one or more alkyl, cycloalkyl, alkoxy, aryl, hydroxy, heteroaryl, heteroalkyl, heterocyclyl, halo, pseudohal
  • the cancer can be multiple myeloma.
  • the cancer can be another B-lymphocyte lineage disorder, such as MGUS, amyloidosis or a leukemia or lymphoma.
  • the cancer can be a solid tumor.
  • the symptoms or disorders associated with multiple myeloma include one or more of the following: production of monoclonal proteins in serum and/or urine, destructive boney lesions, renal failure, hypercalcemia, loss of appetite, fatigue, muscle weakness, restlessness, difficulty in thinking or confusion, constipation, increased thirst, increased urine production, nausea and vomiting, pain, e.g., in the lower back and ribs, anemia, primary and recurrent infections, neuropathy, pneumonia, and hyperviscosity of the blood.
  • a cell can be exposed to or contacted with a compound or composition described herein to induce oxidative stress. Exposure or contact can be in vivo or in vitro. Oxidative stress can be monitored using known assays, including fluorescent (e.g. FACS) assays, e.g., as described below.
  • fluorescent e.g. FACS
  • any of the compounds or compositions can be used to induce cell death or apoptosis in a cell, e.g., a cancerous cell; apoptosis/cell death can be monitored as described herein or by standard assays known to those having ordinary skill in the art.
  • Reagents Chaetocin, doxorubicin, dexamethasone, apicidin, trichostatin A, reduced glutathione, N-acetyl cysteine, H 2 O 2 , aphidicolin, DRB (5,6-dichloro-l-beta-D- ribofuranosylbenzimidazole), and cycloheximide were purchased from Sigma (St. Louis, MO); and tetramethylrhodamine methyl ester (TMRM) from Invitrogen (Carlsbad, CA).
  • TMRM tetramethylrhodamine methyl ester
  • Cell culture Cells were cultured in the following media: A549 (obtained from ATCC, Chicago IL) in RPMI 1640 containing 5% (v/v) FBS; myeloma cell lines KAS6/1 0CI-MY5, MM1S/4 and RPMI 8226 in RPMI 1640 containing 10% FBS; patient bone marrow cells in MEM containing 20% FBS; KAS6/1 cells were supplemented with 1 ng/mL IL-6. All media contained 100 LVmL penicillin G, 100 ⁇ g/mL streptomycin and 2 mM 1-glutamine. Cells lines were passaged twice weekly and maintained at 37°C in an atmosphere containing 95% air-5% CO 2 (v/v).
  • Patient bone marrow cells were collected via posterior superior iliac crest bone marrow aspiration under local anesthesia in accord with approved Mayo Clinic IRB protocols.
  • Patient bone marrow leukocytes were divided into myeloma (CD 138+) and non-myeloma/normal leukocyte (CD 138-) fractions employing sorting using magnetic bead technology in kit form (MACS CD 138 microbeads, Miltenyi Biotech, Auburn, CA). Sorted cells were plated in a 96-well tissue culture plate at a concentration of 5x10 5 cells and dosed with indicated drug concentrations for 24 hours. Survival was assessed using a trypan blue exclusion assay.
  • FACS analyses assessing the effects of chaetocin on marrow cell populations was accomplished using annexin V (Caltag Laboratories, Burlingame, CA) and 7 amino- actinomycin D (7-AAD, Calbiochem, San Diego, CA) staining to identify viable, apoptotic and dead cells.
  • annexin binding buffer 0.15 M NaCl, 0.0033 M CaCl 2 HEPES buffer, pH 7.4; IXlO 6 cells/100 ⁇ L
  • ABB annexin binding buffer
  • IXlO 6 cells/100 ⁇ L was added to each of two tubes along with 5 ⁇ L each anti-annexin V fitc, anti-CD38 pe (phycoerythrin), and anti-CD45 ape in one tube; and anti-CD56 fitc, anti-CD38 pe, and anti-CD45 ape in the second.
  • Multivariate analysis using the typical CD45-, bright CD38 gating and FSC vs. SSC identified the viable, apoptotic and dead fractions of examined cell populations.
  • B-cell chronic lymphocytic leukemia (B-CLL) cells were obtained from patients with persistent lymphocytosis of >5000 lymphocytes/mm 3 and a CD5+, dim surface Ig expression and monoclonal K or ⁇ expression.
  • Peripheral blood mononuclear cells (PBMCs) isolated from heparinized blood by Ficoll (Gallard-Schlesinger Industries, Inc., Plainview, NY) density gradient centrifugation were washed twice with normal saline, counted using a Vi Cell XR cell viability analyzer (Beckman Coulter, Fullerton, CA), resuspended to 100 million/mL in PBS with 2% FBS; and the B-cell population was isolated using the Human B Cell Enrichment Kit (without CD43 depletion; StemCell Technologies, Vancouver, BC, Canada) in conjunction with a RoboSep Fully Automated Cell Separator (StemCell Technologies).
  • PBMCs Peripheral blood mononucle
  • Apoptosis was assessed using transmission electron microscopy and Hoechst 33258 staining using fluorescence microscopy by examining cells for apoptotic morphological changes, expressing number of apoptotic cells at a percentage of 200 total counted cells, as previously described (Bible KC and Kaufmann SH, Flavopiridol (NSC 649890, L86-8275): A cytotoxic flavone that induces death in non-cycling A549 human lung carcinoma cells. Cancer Res. 1996;56:4856-4861).
  • Immunob lotting Cells grown in suspension culture at densities of 3-6 x 10 5 cells/mL and treated as indicated were washed three times with PBS, lysed in lysis buffer (50 mM Tris-HCl pH 8.0, 400 mM NaCl, 0.5% NP-40, 10% glycerol, ImM EDTA supplemented immediately before use with 10 ng/ ⁇ L pepstatin A, 500 ⁇ M PMSF, 10 ng/ ⁇ L leupeptin, 10 ng/ ⁇ L aprotinin, and 200 ⁇ M sodium orthovanidate), and processed for SDS-polyacrylamide gel electrophoresis and subsequent immunoblotting for acetylated histone H3 (Cell Signaling Technology, Beverly, MA), poly ADP-ribose polymerase (PARP; BD PharMingen, San Diego, CA) and actin (Sigma, St. Louis, MO).
  • lysis buffer 50 mM Tris-HCl pH 8.0, 400 mM Na
  • Tumor cells treated with indicated chaetocin concentrations were washed twice with ice cold PBS, immediately solubilized in 0.5 M perchloric acid, and evaluated "real time" via HPLC using Beckman System Gold Wunsch Software with a dual pump 125 gradient pump system, 507e autosampler, 168 diode array detector, and Beckman Ultrasphere ODS column (4.6 mm xl5 mm x 7 ⁇ m) using the following elution profile: 100% water to 100% methanol linear gradient over 40 min followed by a 10 min period of elution with 100% methanol.
  • Cellular oxidative stress was assessed utilizing 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate or hydroethidium (Molecular Probes/Invitrogen, Carlsbad, CA) as cell permeable fluorescent probes and FACS analyses. Briefly, cells were treated with the indicated drug for 24 hours, incubated in media containing 5.4 ⁇ g/ml probe at 37 0 C for 15 min, sedimented and resuspended in PBS before flow microfluorimetry (FACScan flow cytometer; Becton Dickinson, Mountain View, CA) with a 488 nm laser. Fluorescence emission was observed through a 530/30 nm filter, and 20,000 events were analyzed using CellQuest software (Verity Software House, Topsham, ME).
  • mice utilized the following formulation: 0.06 mg/mL chaetocin in 20%PEG400 and 79.14% 0.9N NaCl with 0.86% DMSO as a cosolvent. This system retains in vitro activity in cell culture models.
  • Reagents Chaetocin, ⁇ -Nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt hydrate (NADPH), rat liver thioredoxin reductase, 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB), oxidized glutathione (GSSG), bovine insulin, gliotoxin, thioredoxin reductase assay kit, anti-actin antibody and CelLytic lysis reagent were purchased from Sigma (St. Louis, MO); yeast glutathione reductase and Complete Protease Inhibitor Tablets from Roche (Indianapolis, IN); oxidized E.
  • HeLa cells were obtained from ATCC.
  • DTNB method Cell-free thioredoxin reductase activity was assayed in 100 mM potassium phosphate (pH 7.0), 10 mM EDTA according to the Sigma kit protocol. Final concentrations were 0.0005 U/ ⁇ L of enzyme and 0.24 mM NADPH in the presence of chaetocin as indicated in a 100 ⁇ L reaction. The reaction was started by the addition of DTNB (3 mM) and the change in absorbance at 405 nm was monitored in a plate reader. Activity was calculated as the increase in absorbance between 2 and 5 min after DTNB addition.
  • Glutathione Reductase Activity Assay Cell-free glutathione reductase activity was assayed in 100 mM potassium phosphate (pH 7.0), 10 mM EDTA. The 200 ⁇ L reaction mixture comprised 0.00006 U/ ⁇ L of enzyme, 0.75 mM DTNB, 0.1 mM NADPH and varying concentrations of chaetocin as indicated. The reaction was started by addition of oxidized glutathione (1 mM) and was monitored in a plate reader at 405 nm. Activity was calculated as the increase in absorbance between 1 and 3 min after glutathione addition.
  • Thioredoxin Reductase Activity Assay (gel-based oxidation state of thioredoxin method): Reduction of thioredoxin by thioredoxin reductase was measured by the decrease in electrophoretic mobility caused by covalent modification of thioredoxin (by AMS) when the disulfide is reduced.
  • the reaction mix contained 100 mM potassium phosphate (pH 7.0), 10 mM EDTA, 0.24 mM NADPH, chaetocin or other compounds as indicated, 50 ⁇ M oxidized thioredoxin and 0.0002 U/ ⁇ L thioredoxin reductase (except for the initial rate K 1 experiment, which contained 0.00005 U/ ⁇ L thioredoxin reductase).
  • a 5 ⁇ L sample was removed and immediately added to 5 ⁇ L of 30 mM AMS in TE buffer (pH 7.5).
  • the AMS was allowed to react (15 min at 22 0 C) with reduced thioredoxin sulfhydryl groups, 18 then the samples were mixed with non-reducing sample buffer and were electrophoresed on 18% Tris-HCl SDS-PAGE gels. The gels were stained with Coomassie blue and bands were imaged and quantitated using a Syngene InGenius gel documentation system (Frederick, MD).
  • the 100 ⁇ L reaction contained 100 mM potassium phosphate (pH 7.0), 2 mM EDTA, 0.13 mM bovine insulin, 3.9 ⁇ M E. coli thioredoxin and chaetocin as indicated.
  • the reaction was initiated by addition of 0.33 mM DTT, and turbidity was monitored at 620 nm in a plate reader. The initial linear rate was calculated based on the slope of the line after an increase in absorbance (indicating precipitation of insulin) started to occur. 19
  • the 100 ⁇ L reaction consisted of 100 mM potassium phosphate (pH 7.0), 10 mM EDTA, 0.0004 U/ ⁇ L thioredoxin reductase and chaetocin or other compounds as indicated.
  • the assay was carried out in a 96-well quartz plate and the oxidation of NADPH was measured as a change in absorption at 340 nm.
  • the velocity versus concentration data were then analyzed using SigmaP lot's Enzyme Kinetics 1.3 program (Systat Software, San Jose, CA).
  • Fractions were step- eluted off the Zip Tip using 10 ⁇ l of 1% acetic acid in water containing 20, 30, 40, or 60% acetonitrile. Collected fractions were injected by loop injection (2 ⁇ l) directly into the mass spectrometer using a mobile phase of 30% acetonitrile: 69% water: 1% acetic acid at 5 ⁇ l/min. Mass spectra were collected on an Agilent Technologies LC/ MSD-TOF mass spectrometer in positive electrospray ionization mode over a m/z range of 400 to 1500. The capillary, fragmentor, skimmer, and OCT RF voltages (3500, 185, 60, 200 volts, respectively) were optimized to enhance signal and minimize instrument fragmentation.
  • HeLa cells were cultured in RPMI 1640 containing 5% FBS and 2 mM L-glutamine. Cells were passaged twice weekly and maintained in 37 0 C in an atmosphere containing 95% air-5% CO 2 (vol/vol).
  • 10 5 cells per well were plated in 12-well plates and were transfected with 1 ⁇ g pcDNA or pcDNA-Trx 20 ' 21 using standard Lipofectamine-Plus procedures. Transfection efficiency based on cells transfected with GFP was 80%. Twenty four hrs after transfection the cells were treated with either DMSO, 100 nM chaetocin or 100 nM doxorubicin for 24 hrs.
  • Chaetocin has structural similarities to histone deacetylase inhibitors (HDACIs), yet does not appreciably alter cellular levels of acetylated histone H3 at cytotoxic concentrations.
  • HDACIs histone deacetylase inhibitors
  • chaetocin bears a high degree of structural similarity to the acetylated histone lysine moiety mimicked by many HDACIs ((Colletti SL, Myers RW, Darkin- Rattray SJ, et al, Broad spectrum antiprotozoal agents that inhibit histone deacetylase: structure-activity relationships of apicidin, Part 1 : Bioorg Med Chem Lett. 2001 ; 11 : 107- 11); Fig.
  • HDAC inhibition accompanying chaetocin-induced cytotoxicity was probed.
  • treatment of A549 human small cell lung cancer cells with chaetocin produced increased acetylated histone H3 at high nanomolar concentrations (Fig. ID)
  • treatment of U266, KAS 6/1 or 0CI-MY5 human myeloma cells had no effects on cellular levels of acetylated histone H3 (Fig. IE and data not shown).
  • chaetocin-induced HDAC inhibition was observed in some cell lines, it was not observed in tested myeloma lines at cytotoxic concentrations.
  • Chaetocin kills myeloma cells in vitro via induction of morphological apoptosis accompanied by DNA ladder formation and PARP cleavage.
  • In vitro anti-myeloma activity of chaetocin was associated with induction of apoptotic morphological changes as assessed by electron microscopy (Fig. 2A), Hoechst 33258 staining (Fig. 2B), loss of mitochondrial membrane potential (Fig. 2B), PARP cleavage (Fig. 2C), and DNA ladder formation (Fig. 2D).
  • Chaetocin selectively kills freshly collected sorted patient CD 138+ myeloma cells with superior efficacy to the commonly utilized anti-myeloma agents dexamethasone and doxorubicin.
  • Rigorous studies were undertaken employing freshly collected sorted CD 138+ myeloma cells from 12 patients in accord with an approved IRB protocol, using matched negatively sorted patient CD138- bone marrow leukocytes as controls.
  • chaetocin demonstrated dramatic anti-myeloma activity while largely sparing matched (CD138-) normal bone marrow leukocytes (Fig. 3A-C; representative results from three patients shown).
  • the anti-myeloma effects of chaetocin were uniformly superior to those produced by the first- line anti-myeloma agents doxorubicin and dexamethasone under identical conditions (Fig. 3A-C). Furthermore, the potent and selective anti-myeloma effects of chaetocin were observed in samples obtained from patients afflicted with a broad range of different myeloma subtypes, including smoldering myeloma and heavily treated myeloma post-peripheral blood stem cell transplantation; as well as in myeloma cells demonstrating a wide array of cytogenetic abnormalities.
  • chaetocin appears not to be cross resistant to dexamethasone- resistant myeloma cell lines or patient samples.
  • RPMI 8226 paired doxorubicin-sensitive and doxorubicin-resistant myeloma cells were examined, there was moderate cross resistance to chaetocin (data not shown).
  • PgP P-glycoprotein
  • Chaetocin potently kills patient myeloma cells when treated in mixed bone marrow cultures. Over concern that the observed cytotoxic effects of chaetocin might reflect an artifact seen only in sorted myeloma cells, chaetocin-induced cytotoxicity in myeloma cells and neutrophils/monocytes treated in mixed bone marrow culture was evaluated. Unsorted bone marrow cells obtained from relapsed myeloma patients were treated with chaetocin (100 nM) or diluent in mixed culture (24 hours), with the cytotoxic effects of chaetocin evaluated using multi-channel FACS analyses. Consistent with the selectivity observed in sorted cells, chaetocin also killed myeloma cells with selectivity in comparison to control patient neutrophils/monocytes in this mixed culture system (Fig. 4A-C).
  • Chaetocin has in vivo anti-myeloma activity. Preliminary in vivo experiments using chaetocin (0.25 mg/kg intraperitoneally twice weekly, RPMI 8226 SCID mouse model; flank xenografts) demonstrated antiproliferative activity (p ⁇ 0.05), with T/C values in the 50-60% range in response to three weeks of treatment of established tumors (Fig. 4D).
  • Glutathione pretreatment attenuates chaetocin-induced cytotoxicity and impairs cellular accumulation of chaetocin.
  • glutathione the primary intracellular reductant
  • NAC N-acetyl cysteine
  • aphidicolin an inhibitor of DNA synthesis
  • DRB an inhibitor of RNA synthesis
  • cycloheximide an inhibitor of protein synthesis
  • Differential chaetocin-induced cytotoxicity observed between patient myeloma cells and paired normal patient leukocytes may be attributable to a relative hypersensitivity of myeloma cells to imposed oxidative stress. Having found no indication that the heightened cytotoxicity of chaetocin in myeloma cells in comparison to matched normal leukocytes might be attributable to increased intracellular accumulation of chaetocin in the more sensitive myeloma cells, the inherent susceptibility of myeloma cells to the cytotoxic effects of oxidative stressors was examined.
  • the observed selective cytotoxicity of chaetocin in myeloma cells therefore appears, at least in part, to be attributable to a generally heightened susceptibility of myeloma cells to oxidative stressors.
  • Chaetocin has potent and selective anti-cancer activity in solid tumor cell lines. Many solid tumor cell lines are readily killed by chaetocin (Fig. 10). Furthermore, chaetocin has been demonstrated to kill thyroid cancer cell lines more readily (selectively) than normal thyrocytes (normal thyroid cells, Fig. 12). This indicates that chaetocin has selectivity for cancerous cells in at least some solid tumor models.
  • Chaetocin is effective in killing even highly chemotherapy-resistant cancer cells and cell lines. Having seen that chaetocin readily kills freshly collected patient myeloma cells that are largely resistant to dexamethasone or doxorubicin (see Fig. 3A-C), the effects of chaetocin in dexamethasone- or doxorubicin-resistant myeloma cell lines were assessed. In particular, dexamethasone -resistant MMIR L cells were found to be equally sensitive to chaetocin-induced cytotoxicity as were dexamethasone-sensitive MMlS cells, despite maintaining a high level of resistance to dexamethasone (Fig.
  • Chaetocin inhibits thioredoxin reductase but not glutathione reductase or thioredoxin. Since chaetocin (FIG. 8, FIG. 14A) contains two disulfide bonds and is known to induce oxidative stress in cancer cell, as shown above, chaetocin might interact with oxidative stress-related proteins that rely upon disulfide bond redox cycling for activity. Initial experiments showed that chaetocin inhibited TrxRl -initiated turnover of the synthetic substrate DTNB (Reaction 1) in a cell-free assay in a dose-responsive manner (FIG. 14), with an IC 50 of about 4 ⁇ M.
  • TrxRl 1 TrxRl 1 ) DTNB + NADPH + H ⁇ 2TNB + NADP
  • TrxRl is a major downstream effector substrate of TrxRl - and because Trx itself is a disulfide-containing reductase. Since chaetocin inhibits TrxRl activity, however, an activity assay based on insulin reduction (Reaction 3) was utilized that did not rely on the coupled TrxRl/Trx reaction.
  • Chaetocin and related thiodioxopiperazines inhibit the reduction of thioredoxin by thioredoxin reductase.
  • DTNB is not the native substrate for thioredoxin reductase
  • chaetocin might also impair the ability of thioredoxin reductase to reduce its native substrate, thioredoxin (Reaction 4).
  • a novel gel-based kinetics assay was developed to resolve the oxidized and reduced forms of Trx by rapid covalent modification of the free sulfhydryl groups of Trx with AMS. Using this method, chaetocin was indeed also found to inhibit the ability of TrxRl to reduce Trx (FIG.
  • Chaetocin and related thiodioxopiperazines serve as substrates for thioredoxin reductase. Chaetocin, which itself contains two disulfides, might inhibit thioredoxin reductase by serving as a competative substrate for TrxRl . Consistent with this possibility, NADPH is indeed oxidized over time when chaetocin is substituted for Trx in Reaction 4, with a K m for chaetocin of 4.6 ⁇ 0.6 ⁇ M; indicative of substrate functionality (FIG. 16A).
  • Trx the K m for Trx in this same assay is 104.7 ⁇ 26 ⁇ M, almost 25 times higher than that of chaetocin, intimating that chaetocin effectively serves as a more efficient substrate for TrxRl than its native substrate, Trx - consistent with the hypothesis that the ability of chaetocin to serve as a TrxRl inhibitor (FIG. 14) most likely relates to its function as a competitive TrxRl substrate.
  • the related compounds gliotoxin and chetomin have K m 's of 16.0 ⁇ 5.0 and 16.1 ⁇ 5.4 ⁇ M, respectively (FIG. 18, Table 1), indicating that related thiodioxopiperazines are also TrxRl substrates, but that chaetocin is the highest affinity TrxRl substrate of the series.
  • Transient thioredoxin overexpression attenuates chaetocin-induced cell death.
  • TrxRl substrate Trx was transiently overexpressed, since Trx is the principal downstream enzyme affected by inactivation of TrxRl and thereby ultimately functions as the primarily affected cellular ROS-scavenger.
  • Trx overexpression significantly attenuated chaetocin-induced cell death (FIG. 17A), consistent with a linkage between the ability of chaetocin to inhibit the reduction of Trx by TrxRl and its cytotoxic effects.
  • myeloma is an incurable cancer characterized by the clonal proliferation of B-cell lineage plasma cells resulting in the production of monoclonal proteins in serum and/or urine, destructive boney lesions, and the deaths of about 12,000 individuals in the U.S. alone annually.
  • increasing numbers of therapeutics are becoming available to treat this disease— with the potential for significant symptom palliation, induction of disease responses and prolongation of disease-free survival— available therapeutic approaches including peripheral blood stem cell transplantation and newer agents have, unfortunately, had only modest impact on patient overall survival in several randomized trials. As a consequence, there is still a need for improved anti-myeloma therapies.
  • chaetocin represents a promising agent for further development as an anti-myeloma therapeutic. Not only does chaetocin have potent in vitro anti-myeloma activity (Figs. IB and C, 2), but also striking ex vivo potency and selectivity (Fig. 3A-C and 4); as well as in vivo efficacy (Fig 4D). Moreover, studies of chaetocin in dexamethasone- or doxorubicin-resistant myeloma cell lines indicated that several highly drug-resistance myeloma lines were largely non-cross-resistant to chaetocin (Fig. 13).
  • chaetocin exhibits striking selectivity in killing myeloma cells even in comparison to closely lineage-related normal and neoplastic B-lymphocytes (Fig. 3D and E). Additionally, the striking anti-myeloma activity of chaetocin was observed in samples obtained from patients afflicted with many types of myeloma, including smoldering myeloma as well as heavily pretreated myeloma patients who had previously undergone peripheral blood stem cell transplantation.
  • chaetocin-induced cytotoxicity may rely upon both intracellular drug accumulation (Fig. 5) and upon induction of cytotoxic oxidative stress upon cell entry (Figs. 7 A-F).
  • the ability of chaetocin to selectively kill myeloma cells appears may be based upon a generally increased susceptibility of myeloma cells to oxidative stressors (Fig. 7 G, H).
  • chaetocin While not being bound by theory, the present studies of chaetocin also indicate that intact chaetocin disulfide bonds may be required for its cellular entry, and therefore for its cytotoxicity (Fig. 4D). Unlike gliotoxin, intracellular reduction of chaetocin disulfides does not appear to take place to any appreciable extent (Fig. 5A). It therefore appears that intracellular reduction of thiodioxopiperazines disulfide bonds may not be required for the maintenance of high intracellular thiodioxopiperazine levels.
  • thiodioxopiperazine disulfides appear not to be directly responsible for cytotoxicity, as the gliotoxin disulfide apparently exists in the reduced state intracellularly, yet gliotoxin is still cytotoxic.
  • thiodioxopiperazine structural moieties other than intact disulfides may be important in the infliction of cytotoxicity once cellular entry is otherwise secured.
  • the quinone-like carbonyl residues of chaetocin may be important in the induction of cellular oxidative stress, much akin to that imposed by many true quinines.
  • the disulfides of thiodioxopiperazines may represent reactive sulfur species that are involved in the imposition of observed oxidative stress.
  • the results herein demonstrate that the thiodioxopiperazine natural product chaetocin has potent and selective in vitro, ex vivo and in vivo anti- myeloma activity that appears to require both intact chaetocin disulfides to facilitate intracellular accumulation as well as the infliction of cellular oxidative stress upon cell entry.
  • TrxRl a specific molecular target
  • TrxRl inhibition by chaetocin also linking TrxRl inhibition by chaetocin to its anticancer effects (FIG. 17).
  • TrxRl a specific molecular target
  • chaetocin might otherwise appear to be a classical inhibitor of TrxRl (FIGs. 14 and 15)
  • TrxRl K m TrxRl K m than that of the TrxRl native substrate Trx
  • TrxRl TrxRl native substrate Trx
  • chaetocin effectively spends more time associated with the enzyme, consequently serving as a noncovalent TrxRl inhibitor.
  • TrxRl regains its activity at high chaetocin concentrations at later time points (FIG. 15 A and B), when chaetocin is completely reduced by TrxRl and therefore no longer capable of TrxRl inhibition as demonstrated for related compounds lacking intact bridged disulfide bonds (FIG.
  • TrxRl contains two redox sites, a Cys59 - Cys64 active site pair, and a selenoCys 496' - Cys495' pair in the C-terminal region that interacts with the active site cysteine pair.
  • 3 Glutatione reductase and Trx each contain only solitary active sites, a cysteine-cysteine pair.
  • chaetocin Based on the ability of chaetocin to act as an substrate/inhibitor for TrxRl but not G.R. or Trx, it is interesting to postulate that chaetocin might primarily interact with the C-terminal selenoCys 496' - Cys495'.
  • TrxRl mutants lacking the selenocysteine active site would be required to further examine this possibility.
  • TrxRl contains two active sites, it is intriguing that the initial rate (v) versus concentration [S] kinetics plots were best fit by the Hill equation for chaetocin and Trx (FIG. 19). This sigmoidal v by [S] plot often indicates cooperative binding of substrate to the active site. This behavior is most common for substrates interacting with multimeric enzymes containing several interacting active sites, and has been described for TrxRl family members but not specifically for TrxRl itself to the best of the inventors' knowledge.
  • TrxRl by inhibiting TrxRl, chaetocin consequently attenuates otherwise normal levels of TrxRl redox cycling of its major downstream effector Trx (FIG. 15), thereby apparently compromising cellular ROS mitigation capacity, and thereby lending an explanation for the increased cellular ROS observed accompanying the exposure of cancer cells to chaetocin.
  • Trx overexpression the observation that chaetocin-induced cytotoxicity is attenuated by Trx overexpression in cancer cells (FIG. 17A) importantly establishes a potential linkage between TrxRl inhibition by chaetocin and chaetocin-induced ROS and anticancer activity. Because doxorubicin-induced cytotoxicity was unaffected by Trx overexpression (FIG.
  • TrxRl overexpression may in part represent an adaptive mechanism facilitating mitigation of otherwise cytotoxic higher basal ROS levels characteristic of many cancers, thereby making TrxRl overexpression required for survival in these cancers.
  • TrxRl may represent especially attractive target-directed therapeutics for TrxRl - overexpressing neoplasms.
  • TrxRl overexpression might be usable as a biomarker to define cancers most likely to respond to chaetocin therapy.
  • thioredoxin reductase is a cytotoxic molecular target of chaetocin of potential relevance and importance to its selective anticancer effects.
  • the data support a model (FIG. 17B) whereby chaetocin serves as a potent competitive substrate for the redox cycling enzyme thioredoxin reductase, competing with thioredoxin for reduction by TrxRl, and thereby serving to deplete levels of reduced cellular Trx, the critical ROS remediation substrate and downstream effector of TrxRl.
  • TrxRl competitively inhibit TrxRl - intimating that thiodioxopiperazines as a class may target TrxRl.

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Abstract

La présente invention concerne des compositions et des procédés permettant de traiter le cancer, y compris des cancers hématologiques tels que le myélome multiple. Dans certains modes de réalisation, il est possible d'utiliser la chaetocine, la chaetomine, ou la gliotoxine pour le traitement ou l'amélioration d'un ou de plusieurs symptômes ou troubles associés au myélome multiple.
PCT/US2007/080588 2006-10-05 2007-10-05 Procédés et compositions permettant de traiter le cancer Ceased WO2008112014A1 (fr)

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US8653080B2 (en) 2009-01-14 2014-02-18 Salk Institute For Biological Studies Methods for screening and compounds that protect against amyloid diseases
CN103717222A (zh) * 2011-02-24 2014-04-09 浙江大学 桥二硫二氧代哌嗪及其在治疗癌症方面的用途
US9856273B2 (en) 2012-10-22 2018-01-02 City Of Hope ETP derivatives
EP2909214A4 (fr) * 2012-10-22 2016-04-20 Hope City Dérivés d'étoposide
US9527868B2 (en) 2012-10-22 2016-12-27 City Of Hope ETP derivatives
CN104870452A (zh) * 2012-10-22 2015-08-26 希望之城 Etp衍生物
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CN108440571A (zh) * 2012-10-22 2018-08-24 希望之城 Etp衍生物
US10246471B2 (en) 2012-10-22 2019-04-02 City Of Hope ETP derivatives
CN106397459A (zh) * 2016-08-31 2017-02-15 中国科学院海洋研究所 硫代二酮哌嗪类化合物及其应用
CN106397459B (zh) * 2016-08-31 2018-07-13 中国科学院海洋研究所 硫代二酮哌嗪类化合物及其应用
US11584760B2 (en) 2016-09-15 2023-02-21 City Of Hope Dithio ETP derivatives
CN116473977A (zh) * 2023-05-12 2023-07-25 南充市中心医院 毛壳素在制备治疗食管鳞癌的药物中的应用

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