US20190117602A1 - Prevention and reversal of inflammation induced dna damage - Google Patents

Prevention and reversal of inflammation induced dna damage Download PDF

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US20190117602A1
US20190117602A1 US16/092,816 US201816092816A US2019117602A1 US 20190117602 A1 US20190117602 A1 US 20190117602A1 US 201816092816 A US201816092816 A US 201816092816A US 2019117602 A1 US2019117602 A1 US 2019117602A1
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ape1
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apx3330
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Mark R. Kelley
Jill Fehrenbacher
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Indiana University Research and Technology Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/282—Platinum compounds

Definitions

  • the present disclosure relates generally to methods of reducing neuronal sensitivity, thereby reducing inflammation and chronic pain. Particularly, it has been found herein that by enhancing the DNA base excision repair (BER) pathway, through the administration of APX3330, neuronal sensitivity to inflammatory mediators is reduced, thereby alleviating inflammatory or chronic pain.
  • BER DNA base excision repair
  • Inflammatory mediators released from damaged tissue and immune cells during damage, can have acute and chronic effects on the sensitivity of primary sensory neurons.
  • Prostaglandins, bradykinin, histamine, serotonin, tryptases, cytokines, and ATP can alter the sensitivity of sensory neurons to various stimuli via posttranslational modifications of ion channels that contribute to the depolarization of sensory neurons (see Richardson and Vasko, 2002).
  • These inflammatory mediators enhance kinase activity, resulting in the phosphorylation and modulation of ion channels to alter neuronal sensitivity.
  • Peripheral sensitization is a key component of inflammatory diseases and chronic pain syndromes.
  • This sensitization manifests as hyperalgesia and allodynia in humans and as hypernociception in animal models of pain.
  • acute hypersensitivity after injury is an important component of the inflammatory response that aids in protecting the injury, there is oftentimes a maintenance of this hypersensitivity beyond the time required for tissue repair.
  • ROS reactive oxygen
  • RNS nitrogen species
  • the repair of DNA damage is critical for the maintenance of neuronal homeostasis (Brooks, 2002, McMurray, 2005, Fishel et al., 2007a, Hetman et al., 2010), as endogenous metabolic activity, oxidative stress secondary to injury (Kruman and Schwartz, 2008), environmental toxins, (Kisby et al., 1999) and drugs (Ahles and Saykin, 2007) all can cause neuronal DNA damage.
  • Neurons contain the major DNA repair pathways including base excision repair (BER), nucleotide excision repair, mismatch repair, direct damage repair, and nonhomologous end-joining or homologous recombination (Fishel et al., 2007b, Barzilai et al., 2008, Fortini and Dogliotti, 2010).
  • the BER pathway repairs DNA damage in the nucleus and mitochondria, caused by oxidative damage to bases, alkylation of bases, or deamination, and is likely the most important repair pathway for protecting neurons.
  • the first step in BER is removal of the incorrect or damaged base by a DNA glycosylase.
  • the second step in the BER pathway involves the enzyme APE1, which hydrolyzes the phosphodiester backbone immediately 5′ to an apurinic/apyrimidinic (AP) site. This generates a normal 3′-hydroxyl group and an abasic deoxyribose-5-phosphate, which is processed by subsequent enzymes of the BER pathway.
  • APE1 hydrolyzes the phosphodiester backbone immediately 5′ to an apurinic/apyrimidinic (AP) site. This generates a normal 3′-hydroxyl group and an abasic deoxyribose-5-phosphate, which is processed by subsequent enzymes of the BER pathway.
  • the present disclosure provides insight into the pathway by which inflammatory mediators sustain changes in neuronal sensitivity and highlights the enhancement of neuronal DNA repair as a pharmacological target to alleviate inflammatory and/or chronic pain. Further, the present disclosure provides a compound, APX3330, to enhance DNA repair and reduce neuronal sensitivity.
  • the present disclosure relates generally to methods of reducing neuronal sensitivity, thereby reducing inflammatory and chronic pain. Particularly, it has been found herein that by enhancing the DNA base excision repair (BER) pathway, through the administration of APX3330 (and/or analogs thereof), neuronal sensitivity to inflammatory mediators is reduced, thereby alleviating inflammatory or chronic pain.
  • BER DNA base excision repair
  • the present disclosure is directed to a method of reducing neuronal sensitivity in a subject in need thereof.
  • the method comprises administering to the subject an effective amount of an apurinic/apyrimidinic endonuclease 1 redox factor 1 (APE1/Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, which selectively inhibits the amino terminal portion of APE1.
  • APE1/Ref-1 apurinic/apyrimidinic endonuclease 1 redox factor 1
  • the present disclosure is directed to a method of treating inflammation and chronic pain in a subject in need thereof.
  • the method comprises administering to the subject an effective amount of an apurinic/apyrimidinic endonuclease 1 redox factor 1 (APE1/Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, which selectively inhibits the amino terminal portion of APE1.
  • APE1/Ref-1 apurinic/apyrimidinic endonuclease 1 redox factor 1
  • the present disclosure is directed to a method of enhancing neuronal DNA repair function in a subject in need thereof.
  • the method comprises administering to the subject an effective amount of an apurinic/apyrimidinic endonuclease 1 redox factor 1 (APE1/Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, which selectively inhibits the amino terminal portion of APE1.
  • APE1/Ref-1 apurinic/apyrimidinic endonuclease 1 redox factor 1
  • FIGS. 1A-1C shows that DNA damage is enhanced in the lumbar DRG following hindpaw inflammation.
  • FIG. 1A is a representative western blot of pH2A.X and vinculin (loading control) expression in contralateral and ipsilateral L4/L5 DRG 5 days following unilateral CFA injection into the rat hindpaw.
  • FIG. 1B depicts the mean ⁇ SEM of the density of pH2A.X from 6 experiments normalized to the amount of vinculin.
  • An * indicates a statistically significant increase in the DRG ipsilateral to CFA injection compared to those contralateral to the injection (p ⁇ 0.05, t-test).
  • FIG. 1C are photomicrographs (20 ⁇ ) of pH2A.X in L5 DRG from a rat 5 days after CFA injection. Green fluorescence indicates the immunoreactivity to pH2A.X.
  • FIGS. 2A & 2B show that DNA damage is enhanced in neuronal cultures in a time-dependent manner following exposure to inflammatory mediators.
  • FIG. 2A are representative western blots for pH2A.X and vinculin (loading control) from cultures grown in the absence or presence of LPS or MCP-1 for the indicated time periods.
  • FIG. 2B shows the mean ⁇ SEM of pH2A.X band density normalized to that of vinculin following treatment with 1 ⁇ g/ml LPS (light bars) or 100 ng/ml MCP-1 (dark bars).
  • FIG. 3 depicts that the CGRP release from neuronal cultures was altered following exposure to inflammatory mediators.
  • Columns represent the mean ⁇ SEM of CGRP release stimulated by a 10-minute exposure to 30 nM capsaicin following a 24-hour exposure to increasing concentrations of LPS (light bars) or MCP-1 (dark bars).
  • An * indicates a significant difference from release in the absence of LPS or MCP-1, one-way ANOVA with Dunnett's posttest, p ⁇ 0.05.
  • FIGS. 4A & 4B depict that the changes in DNA damage and stimulated CGRP release following exposure to LPS or MCP-1 were reversed by antagonists to the TLR4 (LPS) and CCR2 (MCP-1 and LPS).
  • Columns represent the mean ⁇ SEM of pH2A.X expression ( FIG. 4A ) or CGRP release stimulated by a 10-minute exposure to 30 nM capsaicin ( FIG. 4B ) following a 24 hr exposure to 3 ⁇ g/ml LPS (light bars) or 0.3 ⁇ g/ml MCP-1 (dark bars) in the absence or presence of LPS-RS or RS 50493, as indicated.
  • An * indicates a significant difference from DNA damage or release in the absence of LPS or MCP-1, one-way ANOVA with Dunnett's posttest, p ⁇ 0.05.
  • FIGS. 5A-5D depict that the effects of LPS to include DNA damage and inhibit CGRP release were reversed by increasing APE1-mediated DNA repair.
  • FIG. 5A shows the treatment schema.
  • FIG. 5B are representative western blots for pH2A.X, APE1, HA tag and vinculin (loading control) from cultures grown in the absence or presence of LPS for 24 hours following the indicated pretreatments.
  • FIG. 5C depicts pH2A.X densitometry. Each column represents the mean ⁇ SEM of pH2A.X band density normalized to that of vinculin induced by treatment with 3 ⁇ g/ml LPS following the indicated pretreatments in conjunction with SCsiRNA (light bars) or APE1siRNA (dark bars).
  • FIG. 5D depicts CGRP release. Each column represents the mean ⁇ SEM of CGRP release (expressed as % of total content) stimulated by capsaicin following treatment with 3 ⁇ g/ml LPS in the absence and presence of APE1 overexpression, as indicated, in conjunction with SCsiRNA (light bars) or APE1siRNA (dark bars).
  • An * indicates a significant difference in release compared to SCsiRNA-treated vector control, two-way ANOVA with Dunnett's multiple comparisons posttest, p ⁇ 0.05.
  • FIGS. 6A-6D depict that the effects of MCP-1 to induce DNA damage and augment CGRP release were reversed by increasing APE1-mediated DNA repair.
  • FIG. 6A depicts the treatment schema.
  • FIG. 6B are representative western blots for pH2A.X., APE1, HA tag and vinculin (loading control) from cultures grown in the absence or presence of MCP-1 for 24 hours following the indicated pretreatments.
  • FIG. 6C depicts pH2A.X densitometry. Each column represents the mean ⁇ SEM of pH2A.X band density normalized to that of vinculin induced by treatment with 3 ⁇ g/ml MCP-1 following the indicated pretreatments in conjunction with SCsiRNA (light bars) or APE1siRNA (dark bars).
  • FIG. 5D depicts CGRP release. Each column represents the mean ⁇ SEM of CGRP release (expressed as % of total content) stimulated by capsaicin following treatment with 3 ⁇ g/ml MCP-1 in the absence and presence of APE1 overexpression, as indicated, in conjunction with SCsiRNA (light bars) or APE1siRNA (dark bars).
  • An * indicates a significant difference in release compared to SCsiRNA-treated vector control, two-way ANOVA with Dunnett's multiple comparisons posttest, p ⁇ 0.05.
  • FIGS. 7A-7E depict the differential role of Ref-1/APE1 redox inhibition in sensory neurons vs. tumor cells.
  • FIG. 7A shows that, in tumor cells, Ref-1/APE1 redox inhibition has multiple downstream effects on tumor growth, survival, migration and tumor inflammation.
  • FIG. 7B shows that, in sensory neuron cells such as DRG neurons, the addition of APX3330 does not have a negative effect on the cells and promotes survival and functional protection through enhancement of Ref-1/APE1 DNA repair activity against oxidative DNA damaging agents (e.g. cisplatin, oxaliplatin) that invoked the DNA BER pathway.
  • oxidative DNA damaging agents e.g. cisplatin, oxaliplatin
  • FIG. 7C provides the treatment paradigm for investigation of the effects of cisplatin and APX3330 on DNA damage within DRG.
  • FIG. 7D are representative blots demonstrating pH2A.X immunoreactivity at D24 and D31.
  • FIG. 7E depicts the quantification of pH2A.X immunoreactivity.
  • An * indicates statistical significance between D18 and D24 ( FIG. 7E ) as determined by a one-way ANOVA with Tukey's posttest with p ⁇ 0.05.
  • a ⁇ indicates statistical significance between Veh/Veh group and the Veh/Cis group ( FIG. 7E ) as determined by a two-way ANOVA with Bonferroni's posttest with p ⁇ 0.05.
  • FIGS. 8A & 8B depict new chemical entities (NCE); E3330 analogs.
  • FIG. 8A is a schematic of E3330 and new compounds. Groups that were investigated include the Quinone series (A), 3-Position series (B), Alkyl sidechain series (C), and Carboxylic Acid/Amine series (D).
  • FIG. 8B depicts current new analogs with more potent Ref-1 redox inhibition.
  • FIGS. 9A-9D depict the identification and characterization of chemical analogues to APX3330 (E3330) targeting APE1 for the prevention of chemotherapy-induced peripheral neuropathy (CIPN).
  • FIG. 9A depicts results from redox inhibition assays of APX3330 and its chemical analogues.
  • FIG. 9B depicts the inhibition of NF ⁇ B binding of APX3330 and its chemical analogues.
  • FIG. 9C depicts the tumor cell killing ability of APX3330 and its analogues in a IMR32 cell line.
  • FIG. 9D depicts the tumor cell killing ability of APX3330 and its analogues in a SK-N-SH cell line.
  • FIG. 10 depicts EMSA and transactivation data of APX3330 and its chemical analogues.
  • FIGS. 11A & 11B depicts the pharmacokinetic profile of APX2009 in IMR32 cells ( FIG. 11A ) and in SK-N-SH cells ( FIG. 11B ).
  • FIGS. 12A & 12B depict that pretreatment with E3330 and APX2009, but not APX2007 or APX2032, attenuated cisplatin-induced cell death in sensory neuronal cultures.
  • FIG. 12A depicts survival of cells from cultures treated with various concentrations of drugs as indicated for 24 hours. Each column represents the mean ⁇ SEM of percent. Cell viability as measured by trypan blue exclusion was determined on day 14 in culture from 3 independent harvests. An * indicates significant difference in survival in after drug treatment compared to no drug treatment using ANOVA and Tukey's post hoc test.
  • FIG. 12A depicts survival of cells from cultures treated with various concentrations of drugs as indicated for 24 hours. Each column represents the mean ⁇ SEM of percent. Cell viability as measured by trypan blue exclusion was determined on day 14 in culture from 3 independent harvests. An * indicates significant difference in survival in after drug treatment compared to no drug treatment using ANOVA and Tukey's post hoc test.
  • FIG. 12A depicts survival of cells from cultures treated with various concentration
  • 12B depicts neuronal cultures exposed to vehicle (DMSO) or to 20 ⁇ M of E3330, APX2007, APX2009 or APX2032 APX drugs (as indicated) for 72 hours and to various concentrations of cisplatin for 24 hours.
  • DMSO vehicle
  • Each column represents the mean ⁇ SEM of the percent survival of cells as measured by trypan blue exclusion.
  • An * indicates significant difference in cultures not treated with cisplatin compared to cultures treated with the drug using ANOVA and Tukey's post hoc test.
  • FIGS. 13A-13D depict results of DNA repair assays of APX3330 chemical analogues (Inhibitor III ( FIG. 13A ); APX2007 ( FIG. 13B ); APX2009 ( FIG. 13C ; APX 2032 ( FIG. 13D )).
  • FIGS. 14A & 14B show that E3330 and APX2009 did not alter CGRP release from sensory neurons in culture, but attenuated the cisplatin-induced reduction in capsaicin-evoked release of CGRP.
  • Each column represents the mean ⁇ SEM of basal release (open columns) or capsaicin-stimulated release (shaded columns) of CGRP in fmol/well/min.
  • FIG. 14A depicts cultures exposed to medium or to 10 or 20 ⁇ M of the various drugs (as indicated) for 72 hours prior to release experiments.
  • FIG. 14B depicts cultures exposed to medium or to 10 or 20 ⁇ M of the various drugs (as indicated) for 72 hours and to cisplatin for 24 hours prior to release experiments.
  • An * indicates a significant difference in capsaicin-stimulated release compared to untreated cells using ANOVA and Tukey's post hoc test.
  • FIG. 15 shows that APX2009, but not APX2007 or APX2032, attenuated the cisplatin-induced phosphorylation of H2AX in sensory neuronal cultures.
  • the top panel shows representative Western blots of phospho-H2AX (pH2AX) and vinculin from cultures prior to and after 24 and 48 hours of exposure to 10 ⁇ M cisplatin. Cultures were exposed to DMSO as a vehicle control or to 20 ⁇ M APX2007, APX2009 or APX2032 for 72 hours before and during cisplatin treatment as indicated.
  • the bottom panel represents the mean ⁇ SEM of the densitometry of pH2AX expression normalized to vinculin from 3 independent experiments. An * indicates a statistically significant increase in pH2AX density in cells treated with cisplatin, whereas a indicates a significant change by drug compared to DMSO controls at the same time points using ANOVA and Tukey's post hoc test.
  • FIGS. 16A-16C show that APX2009 attenuated the oxaliplatin-induced toxicity of sensory neurons in culture.
  • FIG. 16A shows percent cells surviving after a 24 hour exposure to various concentrations of oxaliplatin. Each column represents the mean ⁇ SEM of percent cells surviving as measured by trypan blue exclusion after a 24 hour exposure to various concentrations of oxaliplatin as indicated. Cultures are treated for 72 hours with DMSO as a vehicle control (left) 10 ⁇ M APX2009 (center) or 20 ⁇ M APX2009 (right).
  • FIG. 16B depicts basal release of CGRP (open columns) or release stimulated by 30 nM capsaicin (shaded columns) in fmol/well/min.
  • FIG. 16C the top panel shows representative Western blots of phospho-H2AX (pH2AX) and vinculin from cultures prior to and after 24 and 48 hours of exposure to 30 ⁇ M oxaliplatin and DMSO or 20 ⁇ M APX2009 for 72 hours before and during cisplatin treatment as indicated.
  • the bottom panel represents the mean ⁇ SEM of the densitometry of pH2AX expression normalized to vinculin from 3 independent experiments.
  • An * indicates a statistically significant difference on oxaliplatin treated cultures compared to controls using ANOVA and Tukey's post hoc test.
  • FIGS. 17A-17C depict tumor, but not CAF, cell killing by APX2009 in PDAC 3D model.
  • FIGS. 17A & 17B depicts Pa03C (tumor cells (transduced with TdTomato) grown in 3D cultures in the presence and absence of CAFs (transduced with EGFP). Tumor cells alone and tumor cells with CAFs in spheroids are shown.
  • the middle and right quantitation graphs in FIGS. 17A & 17B show the tumor (middle) vs. CAF (right) intensity ( FIG. 17A ) and area ( FIG. 17B ).
  • Spheroids were treated with APX2009 and the area of intensity ( FIG. 17A ) and area ( FIG.
  • FIG. 17B Differences were determined using both Student's t test (vehicle control vs drug treatment at each dose) and one-way ANOVA and statistical differences were observed for the tumor alone or tumor co-cultured with CAFs (*p ⁇ 0.05, ** p ⁇ 0.01, ***p ⁇ 0.001). No differences were observed in CAFs treated with APX2009 from control.
  • the present disclosure relates generally to methods of reducing neuronal sensitivity, thereby reducing inflammation and chronic pain. Particularly, it has been found herein that by enhancing the DNA base excision repair (BER) pathway, through the administration of APX3330, neuronal sensitivity to inflammatory mediators is reduced, thereby alleviating inflammatory or chronic pain.
  • BER DNA base excision repair
  • the present disclosure includes administering to a subject in need thereof an effective amount of an APE1 inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, the APE1 inhibitor capable of interacting with the APE1 protein such to cause unfolding of the APE1 protein, inhibiting the ability of APE1 to interact with other proteins in the neurons or to perform its redox signaling function.
  • the APE1 inhibitor is 3-[(5-(2,3-dimethoxy-6-methyl1,4-benzoquinoyl)]-2-nonyl-2-proprionic acid, (hereinafter “E3330” or “3330” or “APX3330”), and/or its analogs (e.g., [(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylidene]-N,N-diethylpentanamide] (hereinafter “APX2009”), (2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronapthalen-2-yl)methylidene]-N,N-dimethylpentanamide] (hereinafter “APX2007”), (2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronapthalen-2-yl)
  • APX3330 may be found in Abe et al., U.S. Pat. No. 5,210,239, and information on APX2009 may be found in Kelley et al., J Pharmacol Exp Ther. 2016 November, 359(2): 300-309, each incorporated herein by reference to the extent they are consistent herewith.
  • APX3330 inhibits APE1 protein from interacting with other proteins in the neurons.
  • This interaction inhibition allows for APE1 to be free to perform enhanced DNA repair functions at an oxidized or abasic site in damaged DNA (damaged by inflammatory and other effectors of neuronal pain pathway induction).
  • peripheral inflammation induces DNA damage in the soma of neurons of the lumbar DRG and recapitulates this DNA damage in DRG cultures exposed to the inflammatory mediators, LPS or MCP-1.
  • DNA damage mediates changes in neuronal sensitivity, as determined by capsaicin-stimulated neuropeptide release by exogenously enhancing DNA repair via the overexpression of the enzyme APE1.
  • the present disclosure thereby identifies a pathway by which inflammatory mediators sustain changes in neuronal sensitivity and highlights the enhancement of neuronal DNA repair as a pharmacological target to alleviate inflammatory or chronic pain.
  • the administration of APX3330 can help to prevent or reduce the effects of chemotherapy-induced peripheral neuropathy (CIPN).
  • CIPN chemotherapy-induced peripheral neuropathy
  • Chemotherapy-induced peripheral neuropathy (CIPN) is a potentially debilitating side effect of a number of chemotherapeutic agents.
  • the major symptoms of these neuropathies including allodynia, increased sensitivity to cold, loss of proprioception, loss of touch, reduced tendon reflexes and pain, are largely characterized by alterations in peripheral sensory function, suggesting that sensory neurons are a major target of the toxicity.
  • Suitable dosages of the APE1 inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, for use in the methods of the present disclosure will depend upon a number of factors including, for example, age and weight of an individual, severity of inflammatory or chronic pain, nature of a composition, route of administration and combinations thereof.
  • a suitable dosage can be readily determined by one skilled in the art such as, for example, a physician, a veterinarian, a scientist, and other medical and research professionals.
  • a low dosage can be increased until reaching the desired treatment outcome or result.
  • a high dosage can be decreased until reaching a minimum dosage needed to achieve the desired treatment outcome or result.
  • the APE1/Ref-1 inhibitor is APX3330, and the subject is administered from about 5 ⁇ M to about 50 ⁇ M APX3330.
  • the APE1 inhibitor is administered via a composition that includes the APE1 inhibitor and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may be, for example, excipients, vehicles, diluents, and combinations thereof.
  • the compositions may be formulated as tablets, capsules, granules, powders, or syrups; or for parenteral administration, they may be formulated as injections (intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intravitreal), drop infusion preparations, or suppositories.
  • compositions can be prepared by conventional means, and, if desired, the active compound (e.g., APX3330) may be mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent, a coating agent, or combinations thereof.
  • the active compound e.g., APX3330
  • any conventional additive such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent, a coating agent, or combinations thereof.
  • the pharmaceutical compositions of the present disclosure can further include additional known therapeutic agents, drugs, modifications of the synthetic compounds into prodrugs, and the like for alleviating, mediating, preventing, and treating the diseases, disorders, and conditions described herein.
  • the APE1 inhibitor can be administered with one or more of platinum drugs (e.g., cisplatin, oxaliplatin carboplatin), taxanes (e.g., paclitaxel, docetaxel, cabazitaxel), doxorubicin, alkaloids (e.g., vincristine, vinblastine, etoposide) thalidomide, lenolidomide, pomalidomide, bortexomib, carfilzomib, eribulin, or ionizing radiation.
  • platinum drugs e.g., cisplatin, oxaliplatin carboplatin
  • taxanes e.g., paclitaxel, docetaxel, cabazitaxe
  • compositions including the APE1 inhibitor and/or pharmaceutical carriers used in the methods of the present disclosure can be administered to a subset of individuals in need.
  • an “individual in need” refers to an individual at risk for or having inflammatory and/or chronic pain, or an individual at risk for or having a disease or disorder associated with inflammation and/or chronic pain (e.g., obesity, diabetes, asthma, arthritis (osteoarthritis, rheumatoid arthritis, psoriatic arthritis) chronic periodontitis, ulcerative colitis, Crohn's disease, chronic sinusitis, chronic active hepatitis, chronic peptic ulcer, diverticulitis, fibromyalgia, irritable bowel syndrome, Alzheimer's, Parkinson's disease, atherosclerosis, and tuberculosis).
  • a disease or disorder associated with inflammation and/or chronic pain e.g., obesity, diabetes, asthma, arthritis (osteoarthritis, rheumatoid arthritis, psoriatic arthritis) chronic periodon
  • an “individual in need” is also used herein to refer to an individual at risk for or diagnosed by a medical professional as having inflammatory or chronic pain.
  • the methods disclosed herein are directed to a subset of the general population such that, in these embodiments, not all of the general population may benefit from the methods.
  • the method embodiments of the present disclosure are directed to specific subsets or subclasses of identified individuals (that is, the subset or subclass of individuals “in need” of assistance in addressing one or more specific conditions noted herein), not all individuals will fall within the subset or subclass of individuals as described herein.
  • the individual in need is a human.
  • the individual in need can also be, for example, a research animal such as, for example, a non-human primate, a mouse, a rat, a rabbit, a cow, a pig, and other types of research animals known to those skilled in the art.
  • a research animal such as, for example, a non-human primate, a mouse, a rat, a rabbit, a cow, a pig, and other types of research animals known to those skilled in the art.
  • tissue culture supplies were obtained from Thermo Fisher Scientific (Waltham, Mass.).
  • Poly-D-lysine, laminin, mouse monoclonal anti-vinculin antibody, 1-methyl-2-pyrrolidone (MPL), complete Freund's adjuvant (CFA), and routine chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.).
  • Nerve growth factor was purchased from Envigo (Indianapolis, Ind.) and Normocin from Invivogen (San Diego, Calif.).
  • Neuroporter was purchased from Genlantis (San Diego, Calif.).
  • Mouse monoclonal antihuman APE1 antibodies were raised in the laboratory and available from Novus Biologicals (Littleton, Colo.), mouse monoclonal anti-phospho-H2AX antibody was from EMD Millipore (Billerica, Mass.), and anti-Hemagglutinin (HA) antibody conjugated to horseradish peroxidase was purchased from Miltenyi Biotec (San Diego, Calif.). Chemiluminescence secondary antibodies were obtained from Roche Diagnostics Corp. (Indianapolis, Ind.).
  • APX3330 (also referred to herein as “E3330”) was synthesized per previous publications (e.g., J Med Chem. 2010 Feb. 11; 53(3): 1200-1210), dissolved in N,N-dimethylformamide (Sigma-Aldrich) and stored as a 40 mM stock at ⁇ 80° C.
  • LPS Lipopolysaccharides
  • Escherichia coli 0111:B4 was purchased from Sigma-Aldrich Inc. (St. Louis, Mo.), dissolved in MPL and stored as a 50 mM at ⁇ 20° C. for a month.
  • Recombinant rat CCL2/MCP-1 protein was purchased from R&D Systems (Minneapolis, Minn.), dissolved in PBS and stored at ⁇ 20° C. for up to a month.
  • the TLR4 antagonist, LPS-RS was purchased from Invivogen, dissolved in MPL and stored at ⁇ 80° C.
  • the CCR2 antagonist, RS 504393 was purchased from Sigma-Aldrich Inc. (St. Louis, Mo.), dissolved in MPL and stored 20° C. for a month. Before drug treatment, the stocks were diluted in F-12 growth medium and added to cultures and incubated for 2-96 hours as indicated. The Animal Care and Use Committee at Indiana University School of Medicine, Indianapolis, Ind. approved all procedures used in this Example.
  • Rats were anesthetized briefly with isoflurane and injected subcutaneously with 150 ⁇ l of a 1:1 (v/v) solution of CFA and 0.9% saline into the plantar surface of the right hind paw. Inflammation was confirmed by redness and swelling; only animals with an increase in the injected paw thickness of 3.5 mm or greater were used in experiments.
  • DRG Dorsal root ganglia
  • Cells were maintained in F-12 media supplemented with 10% horse serum, 2 mM glutamine, 100 ng/ml Normocin, 50 ng/ml penicillin, 50 ng/ml streptomycin, 50 ⁇ M 5-fluoro-2′-deoxyuridine, 150 ⁇ M uridine, and 30 ng/ml of NGF in 3% CO 2 at 37° C. Growth medium was changed every other day.
  • APE1siRNA Small interfering RNAs to APE1
  • SCsiRNA scrambled siRNA
  • Opti-MEM 1 media containing 100 nM of APE1siRNA (5′-GUCUGGUAAGACUGGAGUACC-3′ (SEQ ID NO:1)) or SCsiRNA (5′-CCAUGAGGUCAGCAUGGUCUG-3′(SEQ ID NO:2)); (Vasko et al., 2005)) and 10 ⁇ l of the transfecting reagent, Neuroporter.
  • 0.5 ml of the growth media without antibiotics was added to each well, and after an additional 24 hours the media containing siRNA was replaced with normal growth media.
  • Lentiviral constructs containing (1) the CMV promoter, HA-tagged APE1, IRES, and enhanced green fluorescent protein (EGFP); or (2) CMV, IRES, and EGFP were developed.
  • DNA sequencing confirmed the constructs in the pLenti6-R4R2-V5 plasmid containing WT-, C65-, or 226+177-APE1-IRES-EGFP.
  • DRG cells were cultured 5 days before 150 pfu/cell of the lentivirus was added to the media. Two days later, the virus was removed and the cells grown an additional 5 days in regular media.
  • APE1 expression was selectively reduced in the neuronal cultures with siRNA to rat APE1 mRNA and added back human APE1 transgenes that are not affected by the rat siRNA since the human APE1 homolog has a different nucleic acid sequence at the binding site (Vasko et al., 2005).
  • Tissues or cells were harvested, lysed in RIPA buffer (Santa Cruz Biotechnology; Santa Cruz, Calif., USA), sonicated, and cleared of cellular debris by centrifuging at 4000 RPM for 2 minutes. Protein was quantified using Lowry assay, and electrophoresed in a 12% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to a PVDF membrane, and blocked with Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% nonfat dry milk for 1 hour at room temperature while gently agitating.
  • RIPA buffer Purified using Lowry assay, and electrophoresed in a 12% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to a PVDF membrane, and blocked with Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% nonfat dry milk for 1 hour at room temperature while gently agitating.
  • TST Tris-buffered saline
  • Mouse monoclonal antihuman Ape1 antibodies (1:1000), mouse monoclonal anti-phospho H2AX antibodies (1:1000), mouse monoclonal anti-vinculin antibody (1:1000), and anti-Hemagglutinin (HA) antibody were added to the blocking solution and incubated for 2 hours at room temperature while gently agitating. Antibody binding was detected following appropriate secondary antibody methods using chemiluminescence. The density of the bands was measured using Quantity One software from Bio-Rad (Hercules, Calif.) and data expressed as density normalized to vinculin.
  • HEPES buffer consisting of (in mM) 25 HEPES, 135 NaCl, 3.5 KCl, 2.5 CaCl 2 , 1 MgCl 2 , 3.3 D-glucose, and 0.1% bovine serum albumin, pH 7.4 and maintained at 37° C. They were then incubated for successive 10-minute intervals with 0.4 ml of HEPES buffer alone (basal release), with buffer containing 30 nM capsaicin, then with buffer alone (to assess return to basal release).
  • the buffer was removed and the amount of immunoreactive CGRP in each sample was measured using radioimmunoassay as previously described (Chen et al., 1996).
  • the cells in each well were in 0.4 ml of 0.1 M HCl 10 minutes and an aliquot taken to measure total CGRP content in the cultures using radioimmunoassay. Total content (fmol/well) was calculated by adding the total amount released in all incubations to the amount measured in the cells. The release data is calculated as fmol released/well/10 minutes.
  • TLR4 receptor pathway and CCR2 receptor pathway The cognate receptor pathways that are activated by LPS and MCP-1 are the TLR4 receptor pathway and the CCR2 receptor pathway, respectively (Charo et al., 1994, Poltorak et al., 1998); however, there have been recent reports that these inflammatory agents may modulate other targets (Meseguer et al., 2014). Therefore, it was determined whether blocking the activation of the TLR4 and CCR2 inhibited the effects of the inflammatory mediators to enhance pH2A.X expression and alter neuronal sensitivity by performing experiments in the presence of the TLR4 antagonist, LPS-RS (2 ⁇ gimp, or the CCR2 antagonist, RS 50493 (1 ⁇ M), respectively.
  • Neurons contain the major DNA repair pathways including BER, nucleotide excision repair, mismatch repair, direct damage repair, and nonhomologous end-joining or homologous recombination (Fishel et al., 2007b, Barzilai et al., 2008, Fortini and Dogliotti, 2010).
  • the BER pathway repairs DNA damage in the nucleus and in mitochondria that is caused by oxidative damage to bases, alkylation of bases, or deamination and is likely the most important repair pathway for protecting neurons (see Fishel et al., 2007b). It was next examined whether enhancing or diminishing the activity of APE1, a critical enzyme in the BER pathway, altered the DNA damage and changes in neuronal sensitivity induced by LPS and MCP-1 treatment.
  • cultures were treated as illustrated in FIG. 5A .
  • Cultures were transfected with SCsiRNA or APE1siRNA on days 4-6 in culture and then exposed to lentivirus containing expression constructs for vector control, wildtype APE1, C65 APE1, or 226/117 APE1 on days 6-8 in culture.
  • the C65 APE1 mutant has impaired redox function whereas the 226/117 APE1 mutant has impaired DNA repair function (Izumi et al., 2004, Luo et al., 2008).
  • the neurons were treated with E3330 on days 10-14 days in culture.
  • cultures were treated with LPS (3 ⁇ g/ml) for the 24 hours immediately prior to experiments.
  • LPS (3 ng/ml) treatment attenuated the release of CGRP stimulated by capsaicin ( FIG. 5D ).
  • the stimulated release of CGRP from vehicle-treated wells was 10.4 ⁇ 0.6% of total content, whereas release from cells treated with LPS for 24 hours was decreased to 7.1 ⁇ 0.6% of total content.
  • Exogenous expression of either wildtype APE1 or C65 APE1 (repair-competent) reversed the effects of LPS, so that the stimulated release of CGRP was 10.4 ⁇ 1.1 and 10.5 ⁇ 0.4% of total content in the presence of APE1 wildtype and C65 mutant, respectively.
  • FIGS. 5A-5D APE1 expression and activity was manipulated and then cultures were treated with MCP-1 (0.3 ng/ml) for the 24 hours immediately prior to experiments ( FIG. 6A ).
  • MCP-1 0.3 ng/ml
  • SCsiRNA-treated sensory neurons exposed to MCP-1 had enhanced expression of pH2A.X ( FIGS. 6B and 6C ).
  • Increasing the exogenous expression of wildtype or repair-competent APE1 prevented the ability of MCP-1 to increase pH2A.X; levels of pH2A.X were decreased to 36.9 and 33.6% of the MCP-1-induced increase in the presence of wildtype and C65 APE1, respectively.
  • MCP-1 (0.3 ⁇ g/ml) enhanced the release of CGRP. This enhancement was not observed when APE1 expression was enhanced exogenously with either the wildtype APE1 or a repair-competent APE1 (C65 APE1). Exogenous expression of the repair-deficient APE1 (226/177); however, did not prevent the MCP-1 induced sensitization of CGRP release ( FIG. 6D , light gray columns). In cultures treated with APE1siRNA, MCP-1 treatment caused a decrease in CGRP release, suggesting that the response to MCP-1 is shifted leftwards, based on the concentration response curve presented in FIG. 3 , in cultures with reduced DNA repair activity.
  • the signaling pathways by which inflammation alters the sensitivity of primary afferent neurons have been investigated extensively and include posttranslational modifications to reversibly alter the function of receptors, ion channels, or associated regulatory proteins and transcriptional regulation to alter the expression of receptors, ion channels, or neurotransmitters or to induce novel expression of these proteins to modulate the phenotype of sensory neurons (Neumann et al., 1996).
  • neuronal cultures derived from DRG were utilized. The cultures were treated with the TLR4 or CCR2 ligands, LPS or MCP-1/CCL2, respectively, to mimic the effects of inflammation on neurons in culture.
  • LPS is expressed on the outer membrane of gram negative bacteria, including the inactivated Mycobacterium tuberculosis present in complete Freund's adjuvant used in the in vivo inflammation studies and is an exogenous ligand for the TLR4 receptor. LPS enhances the expression of TNF ⁇ , IL-1 ⁇ , COX-2 and MCP-1 in sensory neurons (Tse et al., 2014, Miller et al., 2015), thus recapitulating the activation of multiple pathways elicited by inflammation.
  • LPS acutely enhances the sensitivity of sensory neurons as demonstrated by nociceptive behaviors following injection into the hindpaw of rodents (Ferreira et al., 1993, Calil et al., 2014) and by in vitro experiments, where LPS enhances the excitability and exocytotic activity of sensory neurons (Hou and Wang, 2001, Diogenes et al., 2011, Meseguer et al., 2014).
  • MCP-1 is a cytokine that is upregulated in DRG by inflammation (Jeon et al., 2008), and released from DRG or dorsal spinal cord via stimulation of sensory neurons (Dansereau et al., 2008).
  • MCP-1 exposure has been shown to upregulate the neuronal expression of TRPV1 and NaV1.8 (Kao et al., 2012), potentially mediated by the activation of NF ⁇ B (Tse et al., 2014, Zhao et al., 2014).
  • MCP-1 also enhances the sensitivity of sensory neurons via posttranslational modifications, as evidenced by an increase in nociceptive behaviors following hindpaw injection (Dansereau et al., 2008) and by a direct stimulation of CGRP from cultures derived from neonatal DRG (Qin et al., 2005).
  • MCP-1 is a ligand for the CCR2 receptor.
  • the CCR2 is not expressed in DRG neurons derived from na ⁇ ve animals, the CCR2 is expressed in DRG following inflammation or nerve injury (White et al., 2005, Miller et al., 2012, Zhang et al., 2013). Furthermore, the CCR2 is functionally active in cultures derived from DRG (Qin et al., 2005, Kao et al., 2012).
  • inflammation In addition to the activation of kinases and transcription factors to elicit hypersensitivity, inflammation also enhances the generation of reactive oxygen and nitrogen species, which play a role in mediating changes in neuronal sensitivity. Inflammatory mediators enhance the production of ROS/RNS via enzymatic (NADPH oxidase) and autooxidation reactions (via metabolism-induced increases in electron transport chain leakage) (Bauerova and Bezek, 1999, Babior, 2000, Remans et al., 2005, Ibi et al., 2008).
  • NADPH oxidase enzymatic
  • autooxidation reactions via metabolism-induced increases in electron transport chain leakage
  • ROS/RNS function as agonists for the TRPV1 and TRPA1 channels (Andersson et al., 2008, Sawada et al., 2008, Keeble et al., 2009, Ito et al., 2013, Lin et al., 2015).
  • an intracellular increase in free radical moieties can lead to the oxidation of molecules, including nucleic acids, proteins, and lipids, leading to potentially serious consequences for sensory neurons. It was recently demonstrated that DNA damage was a causative factor in altering the sensitivity of neurons following treatment with cisplatin (REF).
  • ROS/RNS can be produced by endogenous metabolic activity, oxidative stress secondary to injury (Kruman and Schwartz, 2008), environmental toxins, (Kisby et al., 1999) and drugs (Ahles and Saykin, 2007) and because ROS/RNS elicits oxidative DNA damage, sensory neurons have endogenous antioxidant mechanisms to combat excessive production of ROS/RNS. In the event that the free radical moieties overwhelm the endogenous antioxidants, sensory neurons also have DNA repair mechanisms to repair oxidative DNA damage.
  • BER involves several steps to repair a DNA lesion, including removal of the oxidatively damaged base by a DNA glycosylase to create an apurinic/apyrimidinic site (AP-site), cleavage of the DNA backbone by apurinic/apyrimidinic endonuclease 1/redox factor (APE1/Ref-1 or APE1) to produce a 3′-OH terminus in preparation for a DNA polymerase and ligase to insert a new base and ligate the DNA backbone, respectively.
  • APE1/Ref-1 or APE1 apurinic/apyrimidinic endonuclease 1/redox factor
  • Failure to repair oxidative DNA damage can result in mutations, obstruction of DNA replication, and genetic instability.
  • the importance of the BER pathway, specifically the activity of APE1, in protecting isolated sensory neurons from the toxic effects of anticancer treatment has been examined. Reducing the expression of APE1 increases the neurotoxicity produced by cisplatin exposure, whereas, augmenting the activity of APE1 lessened the neurotoxicity (Vasko et al., 2005, Jiang et al., 2008b, Jiang et al., 2009, Kelley et al., 2014).
  • the enzyme also has activity to modulate the redox status of transcription factors to regulate their function (REF).
  • ROS/RNS Reactive oxygen and nitrogen species can be generated by multiple sources: a major driver of ROS/RNS generation is respiratory chain activity in the mitochondria, yet non-mitochondrial ROS/RNS can be produced by enzymes such as NADPH oxidase, xanthine oxidase, cyclooxygenase, cytochrome p450, and lipoxygenase (Sauer et al., 2001, Holmstrom and Finkel, 2014).
  • inflammation could contribute to functional changes in neurons that are reproducible and that enhanced DNA repair could reverse the functional changes in neurons induced by the damage.
  • Oxidative damage to DNA is known to alter the ability of transcription factors to recognize and bind promoter regions (Ziel et al., 2004, Gillespie et al., 2009, Pastukh et al., 2015), thus the DNA damage induced by inflammation might be reproducible because of damage to promoter/repressor regions of genes or transcription factors that are already activated by inflammation (Ruchko et al., 2009).
  • the present disclosure demonstrates that inflammation or exposure to inflammatory mediators elicits DNA damage in sensory neurons. By enhancing base excision repair, it is demonstrated that this DNA damage mediates the maintenance of neuronal hypersensitivity induced by inflammatory mediators.
  • APX3330 was analyzed for its effects on DNA repair activity.
  • Neuroblastoma cells were implanted subcutaneously into the right flanks of 6-wk old male NSG mice and allowed to proliferate until tumor volumes ⁇ 150 mm 3 . Mice were then randomized for treatment with cisplatin ⁇ APX3330 treatment. Cisplatin and APX3330 were administered concurrently for 3 weeks (Day 0-Day 17) and endpoints of neuronal toxicity were assessed within the DRG of mice at several time points following the last dose of cisplatin.
  • APX3330 When isolated sensory neurons were exposed to APX3330, a concentration-dependent increase in Ref-1/APE1 endonuclease activity occurred, which is not observed in tumor cells.
  • APX3330 is a targeted inhibitor of Ref-1/APE1's redox function, it appears that, in the setting of sensory neurons, it can also enhance the protein's DNA repair (AP endonuclease) activity ( FIGS. 7A-7E ).
  • APX3330 causes the protein to unfold over time. This unfolding primarily alters the amino end of Ref-1/APE1, affecting its interactions with downstream transcription factor targets by perturbing the equilibrium of the protein's folded/unfolded states and facilitating repair activity. This disengagement of Ref-1/APE1 from its Ref-1/APE1 redox activity could enhance Ref-1/APE1 repair endonuclease activity.
  • a critical property of any putative therapeutic for neurotoxicity is that it will not compromise the anticancer function of the treatment(s) administered.
  • the enhancement of DNA repair activity by APX3330 was not observed in mitotic cells. It has been previously shown that APX3330 negatively affects the growth and/or survival of tumor cell lines, patient-derived cell lines, and tumors in animal models. Therefore, it is possible that APX3330 could protect postmitotic cells without altering the effects of anticancer drugs on tumor cells ( FIGS. 7C-7E ). Additionally, APX3330 did not affect cisplatin or oxaliplatin's tumor-killing efficacy in vivo, yet it protects DRG neurons from oxidative DNA damage (data not shown).
  • APX3330 analogs were analoyzed for their ability to protect against neurotoxicity-induced by cisplatin or oxaliplatin while not diminishing the anti-tumor effect of the platinum. Also, the analog APX2009 was assessted for its anti-tumor effects in neuroblastoma cell lines as well as in a 3D spheroid pancreatic tumor model.
  • Mouse monoclonal anti-phospho-H2AX antibodies were from EMD Millipore (Billerica, Mass.) and ⁇ -Actin monoclonal antibody from Thermo Fisher Scientific (Fremont, Calif.). Chemiluminescence secondary antibodies were from Roche Diagnostics Corp. (Indianapolis, Ind.).
  • Cisplatin was purchased from Sigma-Aldrich Inc. (St. Louis, Mo.), and oxaliplatin was purchased from LKT Laboratories, Inc. Cisplatin was initially dissolved in N,N-dimethylformamide (Sigma-Aldrich) and stored as a 40 mM solution at ⁇ 80° C. and oxaliplatin dissolved in PBS and stored as a 5 mM stock at ⁇ 80° C. Before drug treatment, the stocks were diluted in F-12 growth medium and added to cultures and exposed for 24-72 hours. The Animal Care and Use Committee at Indiana University School of Medicine, Indianapolis, Ind. approved all procedures used in these studies.
  • iodolawsone in a subsequent reaction is treated with methacrylic acid or 2-propylacrylic acid, with oxalyl chloride and the corresponding amine, and with sodium methoxide in methanol to yield (2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylidene]-N,N-dimethylpentanamide (APX2007), (2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylidene]-N,N-diethylpentanamide (APX2009), and (2E)-2-(3-methoxy-1,4-dioxo-1,4-dihydro naphthalen-2-yl)-N,N,2-trimethylprop-2-enamide (APX2032).
  • Marvin was used for drawing, displaying and characterizing chemical structures, substructures and reactions, Marvin 15.8.24.0, 2015, ChemAxon (http://www.chemaxon.com). Calculator Plugins were used for structure property prediction, Marvin 15.8.24.0, 2015, ChemAxon (www.chemaxon.com).
  • Molecular modeling was performed using the Open Eye Scientific software OMEGA (OMEGA 2.5.1.4) (Hawkins et al., 2010) and ROCS (ROCS 3.2.1.4: OpenEye Scientific Software, Santa Fe, N. Mex. www.eyesopen.com) (Hawkins et al., 2007).
  • Molecular visualization was performed using the Open Eye Scientific software VIDA (OpenEye Scientific Software, Santa Fe, N. Mex. www.eyesopen.com).
  • Cells were maintained in F-12 media supplemented with 10% horse serum, 2 mM glutamine, 100 ⁇ g/ml NormocinTM, 50 ⁇ g/ml penicillin, 50 ⁇ g/ml streptomycin, 50 ⁇ M 5-fluoro-2′-deoxyuridine (Invitrogen), 150 ⁇ M uridine, and 30 ng/ml of NGF in 3% CO2 at 37° C. Growth medium was changed every other day. Experiments were performed after cells were maintained in culture for 12-14 days.
  • the IMR32 and SK-N-SH cell lines were obtained from the American Type Culture Collection and grown in RPMI-1640 supplemented with 10% FBS. Cell line identity was confirmed by DNA finger print analysis (IDEXX BioResearch) for species and base-line short-tandem repeat analysis testing. All cell lines were 100% human and a 9-marker short-tandem repeat analysis is on file.
  • Cells were seeded in 96-well plates (IMR32: 1000 cells/well; SK-N-SH: 3000 cells/well) and treated for 5 days with APX2007, APX2009, APX2032, or APX3330 (also referred to herein as “E3330”).
  • Final DMSO concentration was ⁇ 0.1%.
  • Cell viability was determined using the methylene blue assay as previously described (Tonsing-Carter et al., 2015). Each experiment was performed in triplicate and repeated three times. The percent viabilities, normalized to the control, were graphed and ED 50 values determined using the Chou-Talalay method (Chou and Talalay, 1984).
  • Immunoblotting was performed as previously described (Kelley et al., 2014). Briefly, cells were lysed in RIPA buffer (Santa Cruz Biotechnology; Santa Cruz, Calif., USA) and protein was quantified using the Lowey assay. Proteins were separated by electrophoresis on a 4-12% SDS-polyacrylamide gel. The gel was transferred to a PVDF membrane and incubated overnight at 4° C. in Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% nonfat dry milk while gently agitating.
  • Mouse monoclonal antihuman Ape1 antibodies (1:500), mouse monoclonal anti-phospho H2AX antibodies (1:1000), or ⁇ -Actin monoclonal antibody (1:1000) were added to the blocking solution and incubated overnight at room temperature while gently agitating. Antibody binding was detected following appropriate secondary antibody methods using chemiluminescence. The density of the bands was measured using QUALITYONE® software from Bio-Rad (Hercules, Calif.) and data expressed as density normalized to actin.
  • HEPES buffer consisting of (in mM) 25 HEPES, 135 NaCl, 3.5 KCl, 2.5 CaCl 2 , 1 MgCl 2 , 3.3 D-glucose, and 0.1% bovine serum albumin, pH 7.4 and maintained at 37° C. They then were incubated for successive 10-minute intervals with 0.4 ml of HEPES buffer alone (basal release), with buffer containing 30 nM capsaicin, then with buffer alone (to assess return to basal release).
  • CGRP immunoreactive calcitonin-gene related peptide
  • APE1 repair activity assay was performed in a plate assay using two annealed oligonucleotides (5′-6-FAM-GCCCCC*GGGGACGTACGATATCCCGCTCC-3′ (SEQ ID NO:3) and 3′-Q-CGGGGGCCCCCTGCATGCTATAGGGCGAGG-5′ (SEQ ID NO:4)) containing a quencher on one strand and a fluorescent 6-FAM label with tetrahydrofuran as an AP site mimic. Oligo cleavage at the AP mimic site results in 6-FAM release and detection. The fluorescence was read at five, one-minute intervals using a Tecan Ultra plate reader (Chemical Genomics Core, Indiana University School of Medicine). The rate of the reaction was used to determine the change in APE1 repair activity as compared to the vehicle control.
  • EMSAs were performed as described (Luo et al., 2012). Purified APE1 was reduced with 1.0 mM DTT for 10 minutes and diluted to a final concentration of 0.006 mM with 0.02 mM DTT in PBS. Reduced APE1 was added to EMSA reaction buffer (10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM MgCl 2 , 1 mM EDTA, 5% [vol/vol] glycerol) with 2 mL 0.007 mM protein mixture (1:1) of purified truncated c-Jun and c-Fos proteins containing DNA-binding domain and leucine zipper and incubated for 30 minutes at room temperature. The EMSA assay was performed as previously described (Luo et al., 2008; Nyland et al., 2010; Kelley et al., 2011; Luo et al., 2012).
  • Reporter assays were performed as previously described (Georgiadis et al., 2008; Kelley et al., 2011; Cardoso et al., 2012b; Luo et al., 2012).
  • Cells were transfected with NF- ⁇ B-Luciferase construct containing an NF- ⁇ B-response promoter and driving the expression of a luciferase gene and a Renilla luciferase control reporter vector pRL-CMV. After a 24-hour transfection period, cells were lysed, and Firefly and Renilla luciferase activities were assayed using Renilla luciferase activity for normalization. All of the transfection experiments were performed in triplicate and repeated at least three times in independent experiments. Data are expressed as mean ⁇ standard error from a representative experiment, and Student's t tests were performed.
  • TdTomato-labeled PDAC cells and EGFP-labeled CAFs are resuspended in colorless DMEM media containing 3% Reduced Growth Factor Matrigel (BD Biosciences) and 5% FBS at a cell ratio of 1:4 (tumor:CAF) and fed on days 4 and 8 following plating. Both cell populations are quantitated for intensity and area via Thermo ArrayScan at day 12 of co-culture.
  • PK studies were performed in the IU Simon Cancer Center Clinical Pharmacology Analytical Core (CPAC), as previously described for E3330 (Fishel et al., 2011) and standards for the compounds used. P450 metabolism studies using human microsomes were also performed in CPAC directed by Dr. David Jones.
  • CPAC Clinical Pharmacology Analytical Core
  • E3330 A number of analogs of E3330 were synthesized by replacing the core dimethoxybenzoquinone (A) with a napthoquinone ring, the methyl group (B) on the ring structure with various halogens or hydrogen, and shortening the carbon chain (C) on the double bond to modulate activity ( FIG. 8A ).
  • the carboxylic acid moiety (D) was modified in concert with shortening the carbon chain (C) on the double bond. These changes modified two physical properties of the structure.
  • E3330 exists as a charged molecule at physiological pH. Amide derivatives of the carboxylic acid (D), which are not a charged supporting chemical feature were prepared.
  • E3330 has a very lipophilic carbon chain, which is believed to be a modifiable feature.
  • the new structures have significantly shorter carbon chains (C) on the double bond and are therefore less lipophilic.
  • C carbon chains
  • Detailed synthesis data can be found in U.S. Pat. No. 9,089,605, which is hereby incorporated by reference to the extent it is consistent herewith.
  • Three new structures from the compounds made ( FIG. 8B ) were analyzed in redox APE1 electrophoretic mobility shift assay (EMSA) studies to determine which compounds affect the redox function of APE1.
  • the compounds had redox inhibition IC 50 s of: APX2007 2 ⁇ M, APX2009 1 ⁇ M, and APX2032 1 uM ( FIG. 9A ).
  • E3330 has been previously presented and has an IC 50 of 25 uM in similar assays.
  • Reporter transactivation assays were performed to verify the new compounds as effective in cells and hitting their target APE1 which, in this assay, regulates NF ⁇ kB function.
  • all three compounds, APX2007, APX2009 and APX2032 demonstrated similar inhibition of NF ⁇ B binding to the reporter construct with an IC50 of 7 ⁇ M, while E3330 has an activity of 45 ⁇ M ( FIG. 9B ).
  • the ED 50 for tumor cell killing was determined in two neuroblastoma cell lines, IMR32 (p53 wt, MYCN amplified) and SK-N-SH (p53 wt, MYCN non-amplified) ( FIGS. 9C & 9D ).
  • All three compounds had a reduced ED 50 compared to E3330; 7-10 fold greater in IMR32 cells and 4-6 fold greater in SK-N-SH cells ( FIGS. 9C & 9D ).
  • the enhanced tumor cell killing data is consistent with the increased efficacy of the compounds on APE1 function as demonstrated by EMSA and transactivation data in FIG. 10 .
  • the pharmacokinetic profile of APX2009 was also assessed. As shown in FIGS. 11A & 11B , the half-life of APX2009 is 25.8 hours compared to 3.6 hours for E3330, or an approximate 7-fold half-life increase. Additionally, using human microsomes in a P450 metabolism analysis, APX2009 had a 173 vs 20-minute half-life or an 8.7-fold increase ( FIGS. 11A & 11B ).
  • DNA repair activity assays were performed as previously described (Bapat et al., 2010). As shown in FIGS. 13A-13D , only APX2009 demonstrated a stimulation of APE1 repair activity in this assay and in the nanomolar range, a significant increase in activity compared to E3330 ( FIGS. 11A & 11B ). APX2007 and APX2032 had no effect, either for stimulation or inhibition of APE1 endonuclease activity.
  • This cisplatin-induced cell death was blocked by exposing neuronal cultures to E3330 (20)(M) or to APX2009 (20 ⁇ M) for 48 hours prior to and throughout the cisplatin treatment ( FIG. 12B ).
  • pretreatment with 20 ⁇ M of APX2007 or APX2032 did not attenuate the cisplatin-induced cell death, with the combination of APX2032 and cisplatin (100 ⁇ M) reducing cell viability to 9 ⁇ 9% ( FIG. 12B ). Therefore, APX2009 protects sensory neuronal cultures against cisplatin-induced cell death at all dose levels used, whereas APX2007 and APX2032 caused cell killing at high dose (100 ⁇ M).
  • E3330 analogs could attenuate a functional endpoint of cisplatin-induced neurotoxicity, i.e., the decrease in capsaicin-evoked release of CGRP.
  • E3330 (20 ⁇ M) or APX2009 (10 or 20 ⁇ M) for 72 hours and CGRP release examined, there was no significant change in either basal (resting) release or release stimulated by 30 nM capsaicin when compared untreated cells ( FIG. 14A ).
  • APX2009 was prioritized for use in subsequent studies with another platinum agent, oxaliplatin.
  • Cisplatin and oxaliplatin both produce significant levels of ROS in cells, with cisplatin producing higher levels.
  • the DNA cross-links produced by these two agent differ: with cisplatin producing Pt-1-2-d(GpG) intrastrand DNA crosslinks while oxaliplatin creates predominantly Pt-1-3 d(ApG) interstrand DNA crosslinks.
  • APX2009 also significantly reduced the phosphorylation of H2AX after 24 and 48 hr treatments of oxaliplatin ( FIG. 16C ), indicating that its neuroprotective effects may be due to reduced DNA damage.
  • E3330 analogs were capable of tumor cell killing similar to what has been observed with E3330.
  • a three-dimensional co-culture model of pancreatic cancer was used as an ex vivo system that included both low passage patient-derived tumor cells and cancer-associated fibroblasts.
  • the effects of APX2009-induced cytotoxicity on the area and intensity of both tumor cells alone and in co-culture with CAFs were assessed.
  • Spheroids composed of patient-derived PDAC cells (Pa03C—labeled red) and CAF19 cells (labeled green) were treated with APX2009, and the area and intensity of red and green fluorescence were evaluated separately as markers for each cell type ( FIGS. 17A-17C ).
  • CAFs were not significantly affected by APX2009 treatment, again suggesting that non-tumorigenic cells can tolerate the effects of APE1 inhibition more than tumor cells. This data is similar to what is observed with E3330, but being effective at lower dose levels, validating APX2009 as a potential PDAC therapeutic agent while also showing CIPN protective indications.

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