Iron chelators in tumor therapy

Abstract

The present invention relates to a pharmaceutically compatible iron chelator or a prodrug thereof for use in treating and/or preventing cancer in a subject suspected or known to comprise hypoxic cancer cells, and use in treatment and/or prevention of a human papillomavirus (HPV) related lesion. The present invention further relates to a use of an iron chelator or prodrug thereof for inducing senescence in a cancer cell, preferably a hypoxic cancer cell; and to a method for inducing an irreversible proliferation arrest in cancer cells comprising a) contacting said cancer cells with an iron chelator or prodrug thereof and, thereby, b) inducing an irreversible proliferation arrest in said cancer cells.

Claims

1. A method for treating cancer in a subject comprising contacting hypoxic cancer cells in the subject with a pharmaceutically compatible iron chelator or a prodrug thereof, wherein the subject is suspected or known to comprise hypoxic cancer cells, wherein the pharmaceutically compatible iron chelator or prodrug thereof comprises a structure (I) ##STR00003## wherein: n is 0, 1, 2, 3, 4, 5, or 6; R.sup.1 is H, optionally substituted carbonyl, optionally substituted phosphoryl, or optionally substituted sulfonyl; R.sup.2 is selected from cyclohexyl, H, C1-C6 alkyl, C5-C8 cycloalkyl, C1-C6 alkoxy, and halogen; R.sup.4 is selected from methyl, H, C1-C6 alkyl, C5-C8 cycloalkyl, C1-C6 alkoxy, and halogen; and R.sup.3 and R.sup.5 are independently selected from H, C1-C6 alkyl, C5-C8 cycloalkyl, C1-C6 alkoxy, and halogen.

2. The method of claim 1, wherein the pharmaceutically compatible iron chelator is ciclopirox (2(1H)-Pyridinone, 6-cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one) or ciclopirox olamine.

3. The method of claim 1, wherein the hypoxic cancer cells are slowly proliferating or non-dividing hypoxic cancer cells.

4. The method of claim 3, wherein the hypoxic cancer cells are cancer cells under an oxygen saturation of at most about 5%, at most about 3%, or at most about 1%.

5. The method of claim 1, wherein the cancer forms at least one tumor mass having a diameter of at least about 1 mm, at least about 5 mm, or at least about 10 mm.

6. The method of claim 1, wherein the treating comprises administration of at least one further anticancer therapy, and optionally wherein the further anticancer therapy is radiotherapy, chemotherapy, anti-hormone therapy, targeted therapy, immunotherapy, or any combination thereof.

7. A method for inducing an irreversible proliferation arrest in hypoxic cancer cells comprising: (a) contacting the hypoxic cancer cells with an iron chelator or prodrug thereof and, thereby, (b) inducing an irreversible proliferation arrest in the hypoxic cancer cells, wherein the iron chelator or prodrug thereof comprises a structure (I) ##STR00004## wherein: n is 0, 1, 2, 3, 4, 5, or 6; R.sup.1 is H, optionally substituted carbonyl, optionally substituted phosphoryl, or optionally substituted sulfonyl; R.sup.2 is selected from cyclohexyl, H, C1-C6 alkyl, C5-C8 cycloalkyl, C1-C6 alkoxy, and halogen; R.sup.4 is selected from methyl, H, C1-C6 alkyl, C5-C8 cycloalkyl, C1-C6 alkoxy, and halogen; and R.sup.3 and R.sup.5 are independently selected from H, C1-C6 alkyl, C5-C8 cycloalkyl, C1-C6 alkoxy, and halogen.

8. The method of claim 7, wherein the pharmaceutically compatible iron chelator is ciclopirox (2(1H)-Pyridinone, 6-cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one) or ciclopirox olamine.

9. The method of claim 1, wherein the cancer is a solid cancer, a metastasis, or a relapse thereof.

Description

FIGURE LEGENDS

(1) FIG. 1: Senescence assays (A) and colony formation assays (B) of HeLa cells after treatment with 10 ?M CPX or solvent control EtOH.

(2) FIG. 2: Senescence assays (A) and colony formation assays (B) of HPV18-positive HeLa and HPV16-positive SiHa cells under hypoxic conditions (1% O.sub.2).

(3) FIG. 3: Western blots of the indicated proteins in extracts from cells of Example 3.

(4) FIG. 4: Senescence assays (A) and colony formation assays (B) in the presence of CPX and/or rapamycin.

(5) FIG. 5: Effect of CPX on HPV E6 and E7 expression: (A) E6 and E7 western blots on protein extracts of cells of Example 6; (B) relative E6/E7 mRNA quantification; (C) effect of iron or zink substitution of E7 and ferritin expression (western blot).

(6) FIG. 6: Other iron chelators also repress HPV E6 and E7: DFO and BP also reduce both HPV18 and HPV16 E6 and E7 protein levels, indicating that the repression is linked to the deprivation of intracellular iron levels.

(7) FIG. 7: Colony formation assays of HeLa cells after treatment with CPX and etoposide, cisplatin or irradiation under normoxia or hypoxia: Colony formation assay of HeLa cells after treatment with CPX under normoxia and hypoxia for 4d (A), treatment with CPX in combination with 5 ?M etoposide (B), 5 ?M CDDP (cisplatin, C) or irradiation with 10 Gy (D) under normoxia and hypoxia.

(8) FIG. 8: Combinatorial treatments of 3-dimensional cell aggregates (spheroids) with CPX in combination with etoposide or cisplatin under normoxic conditions. (A) HeLa spheroid growth after treatment with 10 ?M CPX, 5 ?M etoposide or a combination of both. (B) HeLa spheroid growth after treatment with 10 ?M CPX, 5 ?M CDDP (cisplatin) or a combination of both.

(9) FIG. 9: CPX also induces apoptosis under normoxic conditions. Depending on treatment duration, senescence or apoptosis is induced. 48 h treatment leads to the induction of senescence. 5 d treatment (A and B) induces apoptosis and hence no colonies grow out after release in colony formation assays. C: activation of caspase 3/7 (apoptosis marker) upon CPX treatment; D: 72 h treatment with CPX, cells were stained with the Annexin V and PI: Annexin V positive, PI negative cells represent early apoptotic cells; Annexin V positive, PI positive cells represent late apoptotic cells.

(10) FIG. 10: TUNEL assay (bright cells=apoptotic cells) under normoxic conditions with the indicated cell lines; A: time kinetic of 10 ?M CPX; B: dose kinetic of 96 h CPX treatment.

(11) FIG. 11: CPX induces DNA damage (gamma-H2AX=DNA damage marker) under normoxic conditions.

(12) FIG. 12: CPX can induce apoptosis under hypoxic conditions. A) TUNEL assay (green cells=apoptotic cells), EtOH=solvent control. B) Colony Formation Assay upon CPX treatment of hypoxic HeLa and SiHa cells.

(13) The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1: METHODS

(14) Cell Culture

(15) HPV18-positive HeLa cells were bought from American Type Culture Collection (ATCC).

(16) Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% penicillin (100 U/ml), streptomycin (100 ?g/ml) and 2 mM L-glutamine under normoxic (37? C., 21% O.sub.2, 5% CO.sub.2) conditions.

(17) Iron Chelators

(18) For all experiments CPX olamine compound from Sigma-Aldrich (C0415-1G) was utilized, stored at room temperature. The powder was diluted in 100% EtOH and prepared freshly for every experiment. Therefore, EtOH was used as a solvent control in experiments performed with CPX. Stock solutions of 10 mM were prepared and diluted 1:1000 in medium for the treatment of cells. DFO from Sigma-Aldrich was used as a deferoxamine mesylate salt. DFO was stored as a powder at ?20? C. and diluted in distilled H.sub.2O to a stock solution of 100 mM, which can be stored at ?20? C. for 7 days. For the treatment of cells DFO stock was diluted 1:1000 in medium. BP from Sigma-Aldrich was dissolved in ethanol (100 mM, stable for several months) and used for the treatment of cells with a final concentration of 100 ?M.

(19) Cell Treatment

(20) Cells were seeded in 6 cm (21 cm2) Greiner cell culture dishes containing 3 ml DMEM medium to reach 20-30% confluency after 24 h. Then the medium was exchanged for medium containing iron chelators. In some experiments CPX was pre-incubated with either iron- or zinc-containing agents. EtOH and 10 ?M CPX were either pre-incubated with H.sub.2O, 6.67 ?M ferric ammonium citrate (FAC), iron sulfate (FeSO4), zinc chloride (ZnCl2) or zinc sulfate (ZnSO4) for 10 min prior to cell treatment.

(21) Cell Counting

(22) For cell counting experiments and cell growth analysis cell numbers were determined in duplicates by trypan blue technique using Countess? Automated Cell Counter. Furthermore the IncyCyte S3 system was used to determine cell proliferation.

(23) Colony Formation Assay (CFA)

(24) The ability to form colonies of a treated cell comparable to an untreated cell after iron chelation was tested performing a colony formation assay (CFA). In order to do this, cells were fixed and cell nuclei stained with crystal violet (hexamethyl pararosaniline chloride). After treatment cells were splitted 1:100 and 1:200 into new dishes and cultured for another 5-10 days at 37? C. in normal DMEM to display the amount of still growing cells after treatment. Subsequently, cells were fixed and stained with 350 ?l formaldehyde-crystal violet for 3 min and washed with H.sub.2O. For quantification with the photometer, cells were discoloured with 33% acidic acid. Images were made with Epson Perfection 4990 Photo Scanner.

(25) Senescence Assay

(26) Treated cells were stained for senescence-associated beta-galactosidase (SA-beta-gal) activity at pH 6. Dimri et. al. could show that the marker SA-beta-gal is only expressed in senescent fibroblasts and keratinocytes, excluding pre-senescent, quiescent and differentiated cells (Dimri et al. 1995). The activity of SA-beta-gal can be determined by adding 5-Brom-4-chlor-3-indoxyl-beta-D-galactopyranosid (X-gal), which is then hydrolyzed by the galactosidase enzyme into galactose and 5-bromo-4-chloro-3-hydroxyindole. Dimerization of the latter followed by oxidation results in the formation of an insoluble blue product 5,5-dibromo-4,4-dichloro-indigo, which can be detected by a brightfield microscope. After treatment with iron chelators, the cells were splitted usually 1:2, 1:5 and 1:10 and cultured for 3-4 days in normal DMEM. Then the cells were washed, fixed with 1% formaldehyde/0.2% glutaraldehyde in PBS for 3 min followed by another wash with PBS. Fixed cells were incubated with 1.5 ml in senescence assay buffer mix overnight at 37? C. Then cells were washed again with PBS and SA-beta-gal positivity was detected under a brightfield microscope. Cells that are positive for SA-?-gal activity are blue.

EXAMPLE 2: SENESCENCE AND COLONY FORMATION CAPACITY OF HELA CELLS AFTER TREATMENT CPX

(27) In general, E6/E7 repression leads to the induction of senescence. Under hypoxia, however, the impaired mTOR pathway prevents efficient senescence induction and cells escape from anti-proliferative treatments like chemotherapy or radiotherapy. Ciclopirox (CPX) or EtOH were incubated with either H.sub.2O, 6.67 ?M FeSO.sub.4, FAC, ZnSO.sub.4 or ZnCl.sub.2 for 10 min prior to addition on HeLa cells. CPX induces senescence, indicated by the dark cells in the senescence assays (FIG. 1A) and induction of senescence by CPX reduces colony forming capacity (FIG. 1B). The effect of CPX can be rescued by the addition of iron (FeSO.sub.4 or FAC), but not zinc (ZnSO.sub.4 or ZnCl.sub.2), indicating that the effects are a result of iron depletion. Thus, the iron chelator CPX induces senescence under normoxia which can be reverted by the addition of iron (but not zinc), indicating that it is caused by the loss of iron.

EXAMPLE 3: SENESCENCE AND COLONY FORMATION CAPACITY OF HPV18-POSITIVE HELA AND HPV16-POSITIVE SIHA CELLS UNDER HYPOXIC CONDITIONS

(28) Cells were cultured for 24 h under hypoxic conditions (1% 02) and then treated with 10 ?M CPX or solvent control EtOH for another 72 h under hypoxic conditions. CPX treated cells still undergo senescence even under hypoxia (FIG. 2A) and the colony forming capacity is impaired (FIG. 2B). Thus, interestingly (and despite E6/E7 inhibition under hypoxia) CPX can induce senescence and reduce colony forming capacity even under hypoxic conditions

EXAMPLE 4: EFFECT OF CPX ON MTOR SIGNALING

(29) In the same experimental setup as in Example 3, proteins were extracted at the end of CPX treatment. Under hypoxic conditions mTOR signaling is impaired as indicated by the lack of phosphorylated substrates of the active mTOR pathway (FIG. 3A, P-S6K and P-4E-BP-1). CPX treatment under hypoxia does not reconstitute mTOR signaling in HeLa and SiHa cells. Notably, CPX acts as an mTOR inhibitor, since under normoxic conditions protein levels of P-S6K and P-4E-BP-1 are decreased (similar to treatment with the mTOR inhibitor rapamycin, FIG. 3B). Thus, CPX does not reconstitute mTOR signaling, but rather acts as an mTOR inhibitor.

EXAMPLE 5: CPX INDUCES SENESCENCE INDEPENDENT OF ACTIVE MTOR SIGNALING

(30) HeLa cells were treated simultaneously with 10 ?M CPX and 50 nM rapamycin (a chemical mTOR inhibitor) for 48 h. Even in the presence of rapamycin, CPX still induces senescence (FIG. 4A) and reduces the colony forming capacity (FIG. 4B), Thus, CPX-induced senescence is mTOR independent. Proteins that show the downregulation of phosphorylated substrates of the active mTOR signaling upon rapamycin treatment are displayed in FIG. 3B.

EXAMPLE 6: EFFECT OF CPX ON HPV E6/E7 EXPRESSION

(31) HPV18-positive HeLa cells were treated with different concentrations of CPX for 24, 48 and 72 h. Immunoblot analyses show that CPX decreases E6 and E7 protein levels in a time- and dose-dependent manner (FIG. 5A). E6/E7 are also downregulated at the mRNA level (FIG. 5B, qPCR analyses). Furthermore the repression of E6/E7 is linked to the depletion of iron, since the pre-incubation of 10 ?M CPX with 6.67 ?M iron (FeSO4 and FAC) but not zinc (ZnSO4 and ZnCl2) can prevent the repression of E7 (FIG. 5C). CPX reduces HPV E6/E7 on protein and mRNA level in an iron-dependent manner. Accordingly, like senescence induction, oncogene repression by CPX is linked to iron deprivation, since saturation of CPX with iron (but not zinc) inhibits the repression.

EXAMPLE 7: OTHER IRON CHELATORS

(32) Other clinical iron chelators, Deferoxamin (DFO) and Bipyridil (BP) also repress HPV E6 and E7: DFO and BP also reduce both HPV18 and HPV16 E6 and E7 protein levels, indicating that the repression is linked to the deprivation of intracellular iron levels (FIG. 6)

EXAMPLE 8: DOUBLE TREATMENT WITH CPX AND CANCER THERAPEUTICS

(33) HeLa cells were treated with 10 ?M CPX or EtOH (FIG. 7A) or in combination with the chemotherapeutic drug etoposide (5 ?M) for 72 h at 210% or 1% 02 (FIG. 7B). After treatment colonies were stained with crystal violet. The results of treatment with cisplatin or irradiation with 10 Gy instead of etoposide treatment are shown in FIGS. 7C and D. The colony formation assays after combinatorial treatments of CPX with either etoposide, cisplation or irradiation therapy show that CPX can target the cells that escape from chemotherapy/radiotherapy under hypoxia, but also under normoxia.

EXAMPLE 9: EFFECT OF CPX ON 3D CELL SPHEROIDS

(34) Generation and treatment of spheroids: 5000 cells (HeLa) were seeded in 200 ?l DMEM+30% methylcellulose stock solution in a low attachment U-bottom 96-well plate. After 3 days, half of the medium was removed and replaced with media containing drugs or solvent control. After 3 days half of the medium was replaced with fresh medium plus drugs. Spheroid size was monitored using the IncuCyte S3 live imaging system. As shown in FIG. 8, there is a clear inhibition of 3D spheroid growth by CPX. There is also a cooperative effect of CPX+chemotherapy in inhibiting the growth of 3D cell spheroids already under normoxic conditions.

EXAMPLE 10: EFFECT OF CPX UNDER NORMOXIC CONDITIONS

(35) While inducing senescence in cancer cells under normoxia and after short-term incubation (Example 2), CPX was found to induce apoptosis after longer-term incubation, e.g. after 3 to 4 days (FIGS. 9 and 10). Moreover, it was surprisingly found tat CPX can also induce DNA damage in cancer cells (FIG. 11).

EXAMPLE 11: INDUCTION OF APOPTOSIS BY CPX UNDER ANAEROBIC CONDITIONS

(36) As shown in FIG. 12, CPX can also induce apoptosis after 3 to 4 days of anaerobic incubation in cancer cells.

(37) The following non-standard literature was cited: 1. de Martel et al. Int J Cancer, 2017. 141(4): p. 664-670. 2. Dyson et al., Science, 1989, 243(4893): p. 934-7. 3. Martinez-Zapien et al., Nature, 2016, 529(7587): p. 541-5. 4. Hall and Alexander, J Viral, 2003, 77(10): p. 6066-9. 5. Vaupel et al., Antioxid Redox Signal, 2007, 9(8): p. 1221-35. 6. Vaupel and Mayer, Cancer Metastasis Rev, 2007, 26(2): p. 225-39. 7. Hoppe-Seyler et al., Proceedings of the National Academy of Sciences, 2017, 114(6): p. E990-E998. 8. Overgaard J Clin Oncol, 2007, 25(26): p. 4066-74. 9. Hili et al., Sem ln Radial Oncol, 2015, 25(4):p. 260-72. 10. Manoochehri Khoshinani et al., Cancer Invest, 2016, 34(10): p. 536-545. 11. Torti and Torti, Nat Rev Cancer, 2013, 13(5): p. 342-55. 12. Sanvisens et al. Biomed J, 2013, 36(2): p. 51-8. 13. Waris and Ahsan, Journal of Carcinogenesis, 2006, 5: p. 14-14. 14. Shen and Huang, Curr Pharm Des, 2016, 22(28): p. 4443-50. 15. Zhou et al., Journal international du cancer, 2010, 127(10): p. 2467-2477. 16. Eberhard et al., Blood, 2009, 114(14): p. 3064-3073. 17. Clement et al., Int J. Cancer, 100: 491-498, 2002 18. Song et al., Cancer Res. 71: 7628-7639, 2011 19. Laberge et al., Nat. Cell Biol. 17: 1049-1061