Patent Application: US-201715625337-A

Abstract:
a cell permeable iron chelator , optionally in combination with an autophagy inhibiting agent , is used for treating a solid cancer tumour in a person . a preferred chelator is an alkyl substituted n -- n -- hydrazine . a preferred autophagy inhibiting agent is chloroquine . also disclosed is a pharmaceutical composition comprising iron chelator , pharmaceutically acceptable carrier and , optionally , autophagy inhibiting agent ; and a method of treating cancer by administering cancer combating - effective amount of the iron chelator or the combination of iron chelator and autophagy inhibiting agent .

Description:
compounds of the invention were obtained from compound libraries . they can be prepared according to methods described in the literature , such as in wo 02 / 089809 , or by their non - inventive modifications . the compounds were dissolved in dmso . a final concentration of 0 . 5 % dmso was reached in cell cultures . hct116 colon carcinoma cells were maintained in mccoy &# 39 ; s 5a modified medium / 10 % fetal calf serum at 37 ° c . in 5 % co 2 . mcs were prepared using a modification of our previously described method ( 12 ). a cell suspension containing 10 , 000 cells ( 200 μl ) was added to each well of poly - hema coated 96 well plates . the wells were then overfilled by adding an additional 170 μl media to acquire a convex surface curvature . plasticine spacers ( 3 mm ) were placed in the corners of each plate to prevent the lids from touching the media . the plates were then inverted in order to allow the cells to sediment to the liquid / air interface and incubated in gentle shaking . after 24 hrs incubation the plates were returned to normal . first excess media was removed by aspiration and then plasticine spacers . the plates were incubated for 4 days prior to drug treatment . after 24 hours of drug treatment , np40 was added to the culture medium to a concentration of 0 . 1 % to extract caspase - cleaved k18 from mcs and to include material released to the medium from dead cells . caspase cleaved keratin - 18 ( k18 - asp396 ) was determined using 25 ml medium / extract using the m30 cytodeath elisa assay ( a variant of the m30 - apoptosense ® elisa ( 13 ) developed for in - vitro use ( peviva ab , bromma , sweden )). viability measurements were performed by the acid phosphatase ( aph ) method described by friedrich et al . ( 14 ). background activity was subtracted . htert - rpe1 cells were obtained from clontech laboratories , mountain view , calif . htert - rpe1 is an immortalized human retinal epithelial cell line that stably expressed human telomerase reverse transcriptase ( htert ). the fluorescence microscope arrayscan v hcs system ( cellomics inc ., pittsburgh , pa ., usa ) was used to determine edu incorporation . before addition of test compounds hct116 cells were seeded into 96 - well plates ( perkinelmer inc ., wellesley , mass ., usa ) and left to attach over night . cells were treated with cb21 for 24 h or with vehicle control . cells were stained using click - it edu hcs assay ( c 10354 , invitrogen , molecular probes inc , or , usa ) according to the manufacturer &# 39 ; s instructions . processed plates were loaded in the arrayscan and analyzed . images were acquired for each fluorescence channel , using suitable filters with 10 × objective and in each well at least 1000 cells were analyzed . average total intensity in the bdu channel was measured . results are shown as average of two independent experiments , each performed in duplicate wells and shown as mean ± sd . mcs produced by the hanging drop method in 96 well plates were fixed in paraformaldehyde , dehydrated , embedded in paraffin and sectioned . each sample contained 32 mcs ( mcs from each 96 well plate were pooled into 3 groups ). the sections were deparaffinized with xylene , rehydrated and microwaved , and then incubated overnight with the monoclonal primary antibodies diluted in 1 % ( weight / volume ) bovine serum albumin and visualized by standard avidin - biotin - peroxidase complex technique ( vector laboratories , burlingame , calif ., usa ). counterstaining was performed with mayer &# 39 ; s haematoxylin . antibody mib - 1 ( against the nuclear proliferation - associated antigen ki67 ) was obtained from immunotech sa , marseille , france and used at a dilution of 1 : 150 ; antibody against active caspase - 3 was obtained from pharmingen and used at a dilution of 1 : 50 . cell extract proteins were resolved by tris - acetate page gels ( invitrogen , carlsbad , calif .) and transferred onto a polyvinylidene difluoride ( pvdf ) membrane . the membranes were incubated overnight with antibodies , washed and incubated with hrp - conjugated anti - rabbit ig ( amersham biosciences , little chalfont , uk ) for 1 h . peroxidase activity was developed by supersignal west pico ( pierce biotechnology , rockford , ill .) according to manufacturer &# 39 ; s instructions . the connectivity map ( cmap ) ( www . broad . mit . edu / cmap ) build 02 contains genome - wide expression data for 1300 compounds ( 6100 instances , including replicates , different doses and cell lines ). the original protocol using mcf - 7 breast cancer cells as described by lamb et al ( 15 ) was followed . cells were plated in 6 - well plates at a density of 0 . 4 × 106 cells per well and left to attach for 24 h , followed by exposure to nsc76022 , nsc620358 or nsc647889 at a final concentration of 10 μm , or to vehicle control ( dmso ). after 6 h treatment , the cells were washed with pbs . total rna was prepared using rneasy miniprep kit ( qiagen , chatsworth , calif .,). starting from two micrograms of total rna , gene expression analysis was performed using genome u133 plus 2 . 0 arrays according to the genechip expression analysis technical manual ( rev . 5 , affymetrix inc ., santa clara , calif .). raw data was normalized with mass ( affymetrix ) and gene expression ratios for drug treated vs . vehicle control cells were calculated to generate lists of regulated genes . filter criteria were present call for all genes in the treated cell line and an expression cut - off of at least 100 arbitrary expression units . for cmap compatibility reasons only probes present on hg u133a were used . to retrieve a ranked compound list the 40 most up and down regulated genes ( i . e . probes ) for each compound were uploaded into the cmap and compared with the 6100 instances in the cmap database . measurement of respiration was performed as described ( 16 ). succinate ( 5 mm ) in the presence of rotenone ( 2 mm ), malate + pyruvate ( 5 mm each ) and tmpd ( 0 . 5 mm )+ ascorbate ( 1 mm ) were used as mitochondrial substrates . changes in the oxygen concentration were monitored with an oxygen electrode ( hansatech instruments , norfolk , uk ) and analyzed with the oxygraphplus software ( hansatech instruments , norfolk , uk ). basal v4 respiration in cells was estimated in the presence of 1 m atractyloside , which blocks adp entry into mitochondria . when hct116 tumours in scid mice had grown to a size of 200 mm 3 the mice were injected with drugs i . p ., and tumour size measured daily . example 1 . the compound of the invention induces apoptosis and reduces viability of mcs treatment of mcs with cb21 for 6 h followed by incubation for 96 h in drug - free medium resulted in mcs of smaller size with central areas of necrosis ( fig2 a ). caspase - 3 induction was modest compared to nsc647889 . importantly , treatment of mcs for 6 hours with cb21 reduced the clonogenicity to & lt ; 5 % ( fig2 b ). the decrease in clonogenicity was stronger than that observed for cisplatin , irinotecan and doxorubicin ( despite the use of concentrations of 5 - 10 μm ; & gt ; 10 - fold the ic 50 of these compounds in monolayer cultures ). treatment of monolayer hct116 cells with cb21 resulted in a slight increase in cell numbers between 0 to 24 h , followed by loss of cells ( fig2 c ). examination of 5 - ethynyl - 2 ′- deoxyuridine incorporation ( edu ) in cb21 - treated cells showed that dna synthesis was almost completely abrogated at 24 hours ( fig2 d , 2 e ). cb21 was equally effective on cells where the p53 tumour suppressor gene had been disrupted as on cells expressing wt p53 ( fig2 b ). the response of immortalized human epithelial cells ( htert - rpe1 cells ) differed from that of hct116 cells . the growth of these cells was arrested , but cell numbers were not reduced ( fig2 f ). when htert - rpe1 cells were plated at high density ( 70 , 000 cells / well ), essentially no cell loss was observed after treatment with cb21 ( fig2 g ). this difference in response to cb21 between hct116 and htert - rpe1 cells is shown in fig2 h . example 2 . the compound of the invention is a cell permeable iron chelator to generate hypotheses regarding the mechanism of action of cb21 , the connectivity map ( cmap ) ( 15 ), a compendium of gene expression signatures from drug - treated cell lines , was used . the changes in gene expression elicited by cb21 were most similar to those of ciclopiroxolamine ( cpx ), an antimycotic agent with iron chelating capacity ( 17 ) ( fig3 a ). to test whether the cytotoxic activity of cb21 was dependent on iron depletion , iron chloride was added to hct116 cells prior to the addition of cb21 . iron chloride was found to totally abrogate the effect of cb21 ( fig3 b ), both on hct116 cells expressing wtp53 as on hct116 cells where the p53 gene has been disrupted . the anti - proliferative activity of cb21 was compared with that of other known iron chelators . cb21 was found to be more potent than vlx50 , deferasirox , ciclopiroxolamine , deferoxamine ( fig3 c ). structure - activity relationships were examined by use of a number of structurally related compounds ( fig3 d , 3 e ). these studies showed that cb21 was the most effective compound in both monolayer and mcs cultures . example 3 . the compound of the invention induces a widespread autophagic response the anti - tumourigenic activity of iron chelators is generally attributed to inhibition of ribonucleotide reductase , leading to inhibition of cell proliferation ( 18 ). mcss contain mostly non - proliferating cells . the finding of induction of cytotoxic effects on mcss by the iron chelator cb21 thus was unexpected . the mechanism ( s ) of action was studied in more detail . visual inspection of cb21 - treated cells revealed that cells contained multiple large cytoplasmic vesicles ( fig4 a ). these vesicles stained positively with an antibody to microtubule associated protein 1 light chain 3 ( lc3 ), suggesting that they were associated with autophagy . lc3 staining was observed at 24 h and was stronger at 42 h ( fig4 b ). western blot analysis showed that treatment of hct116 monolayer cells with cb21 induced a strong increase in the levels of both lc3 - i and lc3 - ii ( fig4 c ). lc3 - ii ( the pe - conjugated form of lc3 ) is a protein marker regarded to be most reliably associated with autophagosomes ( 19 ). lc3 - ii levels were strongly induced by cb21 also in hct116 mcs ( fig4 c ). cb21 also induced lc3 - ii in htert - rpe1 cells , but the level of induction was much weaker compared to hct116 cells ( fig4 d ). this result shows that the extent of cb21 - induced autophagy correlates to the cytotoxic effect of the compound in these two cell types . hct116 cells were treated with cytotoxic concentrations of different iron chelators for 24 h . induction of lc3 - i and lc3 - ii was observed in all instances , showing that lc3 induction was a general effect of iron chelators ( fig4 e ). induction of lc3 - i and - ii by iron chelators was much stronger compared to that observed after treatment with rapamycin or nvp - bez235 ( no induction observed at 24 h , weak induction at 6 h ). for examination of whether cb21 is able to induce cellular changes in the inner core cells of the mcs , hct116 mcs were treated with cb21 for 6 h , washed and incubated for different time periods , fixed , sectioned , and examined by electron microscopy . large vesicles were observed in cells starting at 24 h after treatment ( fig4 f ). notably , a common feature of cb21 - treated mcs was the early appearance of enlarged and swollen mitochondria ( fig4 f ). most importantly , massive vacuolization occurred in a time - dependent manner not only in cells in the mcs periphery but also in the center of the mcs ( fig4 f ). it was concluded that cb21 induces the formation of vesicles in the cells of the central cores of mcs , found to be resistant to apoptosis , and that this response was associated with loss of viability of these cells . example 4 . blocking autophagy or autophagosome - lysosome fusion enhances cell death by the compound of the invention autophagy is generally considered to be a survival response to stress conditions but may also be a mechanism of programmed cell death ( 20 , 21 ). to examine the effects of different inhibitors of autophagy on cb21 - induced cell death , the cytotoxic effect of cb21 was potentiated by 3 - ma ( fig5 a ), a pi3k inhibitor commonly used as an inhibitor of autophagy . next a knock - down of beclin / atg6 using sirna was performed . this resulted in almost complete knock - down of the expression of this protein . beclin / atg6 knock - down reduced the viability of hct116 cells by ˜ 50 %; viability was further reduced by cb21 toxicity ( fig5 b ). examination of the effect of bafilomycin a , an antibiotic that prevents fusions of autophagosomes and lysosomes , demonstrated that bafilomycin a suppresses the appearance of large cytoplasmic lc3 - ii positive vesicles in hct116 cells ( fig5 c ). whereas bafilomycin a induced cytotoxicity at ˜ 72 hours , the combination of bafilomycin a and cb21 cytotoxicity was observed earlier (˜ 48 hours ) ( fig5 d ). these findings show that inhibition of autophagy potentiates the cytotoxic effect of the compound of the invention , and that the large vesicles observed in cb21 treated cells are caused by the fusion of autophagosomes and lysosomes . chloroquine ( cq ) is a lysosomotropic agent widely used to inhibit the maturation of autophagosomes into degradative autolysosomes ( 22 ). cq has no effect on its own on the proliferation of hct116 cells . the combination of cq and cb21 resulted in a strong potentiation of cell death on monolayer hct116 cells ( fig5 e ). examination of cytotoxicity to mcs of the combination of cq and cb21 revealed an effect potentiated in comparison with the effect of either cq or cb21 on mcs ( fig5 f ). cb21 induced a number of hypoxia responsive genes and also a number of genes known to be regulated by p53 ( fig6 a ). induction of hif - 1a and p53 protein levels by western blotting ( fig6 b ) was confirmed . a large induction was also observed using a reporter cell line where gfp is regulated by the hif - 1a promoter ( fig6 c ). among different genes strongly induced by cb21 was noted the gene encoding the bh3 - only protein bnip3 . bnip3 is a known target of hif - 1 a ( 23 ). bnip3 expression has been reported to induce extensive cytoplasmic vacuolization and autophagy ( 24 ). cb21 was found to induce the expression of bnip3 protein in hct116 cells ( fig6 d ). bnip3 expression was , however , also strongly induced by cb21 in htert - rpe1 cells ( fig6 d ). this finding is not consistent with bnip3 being a presumed mechanism of cb21 - induced autophagy . knock - down of bnip3 using sirna did not decrease induction of lc3 - ii and cell death by cb21 ( fig6 e ). example 6 . the compound of the invention inhibits oxygen consumption and decreases mtor activity the results described above suggest that autophagy is induced as an attempt to rescue cells from toxic insults induced by cb21 . since a number of key proteins involved in cellular energy metabolism contain fe — s complexes ( 25 ), the present inventors hypothesized that iron chelation by cb21 might lead to disturbances in cell metabolism that would trigger autophagy . to evaluate this hypothesis the effect of cb21 on intracellular levels of atp was examined . however , no decrease of intercellular atp levels at concentrations that induce autophagy could be observed nor could an induction of the phosphorylation of ampk ( amp - activated protein kinase ) be detected ( not shown ). next , a possible affectation of glucose transport by cb21 was followed by flow cytometry using the fluorescent d - glucose analog 2 -[ n -( 7 - nitrobenz - 2 - oxa - 1 , 3 - diazol - 4 - yl )- amino ]- 2 - deoxy - d - glucose ( 2 - nbdg ). as shown in fig7 a , a 25 % increase in 2 - nbdg uptake was observed in cb21 - treated cells . examination of the effect of cb21 on cellular oxygen consumption revealed that in hct116 monolayer cultures , v3 ( state 3 ) and vu ( uncoupled ) respiration did significantly decrease ( p & lt ; 0 . 05 ) after 6 hours of cb21 treatment ( fig7 b ). to examine whether respiration in tumour cells in the mcs is affected by cb21 an indirect approach was used . it is known that inhibition of mitochondrial respiration leads to increased tissue oxygen tension , which can be visualized as decreased pimonidazole staining ( 26 ). it was found indeed that the area of sectioned mcs staining positive with pimonidazole was ˜ 50 % of control in cb21 - treated mcss ( fig6 c ; table ). the effect was observed after 3 hours of drug exposure and persisted at 24 hours ( fig7 c ). as a control , hct116 monolayer cultures were treated with the mitochondrial uncoupling agent carbonylcyanide - 3 - chlorophenylhydrazone , cccp , known to increase oxygen consumption . as expected , mcs treated with cccp displayed a larger of area of pimonidazole staining ( fig7 c ; table ). the mammalian target of rapamycin ( mtor ) is a serine / threonine kinase regulating cell growth in response to nutrient status . it is well established that metabolic stress affects the activity of the mtor pathway ( 27 ). the mtor pathway regulates mitochondrial oxygen consumption and oxidative capacity ( 28 , 29 ). in order to determine whether the decreased oxygen consumption observed after cb21 treatment was associated with mtor inhibition , phosphorylation of the mtor substrate 4ebp1 was examined . as shown in fig7 d , 4ebp1 phosphorylation is inhibited by cb21 . the decrease in phosphorylation is associated with an increased akt - phosphorylation . inhibition of mtorc1 is known to release a negative feedback loop involving , resulting in strong akt activation ( 30 ). to test whether mtor inhibition would reduce oxygen consumption , hct116 mcs was treated with rapamycin ( a specific pharmacological inhibitor of mtor - raptor complex formation ) and stained sections with pimonidazole . a reduction of pimonidazole staining was observed , although not as strong as with cb21 ( fig7 c ; table 1 ). these findings prompted examination of whether direct inhibition of mtor does lead to similar effects as by cb21 . for these experiments was used the dual pi3k / mtor inhibitor nvp - bez235 , a compound in clinical trials . importantly , nvp - bez235 was found to decrease 4ebp1 phosphorylation in hct116 cells grown both under monolayer or mcs conditions ( fig7 d ). in contrast to cb21 , nbpbez235 did not affect the viability of the cells in the core of the mcs . example 7 . cell death induced by the compound of the invention is enhanced by glucose starvation approximately 50 % of cellular atp production in tumour cells is by oxidative phosphorylation ( 31 ). oxygen consumption has been reported to decrease in the interior regions of tumour mcs , possibly as a consequence of decreased proliferative activity ( 32 , 33 ). other investigators have found that oxygen consumption is rather uniform in viable regions of mcs ( 34 ); it has been reported that fibroblast clones at the same stage of transformation may have quite distinct metabolic activity in mcs culture ( 33 ). even in the event of low cellular oxygen consumption in the cells of the central core , a further decrease induced by cb21 is expected to lead to an increased dependence of glucose . whereas monolayer cells may compensate increased glucose dependence by increased uptake ( as shown in fig8 a ), glucose will be limiting in mcs . as shown in fig8 a , hct116 mcs core cells are dependent on glucose for survival : glucose depletion leads necrosis of central areas , an effect is reminiscent of that of cb21 ( fig2 ). based on these considerations it was tested whether glucose starvation increases the sensitivity of hct116 monolayer cells to cb21 . this was indeed the case : glucose starvation decreased cell viability and increased apoptosis by cb21 . these findings are likely to at least partly explain the sensitivity of central core cells to cb21 . the in - vivo anti - tumour activity by cb21 was examined in the hct116 model . tumours were allowed to grow to a size of 0 . 2 ml and then treated with cb21 . a clear anti - tumour effect of the compound cb21 was observed ( fig1 ). a number of iron chelators have been developed that exhibit anti - tumour activity , including triapine ( 35 ), tachpyr ( 36 ) and trensox ( 37 ). iron is important for many metabolic reactions , including the formation of deoxyribonucleotides from ribonucleotides by ribonucleotide reductase ( 38 ). in the absence of fe , cells cannot progress from the g1 to the s phase of the cell cycle , explaining the anti - proliferative action of cb21 on both hct116 and htert - rpe1 cells observed . since iron chelators are principally regarded as specific to proliferating cells , the identification of an iron chelator in a screen for agents that show cytotoxicity on mcs was not anticipated . further investigations revealed possible mechanisms for the effects of cb21 on non - proliferating cells in mcs cores . it was furthermore found that cb21 decreased oxygen consumption of hct116 and htert - rpe1 cells , as observed both by direct measurement and by using pimonidazole staining of mcs . it is known that mitochondrial oxygen consumption and oxidative capacity is regulated by the mtor pathway ( 28 , 29 ). it has also been reported that the iron chelator deferasirox inhibits mtor signaling ( 39 ). cb21 was indeed found to inhibit phosphorylation of 4ebp1 and to lead to a upregulated akt phosphorylation . the inhibition of mtor signaling by deferasirox has been ascribed to induction of redd1 ( also referred to rtp801 ), a gene induced by hypoxia , which in turn activates the tsc2 protein ( 40 ). it is conceivable that the effect of cb21 on oxygen consumption is at least partly mediated by this mechanism . another possibility is that metabolic stress induced by iron depletion affects the activity of the mtor pathway by some other mechanism . another effect of cb21 , shared by other iron chelators ( 41 ), is the induction of lc3 positive cytoplasmic vesicles and lc3 - ii protein . lc3 induction was found to be much less pronounced in htert - rpe1 cells . the induction of lc3 - ii by iron chelators was significantly stronger than that observed with mtor inhibitors , suggesting that lc3 - ii induction was not mediated exclusively by mtor inhibition . it was found that the pi3k / mtor inhibitor nvp - bez235 does not induce detectable cytotoxic effects on cells in hct116 cores ( hernlund et at ., unpublished ), again suggesting mtor inhibition not being the only factor responsible for autophagy induction and cell death by cb21 . the decreased oxygen consumption observed after cb21 treatment should lead to increased dependence of glucose for atp production , similarly to what has been reported for rapamycin ( 42 ). this proposition seems to be confirmed by the observation that the population of cells present in the mcs core showed signs of constitutive er stress ( grp78 positive ), a condition probably induced by hypoxia and limited glucose supply . glucose starvation of mcs induced cell death of the core cells , consistent with the concept that survival of this cell population is dependent on glucose . the increased dependence of glucose observed after treatment with cb21 is very likely contributing to cell death of the population of core cells . apoptosis did not appear to be the main mechanism of cell death by cb21 , as evidenced by weak caspase - 3 induction compared to the strong induction of caspase - 3 in peripheral cells ( not shown ). conditions of poor cellular energy status may lead to resistance to apoptosis ( also explaining the resistance of core cells to nsc647889 - induced apoptosis ( not shown ). it seems that cb21 induces increased glucose dependence of hct116 cells , and that this leads to decreased viability of hypoxic cells in mcs cores . autophagy is a catabolic degradation response to metabolic stress , which strives to maintain homeostasis through degradation of proteins and organelles . pi3k - akt - mtor , lkb1 - ampk - mtor and p53 are the main regulators of the autophagic pathway . autophagy is believed to be involved in mediating resistance of cancer cells to anticancer therapy and to be an attractive therapeutic target in anticancer drug resistance ( 20 , 43 ). cb21 induced a remarkable autophagic response , characterized be strong lc3 - i and - ii induction . the present invention reveals inhibition of autophagy to potentiate the cytotoxicity of cb21 . 1 . tannock i f et al . limited penetration of anticancer drugs through tumour tissue : a potential cause of resistance of solid tumours to chemotherapy . clin cancer res 2002 ; 8 : 878 - 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