Patent Application: US-201214006277-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 . cell culture , generation of mcs and screening . 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 ). evaluation of dna synthesis . 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 ( c10354 , invitrogen , molecular probes inc , oreg ., 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 . immunological assays . 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 . western blotting . 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 . connectivity map . 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 mas5 ( 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 . oxygen consumption . 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 . treatment of mouse xenografts . 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 . 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 ic50 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 . 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 . 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 . 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 . 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 - la 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 - la 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 - ll and cell death by cb21 ( fig6 e ). 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 . 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 if 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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