Patent Application: US-26340202-A

Abstract:
treatment of normal , non - cancerous , animal cells with an agent that depletes polyamines within the cells results in an inhibition of apoptosis when the cells are exposed to an inducer of apoptosis . this inhibition of apoptosis is not observed , or is observed to a lesser extent , in similarly treated cancerous cells . the method of the invention is useful in obtaining preferential killing of cancer cells , as opposed to normal cells , due to anti - cancer therapy .

Description:
in a preferred embodiment , the invention is a method for preferentially inhibiting apoptosis in non - neoplastic cells compared to neoplastic cells that are exposed to a similar apoptosis - inducing event . the method of the invention inhibits apoptosis in non - cancerous cells while not inhibiting , or inhibiting to a lesser extent , apoptosis in cancer cells . the cells , normal and / or cancer cells , may be within the body of an animal , such as a human or veterinary patient , or may be outside of the body of an animal , such as in a cell culture or a surgically removed biopsy sample . in accordance with the preferred method of the invention , an inhibitor of odc is administered to the cells , the level of polyamines , especially that of putrescine , in the cells is depleted , and apoptosis is inhibited in normal cells and is not inhibited , or is inhibited to a lesser extent , in cancer cells . the inhibitor of odc may be any organic or inorganic molecule that prevents odc from catalyzing the formation of putrescine . preferably , the inhibitor of odc is dfmo . however , any inhibitor of odc may be employed in accordance with the preferred embodiment of the invention , so long as the inhibitor does not itself cause death of normal cells . preferably , each of the three polyamines , putrescine , spermidine , and spermine , are depleted from the cell . however , in certain instances and in accordance with the invention , only put and spd levels may be reduced , with retention of the pre - treatment level of spm . depletion of polyamines , as used in this specification , refers to depletion of measurable polyamines within the cell . in the examples that follow , apoptosis in normal cells , with and without inhibition of odc , is illustrated in a limited number of cell types , including normal rat epithelial cells . it is understood , however , that the invention is applicable to cells other than rat cells , such as human , dog , or cat cells . for example , the method of the invention may be applied to neoplastic and non - neoplastic cells of ectodermal , mesodermal , or endodermal origin from any animal species . in accordance with the method of the invention , the odc - inhibiting agent may be administered to a patient in need of therapy by any suitable route for the particular agent . for example , a chemical odc inhibitor may be administered orally , by intramuscular , subcutaneous , or intravascular injection , or by administration directly into a tissue , such as by direct injection into a tumor and / or surrounding non - neoplastic tissues . other suitable means of administering a chemical odc inhibitor to a patient include ophthalmic application , suppositories , intradermal administration , and transmucosal administration , such as by intranasal droplets , lozenges or a gargle . in accordance with the invention , the odc inhibitor , such as dfmo , is administered to a cell or a patient in an amount effective to inhibit the catalytic action of odc in the first rate - limiting step in intracellular polyamine biosynthesis and in an amount below that which will result in death of the cell or of the patient . as an example , a suitable range of dfmo for administration to a mammal , such as a human , is preferably between 10 mg / kg to 200mg / kg per day . however , if desired , amounts higher or lower than this range may be administered , so long as the amount of dfmo administered is high enough to inhibit odc and not so high as to seriously injure or kill the cell or patient . in human patients , the dfmo may be administered orally in a tablet or capsule form containing 100 to 500 mg of dfmo , which is administered 1 to 10 times daily to achieve the desired effect . the examples that follow are illustrative of the invention and are not to be construed as limiting the invention . iec - 6 cells , having the atcc designation crl - 1592 , were obtained from the american type culture collection at passage 13 . the iec - 6 cells are normal , non - cancerous intestinal epithelial cells derived from fetal rat crypt cells . these cells are non - tumorigenic and retain the undifferentiated character of epithelial stem cells . the iec - 6 cell stock was maintained in t - 150 flasks in a humidified , 37 ° c . incubator in an atmosphere of 90 : 10 air : co2 . the medium used was dulbecco &# 39 ; s modified eagle medium ( dmem ) ( gibco brl , grand island , n . y .) with 5 % heat inactivated fetal bovine serum ( fbs ) ( sigma , st . louis , mo .) and 10 μg insulin and 50 μg gentamicin sulfate per ml . the stock was passaged weekly at 1 : 10 , fed 3 times per week , and passages 15 - 20 were used . the cells were plated on day 0 at a density of 6 . 25 × 10 4 cells / cm 2 in t - 150 flasks in dmem / dfbs ( dialyzed fetal bovine serum with 10 , 000 molecular weight cut - off ) ( sigma , st . louis , mo .) with or without the treatment compound or compounds described in the examples that follow . the cells were fed on day 2 . on day 3 , medium was removed and replaced with serum - free medium . on day 4 , camptothecin ( sigma , st . louis , mo .) in a dmso vehicle was added to a concentration of 20 μm into the serum - free medium containing the cells of example 1 for 3 to 18 hours , with the vehicle ( dmso ) added to controls . detachment - induced cell death ( dicd ) is a well recognized form of apoptosis in anchorage - dependent cell types , such as intestinal epithelial cells . floating cells were poured into a 25 ml tube and the monolayer was washed once with hbss without calcium and magnesium . this wash was then combined into the tube with the floating cells . attached cells were taken up with 0 . 05 % trypsin plus 0 . 53 mm edta , followed by one wash of the flask with dmem / 5 % fbs . cell counts were determined separately for floating and attached cells by counting on a model zf coulter counter . floating cells were expressed as a percentage of the total cell count obtained by combining the number of floating and attached cells . as shown in fig1 a , there was an 8 - fold increase in the number of floating cells in the camptothecin treated group as compared to the dmso control group within 6 hours of treatment . in a separate flask , following camptothecin or dmso addition , the cells were harvested after 16 hours in order to obtain a sufficient number of floating cells for analysis . floating cells were then poured off to be counted and were combined with one wash with cold dpbs ( sigma , st . louis , mo .). the attached cells were then harvested for determination of caspase activity . 10 ml of dpbs was added to the flask and the monolayer was scraped and collected into a 25 ml tube . the flask was washed once with 10 ml of dpbs and combined into the 25 ml tube . the cells were pelleted by centrifugation at 800 × g for 5 minutes . the supernatant was discarded and the pellet was resuspended in 1 ml of cold dpbs and transferred into a microfuge tube . the cells were pelleted by centrifugation at 10 , 000 × g at 4 ° c . for 10 minutes . the supernatant was discarded and the cells were lysed in 100 ml of ice cold cell lysis buffer ( 50 mm hepes , ph 7 . 4 , 0 . 1 % chaps , 1 mm dtt , 0 . 1 mm edta , 0 . 1 % np40 ) ( sigma , st . louis , mo .). the assay for caspase activity was carried out in a 96 - well plate . into each well was placed 20 μl of cell lysate , 70 μl of assay buffer ( 50 mm hepes , ph 7 . 4 , 0 . 1 % chaps , 100 mm nacl , 10 mm dtt , 1 mm edta ) and 10 μl of caspase 3 calorimetric substrate ( 2 mm devd - pna prepared in assay buffer , a caspase specific peptide that is conjugated to the chromogen p - nitroanilide ) ( biomol research laboratories , plymouth meeting , pa .). the 96 - well plate was incubated at 37 ° c . for 2 hours , during which time the caspase in the sample was allowed to cleave the chromophore pna from the substrate molecule . absorbance readings at 405 nm were made after the 2 hour incubation , with the caspase activity being directly proportional to the color reaction . protein was determined for each sample using the bca method ( pierce , rockford , ill .) and a standard curve for p - nitroanilide was carried out . results for caspase activity were expressed as pmol of pna released / mg protein / minute . cells were collected for determining the presence or absence of dna fragmentation . after treatment with camptothecin ( or vehicle ), floating cells were poured off and combined with one dpbs wash of the monolayer . cells were pelleted , by centrifugation at 800 × g for 5 minutes . the supernatant was discarded and the pellet was resuspended in 100 μl of resuspension buffer . the attached cells were collected for dna fragmentation by scraping in dpbs and combining with one wash of the flask with dpbs . cells were pelleted at 800 × g for 5 minutes , the supernatant was discarded and the pellet was resuspended in 100 μl of resuspension buffer . the nucleosomal fragmentation assay was carried out by isolating dna from the cells using the tacs apoptotic dna laddering kit ( trevigen , gaithersburg , md .) and analyzing the labeled dna by agarose gel electrophoresis following the manufacturer &# 39 ; s instructions . as shown in fig1 b and 1c , attached cells showed a 7 - fold increase in caspase 3 activity , but did not show remarkable dna fragmentation . fig1 b and 1c show that detached cells from both control and camptothecin treated groups showed characteristic nucleosomal dna fragments and increased caspase 3 activity . the results establish that camptothecin is an effective inducer of apoptosis in the iec - 6 cells and that caspase 3 activity is well correlated with dna fragmentation associated with apoptosis . because caspase 3 levels indicate the occurrence of apoptosis , caspase 3 activity was used as an indicator of the initiator phase of apoptosis in the examples that follow . iec - 6 cells were grown for 4 days in the presence or absence of dfmo ( marion merrel dow , cincinnati , ohio ) the highly specific inhibitor of odc . dfmo treatment depletes putrescine within 3 hours . depletion of spermidine requires 24 hours and significant depletion ( about 50 %) of spermine requires 3 days . camptothecin - induced apoptosis was determined 6 hours after camptothecin treatment . as shown in fig2 cells treated with dfmo ( polyamine depleted ) were protected against both dmso ( vehicle ) and camptothecin - induced apoptosis . about a 3 - fold decrease in the number of floating cells was observed in polyamine depleted cells as compared to control cells treated with camptothecin , as shown in fig2 a . dfmo treatment also resulted in approximately 50 % lower caspase 3 activity compared to control , as shown in fig2 b . as shown in fig3 a , camptothecin induced apoptosis ( as judged by the number of floating cells ) began within 3 hours and increased progressively up to 18 hours ( from 6 . 2 % at 0 time to 49 . 3 % at 18 hours ) in control cells . the onset of apoptosis was delayed in dfmo treated cells for up to 12 hours ( from 2 . 2 % at 0 time to 9 . 3 % at 12 hours ) and reached maximum of 29 . 1 % at 18 hours . as described in example 2 , attached cells were collected for the determination of caspase 3 activity . caspase 3 activity increased progressively with time in control cells . in contrast , dfmo treated cells did not show remarkable increases in caspase 3 activity for up to 12 hours . in these cells , significant increase in caspase 3 activity was observed only after 18 hours of exposure to camptothecin , as shown in fig3 b . these results indicate that polyamine depletion delays the onset of apoptosis in iec - 6 cells , and suggest that the intracellular polyamine levels may be a critical factor in the regulation of spontaneous apoptosis . as shown in fig4 dfmo again decreased the number of floating cells as compared to control . addition of exogenous 10 μm putrescine along with dfmo restored the number of floating cells to control levels . dna fragmentation was evident in floating cells irrespective of the treatment , but attached cells did not clearly reveal nucleosomal fragments . iec - 6 cells were grown , as described in the above examples , but in the presence of either dfmo ( control ) or diethylglyoxal bis -( guanylhydrazone ) ( degbg ). dfmo inhibits the enzyme ornithine decarboxylase ( odc ), which catalyzes a step in the production of putrescine . degbg blocks the conversion of putrescine to the polyamines spermidine and spermine by inhibiting the enzyme s - adenosyl methionine decarboxylase ( samdc ). intracellular polyamines were analyzed by high performance liquid chromatography . cells were plated in 60 mm dishes at 6 . 25 × 10 4 cells / cm 2 and grown in dmem / dfbs with or without dfmo or degbg . after washing the monolayer three times with ice - cold dpbs , 0 . 5 m perchloric acid was added and the samples were then frozen at − 80 ° c . until ready for extraction , dansylation , and hplc . the standard curve encompassed the range of 0 . 31 - 10 μm . values that fell & gt ; 25 % below the curve were considered not detectable . the level of polyamine was determined by the bradford method , as described in anal . biochem ., 72 : 248 - 254 ( 1976 ). as shown in fig5 a , 5b , and 5 c , degbg significantly increased the level of putrescine in the cells while decreasing the levels of spermidine and spermine . in contrast , control cells grown in the presence of dfmo had decreases in each of the three polyamines . these results establish that inhibition of odc results in depletion of all three polyamines . in contrast , inhibition of samdc leads to an accumulation of putrescine and concomitant depletion of spermidine and spermine . iec - 6 cells were grown in the presence of dmso ( control ), dfmo , or degbg . as shown in fig6 a , dfmo treatment led to a progressive decrease in the percentage of floating cells over days 1 , 2 , and 4 ( 19 . 2 , 12 . 4 , and 2 . 2 %). when compared to control ( 18 . 8 %), dfmo treatment for only 1 day , which results in putrescine depletion , but not spermidine or spermine depletion , did not effect the percentage of floating cells . in contrast , degbg treatment showed a significant increase in the percentage of floating cells on days 1 , 2 , and 4 ( 26 . 6 , 26 . 9 , and 23 . 3 %) as compared to dfmo treated cells , as well as to control cells . caspase 3 activity , as shown in fig6 b , followed the same pattern as that for the number of floating cells . these results show that the accumulation of putrescine is associated with the induction of apoptosis , while depletion of spermidine and spermine leads to the inhibition of apoptosis in iec - 6 cells . total cell protein ( 50 μg ) from cell extracts prepared for caspase 3 assay was separated on 15 % sds - page and transferred to nitrocellulose membranes for western blotting . equal loading of protein was confirmed by staining the nitrocellulose membrane with ponceau s ( sigma , st . louis , mo .). the membrane was then probed with an antibody directed against caspase 3 ( cpp32 ) ( santa cruz biotechnology , santa cruz , calif .). the immunocomplexes were visualized by the enhanced chemiluminescence detection system . as shown in fig7 the level of caspase 3 was not significantly different in polyamine depleted cells ( dfmo ) compared with controls or cells treated with dfmo and putrescine . putrescine was added to cell extracts from dfmo treated cells which were low in caspase 3 activity . increasing putrescine concentration up to 5 μm did not reveal any effect on caspase activity . these data show that the level of intracellular polyamines , rather than caspase activity , is the determinant of the inhibition of apoptosis . studies described in examples 1 to 8 are repeated , except that normal human intestinal cells are substituted for the iec - 6 rat intestinal cells . similar inhibition of apoptosis is seen following treatment with dfmo . studies described in examples 1 to 8 are repeated , except that cancerous human intestinal caco - 2 cells ( atcc no . htb37 ) are substituted for the iec - 6 rat intestinal cell lines . no inhibition of apoptosis is observed following the treatment with dfmo . cancerous human intestinal caco - 2 cells were grown in similar culture media as described in example 1 , except that one group of the cells was grown in a medium not containing dfmo and another group was grown in a medium containing dfmo . after four days of growth , camptothecin at a dose of 20 μm was added to the culture medium of each of the groups . after 6 hours , the number of floating cells in the media of the two groups of cells was determined . the study was repeated using a different inducer of apoptosis , a combination of 20 ng / ml of tumor necrosis factor - α ( tnf - α ) and 25 μg / ml of cycloheximide . the results are shown in table 1 and in fig8 and clearly demonstrate that dfmo did not protect the cancerous cells from apoptosis induced by either of the agents . table i camptothecin control ( no dfmo ): untreated ( no camptothecin ) = 2 . 0 +/− . 09 treated ( camptothecin ) = 2 . 7 +/− . 06 treated ( with dfmo ): untreated ( no camptothecin ) = 3 . 1 +/− . 06 treated ( camptothecin ) = 3 . 7 +/− . 24 tnf - α and cycloheximide control ( no dfmo ): untreated ( no tnf - α + cycloheximide ) = 3 . 3 +/− . 4 treated ( tnf - α + cycloheximide ) = 5 . 7 +/− . 2 treated ( with dfmo ): untreated ( no tnf - α + cycloheximide ) = 4 . 8 +/− . 5 treated ( tnf - α + cycloheximide ) = 6 . 2 +/− . 6 dfmo at a dosage of 100 mg / kg / day is administered orally for four days to an adult dog suffering from a mast cell tumor on one of its rear legs . the tumor is treated in multiple sessions by exposure to co 60 gamma radiation as indicated for this type of tumor . the field of exposure to the radiation exceeds the boundaries of the tumor . following therapy , the tumor is killed and a visually noticeable decrease in death of the surrounding normal tissues is observed compared with that typically observed with this type of therapy . the scientific articles listed in the following list of references are incorporated herein by reference . bowlin , et al ., u . s . pat . no . 5 , 002 , 879 , is incorporated herein by reference . the articles , mccormack et al ., am . j . physiol ., vol . 267 ( cell physiol . 36 ): c706 - 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