Patent Application: US-55060200-A

Abstract:
the present invention relates to a method for decreasing the intracellular , especially nuclear level of β - catenin in an individual and to a method for the prevention or treatment of a disease or condition in an individual , wherein said disease or condition is related to a mutant apc gene or to an elevated level of intracellular β - catenin . furthermore , the invention relates to methods for screening a subject to determine if said subject is a carrier of a mutant apc gene , and to methods for diagnosing an individual &# 39 ; s predisposition for a disease or condition in an individual , said disease or condition being related to a mutant apc gene or to an elevated level of intracellular β - catenin .

Description:
this invention relates to the use of hmr as a therapeutically active compound particularly in diseases , where β - catenin distribution in the cells is abnormal , i . e . maily cytosolic and / or nuclear . in humans such condition is typically seen as familial disease known as familial adenomatous polyposis ( fap ). this condition may be herediatary , but similar conditions may also occur due to environmental factors , which are only poorly known . the animal model , apc - min mutation mouse model , used in this invention , resembles this human condition . β - catenin is one of the proteins , which are linked to the communication of cells with each other . on the other hand , if it is mutated , like in intestinal polyposis , or if it is distributed to cytosolic or nuclear compartments , it may induce cell proliferation and thus promote adenoma development . for the purpose of this invention , hmr or its naturally occurring isomers can be administered by various routes . the suitable administration forms include , for example , oral formulations , parenteral injections including intravenous , intramuscular , intradermal and subcutaneous injection ; and transdermal or rectal formulations . in addition , hmr can be administered as a part of functional food or food incredient or food additive . suitable oral formulations for pharmaceutical purposes include e . g . conventional or slow - release tablets and gelatine capsules . the required dosage of hmr will vary depending on the disease or condition to be treated . for fap , it depends on the adenoma properties and the stage and metastatic dissemination . hmr can be administered preferable once or twice daily . the daily dose is 5 - 100 mg , preferably 30 - 80 mg daily . hmr can be given as tablets or other formulations like gelative capsules alone or mixed in any clinically acceptable non - active ingredients , which are used in the pharmaceutical industry . in addition , hmr can be administered mixed in different food constituents or can be administered as functional food . the invention , the use of hmr in the treatment of fap and other diseases , can be applied based on clinical conditions and symptoms . the exploitation of the invention can also be based on the diagnosis of apc - mutation in each subject by using individual dna mutation assays , or on the diagnosis of β - catenin distribution by polyclonal or monoclonal antibodies against β - catenin or parts of this protein . the dna sequence or the mutant apc gene can be used for screening a subject to determine if said subject is a carrier of a mutant apc gene . the determination can be carried out either as a dna analyse according to well known methods , which include direct dna sequencing of the normal and mutated apc gene , allele specific amplification using the polymerase chain reaction ( pcr ) enabling detection of either normal or mutated apc sequence , or by indirect detection of the normal or mutated apc gene by various molecular biology methods including e . g . pcr - single stranded conformation polymorphism ( sscp )- method or denaturing gradient gel electrophoresis ( dgge ). determination of the normal or mutated apc gene can also be done by using restriction fragment length polymorphism ( rflp )- method , which is particularly suitable for genotyping large number of samples . the determination can also be carried out at the level of rna by analysing rna expressed at tissue level using various methods . allele spesific probes can be designed for hybridization . hybridization can be done e . g . using northern blot , rnase protection assay or in situ hybridization methods . rna derived from the normal or mutated apc gene can also be analysed by converting tissue rna first to cdna and thereafter amplifying cdna by an allele spefic pcr - method and carrying out the analysis as for genomic dna as mentioned above . alternatively , the determination can be carried out as an immunohistochemical method where a sample is contacted with an antibody capable of binding β - catenin or a fragment thereof . an elevated level of intracellular , particularly nuclear , β - catenin is used as an indication of the occurrence of the mutated apc gene or β - catenin gene . the production of antibodies can be done in experimental animals in vivo to obtain polyclonal antibodies or in vitro using cell lines to obtain monoclonal antibodies . the invention will be described more in detail in the following experimental section . the laboratory animal ethics committee of the faculty of agriculture and forestry , university of helsinki approved the study protocol . male c57bl / 6j - min ( multiple intestinal neoplasia ) mice , 5 - 6 weeks of age , were obtained from the jackson laboratory , ( bar habor , me ., usa ). the animals were stratified by body mass and age and assigned randomly to the experimental diets , 8 mice / group , with initial body mass of 21 . 5 g . animals were housed in plastic cages in a temperature - and humidity - controlled animal facility , with 12 - h light / dark cycle . they had a free access to the semisynthetic diets and tap water for five to six weeks . the body weights of the animals were recorded weekly . the high fat ( 40 - energy %) experimental diets were composed in a way that the diets contained similar amounts of protein , carbohydrate and fat per energy ( table 1 ). the fat used in the diets was a mixture of butter , rapeseed oil , and sunflower seed oil providing the intake of saturated , monounsaturated and polyunsaturated fatty acid in the ratio 3 : 2 : 1 . it corresponded the intake of these fatty acids in the western type diet . all diets contain 2 . 5 % ( w / w ) polydisperse β ( 2 → 1 ) fructan , inulin ( raftiline ®, orafti , tienen , belgium ). the rye bran - supplemented diet was prepared by diluting the fiber - free high - fat diet with addition of rye bran at 100 g / kg diet . in this way the nutrient intake per energy was kept constant in all diets . hmr was stored on + 4 ° c . in darkness prior to use . the diets were stored at − 20 ° c . and kept at 4 ° c . only for the use within one week . after the feeding period , the mice were killed by co 2 asphyxiation . the small intestine , caecum and colon were removed , open along the longitudinal axis and rinsed with ice - cold saline . the small intestine was divided into five sections . the caecum and colon were kept together . the small intestine and colon + caecum were then spread flat on a microscope slide and a number ; diameter and location of adenomas were determined with an inverse light microscope with a screen at a magnification of × 2 . 5 . the diameter of adenomas was scored with a mm - scale placed on the screen . the adenomas from each section of the intestine were clipped off as well as the mucosa was scraped off with a microscope slide and snap frozen in liquid nitrogen . tissue samples were homogenized , and the cytosol and particulate fractions extracted as described previously ( a - m pajari et al ., 1998 ) with the addition of the extra centrifugation ( 8 500 × g for 10 min at 4 ° c .) to get the nuclear fraction . rat brain homogenate was used as positive control for β - catenin in immunoblotting analysis . for immunoblotting analysis , 5 ml of the crude extracts were concentrated to { fraction ( 1 / 50 )} volume with millipore ultrafree ®- 4 tubes ( millipore , bedford , mass .). after protein concentration measurement ( bradford , bio - rad protein assay reagent ) the homogenate was mixed with equal volume of sds sample buffer , boiled for 5 min and stored at − 80 ° c . until use . samples ( 30 μg ) and rat brain homogenate ( 10 μg ) were subjected to 10 % sds - page and then transferred to polyvinylidene difluoride ( pvdf ) membranes ( bio - rad laboratories , hercules , calif .) at 210 ma for 2h . the membranes were blocked at 4 ° c . overnight with 1 % nonfat dry milk in phosphate - buffered saline ( pbs ), washed with 0 . 5 % bsa in pbs , incubated with monoclonal mouse anti - β - catenin antibody ( transduction laboratories , lexington , ky .) and alkaline phosphatase - conjugated anti - mouse secondary antibody ( zymed , san francisco , calif .) in 1 % bsa in pbs . β - catenin antibody dose not recognize other catenins . monoclonal mouse anti - β - catenin from santa cruz biotechnology ( santa cruz , calif .) was used to confirm the specificity of the β - catenin signal . β - catenin bands were visualized by colorimetric staining with 5 - bromo - 4 - chloro - 3 - indolyl phosphate and nitroblue tetrazolium substrate mix ( bio - rad laboratories , hercules , calif .). blots were scanned and analyzed on a sharp jx325 scanner with imagemaster ® 1d software , version 2 ( pharmacia biotech , uppsala , sweden ). results in duplicates are expressed as sample band intensity ( optical density of the β - catenin band multiplied by band area ) divided by rat brain band intensity . the differences between the groups were analyzed by non - parametric mann - whitney u test ( spss inc ., version 6 . 1 ). data were considered significant at p & lt ; 0 . 05 . in recent years , considerable efforts have been made to either synthesize or find natural agents that are able to arrest or reverse carcinogenic processes , and thus prevent cancer . in this study , we tested the possible chemopreventive effect of hmr on intestinal adenoma development in apc min mice , an animal model of human familial adenomatous polyposis ( fap ). hmr showed a strong chemopreventive effect in our study by especially preventing the formation of new intestinal adenomas , which was seen as a significantly lower number of adenomas in the mice fed hmr compared with the mice fed the other diets . the mean number of adenomas in the small intestine was only 26 . 6 ± 11 . 0 ( mean ± sd ) in mice fed hmr while it was 39 . 6 ± 8 . 9 ( p = 0 . 031 compared to hmr group ) in inulin fed mice and 36 . 0 ± 7 . 4 ( p = 0 . 049 compared to hmr group ) in inulin / rye fed mice ( fig1 ). hmr did not affect the growth of existing adenomas since there were no differences in mean diameter , adenoma size (% of total ), and distribution of adenomas along the length of the intestine between the diet groups . in the colon and caecum , the incidence of adenomas ( 60 - 75 %) and the number of adenomas ( 0 . 9 - 1 . 3 ) did not differ between the groups . hmr resulted in normalization of β - catenin levels in adenoma tissue of apc min mice , indicating that hmr mediates its chemopreventive effect through the apc - β - catenin pathway . wild type apc protein regulates intracellular β - catenin level and prevents the entrance of β - catenin into the nucleus where excess β - catenin could tiger abnormal gene transcription ( v korinek et al ., 1997 ; pj morin et al ., 1997 ; j beherens et al ., 1996 ). we measure β - catenin levels in cytosolic , particulate and nuclear fractions of both adenoma and surrounding mucosa tissues in the small intestine ( table 2 ). in the cytosolic fraction , β - catenin level in adenoma tissue was significantly elevated ( p = 0 . 008 - 0 . 013 ) in all the diet groups as compared to that of the surrounding mucosa . in the nuclear fraction , β - catenin in the inulin and inulin / rye groups was also significantly higher ( p = 0 . 003 - 0 . 009 ) in the adenoma tissue when compared to the surrounding mucosa . however , hmr was able to restore nuclear β - catenin level of the adenoma tissue to the level found in the surrounding mucosa ( fig2 table 2 ). since there was also a tendency if cytosolic β - catenin to be low in hmr fed mice , it can be speculated that hmr might both enhance degradation of cytosolic β - catenin and prevent β - catenin transport to the nucleus . originally our aim was to see whether hmr and rye ( i . e . matairesinol ) have a similar chemopreventive effect on inulin promoted adenoma formation in apc min mice . even though hmr was quite potent in this respect , rye was not able to overcome the adenoma promotive effect of inulin . one reason for this may be the huge difference in the concentrations of hmr and matairesinol in the diets . estimated intake of hmr was about 20 mg / kg bwt daily and that of matairesinol about 17 μg / kg bwt daily . the exact chemopreventive mechanism of hmr needs further clarification . so far hmr has been shown to be protective in two different studies : in addition to our study with apc min mice , hmr has protected against dmba - induced mammary tumors in rat ( n saarien et al ., in press ). although lignans are classified as phytoestrogens , hmr do not have estrogenic , antiestrogenic or antiandrogenic activity in rats ( n saarien et al ., in press ). the question of safety is important when looking for new chemopreventive compounds . in our study no differences in body weight gain were found between the groups during the course of the experiment . the final body weight ( g ) of the animals were 30 , 30 and 31 . 5 for the inulin , inulin / rye and inulin hmr diet groups , respectively , indicating that the animals grew well . a ) casein was obtained from kainuun osuusmeijeri ( sotkamo , finland ), dextrose from six oy ( helsinki , finland ), mineral and vitamin mix from harlan teklad ( madison , wi ), l - cystine , cholinechloride and tertiary butylhydroxyquinone from yliopiston apteekki ( helsinki , finland ). inulin ( raftiline ®) was from orafti ( tienen , belgium ), hmr from hormos medical ltd , turku and rye bran from melia ( finland ). butter , sunflower oil , and rapeseed oil were from a local market . it will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments , only a few of which are disclosed herein . it will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention . thus , the described embodiments are illustrative and should not be construed as restrictive . ayres d , and loike , j . lignans : chemical , biological and clinical properties . cambridge university press , 1990 . ekman r : analysis of lignans in norway spruce by combined gas chromatography - 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