Patent Abstract:
a method is provided for inhibiting the development of colon tumorigenesis in a mammal by administering to the mammal a pharmacologically effective amount of an isothiocyanate selected from the group consisting of sulforaphane and phenethyl isothiocyanate .

Detailed Description:
as used herein , the abbreviations have the following meanings : itcs , isothiocyanates ; sfn , sulforaphane ; peitc , phenethyl isothiocyanate ; bitc , benzyl isothiocyanate ; afc , aberrant crypt foci ; nac , n - acetylcysteine ; aom , azoxymethane ; nnk , -( 4 - methylnitrosamino )- 1 -( 3 - pyridyl )- 2 - butanone ; cyp , cytochrome p450 ; ndma , n , n - dimethylnitrosamine . all references cited herein are hereby specifically incorporated into this disclosure by reference . the inventors designed a bioassay to examine whether sfn and peitc can inhibit the formation of aberrant crypt foci ( acf ) induced by azoxymethane ( aom ) in fischer rats . acf has been recognized as an early preneoplastic lesion of colon cancer ( 7 - 9 ) and it is generally observed that agents that inhibit colonic acf formation would show chemopreventive activity against colon cancer ( 10 ). the inventors also included in this study the nac conjugates of sfn and peitc since their previous studies showed that certain itc - nac conjugates are promising chemopreventive agents for lung tumorigenesis , probably by a gradual release of the parent itcs via a dissociation reaction ( 11 , 12 ). the structures of sfn and peitc and their nac conjugates are shown below . two treatment regimens in which sfn and peitc or their nac conjugates were administered either after ( post - initiation ) or before ( initiation ) aom treatment were used in the bioassay in order better to define the possible mechanism of action . male f344 rats were from charles river ( kingston , n . y .). they were fed ain - 76a diet ( 5 % corn oil ) and tap water ad libitum . animals were maintained under standard conditions ( 12 - h light / 12 - h dark cycle , 50 % relative humidity at 21 ° c .). at six - weeks of age , they were randomly divided into six per groups as shown in table 1 , except for the control groups 10 to 14 , which consist of three animals . peitc and sfn were purchased from aldrich chemical company ( milwaukee , wis .) and ltk labs , inc . ( st . paul , minn . ), with purity & gt ; 99 % and & gt ; 97 %, respectively . the corresponding nac conjugates were prepared by a published method ( 13 , 26 ). the purity of the conjugates was greater than 97 % as determined by high - performance liquid chromatography . aom obtained from ash - stevens ( detroit , mich .) was dissolved in saline and administered subcutaneously once a week for two weeks . sfn and peitc in corn oil and the nac conjugates in saline ( 10 % dmso ) were given by gavage . groups 2 to 5 were given 5 μmol sfn or peitc or 20 μmol of the nac conjugates , three doses each week for eight weeks , beginning two days after the last dose of aom . groups 6 to 9 were treated with three doses of itc compounds ( 20 μmol itc or 50 μmol itc - nac ) once daily and the last dose was given 2 hr before aom dosing . this dosing regimen was repeated during the second week of aom dosing . groups 10 to 13 were treated with itcs ( 5 μmol / dose ) or the conjugates ( 20 μmol / dose ) only , three times weekly for eight weeks . group 14 served as the control group . the bioassay was terminated at week 10 after the second aom treatment . the colon was processed for microscopic examination and the acf were recorded using a standard procedure described previously ( 14 ). acf were distinguished from the surrounding normal crypts by their increased size , significantly increased distance from lamina to basal surface of cells , and the easily discernable pericryptal zone . for statistical analysis , means were compared among the groups using one - way analysis of variance ( anova ) followed by fisher &# 39 ; s protected t - test . no significant differences in body weights were seen in any of the treated groups compared with the control group , indicating that doses of itcs and the conjugates used did not cause overt toxicity ( see fig1 and 2 ). this bioassay showed that post - treatment by oral administration of sfn and peitc at 5 μmol three doses weekly for eight weeks and their nac conjugates at 20 μmol by the same regimen inhibited the formation of acf . these treatments reduced the total acf from 153 to 100 - 116 ( p & lt ; 0 . 01 ) and multicrypt foci (& gt ; 4 crypts / focus ) from 52 to 27 - 37 ( p & lt ; 0 . 04 ) as shown in table 1 ( groups 1 to 5 ). since the itc conjugates are presumably less toxic than the parent itcs , the doses of the conjugates were 4 times that of sfn and peitc ( 15 , 16 ). however , the inventors did not find significant difference in the inhibition of acf between the itcs and their nac conjugates , suggesting the conjugates themselves are not as active . similar dose - effect relationships were observed previously between parent itcs and their conjugates towards the inhibition of lung tumorigenesis ( 7 ). these results , together with those from the inventors &# 39 ; earlier studies ( 17 , 18 ), suggest that itc conjugates render their inhibitory activity in part by the deconjugation to the parent itcs . although the mechanism of inhibition of acf at the post - initiation phase is not currently clear , recent studies have shown that peitc and related itcs induce p53 - dependent or c - jun kinase - mediated apoptosis in cultured cells ( 19 - 21 ). in addition , nac produced by deconjugation is a known antioxidant which has been recently shown to inhibit mouse fibroblast cell proliferation by blocking cell in g 1 phase ( 22 ). all these activities could have contributed to the inhibition of acf formation during the post - initiation of acf formation . studies by the inventors and others showed that pretreatment of animals with peitc and other related itcs blocked chemical - induced tumorigenicity by inhibiting cytochrome p450 ( cyp ) enzymes responsible for the activation of carcinogens , and consequently reducing dna damage ( 23 - 25 ). the inventors investigated the effects of pretreatment with sfn and peitc on the aom - induced colonic acf formation in fischer rats . the results showed that pretreatment with sfn or peitc significantly decreased total number of acf from 153 to 109 ( p & lt ; 0 . 004 ) or 115 ( p & lt ; 0 . 01 ), respectively , and multicrypt foci (& gt ; 4 crypts ) from 52 to 35 ( p & lt ; 0 . 03 ) for both compounds ( table 1 below ). the inventors have previously shown that peitc and peitc - nac are inhibitors of n , n - dimethylnitrosamine ( ndma ) demethylase ( cyp2e1 ) in rat liver microsomes ( 26 ). like ndma , aom is metabolically activated by cyp2e1 to methyazoxymethanol which can yield a dna methylating species ( 27 ). thus , inhibition of cyp2e1 by these agents in the rat liver may constitute an important mechanism for the inhibition , since cyp enzyme activity in colonic tissue is low compared to that in liver ( 24 ). sfn - nac also reduced total afc to 120 ( p & lt ; 0 . 02 ), however , it had no significant effect on multicrypt foci (& gt ; 4 crypts / focus , 15 % inhibition ). peitc - nac , on the other hand , appeared to enhance acf formation ( total number of acf is 198 , p & lt ; 0 . 002 ; number of multicrypt foci is 74 , p & lt ; 0 . 008 ) ( table 1 ). the adverse effect caused by peitc - nac is unexpected in view of the inhibition by its parent itc . at present , it is not clear as to why there is such a sharp contrast towards acf formation for peitc - nac compared to other itc compounds studied here . in the above study a colon carcinogen , aom , was used to initiate precancerous lesions in the colon ; the dose was considerably higher than the anticipated dose of any carcinogen to which humans would be inadvertently subjected . the chemopreventive agents were administered in two types of treatments . in the first type of treatment , rats were dosed after aom was given with 5 μmole of either sulforaphane ( sfn ) [ 6 . 3 mg / kg ] or phenethyl isothiocyanate ( peitc ) [ 5 . 8 mg / kg ] or 20 μmole of either the n - acetylcysteine conjugate of sfa ( sfn - nac ) [ 48 . 6 mg / kg ] or the n - acetylcysteine conjugate of peitc ( peitc - nac ) [ 46 . 6 mg / kg ] three times per week for eight weeks . in the second treatment regimen , the rats received 20 μmole of the respective itcs or 50 μmole of the n - acetylcysteine conjugates four days per week for two weeks prior to aom administration once each week . symptoms of gastrointestinal toxicity or irritation ( bloody mucus in feces ) were observed very early in the second treatment regimen , suggesting that the dose levels used may have been in excess of a dose safe for human consumption . additional toxicity information regarding peitc is available from a 90 day study in rats , in which 500 , 1500 , and 2500 ppm was administered in the diet ( 35 ). dietary peitc reduced weight gain in male rats at the high dose level by approximately 9 . 5 % ( not significant ). in addition , liver weights of male rats ingesting 1500 and 2500 ppm peitc were significantly elevated . changes in the epithelial lining of the forestomach of both male and female rats at 2500 ppm were also observed . in another study with beagle dogs , peitc was administered daily in gelatin capsules . diarrhea and vomiting , with gastrointestinal irritation , occurred sporadically at dose levels of 2 mg / kg and higher . signs of irritation of the mucosa of the urinary bladder were reported at 4 mg / kg / day and higher . dose related weight loss occurred in both male and female dogs , which became statistically significant in females at 8 mg / kg . no other signs or symptoms of - toxicity were reported ( 36 ). in a preliminary study in humans presently being conducted to determine the efficacy of peitc as a chemopreventive agent for lung cancer in smokers , volunteers have been given 40 mg peitc ( approx . 0 . 6 mg / kg ) in gelatin capsules . no adverse effects from this dose have been observed after repeated dosing every four hours . on the basis of existing toxicity information and efficacy data , the inventors suggest the following safe dose ranges for human consumption : peitc 0 . 6 to 1 . 2 mg / kg body weight peitc - nac 6 . 0 to 12 mg / kg body weight sfn 0 . 55 to 1 . 1 mg / kg body weight sfn - nac 5 . 5 to 11 mg / kg body weight the components of the present invention may be administered as a dietary supplement . for example , they may be administered orally , either alone or with water or another beverage or with a foodstuff . there may be many modifications and variations of the methods and compounds set forth hereinabove . these modifications and variations will not depart from the scope of the invention , if defined by the following claims and equivalents thereof . 1 . lin , h . j ., probst - hensch , n . m ., louie , a . d ., kau , i . h ., witte , j . s ., ingles , s . a ., frankl , h . d ., lee , e . r ., haile , r . w . 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