Patent Application: US-40133709-A

Abstract:
the present invention relates to use of compounds of general formula 1 for anticancerous activity , wherein the said compound being derived semi - synthetically from natural triterpenoic acids known as boswellic acids by the induction of apoptosis thereof cytotoxicity and anti - cancer activity displayed by semi - synthetic analogues of natural triterpenes , known as boswellic acids . these compounds may be used for the treatment of cancer , alone or in combination with pharmaceutically acceptable or other carriers , displaying cytotoxicity and anti - cancer activity for colon , prostrate , liver , breast , central nervous system , leukemia and malignancy of other tissues , including ascites and solid tumors . the cancer cell death is mediated by induction of apoptosis and inhibition of cell proliferation at specific doses .

Description:
the compounds of formula 1 display cytotoxicity and anti - cancer activity against cancers of the colon , prostrate , liver , breast , central nervous system ( cns ), leukemia , as well as malignancies of other tissues , including ascites and solid tumors , wherein the cancer cell death is mediated by induction of apoptosis and inhibition of cell proliferation at specific doses . these semi - synthetic boswellic acid analogues of formula 1 comprise alkyl acylates of a mixture of isomeric structures 3α - hydroxyurs - 12 - ene - 24 - oic acid , 3α - hydroxyolean - 12 - ene - 24 - oic acid , and 11 - keto - 3α - hydroxyurs - 12 - ene - 24 - oic acid , 11 - keto - 3α - hydroxyolean - 12 - ene - 24 - oic acid , and alkyl acylates of a mixture of epimeric structures 3β - hydroxyurs - 12 - ene - 24 - oic acid , 3β - hydroxyolean - 12 - ene - 24 - oic acid , 11 - keto - 3β - hydroxyurs - 12 - ene - 24 - oic acid , and 11 - keto - 3β - hydroxyolean - 12 - ene - 24 - oic acid . these semi - synthetic mixtures are prepared from the natural isolate of boswellic acid ( α and β ) or from semi - synthetic 11 - keto -( α and β )- boswellic acid respectively . in an aspect , the bioactivities of semi synthetic analogues boswellic acids of formula 1 are disclosed herein as cytotoxic and pro - apoptotic activities . as such , these compounds may be useful for the treatment of cancer derived from various tissues . a preparation or formulation is also provided comprising semi - synthetic analogues of boswellic acids for their use as cytotoxicity related anti - cancer agents . the semi - synthetic analogues of formula 1 have been subjected to in vitro cytotoxicity screening in a panel of human cancer cell lines using following methodology . the human cancer cell lines were obtained either from national center for cell science , pune or national cancer institute , fredrick , md ., usa . cells were grown in tissue culture flasks in complete growth medium ( rpmi - 1640 medium with 2 mm glutamine , 100 g / ml streptomycin , ph 7 . 4 , sterilized by filtration and supplemented with 10 % sterilized fetal calf serum and 100 units / ml penicillin before use ) at 37 ° c . in an atmosphere of 5 % co 2 and 90 % relative humidity in a carbon dioxide incubator . the cells at sub - confluent stage were harvested from flask by treatment with trypsin ( 0 . 05 % trypsin in pbs containing 0 . 02 % edta ) and suspended in complete growth medium . cells with cell viability of more than 97 % by trypan blue exclusion technique were used for determination of cytotoxicity . test material were dissolved in dmso ( dimethyl sulphoxide ) to obtain a stock solution of 2 × 10 − 2 m . the stock solution was serially diluted with complete growth medium containing 50 μg / ml of gentamycin to obtain working test solutions of required concentrations . the suspension of human cancer cell lines of required cell density in complete growth medium was prepared and cell suspension ( 100 μl / well ) of each cell line was added to wells of 96 - well tissue culture plate . suitable blanks and controls were also included . the plates were incubated for 24 hrs in a carbon dioxide incubator . the working test solutions of different concentrations ( 100 μl / well ) were added after 24 hr incubation . the plates were further incubated for 48 - hrs after addition of the test materials . after incubation , the cell growth was stopped by gently layering of 50 μl of tca ( 50 % trichloroacetic acid ) on top of the medium in all the wells . the plates were incubated at 4 ° c . for one hour to fix the cells attached to bottom of the wells . liquids of all the wells were gently pipetted out and discarded . the plates were washed five times with distilled water to remove tca , growth medium , low molecular weight metabolites , serum proteins etc . the plates were air dried . cell growth was measured by staining with sulforhodamine b dye ( srb ). the srb solution ( 100 μl , 0 . 4 % in 1 % acetic acid ) was added to each well and the plates were incubated at room temperature for 30 minutes . the unbound srb was quickly removed by washing the wells five times with 1 % acetic acid solution and plates were air dried . tris buffer ( 100 μl , 0 . 01 m , ph 10 . 4 ) was added to all the wells and plates were gently stirred for 5 minutes on a mechanical stirrer . the optical density was recorded on elisa reader at 540 nm . cell growth in the presence of test material was calculated in terms of the percentage of growth inhibition . the in vitro cytotoxicity of four natural boswellic acid i . e . boswellic acid , acetyl boswellic acid , 11 - keto - boswellic acids , acetyl - 11 - ketio - boswellic acid on various cancer cell lines has been examined and typical results are given in the succeeding table . based on their cytotoxicity profile the analogues of formula 1 were studied for inhibition of cell proliferation by using mtt assay method . the semi - synthetic analogues also displayed induction of apotosis in hl - 60 leukemia cells as estimated by flow cytometry . the induction of apoptosis in hl - 60 cells was also validated by generation of dna fragmentation . boswellic acid analogues of formula 1 also displayed early generation of reactive oxygen species as well as endogenous generation of nitric oxide measured by flow cytometric methods . in an animal model , the compound of formula 1 also demonstrated regression of ehrlich ascitic tumor in mice . in vitro cytotoxic acivity of natural boswellic acids on human cancer cell lines a bioactive triterpenoid of formula 1 in the pharmaceutical preparation inhibits human breast mcf - 7 cancer cells growth by 50 % at about 15 μg / ml concentration . a bioactive product of formula 1 comprising triterpenoids kills human cancer cells by induction of apoptosis . a bioactive product of formula 1 kills more than 92 % of prostrate du - 145 and pc - 3 cells at 5 × 10 − 5 m concentration , wherein the prostrate cell lines are selected from du - 145 and pc - 3 cells . a bioactive product of formula 1 kills colon cancer cells up to 96 % at 5 × 10 − 5 m concentration , wherein the colon cancer cell lines are selected from ht - 29 , sw - 620 and colo205 cells . a bioactive product of formula 1 inhibits growth of cancer cells of liver up to 92 % at 5 × 10 − 5 m concentration , wherein the cancer cell line of liver hep2 is selected . the following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention . screening of cell cytotoxicity in a panel of human cancer cell lines in vitro assay of cytotoxicity potential of test materials is a primary standard procedure for seeking lead molecules for development of anti - cancer leads . human cancer cells after trypsinization into single cell suspension were grown in 96 - well culture plate for 24 hr . cells were treated with indicated doses of test analogues and incubated in co 2 incubator for 48 hr . thereafter , cells were stained with sulforhodamine b dye , and the bound dye was eluted to measure the optical density indicating cell growth in elisa reader at 540 nm [ monks et al ., 1991 ]. the od of untreated cells is considered as 100 % while of boswellic acid analogs - treated groups are subtracted from the control group to determine percent inhibition as a measure of cell cytotoxicity . human leukemia cells hl - 60 were grown in suspension in 96 - well culture plate and were incubated with different concentrations of the test analogs for 48 hr . the cells were then incubated with mtt and the mtt - formazon formed is eluted with dmso , and od measured in elisa reader [ shashi et al ., 2006 ]. the intensity of the color formed in the untreated control wells relates to 100 % cell growth . the growth of cells is recorded with different concentrations of the structural analogs , and the concentration that inhibits 50 % cell growth is taken as ic 50 value . the ic50 values in hl - 60 cells were between 10 - 15 μm . concentration related influence of boswellic acid analogs on the relative degree of inducibility of apoptosis during the early events of apoptosis , phospholipid phosphatidyl serine of plasma membrane is externalized , which has very high affinity for annexinv antibody . effect of a single concentration ( 15 μm ) of each boswellic acid analogs on the relative efficiency of induction of apoptosis and necrosis in hl - 60 cells was analyzed by flow cytometry . cells were incubated with each analog for 6 hr and stained with annexin v - fitc / pi . camptothecin at 4 μm was used as positive control . there after , cells were washed and stained with fitc conjugated annexinv antibody and propidium iodide . the cells ( 10 , 000 ) were analysed by flow cytometery ( bd , lsr ) using proquest software . as shown in fig2 , the fraction of cell population in the lower right quadrant indicates apoptotic cells , upper right post - apoptotic and upper left as necrotic populations ( fig2 ). validation of apoptosis by generation of dna fragmentation typical of apoptosis in hl - 60 cells the structural analogs produced programmed cell death ( apoptosis ), a desired therapeutic anticancer drug target in human leukaemia hl - 60 cells . human leukemia cells were grown in culture and exposed to 50 um concentration of each analog for 6 hr . the control group received only the vehicle . cells were similarly treated with camptothecin , 4 μm as positive control . genomic dna was extracted and electrophoresed on agarose gel [ sashi et al ., 2006 ]. influence of boswellic acid analogs on the early generation of reactive oxygen species ( peroxides ) the endogenous generation of peroxides was measured using a non - fluorescent probe dcfh - da which upon entering the cell is deesterified to dcfh which is oxidized by the reactive oxygen species to a fluorescent product dcf that remains entrapped within the cells offering analysis by flow cytometery [ shashi et al , 2006 ]. hl - 60 cells were incubated with different concentrations of compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o for a brief period of 6 hr . thereafter cells were washed and stained for 1 hr with dcfh - da and analysed on flow cytometer . the analog produced dose dependent increase in peroxide positive cell population , being 79 % at 50 μg / ml . the pro - oxidant effect was completely impaired in the presence of anti - oxidant , ascorbate . formation of reactive oxygen and nitrogen species are indicated in the induction of apoptosis . compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o mediated early generation of endogenous nitric oxide measured by flow cytometery measurement of in situ no generation involved the use of a fluorescent probe diaminofluoresceine - 2 - diacetate , which is permeated easily into the cells . once inside the cell it binds no soon it is formed and emits fluorescence [ h . kojima et al , 1998 ]. for this purpose , the hl - 60 cells were incubated with daf - 2 - da for 30 min before being incubated with the indicated concentration of ss - 145 for 4 hr . cells were analyzed on flow cytometer in fl - 1 channel for no . it appears that all the cells formed no within 6 hr irrespective of the concentration of bioactive compound used , because there were no cells in the lower left quadrant as indicated in control cells . interaction of no with superoxide , while both are generated simultaneously , may affect the mitochondrial membrane potential that may be responsible for the activation of apoptosis on account of oxidative stress produced by compound of formula 1 where , r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o . mitochondrial membrane depolarization by a compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o rhodamine - 123 uptake into the mitochondria is driven by mitochondrial transmembrane potential ( ψ mt ) that allows the determination of cell population with active integrated mitochondrial functions . loss of ψ mt would lead to depolarization of mitochondria because of ros and no generation leading to cell death . hl - 60 cells were exposed to ss - 145 for 12 hr and incubated with rh - 123 . cells , 10000 , were acquired for analysis by flow cytometery . the percentage of cells with low rh - 123 fluorescence was calculated from the dot plot statistics . in untreated control cells more than 92 % cells showed rh - 123 fluorescence , which decreased with the treatment of compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o , approx . 42 % at 20 μm concentration . this shows that reactive oxygen / nitrogen species generated by compound of formula 1 bring about oxidative damage to mitochondria ensuing apoptosis . a compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o produced concentration dependent depletion of reduced glutathione contents in hl - 60 cells reduced glutathione content was determined in 3 × 10 6 hl - 60 cells / well / 3 ml medium in a 6 - well plate after incubation with ss - 145 for 6 hr . cells were washed and the reduced glutathione contents were extracted and determined using a standard fluorimetric method [ hissen et al ., 1976 ]. cells were also treated with bso ( 200 μm ) as positive control , and nac ( 5 mm ) as anti - oxidant . influence of compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o on the activation of caspases in leukemia cells caspases are important cysteine - proteases that are involved in the cleavage of dna specifically producing 180 bp dna fragments . the enzymes act through different signaling pathways . caspase - 9 activation involves mitochondrial mediated pathway initiates apoptotic process by recruiting caspase - 3 for the execution of programmed cell death . caspase - 9 activity was determined fluorometrically and caspase - 3 colorimetrically using kits and the instruction provided by the manufacturer ( bd pharmingen , usa ). caspase - 3 activity was determined in both molt - 4 and hl - 60 leukemia cells while caspase - 9 was determined in hl - 60 cells . a compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o was used as the template for other analogs . the analog activated caspase - 3 by about 50 % when cells were incubated with the analog for 6 hrs . the activity was almost abolished in the presence of selective inhibitor demonstrating that the activity is solely of the caspase - 3 . activation of caspase - 3 in leukemia cell lines by boswellic acid analog . caspase - 3 is an executioner enzyme guiding the fragmentation of dna and its activity was stimulated by about 50 % when both leukemia cells were incubated with compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o for 6 hr ( fig8 a ). the specific inhibitor used decreased the activity suggesting that the protease measured is certainly caspase - 3 activity . compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o stimulated strongly the caspase - 9 activity in hl - 60 cells caspase - 9 activity is very sensitive to oxidative stress and as a result its activity is stimulated by 4 - 5 - fold at 50 μm compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o ( fig8 b ) when at this concentration the caspase - 3 was stimulated by about 50 % only . caspase - 9 activations suggests the leakage of cytochrome c from mitochondria and activation of several genes and downstream signal transduction pathways leading to the cleavage of dna mediated by caspase - 9 . effect of compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o on the regression of ehrlich ascitic tumor in mice swiss albino mice of 6 - 8 weeks , weighing 18 - 23 g of single sex were used . for the initiation of experiment , 1 × 10 7 cells of eac ( ehrlich ascitic carcinoma ) cells were transplanted intramuscularly ( i . m .) in 32 mice for the development of solid tumors . the day of tumor transplantation was assigned as day 0 . next day ( day - 1 ) animals were randomly selected and divided into four groups having equal number of mice in each group of similar body wt . groups i and ii were treated with compound of formula 1 where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o at the dose of 50 mg / kg b , wt and 100 mg / kg b . wt . intraperitoneally , respectively . group iii received 0 . 2 ml of 0 . 85 % normal saline and served as normal control while group iv serving as positive control was treated with 5 - fu ( 22 mg / kg b . wt .) intraperitoneally . the total duration of treatment was of 9 days . the evaluation of effect of treatment on tumor was done on 13 th day . ehrlich ascites cells ( 1 × 10 7 ) were injected intramuscularly in the right thigh muscles and on the following day animals started receiving treatment intraperitoneally in 0 . 5 % tween - 20 daily at the indicated doses for 9 days . the growth of tumor and the efficacy of compound of formula 1 , where r 3 = coch 2 ch 2 ch 3 , r 1 = h , r 4 + r 5 = o in inhibiting tumor growth were evaluated on day 13 in terms of the weight of the tumor in treated and control groups count . the determination of solid tumor growth inhibition against eat was calculated as percent tumor growth regression according to the following : ammon , h . p . t . et al ., “ mechanism of antiinflammatory actions of curcumine and boswellic acids ,” j ethnopharmacol . 1993 march ; 38 ( 2 - 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