Patent Application: US-68053008-A

Abstract:
an aspect of the present invention is the use of a preparation containing a phytepsin , more specifically a cyprosin , containing the heterodimer , its n - terminal pro - peptide , the mature n - terminal peptide , and mature c - terminal peptide , as well as other precursor species , processing products , and aggregate species , either isolated or in any combinations of the former , native , extracted and partially purified from flowers of cynara cardunculus , or recombinant , extracted from the supernatant from a culture of saccharomyces cereviseae genetically modified for the heterologous production of cyprosin , for therapeutic applications more precisely for its use as an antitumor agent .

Description:
due to the nature of this invention its detailed description is better achieved through examples . anti - tumour activity of a preparation of native cyprosin containing both structural chains : n - terminal chain ( consisting on the n - terminal pro - peptide and the mature n - terminal ) and c - terminal chain ( mature peptide c - terminal ), isolated and purified from dried cynara cardunculus flowers . the cyprosin preparation was obtained from dried cynara cardunculus flowers as previously described by brodelius et al ., 1995 . the anti tumour activity of the enzyme preparation was evaluated using four human tumour cell lines : an epithelial cell line derived from a carcinoma ( hct116 , atcc ccl - 247 ), an epithelial cell line derived from a fibrosarcoma ( ht1080 , atcc ccl - 121 ), an epithelial cell line derived from a rabdomyosarcoma ( te671 , atcc ccl - 136 ), and an epithelial cell line derived from an adenocarcinoma ( hela , atcc ccl - 2 ™), and two non - tumour cell lines : one consisting of human intestinal ( epithelial ) cells ( fhs74 int , atcc ccl - 241 ) and another consisting of african green monkey kidney epithelial cells ( vero , atcc crl - 1587 ). the tumour cell lines hct116 , ht1080 e te671 were inoculated on basal medium dmem ( cambrex ), supplemented with 5 % foetal bovine serum ( fbs — gibco ). the final concentrations of glucose ( sigma ) and of l - glutamine ( sigma ) were of 4 . 5 g / l e 6 . 0 mm , respectively . the culture medium was supplemented with a 1 % penycillin / streptomycin ( gibco ) solution . the tumour hela cell line was inoculated on dmem ( cambrex ) basal medium , supplemented with 10 % fbs ( gibco ); 2 . 1 g / l sodium bicarbonate ( nahco 3 — sigma ); 1 . 0 mm sodium pyruvate ( c 3 h 3 nao 3 — sigma ); and 0 . 1 mm of a non - essential amino acids solution ( neaa — cambrex ). the final concentrations of glucose ( sigma ) and of l - glutamine ( sigma ) were 1 . 0 g / l and 2 . 0 mm , respectively . the culture medium was supplemented with 1 % penycillin / streptomycin ( gibco ). the non - tumour vero cells were inoculated on basal medium dmem ( cambrex ), supplemented with 10 % foetal bovine serum ( fbs — gibco ) and 3 . 56 mm l - glutamine ( sigma ). the culture medium was also supplemented with 1 % penicillin / streptomycin ( gibco ). the non - tumour cell line fhs74 int was inoculated on hybricare ( atcc ; cat . 46 - x ), supplemented with 10 % foetal bovine serum ( fbs — gibco ); 2 . 1 g / l nahco 3 ( sigma ) solution ; 2 . 0 mm l - glutamine ( sigma ) and 30 ng / ml epidermal growth factor ( egf — sigma ). the medium was supplemented with 1 % penicillin / streptomycin ( gibco ). the cells were propagated in a static culture system operated in batch . the cell concentration and viability were evaluated using the trypan blue exclusion method . table iii presents the specific growth rate ( μ ) and the corresponding doubling time ( dt ) for each cell culture . the ic 50 values were determined for the different cultured cell lines using the sulforhodamine b ( srb ) method . a total volume of 100 μl from each cell line was inoculated in triplicate in 96 well plates . the corresponding densities were estimated based on the specific growth rate of each replicate in such a way that after 24 h of treatment the cell cultures presented approximately 50 % confluence . following this strategy , the inoculum densities obtained for hct116 and hela cells were 3 . 1 × 10 4 cells / cm 2 ; for ht1018 and vero cells were 4 × 10 3 cells / cm 2 ; for te671 cell line were 1 . 6 × 10 4 cells / cm 2 , and for fhs74 int cell line were 2 . 5 × 10 4 cells / cm 2 . the cultures were incubated during 24 h a 37 ° c ., in a 7 % co 2 atmosphere and 90 % humidity . 24 h after inoculation , 100 μl of the a cyprosin preparation were added to each well at decreasing concentrations : 1000 μg / ml ; 100 μg / ml ; 10 μg / ml ; 1 μg / ml ; 0 . 1 μg / ml ; 0 . 01 μg / ml and 0 . 001 μg / ml , for ic 50 calculation . the plates were incubated during 48 h at 37 ° c ., in a 7 % co 2 atmosphere and 90 % humidity . control assays were performed for all cell lines used in the absence of cyprosin preparations . 48 h after addition of the enzyme preparation the cell cultures were observed under the light microscope to register the confluence and the morphological characteristics of the cells . for a cyprosin concentration of 100 μg / ml the differences between the non - tumour fhs74 int cells and hct tumour cells became significant and can be visualized in fig1 . in general , only concentrations of cyprosin preparation ranging between 1000 μg / ml e 100 μg / ml induced differences on the morphology of the different cell lines . the highest concentration ( 1000 μg / ml ) induced lysis in all cell line populations ( tumour and non - tumour ). the enzyme preparation at a concentration of 100 μg / ml induced significant morphological alterations on all tumour cells that became longer and thinner with visible signs of lysis ( fig1 ). in turn , the non - tumour fhs74 int and vero cells , submitted to the same 100 μg / ml enzyme preparation , did not show morphological alterations ( fig1 ). using the method described , no sign of toxicity of the enzyme preparation was observed on the different cell lines assayed for enzyme concentrations below 10 μg / ml . after microscopic observation , all the plate wells were incubated for 1 h at 4 ° c ., with 50 μl of a 50 % ( w / v ) tca solution ( fluka ). the plates were then washed 5 times with distilled water . after the last wash , the plates were dried off and 100 μl of freshly prepared 0 . 4 % ( w / v ) srb ( sigma ) were added to each well . the plates were incubated for 30 minutes at room temperature and protected from light . the srb stain was removed from the cells by washing five times with 250 μl of 1 % acetic acid ( rieldel - de haen ). each plate well was then incubated with 200 μl of a 10 mm trizma base ( fluka ) solution , for 10 minutes , at room temperature , protected from light , under constant shaking . the cells were ruptured and the srb - stained proteins were released . the assay was finished by measuring the absorbance in order to evaluate the relative growth and cell viability upon exposure to the cyprosin preparation and the controls . to calculate the ic 50 values , the incorporation of srb in the cellular proteins (% srb ) was evaluated against the control cells following the equation ( 1 ) were srb e represents the absorbance mean for each concentration of enzymatic preparation , srb b the absorbance mean for the blank assays and srb c the absorbance mean for the control assays : the curves in the graphics of % srb versus logarithm of enzyme concentration ( μg / ml ) were adjusted using the hill function ( 2 ), determined by the biostatistics program prism 5 , for windows ( graphpad software ), where the background and signal parameters are respectively 0 % and 100 %: the graphical representation of the viability of cells stained with srb , related to the logarithm of the cyprosin concentration ( μg / ml ), for each cell line , can be observed in fig2 . the values of the corresponding ic 50 are summarized in table iv : for the studied tumour cell lines , it was observed that hct116 cells are the most sensitive to the antitumour effect of enzyme preparation , while te671 cells are the most resistant . for the non - tumour cell lines , it was observed that fhs74 int cells are more sensitive than vero cells . the fact that tumour cell lines are consistently more susceptible to the cyprosin preparation , which can be demonstrated by their ic 50 values ( five times lower in absolute terms than those obtained for non - tumour cells ) is coherent with the morphological observations . in general , these results represent a tumour cell - specific lethal effect of the native enzyme purified from dried flowers of cynara cardunculus when compared to non - tumour cells submitted to the same concentrations of cyprosin preparations . the results reported show that the potential antitumour cytotoxic effect of the native cyprosin preparation occurs at concentrations up to 1000 μg / ml . antitumour activity of a preparation of recombinant cyprosin , containing the two structural chains : n - terminal chain ( consisting of the n - terminal pro - peptide and the mature n - terminal peptide ), and the c - terminal chain ( consisting of the mature c - terminal peptide ), isolated and purified from the culture medium of a saccharomyces cerevisiae strain transformed with the cypro11 gene . the cyprosin preparation was obtained from the supernatant from a culture of saccharomyces cerevisiae strain ( bj1991 ), transformed with the cypro11 gene coding for cyprosin as previously described ( pais et al ., 2000 ). the antitumour activity of the enzyme preparation was tested on a carcinoma - derived human tumour epithelial cell line ( hct116 , atcc ccl - 247 ), as well as on a non - tumour cell line consisting of epithelial cells from human intestine ( fhs74 int , atcc ccl - 241 ). the tumour cell line hct116 was inoculated on basal medium dmem ( cambrex ), supplemented with foetal bovine serum ( fbs — gibco ). the final concentrations of glucose ( sigma ) and l - glutamine ( sigma ) were 4 . 5 g / l and 6 . 0 mm , respectively . the non - tumour cell line fhs74 int was inoculated on basal medium hybricare ( atcc ; cat . 46 - x ), supplemented with 10 % foetal bovine serum ( fbs — gibco ); 2 . 10 g / l sodium bicarbonate ( nahco 3 ) ( sigma ); 2 . 0 mm l - glutamine ( sigma ), and 30 ng / ml epidermal growth factor ( egf — sigma ). the cells were propagated in a static culture system operated discontinuously . the cell concentration and viability were evaluated using the trypan blue exclusion method . the specific growth rate ( t ) and the doubling time of tumour and non - tumour cell lines , hct116 e fhs74 int respectively , are presented in table iii above ( example i ). like in example i , the morphological analysis of cells was performed by optical microscopy and the determination of ic 50 was done using the sulforhodamine b ( srb ) method . the results of the morphological analysis for cells treated with a 100 μg / ml of enzyme preparation are presented in fig4 . contrasting with the non - tumour cell line fhs74 int , which is not affected by the addition of recombinant cyprosin preparation , the hct cells present clear evidence of lyses 48 h after addition of the enzyme preparation . as in example i , the ic 50 parameters were determined for both cultures after the morphological study . the percent cell viability variation of cells stained with srb related to the logarithm of cyprosin concentration ( μg / ml ), for each cultured cell line , is represented in fig5 the values of ic 50 were 20 . 51 μg / ml for the tumour cell line hct116 and 70 . 50 μg / ml for fhs74 int cell line indicating a three - 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