Patent Application: US-88721506-A

Abstract:
this invention discloses the use of compounds of the azetidinone family in treatment of cholesterol gallstone disease of the biliary tree in mammals . these drugs act by decreasing biliary cholesterol secretion , and at the same time increasing the biliary flow and the hepatic secretion of endogenous compounds into the bile , in turn , these endogenous compounds contribute to inhibiting precipitation and the formation of gallstones in the biliary tree .

Description:
this invention demonstrates that the use of specific inhibitors of cholesterol intestinal absorption that belong to the 2 - azetidinone class , specifically ezetimibe ( sch 58235 ), completely inhibits the development of cholelithiasis in a murine model of the disease on a lithogenic diet . this invention in a preclinical model of cholesterol gallstone disease allows to predict that the selective inhibition of cholesterol intestinal absorption is an effective therapeutic alternative in primary prevention and / or treatment of cholesterol gallstone disease in humans . in addition , as it was discussed in the background section , it is possible to predict that that in humans , the combination of ezetimibe ( sch 58235 ) with inhibitors of cholesterol hepatic neosynthesis ( hmg - coa reductase ), could be more efficient than ezetimibe ( sch 58235 ) alone in the prevention and / or treatment of gallstone disease . experimental animal disease models . spontaneous cholelithiasis is extremely rare in experimental or wild animals , probably because they secrete low levels of cholesterol into the bile and have therefore non - saturated cholesterol bile . however , different animal models ( rabbit , hamster , prairie dog , squirrel and mouse ) develop cholelithiasis similar to the human disease on cholesterol rich plus bile salts ( cholic acid ) and sometimes triglycerides rich experimental diets . the mouse is the best characterized experimental model where the development of the disease is dependent of a diet known as “ lithogenic ” ( 1 . 25 % cholesterol , 0 . 5 % cholic acid , 15 % fat ). under these conditions most of the available mouse strains develop cholesterol cholelithiasis in variable proportions and times ( paigen , 2002 ). it has been established that there are strains more susceptible or more resistant to develop cholelithiasis , which could be genetically determined . the mice strains c57l and c57bl / 6 are particularly susceptible to develop cholelithiasis ( 80 - 100 % of the animals develop the disease in 2 - 4 weeks on lithogenic diet ) ( paigen , 2002 ; bouchard et al ., unpublished data communicated in www . jax . org / pub - cgi / phenome ). studies performed in these models during the last years have been vital in the better understanding of the disease and for the discovery of genes that are potentially relevant in generating susceptibility to the illness ( paigen , 2002 ). in this preclinical innovation work we use the mouse strain c57bl / 6 . we used mice c57bl / 6j susceptible to the development of cholelithiasis originally obtained from jackson corp ( bar harbor , me ., usa ) and the colonies were grown in our laboratory of gastroenterology , facultad de medicina , p . universidad católica de chile . the experimental groups were fed a control chow diet low in cholesterol (& lt ; 0 . 02 % cholesterol , prolab rmh3000 , pmi feeds inc ., st . louis , mo .) or a lithogenic diet ( 1 . 25 % cholesterol , 0 . 5 % cholic acid , 15 % fat , td90221 , harlan teklad , madison , wis .) ( amigo et al . 2000 ). these mice were also treated with ezetimibe ( zetia ®, merk / schering plough ) or placebo . in protocol # 1 zetia ® tablets were grinded in a porcelain mortar and dissolved in water to be administered to the mice in one daily dose ( 08 h ) of 5 mg / kg weight by gavage in a final volume of 200 μw ; the control groups were given the same volume of water by gavage without the drug . in protocol # 2 zetia ® tablets were grinded in the same way described above , dissolved in 200 cc 100 % ethanol ( merck ) and added to the lithogenic diet previously grinded and dried for 48 h at room temperature . c57bl / 6j mice received this lithogenic diet plus zetia ® in a estimated dose of 5 - 6 mg / kg / day during 30 days ( the mice consume 4 - 5 gr of diet per day ). the control group received the same grinded lithogenic diet , supplemented with 100 % ethanol ( 200 cc ) and dried for 48 h . during the study the mice were kept at constant room temperature , with day / night cycles of 09 : 00 - 21 : 00 h and 21 : 00 - 09 : 00 h respectively , and free access to food and water . four groups of 8 - 10 week old male mice ( 5 - 10 per group ) were place on the following experimental diets : group 1 - b : control chow diet plus ezetimibe 5 mg / kg / day by gavage ( n = 10 ). group 2 : lithogenic diet plus ezetimibe , 5 - 6 mg / kg / day for 30 days ( n = 5 ). surgery and sample harvest . the groups were maintained for 14 days ( protocol # 1 ) or 30 days ( protocol # 2 ) in the different experimental conditions . during the last three days of the protocols , 24 h depositions were collected from the different groups ( pool ) to determine the fecal excretion of neutral sterols ( cholesterol ) and acidic sterols ( bile salts ). at the end of the time established in the protocols , the mice were anesthetized ( between 08 : 00 - 10 : 00 am ) with an intraperitoneal pentobarbital injection in doses of 4 . 5 mg per 100 gr body weight ( amigo et al ., 2000 ) and subjected to surgery . standardized surgery consists in an extended laparoscopy and visualization of abdominal organs , including liver , biliary tree and gallbladder . first , we performed a resection of the gallbladder ( cholecystectomy ), with the cystic duct previously ligated ( amigo et al ., 2002 ; amigo et al ., 2000 ; amigo et al ., 2003b ). the gallbladder is removed and visually inspected to determine the presence of gallstones . then , the gallbladder bile is extracted to analyze the presence of cholesterol crystals in the fresh samples under the microscope with polarized light ( 20 ×). aliquots are stored at − 20 ° c . for subsequent chemical analysis . afterwards , the common biliary duct is cannulated ( biliary fistula ) with a p - 10 polyethylene cannula to collect bile during 15 minutes , determine bile flow by gravimetry ( 1 g bile = 1 ml ; bile flow is expressed as μl / min / g of mouse liver ) and for posterior chemical analysis of biliary lipids and glutathione , as specified later . the inferior vena cava is punctured with an heparitinized tuberculine syringe to extract 500 - 800 ml of blood . then , the liver is removed , weighted , frozen in liquid nitrogen and stored at − 80 for subsequent analyses . the plasma is separated by centrifugation at 1 , 500 rpm and stored at − 80 ° c . for later analysis . the animal protocols were approved by our institution ethics committee and the animals were treated according to international care rules for experimental animals . determination of lithiasis of the gallbladder and cholesterol crystals in gallbladder bile . the presence or absence of cholesterol gallstones in the mice gallbladder is determined by simple optic inspection , since the gallbladder is translucent and allows to easily detect the presence of particulate structures in its interior ( gallstones or particulate sediments ). in order to determine the presence of typical monohydrate cholesterol crystals , 10 μl of gallbladder bile are placed on a slide and analyzed by optic microscopy under polarized light with magnification of 10 - 20 ×. cholesterol crystals are typically observed as birefringent crystals with the appearance of broken glass ( amigo et al ., 2000 ; miquel et al ., 1998b ; moschetta , bookout and mangelsdorf , 2004 ). intestinal absorption of cholesterol and bile salts . the capacity of ezetimibe to inhibit cholesterol and bile salt absorption was indirectly determined by quantification of neutral sterols ( cholesterol ) and acidic sterols ( bile acids ) present in the feces collected on day 12 , 13 and 14 of the experiment ( three days ). in order to determine cholesterol ( neutral sterols ) fecal excretion , the feces were dried at 50 ° c . for three days and then weighted to obtain an average of fecal excretion per day ( in grams ). the feces were homogenized in a mortar , and 5 ml 2n naoh : methanol , 1 : 1 are added per 100 mg ( dry weight ) aliquots of the homogenate . the sample is vortex and incubated at 60 ° c . for 1 h for saponification , then it is cooled and 5 ml petroleum ether is added followed by vigorous agitation to obtain the separation of the hydrophobic and hydrophilic phases . the samples are then centrifuged at 1 , 000 rpm for 10 minutes and the hydrophobic phase ( upper layer ) is recovered . second and third extractions are performed by adding 5 ml petroleum ether to the hydrophilic phase . the hydrophobic phases are dried under gaseous nitrogen and the lipids are resuspended in 1 ml chloroform . 50 μl aliquots , in duplicate , are dried at room temperature , and total cholesterol is quantified according to the enzymatic method described above . the values obtained are expressed as μmoles cholesterol / day / 100 g of mouse ( miettinen et al ., 1965 ). in order to determine fecal excretion of bile acids the mice feces were collected during three days ( days 12 - 14 ), dried at 50 ° c . for one week and weighted . the feces are then homogenized in a mortar ; ( 14 c ) cholic acid , 200 , 000 cpm is added to 1 g of the homogenate to determine the degree of recovery . the samples are then mixed with nabr , naoh and hcl , dissolved in ethanol and incubated at 120 ° c . for 8 h in a reflux system . the hydrolyzed fecal samples are resuspended in naoh and dried under gaseous n 2 . the pellet obtained is resuspended in ethanol and run through sep - pak columns to eliminate all bile salts contaminants . the bile salts are eluted with a solution methanol : water 85 : 15 and dried under gaseous n 2 . the samples are then resuspended in methanol , aliquots taken in duplicate and bile salts measured using hydroxysteroid dehydrogenase just as they are measured in the bile . the value obtained is normalized with the 14 c cholic acid recovery ( mendez - sanchez et al ., 1998 ). determination of plasma cholesterol , biliary lipids and biliary glutathione . the plasma cholesterol content and the cholesterol , bile salts and phospholipid content in the gallbladder and hepatic bile are determined by enzymatic methods . biliary cholesterol concentration : 500 μl buffer ( tris - hcl ph 7 . 6 , phenol , chlorophenol , triton x - 100 , sodium collate , aminoantipyrine , and the enzymes cholesterol esterase , cholesterol oxidase and peroxidase ) are added to 5 μl hepatic bile or 1 μl gallbladder bile . this reaction mix is incubated for 10 minutes at 37 ° c . and the absorbance quantified at 500 nm . the values obtained are interpolated in a standard cholesterol concentration curve . plasma cholesterol is quantified in the same way from 5 μl plasma ( abell et al ., 1974 ). bile salts concentration is determined by adding 1 ml buffer ( potassium phosphate ph 8 . 0 , hydrazine hydrate , nad and hydroxysteroid dehydrogenase ) to 2 μl hepatic bile or 1 μl gallbladder bile . this reaction mix is incubated at 30 ° c . for 30 minutes and the absorbance quantified at 340 nm . the values obtained are interpolated in a standard taurocholate concentration curve ( talalay , 1960 ). phosphatidylcholine ( phospholipids ) in the bile is determined by adding 1 ml buffer ( tris - hcl ph 7 . 6 , phenol , aminoantipyrine , triton x - 100 , cacl 2 , phopholipase d , choline oxidase and peroxidase ) to 5 μl hepatic bile or 1 μl gallbladder bile . the reaction mix is incubated at 37 ° c . for 10 minutes and the absorbance measured at 500 nm . the values obtained are interpolated in a standard phosphatidylcholine concentration curve ( gurantz et al ., 1981 ). determination of biliary lipids debit . in all the cases presented above , the concentration value in mm is given in terms of debit , indicating the amount of each class of lipid secreted into the bile per time unit in relation to liver or mouse weight . the debit calculation is performed by multiplying the concentration value of each lipid by bile flow . biliary gluthation . biliary gluthation was quantified from the bile obtained by cannulation of the biliary duct for a 5 minute period with tubes containing 5 μl sulfosalicylic acid . duplicate samples were taken from these samples and diluted 1 : 60 in sulfosalicylic acid . the enzymatic reaction for measuring glutathione was done by adding buffer ( sodium phosphate , edta , dntb , nadph ) and the enzyme gsh reductase . the reaction mix was incubated at 37 ° c ., measured at 412 nm , and an enzymatic kinetic curve was obtained using different gst concentrations as standard . the enzymatic activity was transformed into glutathione concentration present in the bile . once the concentration of biliary gluthation was determined , we obtained glutathione debit as it was described earlier for biliary lipids ( anderson , 1985 ). determination of the cholesterol saturation index in gallbladder bile samples . this index was obtained using each biliary lipid concentration ( cholesterol , bile salts and phosphatidylcholine ). from these concentrations we established the total amount of lipids , the phospholipids /( bile salt phospholipids ) and the molar cholesterol percentage . with this data and a table to calculate the saturation index we were able to determine the cholesterol saturation index as it was previously described ( carey , 1978 ). plasma lipoprotein profile . in order to characterize the plasma lipoprotein profile we collected 40 μl of serum from 5 mice in each group ( 200 μl pool for each experimental group ). the samples were loaded into a sepharose 6b column ( fast pressure liquid chromatography , fplc , pharmacia ) were the lipoproteins are separated according to their weight . the column is eluted and fractions collected ( 37 fractions , 300 μl each ). for each fraction the cholesterol content is calculated , which allowed us to establish the distribution of the big ( vldl , idl / ldl ) and small lipoproteins ( hdl ) as follows . to each fraction collected we added 200 μl of the reaction mix for measuring total cholesterol ( without diluting with water ). the enzymatic reaction was done exactly as described for biliary cholesterol . the value obtained for each fraction is given as μg cholesterol per fraction . determination of hepatic cholesterol . the hepatic cholesterol content is quantified from hepatic tissue ( 50 mg ) that is homogenized in chloroform / methanol 2 : 1 . the homogenate is incubated at 50 ° c . for 30 minutes , and then distilled h 2 o is added to separate the phases . the mix is vortexed and incubated at 4 ° c . for two hours and centrifuged at 1 , 000 rpm for 20 minutes . the lower phase ( hydrophobic - chloroform ) is removed and dried under gaseous nitrogen . the lipids on the tube wall are resuspended in chloroform plus triton x - 100 0 . 5 % v / v . four aliquots are taken and dried at room temperature . total cholesterol content was measured to half of the tubes by the enzymatic method described earlier for biliary cholesterol . to the other half , free cholesterol was measured in buffer ( tris - hcl ph 7 . 6 , triton x - 100 , 0 . 2 % phenol , aminoantipyrine ) plus the enzymes cholesterol oxidase and peroxidase . the samples are incubated and quantified in the same way described for total cholesterol . the cholesterol ester content for each sample is calculated as the difference between total cholesterol and free cholesterol . the values obtained in each case are given as mg cholesterol / g liver ( folch et al ., 1957 ). expression of specific bile salts ( ntcp , bsep ), phospholipid ( mdr2 ) and organic anions or glutathione ( mrp2 ); and determination of glutathione synthesis ( gluthamylcystein - synthetase ( ggcs )). western blot analysis . we evaluated protein expression levels of the bile salts transporters ntcp ( sinusoidal pole ) and bsep ( cannicular pole ) by western blotting in total liver membranes or liver plasma membranes from mice treated with the different diets . total membranes were obtained from liver fragments homogenized in tris - hcl ph 7 . 5 , mgcl 2 and sucrose . the homogenates were centrifuged at 1 , 000 g to separate nuclei , and the supernatants were then centrifuged at 100 , 000 g . the pellet , containing the total membranes , was resuspended in the buffer described above and proteins were quantified by the bradford method . aliquots containing 50 μg protein were separated by sds - page and transfer to nitrocellulose . the membranes were incubated in buffer tbs - t ( tris - hcl ph 7 . 5 , nacl and tween - 20 ), milk 5 % w / v plus the antibodies to evaluate the different proteins ( bsep , ntcp ). the secondary antibodies were igg anti rabbit or anti mouse coupled to peroxidase . the protein signals were detected by the ecl method . as internal loading control we used beta - actin , a protein with constitutive expression that does not change its expression levels with the diets and drugs used . total rna extraction . 80 mg hepatic tissue frozen at − 70 ° c . was homogenized in 800 μl solution d , following the method described by chomczynski and sacchi , 1987 . the rna concentration was calculated by measuring the absorbance at 260 nm and its purity by the ratio od 260 / od 280 . the samples are stored at − 20 ° c . rna electrophoresis . 10 μg of total rna are dried under gaseous n 2 or vacuum for 10 minutes . 10 μl loading buffer were added , and the samples heated to 65 ° c ., loaded into a denaturing 1 % agarose / formaldehyde gel and run at 100 volts for 4 hours . the rna integrity was determined by the clear visualization of the 28 s and 18 s ribosomal rnas ( fig8 ). northern blot and hybridization with radioactive probes . the gel with the rna samples is washed with depc h 2 o for 10 minutes to eliminate formaldehyde and then incubated for 20 minutes with 50 mm naoh for partial rna hydrolysis . after a 10 minute wash with depc h 2 o to eliminate naoh , the samples are transferred from the gel to nylon membranes ( hybond - n , amersham pharmacia ) over night by a capillary system . after the transfer , the rna is fixed to the membrane by heating at 80 ° c . for 2 h . probe labeling by “ de novo synthesis ”. for the radioactive labeling of probes specific for mdr2 , mrp2 and gamma - gluthamylcystein - synthetase ( g - gcs ), we used the prime - a - gene labeling system , promega , based on the method developed by feiberg and vogelstein , 1983 . we used a fragment of the gene of interest ( generated by pcr ), dctp - a - p 32 , a mix of unlabeled nucleotides and klenow fragment for the probe labeling . the percentage of incorporated radioactivity into the probe was always over 60 %. membrane hybridization with the labeled probe . the probe was denatured at 100 ° c . for 5 minutes and incubated with the pre - hybridized membrane at 65 ° c . over night . after 2 washes , the membrane was subjected to autoradiography . statistical analysis . the results are presented as mean ± standard deviation . statistical significance among the groups treated with ezetimibe and controls was determined by the non - paired student t test . a value of p & lt ; 0 . 05 was considered significant . 100 % of the mice that received a lithogenic diet and water by gavage for 14 days developed gallstones and presented cholesterol monohydrate crystals in the gallbladder bile . in contrast , none of the mice that received a lithogenic diet plus ezetimibe by gavage for 14 days developed gallstones or cholesterol crystals in the gallbladder ( fig1 ). as it was expected , the mice on a control diet low in cholesterol , either with water or ezetimibe , did not develop cholelithiasis or cholesterol crystals in the bile . this is the crucial result that supports the present invention , the complete inhibition in the generation of cholelithiasis in a preclinical experimental model through the use of ezetimibe , a potent selective blocker of cholesterol intestinal absorption belonging to the 2 - azetinedione family . no significant difference was found in the animals &# 39 ; weight , liver weight and liver / body weight index between the mice on control diet with or without ezetimibe . on lithogenic diet , a significantly increase in liver weight was observed . this increment in liver weight is not significantly different between mice that had not been treated with ezetimibe and those that had been treated ( table 1 ). the concentration of total cholesterol in plasma was similar between the group of mice on the control diet with or without ezetimibe . the concentration of plasma cholesterol increases on a lithogenic diet for 14 similarly in the groups with or without ezetimibe . ( table 1 ). this result is in contrast with other researchers finding where they saw significant differences between mice and high cholesterol diet with and without ezetimibe . however , the mentioned experiments were performed for longer treatment periods ( longer than two months ), what may explain our results . the plasma lipoprotein profile between the experimental groups that received ezetimibe and the controls did not show significant differences ( fig2 ). in order to verify the inhibitory effect of ezetimibe on cholesterol absorption on the control and lithogenic diets used , we quantified hepatic cholesterol content and cholesterol fecal excretion ( neutral sterols ). the fecal excretion of neutral sterols in 24 h , on days 13 - 14 of experimentation was 5 . 36 μmol per day per 100 g mouse on control diet without ezetimibe ; and 19 . 98 μmol per day per 100 g mouse on control diet plus ezetimibe ( 37 % increment ), in accord with previous studies on similar low cholesterol diets . on lithogenic diets , the fecal excretion of neutral sterols ( cholesterol ) increases significantly in both groups ( 8 times ), being 55 % higher in the mice that received ezetimibe compared with those that were not treated with ezetimibe ( table 2 ). the hepatic cholesterol content in mice on control diet was similar between those that received water or ezetimibe , being rather smaller ( 20 %) in the latter . total cholesterol content increased significantly in the mice on lithogenic diet ( 360 %), however , this increment is significantly smaller in the mice on lithogenic diet plus ezetimibe ( 38 %) ( table 1 ). the increase in hepatic cholesterol corresponds to an increase in both cholesterol ester and free cholesterol , being more relevant the esterified cholesterol increase in the mice without ezetimibe . these results confirm that dietary cholesterol that accumulates in the liver on a lithogenic diet is drastically reduced by ezetimibe . ezetimibe does not significantly affect fecal excretion of bile salts ( table 2 ), both on control and lithogenic diets , in agreement with previous studies that demonstrate ezetimibe selectivity in the inhibition of cholesterol intestinal absorption . on the control diet , ezetimibe did not induce significant changes in cholesterol content , bile salts or lecitine in the mice gallbladder bile ; the cholesterol saturation index was also similar in both groups ( table 3 ). as it is already known , the lithogenic diet per se triggers a significant increase in cholesterol content in the gallbladder bile ( 4 - 5 times ), without causing significant changes in bile salts or lecitine ; therefore , the cholesterol saturation index is also increased significantly ( 2 - 3 times ). the treatment with ezetimibe on a lithogenic diet notoriously decreases cholesterol content in the gallbladder bile ( 50 - 60 %), with a concomitant reduction in the cholesterol saturation index ( 50 %). in fact , the cholesterol saturation index and cholesterol content values in the gallbladder bile of mice on lithogenic diet plus ezetimibe , were similar to those obtained by other researchers using murine models with low and high cholesterol diets with and without ezetimibe ( but in the absence of cholic acid and fat ) ( repa et al , 2002 ). the biliary flow and hepatic secretion of lipids was also established in the four experimental groups , representing additional original observations that are part of our invention . on low cholesterol control diet , ezetimibe produces a slight non - significant increase in biliary flow ( 30 - 40 %) ( fig4 ). as it is already known , a lithogenic diet per se significantly increases the biliary flow ( 200 - 300 %), as well as increasing biliary lipid secretion , bile salts , lecitine , and cholesterol ( fig3 ). surprisingly , ezetimibe treatment on lithogenic diet triggered an even greater increment in biliary flow , significantly higher to that of the group lithogenic diet without ezetimibe ( 50 - 60 % larger ). we noticed that this larger increment in biliary flow , was associated to an increase in the secretion of bile salts and phospholipids ( dependent on bile salts secretion ), but did not associate to a greater secretion of biliary cholesterol compared to the group that did not receive ezetimibe ( fig3 ). since biliary flow is mainly dependent on hepatic secretion of bile salts but also on the biliary secretion of other solutes such a glutathione ; we quantified glutathione biliary secretion in the mice on lithogenic diet with or without ezetimibe . we observed a significant increase in glutathione secretion ( 200 %) in the mice treatment with ezetimibe compared with those that received only the lithogenic diet ( fig4 ). these results suggest that , on a lithogenic diet , ezetimibe increases biliary flow by mechanism dependent and independent ( glutathione ) of bile salts , which could be relevant as additional mechanisms that favor cholelithiasis inhibition in this preclinical model . hepatic expression of proteins or their mrnas involved in the transport of cholesterol , bile salts and glucuronized xenobiotics . in order to establish if the differences observed in the biliary flow and in the secretion of biliary lipids and glutathione between the group of mice treated with lithogenic diet and the group on lithogenic diet plus ezetimibe for 14 days could be explained by changes in the hepatic expression of transporters specific for these solutes ( phospholipids , bile salts and organic anions ), we determined their expression levels by western blotting and / or northern blotting . there were no significant differences between the groups in the hepatic expression of sr - bi , ntcp , bsep , mrp2 and ggcs . there was only a slight but statistically significant decrease in the phospholipid transporter mdr2 at the mrna level . however , there was no concomitant decrease in mdr2 protein levels ( data not shown ) or a higher biliary secretion of phospholipids in mice on lithogenic diet plus ezetimibe . therefore , we propose that the increase in biliary flow and in the secretion of bile salts , phospholipids and glutathione in mice on lithogenic diet plus ezetimibe compared to those on lithogenic diet without ezetimibe , is not the result of changes in the expression of specific transporters . these changes could be explained by the subtractive effect of ezetimibe on the liver cholesterol content . it is hypothesized that livers with lower cholesterol content ( cholesterol depleted ) have a larger capacity to transport solutes such as bile salts , lecitine and glutathione than cholesterol overcharged livers . this is an innovative point of view and allows to suggest that ezetimibe could have a protective effect against endogenous ( cholestasis , fatty acids ) or exogenous ( some organic anions ) xenobiotics . since ezetimibe inhibited the formation of gallstones in the 2 week protocol ( 14 days ), we wanted to establish if its effect was maintained for longer diet periods . therefore , we supplied ezetimibe directly in the lithogenic diet ( no by gavage ) during twice the time period ( 30 days ). again , the use of ezetimibe completely inhibited the formation of cholesterol gallstones ( fig5 ). as it was expected , 100 % of the mice on lithogenic diet developed cholelithiasis ; 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