the capacity of drugs to inhibit the bile salt export pump ( bsep ) has been correlated with clinical drug  induced liver injury ( dili ) . furthermore , known inhibitors of bsep and other bile acid ( ba ) transporters have been implicated in the case studies of dili . glibenclamide has been associated with lobular liver injury in the clinic , bosentan has been associated with many cases of hepatotoxicity and carries a black - box warning for hepatotoxicity , and troglitazone was withdrawn from the market due to idiosyncratic dili . currently marketed bsep inhibitors , such as lapatinib , have been given black - box warnings due to issues with liver toxicity . interestingly , bsep inhibitors often have additional proposed mechanisms for hepatotoxicity , and this can often confound our understanding of how these drugs cause dili . understanding the effects of bsep inhibitors on ba metabolism and processing will prove crucial in understanding the importance of bsep inhibition in clinical dili . while much is understood qualitatively about the basics of ba homeostasis , there are still many aspects of ba transport about which little quantitative information exists . for example , regulation of transporter activity by the farnesoid - x receptor ( fxr ) and other nuclear receptors generally responsible for ba synthesis has been demonstrated , and chenodeoxycholic acid ( cdca ) and its amide conjugates have been shown to decrease ba synthesis and trigger fxr . however , to our knowledge , fxr response and transporter activity have not been linked quantitatively to ba concentrations within hepatocytes . secondary bas , such as lithocholic acid ( lca ) and deoxycholic acid ( dca ) , are synthesized in the gut by intestinal bacteria , but to our knowledge , the rates of synthesis and the population variability of this synthesis have not been quantified in any species . transporter kinetic and inhibition studies have used ( or focused on ) taurocholic acid , a metabolically stable model ba , but similar studies rarely have been performed on other bas . furthermore , while certain ba species , such as lca and cdca , are more cytotoxic than other bas , the effect that intrahepatic concentrations of these bas have on hepatocytes remains to be elucidated . while bsep transporter expression variability in humans has been quantified , and underlying diseases have been shown to affect transporter expression , the significance of this variability has not been explored fully . the process of constructing a mathematical model of ba - induced dili can be helpful in determining which data gaps should be filled first for maximum impact . while any mechanistic model of ba - induced dili will be lacking in quantitative accuracy due to the significant data gaps , mechanistic modeling can be used to qualitatively describe elements of the system that would be more or less likely to contribute to ba - mediated dili . in this study , we use dilisym ( www.dilisym.com ) , a mechanistic mathematical model of dili , to explore the nature of ba homeostasis and its disruption by ba transporter inhibitors . dilisym has been used previously to assess the efficacy of different n - acetylcysteine treatment regimens in acetaminophen - overdosed patients and to understand the species differences in methapyrilene toxicity using in vitro metabolism data . for this study , we have constructed a module of ba homeostasis within dilisym for rats and humans ; information on the data used to construct this module is available in the supplementary materials online . we have also collected novel in vivo data from the rats treated with the bsep inhibitor glibenclamide and used these data to ensure the reasonability of the dilisym model . we also compared the human representation in dilisym to serum ba data in humans after acetaminophen overdose . this model was used to explore the changes in intrahepatic ba concentrations that are predicted to occur in humans when a ba transporter inhibitor is administered . finally , a sample population was used to determine which system parameters have the most influence on the magnitude of the intrahepatic ba changes that occur after dosing with a ba transporter inhibitor and would thus be the system components that would be most useful to be illuminated by future experiments . the bsep and na - taurocholate - cotransporting polypeptide inhibitor glibenclamide , when administered orally to rats , causes an increase in total plasma bas ; this increase in ba concentrations is associated with high systemic exposure to glibenclamide , but is not observed at lower exposure levels . figure 1 shows the results from the single - dose studies . glibenclamide is notoriously poorly absorbed ; as such , the systemic exposure for each individual rat is more important than the dose administered . at concentrations below a cmax of 10,000  ng / ml , there was little correspondence between glibenclamide concentrations and serum ba fold change , suggesting that the variability in ba processing among rats exceeds the effects of transporter inhibition at these exposure levels . rats with glibenclamide cmax > 10,000  ng / ml generally displayed a higher concentration of bas , albeit over a wide range . as shown in figure 1 , the simulated population ( simpops ) covers the range of ba increases observed in the experimental rat data at each maximum simulated concentration tested , validating our model . figure 2 summarizes the results of the multiple - dose ( 300 , 750 , and 1,500  mg / kg ) studies where individual ba species were measured . serum data from day 1 are plotted in figure 2 ; each data point represents an individual rat . serum cdca and lca concentrations increased very soon after glibenclamide dosing with a return to the baseline predosing value by the end of 24  h ; serum cdca amide conjugates did not appear to change after glibenclamide dosing . on day 7 , plasma concentrations of glibenclamide did not vary widely in animals treated with the three different dose levels ( data not shown ) ; as a result , a single simulation was conducted on a rat simpops at a simulated drug  exposure level similar to that observed in the rats in vivo . the results of this simulation were compared to the experimental data for lca , cdca , and cdca  amide conjugates in figure 2 . the simulated population showed similar dynamics to those observed in vivo ; the simulated rats exhibited a mild increase in bas between 1 and 2  h after dosing and returned to baseline values by 24  h. for unconjugated cdca , the simulated rat population covered the range of observed responses ; for lca , the simulated responses covered the bulk of the experimental data but missed the highest ba concentrations , while for cdca  amide , our simulated population covered a much wider range than the experimental data , likely due to a wider range of initial cdca  amide concentrations in the rat simpops . figure 2 demonstrates that dilisym provides a good approximation of the range of ba increases that were observed after administration of a transporter inhibitor . as data detailing the effect of ba transporter inhibitors on ba concentrations in humans were scarce , the model was validated in humans by comparing the response of the system after acetaminophen dosing to serum ba data from the clinic . acetaminophen is not a bsep inhibitor , so in this case , ba elevations are due to hepatocyte necrosis . our baseline human model had a similar response to that demonstrated by james et al . , that is , little to no ba elevations in serum when injury was minimal , small but significant increases when there was moderate injury , and large elevations when the injury was severe . the small human simpops also exhibited a similar range of ba concentration changes to the patients reported in the james study . with the model structure validated using the rat and human experimental data , we proceeded to simulate a bsep inhibitor in our baseline human model . the difference in liver ba area under the curves ( aucs ) over the course of simulated drug administration in the presence and absence of the theoretical inhibitor is given in table 1 . in all zones of the liver , increases were observed in the auc of bulk bas , cdca  amide conjugates , and lca  sulfate conjugates . meanwhile , the auc of unconjugated cdca and unconjugated lca actually decreased , while the auc of amide - conjugated lca remained about the same . the changes in ba concentrations with inhibitor as a function of the zonality of the liver were also examined using dilisym ( table 1 ) . in the periportal region , where most bas are concentrated , however , in the centrilobular region , all ba species except for unconjugated lca increased significantly . for cdca amide and lca  sulfate conjugates , the increases in auc were far greater than those predicted in the periportal region . however , the absolute centrilobular auc remained lower than the absolute periportal auc for all ba species . the large model human population was used to understand the sensitivity of the model parameters to ba accumulation . the most important variables for the accumulation of each ba species in simulated human liver are shown in table 2 . since the baseline human model simulations suggested that cdca  amide and lca  sulfate were the most likely ba species to accumulate in the liver , we assessed the correlation between changes in system parameters and increases in cdca  amide and lca  sulfate concentrations . the sensitivity analysis suggested that bsep expression , as represented by cdca  amide canalicular transport vmax , was the most important variable for cdca  amide accumulation , while the magnitude of lca synthesis in the gut was the most important variable for lca  sulfate accumulation . amide accumulation when cdca  amide canalicular efflux vmax changes compared to when the canalicular transport regulation scaling factor changes ( which roughly corresponds to the magnitude of the response of ba transporter expression to fxr activation ) , as seen in figure 4 , is particularly instructive . when cdca  amide canalicular efflux vmax was varied , the baseline cdca  amide concentration of the individual changed ; however , the percentage increase in cdca  amide concentration after administration of the inhibitor was approximately constant across the variable range . in contrast , when the canalicular regulation scaling factor was varied , the baseline concentration of cdca  amide remained the same ( although meal fluctuations were more pronounced with less regulation ) , while the percentage increase in cdca  amide concentration was much higher when canalicular transport regulation was lower . the novel in vivo data included herein demonstrate the importance of considering population variability and individual characteristics in any model of ba homeostasis and its disruption . the variability between rats shrouded the detection of changes in ba due to low concentrations of glibenclamide ( cmax : 50010,000  ng / ml ) and the resultant transporter inhibition . the lack of an apparent exposure / response effect in the individual ba data is due to the population variability . even with higher doses of glibenclamide , when an exposure / response relationship was observed , we found that there was a wide range of responses , as measured by a fold increase in ba concentrations , among the rats with glibenclamide cmax higher than 10,000  ng / ml . few rats reached the higher drug - exposure levels ; further experiments , perhaps using a more readily absorbed formulation of glibenclamide , could help illuminate this effect . the variability in the results also makes model validation difficult , as our ability to reproduce the experimental conditions ( including individual rats ' initial plasma and liver ba concentrations ) within the context of a simulation is challenging ; while our model is reasonably validated , further experimentation in this area will continue to improve our representation . our results suggest that due to the population variability inherent in the ba system , a baseline model is of limited utility for predicting the response of rats ( or patients ) to ba transporter inhibitors . a baseline model is useful for gleaning interesting information about the basic behavior of the system , but quantitative predictions of ba increases and decreases should not be based on the behavior of a baseline ba model . constructing an appropriate population for the human model is , therefore , of the utmost importance for the prediction of ba - mediated dili . to that end , we used the ba model to identify the most important parameters that are likely to vary among individuals . these parameters should be included in the creation of a reasonable population for the prediction of ba toxicity . the model suggests that the end - product ba conjugates are the ba species that are most likely to accumulate in the liver . this is likely due to the fact that the conjugation of bas is fairly rapid in the liver , and these pathways do not easily saturate . previous research has placed the km for ba amidation in the 1 mmol / l range , which is several orders of magnitude higher than typical in vivo unconjugated ba concentrations . as a result of the predicted accumulation of ba conjugates in the liver , ba amide and sulfate conjugates are potential liver toxicophores ; the toxicity of these molecules should be explored and quantified with future experimentation . centrilobular cells , which generally have a lower ba concentration than their periportal counterparts , have a much higher predicted relative accumulation of all ba species . in fact , the auc of unconjugated bas was predicted to increase in the centrilobular zone , even though the same bas ' auc decreased in the periportal zone . despite the differential ba changes in the two zones , the absolute auc of bas in the centrilobular zone was predicted to remain lower than the periportal zone . however , if centrilobular cells are more sensitive to ba toxicity than periportal cells , an increase in ba concentrations could lead to zonal centrilobular toxicity . indeed , some case reports of liver injury due to glibenclamide demonstrate centrilobular injury . the investigation of the differential effects of bas on centrilobular and periportal liver cell toxicity would be another interesting and worthwhile experiment . simulations using a model population provided further insight into the most valuable experiments that could be performed to improve our understanding of ba - mediated dili . among the many system unknowns varied within the model population , the most important variables included lca synthesis in the gut and the regulatory response to ba accumulation . therefore , quantifying lca synthesis in the gut in addition to changes in bsep expression and activity with increasing hepatocyte ba concentrations would be important and impactful experiments . the importance of bsep expression levels to ba accumulation in hepatocytes , for example , could be quantified using existing animal or in vitro models . the modeling also revealed the importance of transporter expression levels on the behavior of the system ; however , it is important to note a key difference between the behavior of the system when these expression levels are varied and its behavior when the regulatory response is varied . when transporter expression levels  especially canalicular transporter levels  are decreased , the baseline ba concentration in the liver increases ; the percentage increase in bas after dosing the inhibitor remains somewhat constant . varying the magnitude of the regulatory response ( fxr activation ) , by contrast , does not have a large effect on baseline ba concentrations in the liver ; however , the percentage increase in bas after inhibitor dosing is highly variable . both transporter expression levels and the magnitude of the regulatory response are expected to vary within a population ; however , only the variability in transporter expression levels may be apparent in untreated individuals . this result suggests both the promise and the limitations of prescreening individuals for certain risk factors before prescribing known bsep inhibitors . it includes ( i ) the synthesis and metabolism of bas in hepatocytes , ( ii ) the basolateral and canalicular active transport of bas , ( iii ) the release of bas from the gallbladder in humans , ( iv ) the synthesis of secondary bas and deconjugation of bas in the gut , ( v ) the recirculation of bas from the gut and subsequent active uptake by the liver , and ( vi ) the regulatory effects of bas on transporter expression and ba synthesis . the model contains representations of lca and cdca and its conjugates , the ba species most frequently linked to toxicity in in vitro experiments , and a  bulk  ba representation that contains the other bas . a more detailed description of the ba homeostasis model and its parameterization is presented in the supplementary materials online . to represent ba dynamics in humans , the model was optimized to the known profile of bas in serum published by trottier et al . , as well as to overall concentrations of bas in the liver measured by setchell and garca - caaveras et al . there were 47 system variables that were either unknown or expected to vary within the human population that were fit to the ba profile ; a list of the variables used in the optimization is provided in the supplementary materials online . the optimization of the model based on the published ba profile was performed in a manner similar to the simpops method outlined in previous publications , wherein a genetic algorithm was used to generate values of variable parameters that lead to model outcomes ( in this case , ba profiles ) that are within the range of experimental data . using this optimization method allowed the selection of a population of 2,400 individuals with reasonable baseline ba concentrations for the large human sample population . a smaller , 10-parameter , 331-individual simpops was also constructed for the purpose of model validation . while the large population was given artificially wide parameter ranges for the purpose of the sensitivity analysis , the small human sample population was constructed with more constrained parameter ranges for the purpose of approximating a plausible population of humans . for example , the transporter vmax ranges included four orders of magnitude in the large population , but were constrained to ranges suggested by transporter expression profiles from meier et al . in the small population . a population of 191 rats using a more limited 11 variable set ( supplementary table s4 online ) was generated . this population was intended to represent rats with both plausible serum ba concentrations and plausible values for the parameters that were varied . the baseline rat model was optimized to the ba profile from the control rats in the present experiment , and the simpops was constructed around this baseline . details on the simulations performed for the model validation and for the model exploration can be found in the supplementary materials online . multivariate analysis on the population sample was performed using jmp 9 from sas ( cary , nc ) . multiple - dose glibenclamide study in rats for the ba - profiling experiments , 16 male 8- to 9-week - old cd-1 rats from charles river laboratories ( raleigh , nc ) weighing between 200 and 300  g were randomized into four groups of four animals each . these groups were administered daily doses of either the vehicle control ( 0.5% hydroxypropylmethylcellulose /0.1% tween 80 in water ) or glibenclamide ( sigma - aldrich , st louis , mo ) in vehicle via oral gavage for 7 days at three dose levels ( 300 , 750 , and 1,500  mg / kg / day ) . rats underwent a viability check twice per day , and detailed clinical observations were taken at least twice over the course of the study . blood was drawn for serum ba profiles at 1 , 3 , 6 , and 24  h after dosing on day 1 and day 7 , and glibenclamide concentrations were measured for toxicokinetic analysis using the same blood samples from day 7 . clinical and anatomic pathology data were collected from the animals , and these results are reported in the supplementary materials online . the serum ba concentrations from day 1 were used for comparison to the simulation results . ba profiling in serum was performed using liquid chromatography  tandem mass spectrometry analysis at glaxosmithkline ( ware , uk ) . bas in liver tissue 24  h after the final dose also were profiled ; results from this analysis , as well as the liquid chromatography  tandem mass spectrometry analytical method , are presented in the supplemental materials online . this study was conducted in accordance with the glaxosmithkline policy on the care , welfare and treatment of laboratory animals and was reviewed by the institutional animal care and use committee . for this experiment , doses of glibenclamide were administered every 24  h for 7 days . systemic exposure data were compared to simulation results for day 7 ; day 1 ba data were compared to simulated ba concentrations on day 1 . short - term glibenclamide studies in rats for the short - term studies , male han wistar rats ( substrain alpkhsdbrlhan : wist ; astrazeneca , macclesfield , uk ) of 1012-week age ( 300400  g ) were used . details on the treatment of the rats used in this study are available in the supplemental materials online . two experiments were performed in which total plasma ba concentrations and glibenclamide plasma concentrations were determined . glibenclamide ( sigma - aldrich ) was formulated as a solution or suspension in hydroxypropyl--cyclodextrin ( acros organics , distributed by fisher scientific , loughborough , uk ) in aqueous 0.2  mol / l na2co3/nahco3 buffer ( ph 10 ) . in the first experiment ( 29 animals ) , four groups of five animals received a single dose via oral gavage of 50 , 250 , and 500  mg / kg glibenclamide or 10% ( w / v ) hydroxypropyl--cyclodextrin vehicle alone , and blood samples were taken at 1 , 6 , and 24  h after dosing ; an additional three animals per glibenclamide - treated group received a single dose of 50 , 250 , and 500  mg / kg glibenclamide , and blood samples were taken at 1 , 3 , 6 , 12 , and 24  h after dosing . in the second experiment ( 20 animals ) , two groups of five animals each received two oral doses of 250 and 500  mg / kg glibenclamide in 20% ( w / v ) hydroxypropyl--cyclodextrin vehicle at 0 and 4  h , and blood samples were taken predose and at 0.5 , 1 , 2 , and 4  h after dosing ; blood samples from an additional five animals per glibenclamide - treated group were taken predose and 1  h after dosing to increase the dataset for this time point .