Hepatocyte Suspensions Research Articles

Construction of three-dimensional vascularized functional human liver tissue using a layer-by-layer cell coating technique

The creation of artificial liver tissue is an active area of research due to the shortage of donors for liver transplantation. Here we investigated whether a simple and efficient cell coating technique developed in our laboratory could be used to generate functional vascularized liver tissue. This technique creates three-dimensional tissue by loading cells sterically onto other cells that have been coated with layer-by-layer (LbL) nanofilms of fibronectin and gelatin, two extracellular matrix proteins. We used this technique to construct homogenous, dense, well-vascularized liver tissue from cryopreserved human primary hepatocytes, human umbilical vein endothelial cells, and normal human dermal fibroblasts. Using LbL cell coating technique resulted in higher cellular function in terms of human albumin production (P<0.01) and cytochrome P450 activity (P<0.01) invitro. Furthermore, after being transplanted subcutaneously into NOD/SCID mice, the vascularized liver tissue showed greater albumin production in the early stage than non-vascularized tissue or a hepatocyte suspension (P<0.01). Histological examination demonstrated that compare to non-vascularized tissue, there were many less-morphologically changed and intact hepatocytes in the vascularized tissue. This cell coating technique would be applicable to the generation of vascularized functional liver tissue for regenerative medicine in the future.

Comparison of the Predictability of Human Hepatic Clearance for Organic Anion Transporting Polypeptide Substrate Drugs Between Different In Vitro–In Vivo Extrapolation Approaches

Prediction of human pharmacokinetic profiles of drug candidates is an essential step toward first-in-human studies. However, it remains difficult to quantitatively predict hepatic clearance, particularly when hepatic uptake is mediated by transporter(s). Using 15 organic anion transporting polypeptide (OATP) substrate drugs, we tested 3 in vitro–in vivo extrapolation (IVIVE) approaches to predict overall hepatic intrinsic clearance in vivo (CLint,all,vivo). IVIVE approaches involved metabolic intrinsic clearance in human liver microsomes (CLint,met) with or without hepatocyte-to-buffer maximum unbound concentration ratio (Kp,uu,max) correction and uptake intrinsic clearance at 37°C (PSinf,37°C) in human hepatocyte suspensions. Kp,uu,max and PSinf,37°C values were determined in 2 hepatocyte batches, and all tested compounds showed temperature-dependent uptake, consistent with the fact of transporter-mediated uptake. CLint,met substantially underestimated CLint,all,vivo. By multiplying CLint,met by Kp,uu,max values, the prediction performance was much improved; however, in vitro–in vivo correlation was poor. Among the IVIVE approaches, PSinf,37°C showed the most robust correlation with CLint,all,vivo, and one of the hepatocyte batches could predict CLint,all,vivo with a minimal empirical scaling factor. These results suggested IVIVE with hepatic uptake clearance determined in hepatocyte suspensions as one of the most practical approaches for predicting CLint,all,vivo of OATP substrate drugs.

Reliable Rate Measurements for Active and Passive Hepatic Uptake Using Plated Human Hepatocytes.

Transporter-mediated hepatic uptake is proven to be the rate-determining step in the systemic clearance of several drugs. Therefore, accurate measurement of active and passive uptake clearances in vitro is critical to facilitate pharmacokinetics and drug-drug interaction predictions. Here, we evaluated the plated human hepatocytes (PHH) and studied the effect of incubation temperature and inhibitor concentration on uptake measurements, in order to reliably estimate hepatic uptake components. Uptake rates measured using PHH, at 37°C without and with rifamycin SV, were comparable with those obtained from suspension hepatocytes and sandwich-cultured hepatocytes for a set of 10-13 compounds. Apparent permeability across monolayers of low-efflux Madin-Darby canine kidney cells was measured at 4, 10, and 37°C. Of the 23 compounds evaluated, 13 compounds showed >2-fold reduction in passive permeability at 4°C compared to 37°C, inferring that low-temperature incubations may underestimate passive uptake. Inhibition studies using transporter-transfected cells suggested that ∼20μM rifamycin SV completely inhibited organic anion-transporting polypeptides (OATPs), while no significant inhibition was noted for other hepatic uptake transporters. On the basis of inhibition profiles, the contribution of active versus passive and OATP versus non-OATP transport to the PHH uptake was discerned for various endogenous substrates and statins. With the exception of fluvastatin, the statins studied were predominantly transported by OATPs in PHH and the non-OATP transporters, such as Na+-taurocholate co-transporting polypeptide, played a minimal role. In conclusion, PHH is useful for uptake measurements, and rifamycin SV employed at different concentrations can reliably estimate active and passive uptake and characterize OATP-dependent active uptake.

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

ABSTRACTEarly prediction of human clearance is often challenging, in particular for the growing number of low-clearance compounds. Long-term in vitro models have been developed which enable sophisticated hepatic drug disposition studies and improved clearance predictions. Here, the cell line HepG2, iPSC-derived hepatocytes (iCell®), the hepatic stem cell line HepaRG™, and human hepatocyte co-cultures (HμREL™ and HepatoPac®) were compared to primary hepatocyte suspension cultures with respect to their key metabolic activities. Similar metabolic activities were found for the long-term models HepaRG™, HμREL™, and HepatoPac® and the short-term suspension cultures when averaged across all 11 enzyme markers, although differences were seen in the activities of CYP2D6 and non-CYP enzymes. For iCell® and HepG2, the metabolic activity was more than tenfold lower. The micropatterned HepatoPac® model was further evaluated with respect to clearance prediction. To assess the in vitro parameters, pharmacokinetic modeling was applied. The determination of intrinsic clearance by nonlinear mixed-effects modeling in a long-term model significantly increased the confidence in the parameter estimation and extended the sensitive range towards 3% of liver blood flow, i.e., >10-fold lower as compared to suspension cultures. For in vitro to in vivo extrapolation, the well-stirred model was used. The micropatterned model gave rise to clearance prediction in man within a twofold error for the majority of low-clearance compounds. Further research is needed to understand whether transporter activity and drug metabolism by non-CYP enzymes, such as UGTs, SULTs, AO, and FMO, is comparable to the in vivo situation in these long-term culture models.

Evaluation of Mitochondrial Respiration in Cultured Rat Hepatocytes.

Mitochondrial dysfunction is a major mechanism whereby drugs can induce liver injury and other serious side effects, such as lactic acidosis and rhabdomyolysis, in some patients. Several in vitro and in vivo investigations can be performed in order to determine if drugs can disturb mitochondrial fatty acid oxidation (FAO) and the oxidative phosphorylation (OXPHOS) process, deplete hepatic mitochondrial DNA (mtDNA), or trigger the opening of the mitochondrial permeability transition pore (MPT). Among these investigations, mitochondrial respiration is a relatively easy test to measure the potential toxicity of a drug. The use of cells instead of isolated mitochondria allows one to test the toxic effect of a parent compound and its metabolites. The use of rat hepatocytes can detect drugs involved in drug-induced liver injuries (DILI). The method consists in measuring oxygen consumption by using a Clark electrode in a chamber containing a suspension of hepatocytes preincubated with drug.

Reversible and time-dependent inhibition (TDI) of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in human hepatocyte suspensions by model inhibitors

Reversible and time-dependent inhibition (TDI) of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in human hepatocyte suspensions by model inhibitors

Use of HμREL Human Coculture System for Prediction of Intrinsic Clearance and Metabolite Formation for Slowly Metabolized Compounds.

Design of slowly metabolized compounds is an important goal in many drug discovery projects. Standard hepatocyte suspension intrinsic clearance (CLint) methods can only provide reliable CLint values above 2.5 μL/min/million cells. A method that permits extended incubation time with maintained performance and metabolic activity of the in vitro system is warranted to allow in vivo clearance predictions and metabolite identification of slowly metabolized drugs. The aim of this study was to evaluate the static HμREL coculture of human hepatocytes with stromal cells to be set up in-house as a standard method for in vivo clearance prediction and metabolite identification of slowly metabolized drugs. Fourteen low CLint compounds were incubated for 3 days, and seven intermediate to high CLint compounds and a cocktail of cytochrome P450 (P450) marker substrates were incubated for 3 h. In vivo clearance was predicted for 20 compounds applying the regression line approach, and HμREL coculture predicted the human intrinsic clearance for 45% of the drugs within 2-fold and 70% of the drugs within 3-fold of the clinical values. CLint values as low as 0.3 μL/min/million hepatocytes were robustly produced, giving 8-fold improved sensitivity of robust low CLint determination, over the cutoff in hepatocyte suspension CLint methods. The CLint values of intermediate to high CLint compounds were at similar levels both in HμREL coculture and in freshly thawed hepatocytes. In the HμREL coculture formation rates for five P450-isoform marker reactions, paracetamol (CYP1A2), 1-OH-bupropion (CYP2B6), 4-OH-diclofenac (CYP2C9), and 1-OH-midazolam (3A4) were within the range of literature values for freshly thawed hepatocytes, whereas 1-OH-bufuralol (CYP2D6) formation rate was lower. Further, both phase I and phase II metabolites were detected and an increased number of metabolites were observed in the HμREL coculture compared to hepatocyte suspension. In conclusion, HμREL coculture can be applied to accurately estimate intrinsic clearance of slowly metabolized drugs and is now utilized as a standard method for in vivo clearance prediction of such compounds in-house.

Determination of Unbound Partition Coefficient and in Vitro-in Vivo Extrapolation for SLC13A Transporter-Mediated Uptake.

Unbound partition coefficient (Kpuu) is important to an understanding of the asymmetric free drug distribution of a compound between cells and medium in vitro, as well as between tissue and plasma in vivo, especially for transporter-mediated processes. Kpuu was determined for a set of compounds from the SLC13A family that are inhibitors and substrates of transporters in hepatocytes and transporter-transfected cell lines. Enantioselectivity was observed, with (R)-enantiomers achieving much higher Kpuu (>4) than the (S)-enantiomers (<1) in human hepatocytes and SLC13A5-transfected human embryonic 293 cells. The intracellular free drug concentration correlated directly with in vitro pharmacological activity rather than the nominal concentration in the assay because of the high Kpuu mediated by SLC13A5 transporter uptake. Delivery of the diacid PF-06649298 directly or via hydrolysis of the ethyl ester prodrug PF-06757303 resulted in quite different Kpuu values in human hepatocytes (Kpuu of 3 for diacid versus 59 for prodrug), which was successfully modeled on the basis of passive diffusion, active uptake, and conversion rate from ester to diacid using a compartmental model. Kpuu values changed with drug concentrations; lower values were observed at higher concentrations possibly owing to a saturation of transporters. Michaelis-Menten constant (Km) of SLC13A5 was estimated to be 24 μM for PF-06649298 in human hepatocytes. In vitro Kpuu obtained from rat suspension hepatocytes supplemented with 4% fatty acid free bovine serum albumin showed good correlation with in vivo Kpuu of liver-to-plasma, illustrating the potential of this approach to predict in vivo Kpuu from in vitro systems.

Hepatocytes as in vitro test system to investigate metabolite patterns of pesticides in farmed rainbow trout and common carp: Comparison between in vivo and in vitro and across species

In vitro tools using isolated primary fish hepatocytes have been proposed as a useful model to study the hepatic metabolism of xenobiotics in fish. In order to evaluate the potential of in vitro fish hepatocyte assays to provide information on in vivo metabolite patterns of pesticides in farmed fish, the present study addressed the following questions: Are in vitro and in vivo metabolite patterns comparable? Are species specific differences of metabolite patterns in vivo reflected in vitro? Are metabolite patterns obtained from cryopreserved hepatocytes comparable to those from freshly isolated cells? Rainbow trout and common carp were dosed orally with feed containing the pesticide methoxychlor (MXC) for 14days. In parallel, in vitro incubations using suspensions of freshly isolated or cryopreserved primary hepatocytes obtained from both species were performed. In vivo and in vitro samples were analyzed by thin-layer chromatography with authentic standards supported by HPLC-MS. Comparable metabolite patterns from a qualitative perspective were observed in liver in vivo and in hepatocyte suspensions in vitro. Species specific differences of MXC metabolite patterns observed between rainbow trout and common carp in vivo were well reflected by experiments with hepatocytes in vitro. Finally, cryopreserved hepatocytes produced comparable metabolite patterns to freshly isolated cells. The results of this study indicate that the in vitro hepatocyte assay could be used to identify metabolite patterns of pesticides in farmed fish and could thus serve as a valuable tool to support in vivo studies as required for pesticides approval according to the EU regulation 1107.

Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse

Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time‒ and bile-acid-concentration‒dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values <50 μM), but only about 20% of the non-sDILI drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune-mediated mechanism, are highly associated with potent inhibition of bile salt transport.

Optimization of specimen preparation from formalin-fixed liver tissues for liver micronucleus assays: Hepatocyte staining with fluorescent dyes

The liver micronucleus (MN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly those that require metabolic activation. For this assay, hepatocytes (HEPs) can be isolated by collagenase treatment but without requirement for in situ liver perfusion. Consequently, the liver MN assay can be integrated into a general repeated-dose (RD) toxicity study. The method is also applicable to liver MN assays involving partial hepatectomy or the use of juvenile rats. Here, we propose an improved method for staining HEPs prepared from formalin-fixed liver tissues for MN assays, without collagenase treatment. HEP suspensions are prepared by treating the tissues with concentrated KOH and a fluorescent dye, SYBR® Gold (SYGO), is used for staining. Visualization of the MN in SYGO-stained HEPs is clearer than with Wright-Giemsa staining. We compared the induction of MN as measured with our new method versus the conventional method using collagenase dispersion. Our method not only enables the integration of the liver MN assay into a general RD toxicity study but also allows it to be conducted retrospectively.

Inter-donor variability of phase I/phase II metabolism of three reference drugs in cryopreserved primary human hepatocytes in suspension and monolayer

Cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) are the most important enzymes for metabolic clearance. Characterization of phase I and phase II metabolism of a given drug in cellular models is therefore important for an adequate interpretation of the role of drug metabolism in toxicity. We investigated phase I (CYP) and phase II (UGT and SULT) metabolism of three drugs related to drug-induced liver injury (DILI), namely acetaminophen (APAP), diclofenac (DF) and tolcapone (TC), in cryopreserved primary human hepatocytes from 5 donors in suspension and monolayer. The general phase II substrate 7-hydroxycoumarin (7-HC) was included for comparison. Our results show that the decrease in CYP, UGT and SULT activity after plating is substrate dependent. As a consequence the phase I/phase II metabolism ratio is significantly affected, with a shift in monolayer towards phase I metabolism for TC and towards phase II metabolism for APAP and DF. Inter-donor variability in drug metabolism is significant, especially in sulfation of 7-HC or APAP. As CYP, UGT and SULT metabolism may lead to bioactivation and/or detoxification of drugs, a changed ratio in phase I/phase II metabolism may have important consequences for metabolism-related toxicity.

OS0105 液液界面を用いた移植用肝細胞選別システムの検討

Hepatocyte transplantation is expected to alternative or bridge treatment to liver transplantation, especially for infants with metabolic disorder. Effectiveness and safety of hepatocyte transplantation is important for clinical efficiency. Particularly, a size of hepatocyte cluster is critical for effectiveness of the treatment. In previous study, we cleared that cluster of hepatocyte have higher ammonia metabolic function than isolated hepatocyte. So it is a necessity that clusters of hepatocyte are selected from hepatocyte suspension for high quality hepatocyte transplantation. In this study, we developed the novel separation method using centrifugal force and Dean Vortices in liquid-liquid two phase flow for hepatocyte transplantation.

In vitro model systems to investigate bile salt export pump (BSEP) activity and drug interactions: A review

The bile salt export pump protein (BSEP), expressed on the canalicular membranes of hepatocytes, is primarily responsible for the biliary excretion of bile salts. The inhibition of BSEP transport activity can lead to an increase in intracellular bile salt levels and liver injury. This review discusses the various in vitro assays currently available for assessing the effect of drugs or other chemical entities to modulate BSEP transport activity. BSEP transporter assays use one of the following platforms: Xenopus laevis oocytes; canalicular membrane vesicles (CMV); BSEP-expressed membrane vesicles; cell lines expressing BSEP; sandwich cultured hepatocytes (SCH); and hepatocytes in suspension. Two of these, BSEP-expressed insect membrane vesicles and sandwich cultured hepatocytes, are the most commonly used assays. BSEP membrane vesicles prepared from transfected insect cells are useful for assessing BSEP inhibition or substrate specificity and exploring mechanisms of BSEP-associated genetic diseases. This model can be applied in a high-throughput format for discovery-drug screening. However, experimental results from use of membrane vesicles may lack physiological relevance and the model does not allow for investigation of in situ metabolism in modulation of BSEP activity. Hepatocyte-based assays that use the SCH format provide results that are generally more physiologically relevant than membrane assays. The SCH model is useful in detailed studies of the biliary excretion of drugs and BSEP inhibition, but due to the complexity of SCH preparation, this model is used primarily for determining biliary clearance and BSEP inhibition in a limited number of compounds. The newly developed hepatocyte in suspension assay avoids many of the complexities of the SCH method. The use of pooled cryopreserved hepatocytes in suspension minimizes genetic variance and individual differences in BSEP activity and also provides the opportunity for higher throughput screening and cross-species comparisons.

Confocal Imaging and Tissue-Specific Fluorescent Probes for Real-Time In Vivo Immunohistochemistry. Proof of the Concept in a Gastric Lymph Node Metastasis Model.

Tumor-specific fluorescent antibodies, which can be recognized at a cellular or tissue level using optical imaging such as confocal laser endomicroscopy (CLE), could provide a means for rapid and accurate tumor diagnosis and staging. The aim of this study was to evaluate the ability of CLE to detect the presence of tagged cells within lymph nodes in an original simulated metastatic model. A solution of indocyanine green containing a suspension of porcine hepatocytes, marked with carboxy-fluorescein-succinimidyl-ester (CFSE), was injected endoscopically in the gastric submucosa of 10 pigs. Fluorescence lymphography using a near-infrared laparoscope was used to identify sentinel and secondary drainage nodes. Additionally, a nonfluorescent gastric and a mesenteric node were identified. Every 5-10min, those nodes were scanned using probe-based or needle-based CLE (pCLE or nCLE). Immunohistochemistry (IHC) using anti-cytokeratin 18 antibodies was subsequently performed to confirm the presence of hepatocytes in the lymph nodes. A total of 36 lymph nodes were analyzed with both CLE probes. Hepatocyte penetration in lymph nodes, as assessed by repeated CLE scanning, took 10-40min after submucosal injection. Concordance between CLE and IHC was 84 and 72% for pCLE and nCLE, respectively. False negatives were partly due to incomplete CFSE labeling of hepatocytes, which could not be recognized by CLE, but were detected with IHC. Real-time CLE analysis effectively recognized the presence in perigastric nodes of marked hepatic cells that had been injected endoscopically in the stomach. Validation studies on tumor-bearing animals using tumor-specific antibodies should be performed.

Prediction of Drug Clearance and Drug-Drug Interactions in Microscale Cultures of Human Hepatocytes

Accurate prediction of in vivo hepatic drug clearance using in vitro assays is important to properly estimate clinical dosing regimens. Clearance of low-turnover compounds is especially difficult to predict using short-lived suspensions of unpooled primary human hepatocytes (PHHs) and functionally declining PHH monolayers. Micropatterned cocultures (MPCCs) of PHHs and 3T3-J2 fibroblasts have been shown previously to display major liver functions for several weeks in vitro. In this study, we first characterized long-term activities of major cytochrome P450 enzymes in MPCCs created from unpooled cryopreserved PHH donors. MPCCs were then used to predict the clearance of 26 drugs that exhibit a wide range of turnover rates in vivo (0.05-19.5 ml/min per kilogram). MPCCs predicted 73, 92, and 96% of drug clearance values for all tested drugs within 2-fold, 3-fold, and 4-fold of in vivo values, respectively. There was good correlation (R(2) = 0.94, slope = 1.05) of predictions between the two PHH donors. On the other hand, suspension hepatocytes and conventional monolayers created from the same donor had significantly reduced predictive capacity (i.e., 30-50% clearance values within 4-fold of in vivo), and were not able to metabolize several drugs. Finally, we modulated drug clearance in MPCCs by inducing or inhibiting P450s. Rifampin-mediated CYP3A4 induction increased midazolam clearance by 73%, while CYP3A4 inhibition with ritonavir decreased midazolam clearance by 79%. Similarly, quinidine-mediated CYP2D6 inhibition reduced clearance of dextromethorphan and desipramine by 71 and 22%, respectively. In conclusion, MPCCs created using cryopreserved unpooled PHHs can be used for drug clearance predictions and to model drug-drug interactions.

Characterization of transporter activities in fresh isolated primary hepatocytes of different species by using fluorescent substrates

Prediction of human pharmacokinetic profiles of drug candidates is an essential step toward first-in-human studies. However, it remains difficult to quantitatively predict hepatic clearance, particularly when hepatic uptake is mediated by transporter(s). Using 15 organic anion transporting polypeptide (OATP) substrate drugs, we tested 3 in vitro–in vivo extrapolation (IVIVE) approaches to predict overall hepatic intrinsic clearance in vivo (CLint,all,vivo). IVIVE approaches involved metabolic intrinsic clearance in human liver microsomes (CLint,met) with or without hepatocyte-to-buffer maximum unbound concentration ratio (Kp,uu,max) correction and uptake intrinsic clearance at 37°C (PSinf,37°C) in human hepatocyte suspensions. Kp,uu,max and PSinf,37°C values were determined in 2 hepatocyte batches, and all tested compounds showed temperature-dependent uptake, consistent with the fact of transporter-mediated uptake. CLint,met substantially underestimated CLint,all,vivo. By multiplying CLint,met by Kp,uu,max values, the prediction performance was much improved; however, in vitro–in vivo correlation was poor. Among the IVIVE approaches, PSinf,37°C showed the most robust correlation with CLint,all,vivo, and one of the hepatocyte batches could predict CLint,all,vivo with a minimal empirical scaling factor. These results suggested IVIVE with hepatic uptake clearance determined in hepatocyte suspensions as one of the most practical approaches for predicting CLint,all,vivo of OATP substrate drugs.

The use of cryopreserved and fresh human hepatocytes and human liver microsomes for the in vitro evaluation of Boswellia serrata extract (BSE) as an inhibitor of CYP450

As the use of dietary supplements increases, herb-drug interaction (HDI) potential should be evaluated. Our in vitro strategy to assess HDI uses an integrated liver system yielding physiologic intracellular concentrations of drugs and herbs. BSE is traditionally used for inflammatory diseases but has no reported clinical HDI. Potent in vitro inhibition (≥65%) across major drug metabolizing enzymes using pooled human liver microsomes (PHLM) have been reported in the literature for levels of BSE as low as 1 µg/mL. This potent level of inhibition led us to question the relevance of the results using PHLM. In PHLM, BSE was a direct inhibitor of CYP2C9 and CYP3A4/5 with IC50= 11 µg/mL and 1.4 µg/mL, respectively; no evidence of time-dependent inhibition of either enzyme. We next evaluated the ability of BSE to inhibit CYP2C9 and CYP3A4/5 in pooled suspensions of human hepatocytes, which have robust metabolism and uptake activities but no biliary efflux capabilities. In this system, considerably less direct inhibition of CYP2C9 was observed (IC50 > 50 µg/mL) for BSE. Finally, to represent the in vivo situation with both transporter and metabolic capabilities integrated together, a sandwich-cultured human hepatocyte (SCHH) system was used to assess potential HDI which would yield physiologic intracellular concentrations. In SCHH, direct inhibition of CYP3A4/5 by BSE was observed resulting in an IC50= 17.2 µg/mL. A maximum 30% decrease in 3-OH-ibuprofen formation (CYP2C9) was observed in SCHH treated with BSE (IC50 > 75 µg/mL). These data suggest that PHLM may provide a worst-case scenario highlighting the need to utilize a more physiologically relevant model that integrates transporter and metabolism to assess HDI potential.

Sex differences in liver toxicity-do female and male human primary hepatocytes react differently to toxicants in vitro?

There is increasing amount of evidence for sex variation in drug efficiency and toxicity profiles. Women are more susceptible than men to acute liver injury from xenobiotics. In general, this is attributed to sex differences at a physiological level as well as differences in pharmacokinetics and pharmacodynamics, but neither of these can give a sufficient explanation for the diverse responses to xenobiotics. Existing data are mainly based on animal models and limited data exist on in vitro sex differences relevant to humans. To date, male and female human hepatocytes have not yet been compared in terms of their responses to hepatotoxic drugs. We investigated whether sex-specific differences in acute hepatotoxicity can be observed in vitro by comparing hepatotoxic drug effects in male and female primary human hepatocytes. Significant sex-related differences were found for certain parameters and individual drugs, showing an overall higher sensitivity of female primary hepatocytes to hepatotoxicants. Moreover, our work demonstrated that high content screening is feasible with pooled primary human hepatocytes in suspension.

Proposal of a novel evaluation index for the effects of shear stress and exposure time on hepatocyte damage.

The purpose of this study was to propose a novel evaluation index for the effects of shear stress level and exposure time on hepatocyte damage. Suspensions of rat hepatocytes (0.5mL) were subjected to shear stress from 1.2 to 3.1Pa for 10min (n=3) using a rheoscope. We counted living and dead cells in photographs taken at 1-min intervals using a digital camera attached to the microscope. Living and dead cells were distinguished using a Trypan blue exclusion test. Under each level of shear stress, at each 1-min time interval, we measured the viability [living-cell number (t)/countable cell number (t)] and the ratio of living cells [RLC: living-cell number (t)/countable cell number in the initial condition]. The effects of shear stress and exposure time on viability and RLC were assessed by multiple regression analysis. As expected, we observed an increase in the number of dead cells and little change in the number of living cells when shear stress was increased. The coefficient of determination (R (2)) to predict the effectiveness of viability and RLC indicated a low to moderate correlation. Viability correlated with shear stress and exposure time (p<0.001); however, RLC only correlated with exposure time of shear stress (p<0.001). In this test condition, viability was strongly related not to living-cell damage but to dead-cell damage. Therefore, we propose RLC as a novel and effective index for investigating the effect of shear stress on living hepatocytes.