8026 Background: Durvalumab improves overall survival (OS) and progression free survival (PFS) when used as consolidation therapy for patients with stage III unresectable non-small cell lung cancer (UR-NSCLC) following chemoradiotherapy (CRT). However, it is uncertain if similar benefits are achieved for patients with PD-L1 positive (1% or greater) and negative (<1%) tumors. We previously found no difference in OS, but no study has yet compared cancer-related overall survival (OS). Methods: Patients with stage III UR-NSCLC on durvalumab following CRT, at any Veterans Health Administration (VHA) facility, from 1/1/17 to 6/30/20, were included. Patients were followed from durvalumab initiation through the earliest of their last VHA visit, loss to follow up, death, or end of study (6/30/25). Electronic health record data were retrospectively collected to determine durvalumab treatment course, OS, and cancer-related OS—as determined by review of each patient’s chart and death certificate. Kaplan-Meier and Cox regression methods were used to compare cancer-related OS. Results: Of the 340 eligible patients, 221 (65%) had PD-L1 positive and 119 (35%) had PD-L1 negative tumors. Groups were similar in age, sex assigned at birth, White race, smoking status, marital status, Charlson score, ECOG 1+ status (78% overall), histology, stage, EGFR, and RAS mutations. Patients with PD-L1 positive and negative tumors had similar median (interquartile range [IQR]) number of durvalumab doses (15 [6-24] vs 13 [5-24], p=0.55), months of durvalumab (8 [3-12] vs 6 [2-12]), and months of follow-up (33 [12-69] vs 28 [11-63], p=0.22). OS was similar for patients with PD-L1 positive and negative tumors in a multivariate model accounting for ECOG 1+ status (HR 0.93, 95% CI 0.70-1.24). Likewise, cancer-related OS was similar in the multivariate model (HR 0.87, 95% CI 0.63-1.21). Median OS was 33.3 months (95% CI 29.8-47.3) for patients with PD-L1 positive tumors and 28.5 months (95% CI 21.6-39.7) for patients with PD-L1 negative tumors. Likewise, median cancer-related OS was 20.4 months (95% CI 16.0-25.4) for patients with PD-L1 positive tumors and 20.6 months (95% CI 15.4-28.5) for patients with PD-L1 negative tumors. The table depicts common causes of death. Conclusions: In this real-world study of VHA patients on durvalumab for stage III UR-NSCLC, OS and cancer-related OS were similar for those with PD-L1 positive and negative tumors. Cause of death. Cause of death PDL1+, n=221 PDL1-, n=119 P-value Disease progression 84% 77% 0.19 Bleeding event 4% 3% 0.75 Cardiac event 14% 14% 1.00 Infection/sepsis 19% 6% 0.01 Intracranial embolism/hemorrhage 2% 3% 0.67 Multiorgan failure 1% 2% 0.56 Thromboembolic event 2% 0% 0.30 Unknown 3% 4% 0.73 Other 9% 19% 0.03

e18793 Background: Clinical trials and real-world studies have documented the benefit of durvalumab consolidation therapy in patients with stage III unresectable non-small cell lung cancer (UR-NSCLC) following chemoradiotherapy (CRT). Methods: Patients with stage III UR-NSCLC on durvalumab following CRT, at any Veterans Health Administration (VHA) facility from 1/1/17 to 6/30/20, were included. Patients were followed from their durvalumab initiation date through the earliest of their last VHA visit, loss to follow up, death, or end of study. Electronic health record and chart review data were retrospectively collected to determine baseline characteristics, number of durvalumab doses, length of follow-up, and overall survival (OS). Chi-square and Wilcoxon rank sum tests were used to compare baseline characteristics for patients with PD-L1 negative (<1%) and positive (1% or greater) tumors. Kaplan-Meier curves were constructed for OS. Cox proportional hazards regression, with controls for divergent baseline characteristics, were used to compare OS between the two groups. Results: Of 935 available patients, 340 had PD-L1 results: 221 had PD-L1 positive (65%) and 119 had PD-L1 negative (35%) tumors. The two groups of patients were largely similar with respect to baseline characteristics, including age, sex at birth, White race, smoking status, marital status, Charlson Comorbidity Index, ECOG status, histology, stage, EGFR, and RAS mutations (Table). Patients with PD-L1 positive and negative tumors had a similar median (interquartile range [IQR]) number of durvalumab doses (15 [6-24] vs 12 [5-23], p=0.38) and length of follow-up (31.5 [12.1-43.5] vs 28.7 [12.2-40.6] months, p=0.43). Kaplan-Meier survival curves were similar for patients with PD-L1 positive and negative tumors: estimated 12-month OS rates (95% CI) of 84% (78%-88%) vs 89% (81%-93%) and 24-month OS rates of 72% (65%-78%) vs 76% (67%-84%); median OS was not reached in patients with PD-L1 positive tumors and was 47.0 months for the PD-L1 negative group. Cox Proportional Hazards regression analysis also demonstrated similar OS for both groups (p=0.64). Conclusions: In this real-world study of a subpopulation of VHA patients with stage III UR-NSCLC and known PD-L1 levels, survival benefits with durvalumab were consistent with those reported in the PACIFIC trial, despite having worse baseline prognostic factors. Patients with PD-L1 positive and negative tumors experienced similar overall survival, including 12- and 24-month OS rates.[Table: see text]

8526 Background: Evidence from the PACIFIC study and real-world data highlight the benefit of durvalumab in patients with stage III unresectable non-small cell lung cancer (UR-NSCLC). However, limited literature exists regarding disparities in durvalumab treatment patterns such as treatment initiation delays (TID), treatment interruptions (TI), number of doses, duration of therapy (DOT), adverse effects (AEs), and treatment discontinuation (TD) in minority populations. Methods: Patients with stage III UR-NSCLC and a self-reported racial identity of Black or White treated with durvalumab following chemoradiotherapy (CRT) at any Veterans Health Administration (VHA) facility from January 1, 2017 to June 30, 2020 were included. Patients were followed from their date of durvalumab initiation through the earliest of their last VHA visit, loss to follow up, death, or end of the study; therefore, all patients had the opportunity to be treated for 12 months. Patients were excluded if durvalumab therapy was ongoing at the end of the study. Patient charts were retrospectively reviewed for baseline characteristics and durvalumab treatment patterns including TID (>42 days from end of CRT to durvalumab start), TI (>28 days between doses), number of doses, DOT, AEs, and TD. Nominal variables were compared using chi-square/Fisher’s exact tests. Continuous variables were compared using Student’s t-tests/Wilcoxon Rank Sum tests. Results: Among 924 patients, Black patients were younger than White patients (median age 67 years [IQR, 63-71] vs. 70 years [IQR, 65-73]; p<0.01), more likely to be current smokers (54% vs. 45%; p=0.03), with more chronic liver disease (22% vs. 9%; p<0.01), but less COPD (63% vs. 72%; p=0.01). Black patients experienced more TI (25% vs. 18%; p=0.03) but TID, number of doses, DOT, and TD were similar between the groups. Black patients were less likely to have an immune-related AE (irAE) (28% vs. 36%; p=0.03) (and less pneumonitis (7% vs. 14%; p<0.01)). Toxicity was the reason for TD in 12% of Black patients vs. 20% of White patients (p=0.01), with no other significant (α < 0.05) differences in reported reasons for TID, TI, or TD between the groups. Conclusions: In this real-world study, Black patients experienced similar TID, number of doses, and DOT as White patients. Black patients were less likely to experience an irAE (including pneumonitis) but experienced more TI; TD were similar but more likely to be from toxicity for White patients. Future research is needed to validate these findings.[Table: see text]

8554 Background: The PD-1/PD-L1 pathway is a mechanism of immune evasion and disruption of this pathway with immune checkpoint inhibitors (ICIs) has shown clinical benefit in multiple malignancies. Based on results from the PACIFIC trial, durvalumab is approved as consolidation therapy in patients (pts) with stage III unresectable non-small cell lung cancer (UR-NSCLC) without progression following concurrent chemoradiotherapy (cCRT). Durvalumab has been used extensively in Veterans Health Administration (VHA) facilities, providing an opportunity to evaluate durvalumab treatment interruptions (TI), treatment discontinuations (TD), and the reasons for these on a national scale. Methods: Patients with stage III UR-NSCLC receiving durvalumab consolidation immunotherapy at the VHA between January 1, 2017 and June 30, 2020 with a minimum follow up for 12 months were included using ICD-10, HCPCS, and J codes and followed from their durvalumab start date through the earliest of last VHA visit, loss to follow up, death, or end of study (excluded if durvalumab therapy was ongoing at the end of the study, because the full treatment course could not be determined). TI were defined as durvalumab infusions separated by >28 days. Reasons for TI and TD are presented descriptively. Durations are reported using medians and interquartile ranges (IQR). Results: 935 pts were included (median age = 69 years; 95% males; 96% current or former smokers; 70% with COPD; histologies [squamous (50%), non-squamous (43%), other/missing (7%)]; and 77% with carboplatin-paclitaxel as their platinum-based CRT). Durvalumab TI were experienced by 19% of pts (median [IQR] number of TI = 1 [1-1], median [IQR] TI duration = 53 days [39-90]). The main reasons for TI were toxicity (8%) and social reasons (3%) (Table). The median duration of treatment (DoT) with durvalumab (TI included) was 9.0 months (IQR 2.9-11.8). Durvalumab TD occurred in 59% of pts. Top reasons for discontinuation across all 935 pts included disease progression (24%) and toxicity (18%) (Table). Conclusions: In this real world analysis of national VHA data, durvalumab DoT was similar to PACIFIC despite having a patient population with worse prognostic factors (e.g. more males, squamous, COPD) with 8% of VHA pts experiencing TI and 18% TD due to toxicity. Patients could benefit from additional efforts to prevent, identify, and manage toxicities in the UR-NSCLC population [Table: see text]

8556 Background: Durvalumab is an FDA-approved immunotherapy for the treatment of adults with UnResectable stage III non-small cell lung cancer (UR-NSCLC) without disease progression following concurrent chemoradiotherapy (CRT). There are limited real-world data regarding Durvalumab treatment initiation delays (TIDs) and reasons for them in the UR-NSCLC population. Methods: Patients with stage III UR-NSCLC receiving consolidation Durvalumab at the Veterans Health Administration (VHA) between January 1, 2017 and June 30, 2020 were selected from the VHA database using ICD-10, HCPCS, and J codes. All had the opportunity to be treated for 12 months and were followed from Durvalumab initiation through the earliest of their last VHA visit, loss to follow up, death, or the study’s end (and excluded if Durvalumab therapy was ongoing at the study’s end). Trained data abstractors determined the occurrence and reasons for TIDs (> 6 weeks from end of CRT to initiation of Durvalumab as in the PACIFIC trial) by chart review. Results: 935 patients were eligible for analysis (median age = 69 years; 95% males; 16% with ECOG performance status >1). TIDs occurred in 39% of the patients (Table). Durvalumab was initiated 61 days (median) from the end of CRT in TID patients vs. 31 days for those without TIDs. There were no significant (α<0.05) differences in age, race, smoking status, histology, or ECOG performance status and no comorbidity differences (except in patients with a history of cerebrovascular accident, for whom TIDs were more likely) between the TID/No-TID patients. Patients without timely post-CRT scans were more likely to have a TID. Of the 367 patients who experienced TIDs, 200 had documented reasons for the delay, consisting of other (not categorized) (28.5%), physician preference (20%), toxicity (11%), patient preference (10.5%), decline in performance status (10%), system issues (9.5%), social reasons (9%), and progression (0.5%). Conclusions: This is one of the largest retrospective cohort studies reporting real-world data in patients with UR-NSCLC receiving Durvalumab. TIDs were associated with increased time to post-CRT scans. This potential issue can be improved with care coordination and involvement of cancer navigators. Additional studies are needed to assess the impact of TIDs on survival outcomes.[Table: see text]

Marketed (bosentan, ambrisentan) and discontinued (sitaxsentan, CI‐1034) endothelin receptor antagonists were examined in the human micropatterned hepatocyte co‐culture (MPCC) model HepatoPac®. Differences across hepatocellular health (cellular adenosine triphosphate/glutathione content), function (urea production/albumin secretion) and taurocholic acid transport (biliary clearance/excretion index) were compared using amiodarone and ciclosporin A as positive controls. Ambrisentan had the weakest potency in all six endpoints, while sitaxsentan, bosentan and CI‐1034 had more potent effects on hepatobiliary transport than health/function endpoints. Normalization to clinical Cmax gave the following relative rank order of safety based on margins for each endpoint: ambrisentan ≥ CI‐1034 ~ bosentan > sitaxsentan. These data suggested impaired hepatobiliary disposition might contribute to a more prominent role in liver injury associated within sensitive human populations exposed to these compounds than direct hepatocellular toxicity. Rat, dog and monkey MPCCs also showed greater sensitivity potential to disrupted hepatobiliary disposition compared with hepatocellular health/functional endpoints. Drug metabolism competency was exhibited across all species. In vivo, rats and dogs appear more resistant to transaminase elevations and/or histological evidence of liver injury caused by these mechanisms even at exceedingly high systemic exposures relative to sensitive humans. Rats and dogs are resistant to hepatobiliary toxicants due to physiological differences in bile composition/handling. Although traditional animal testing provides adequate safety coverage for advancement of novel pharmaceuticals into clinical trials, supplemental assays employing human MPCCs may strengthen weight‐of‐evidence predictions for sensitive human populations. Proving the predictive value of this single impact assessment model in advance of clinical trial information for human liver injury risk is needed across more pharmaceuticals.

There is currently no in vitro technique for assessing plume geometry of nasal sprays under airflow conditions. However, a majority of FDA approved nasal products recommend that patients inhale during actuation. Therefore, a reproducible in vitro test that measures plume angles under physiologically relevant inhalation flow rates would be useful. The purpose of this study was to adapt the recently described Plume Induction Port Evaluator (PIPE) apparatus for nasal sprays under flow and correlate these with nasal cast deposition patterns. Mass Median Plume Angles (MMPAs) of four nasal spray formulations with increasing viscosities were determined using the PIPE apparatus in the absence and presence of airflow. MMPAs were then correlated to drug deposition within 3D printed nasal casts using airflow. We evaluated different inhalation instructions obtained from the package insert of nasal products. MMPAs significantly reduced (narrower angles) when using flow for the three formulations with the lowest viscosities. An increase in the turbinate deposition was observed in the nasal casts when just one of the nostrils was closed during inhalation, except by the highest viscosity formulation. The turbinate deposition numerically correlated with changes in the plume angles observed using PIPE.

Typically, concentration-response curves are based upon nominal inducer concentrations for in-vitro-to-in-vivo extrapolation of CYP3A4 induction. The limitation of this practice is that it assumes the hepatocyte culture model is a static system. We assessed whether correcting for: 1) changes in perpetrator concentration in the induction medium during the incubation period, 2) perpetrator binding to proteins in the induction medium, and 3) nonspecific binding of perpetrator can improve the accuracy of CYP3A4 induction predictions. Of the seven compounds used in this evaluation, significant parent loss and nonspecific binding were observed for rifampicin (29.3-38.3%), pioglitazone (64.3-78.6%), and rosiglitazone (57.1-75.5%). As a result, the free measured EC50 values (EC50u) of pioglitazone, rosiglitazone, and rifampicin were significantly lower than the nominal EC50 values. In general, the accuracy of the induction predictions, using multiple static models, improved when corrections were made for measured medium concentrations, medium protein binding, and nonspecific binding of the perpetrator, as evidenced by 18-29% reductions in the root mean square error. The relative induction score model performed better than the basic static and mechanistic static models, resulting in lower prediction error and no false-positive or false-negative predictions. However, even when the EC50u value was used, the induction prediction for bosentan, which is a substrate of organic anion transporter proteins, was overpredicted by approximately 2-fold. Accounting for the ratio of unbound intracellular concentrations to unbound medium concentrations (Kpuu,in vitro) (0.5-7.5) and the predicted multiple-dose Kpuu,in vivo (0.6) for bosentan resulted in induction predictions within 35% of the observed interaction.

Previously we assessed the inductive response of prototypical inducers in hepatocyte monocultures and the long-term coculture model HepatoPac using cryopreserved hepatocytes from the same donors. We noted that the rifampicin EC50 generated using the HepatoPac model corresponded better to the EC50 based on clinical data compared with data generated in the monoculture system. We postulated that there may be differences in the functioning of uptake transporters between the two systems that may have led to the EC50 difference. In this study, we characterized the functional activity of multiple uptake transporters in the two systems using cryopreserved hepatocytes from the same donors. Our data suggest that uptake transporter activity is higher in HepatoPac compared with the monoculture system. As a follow up to this study, we measured the intracellular concentrations of rifampicin and bosentan, which are known substrates of uptake transporters; we observed significantly higher intracellular concentrations of both compounds in HepatoPac relative to the monoculture system. This finding equated to lower cytochrome P450 isoform 3A4 (CYP3A4) EC50 values in the HepatoPac system compared with the monoculture system for both mRNA and activity. In parallel, no significant EC50 shift was observed for carbamazepine and phenytoin, which are not known to be substrates of uptake transporters. Our data suggest that next generation liver models such as HepatoPac may be a useful in vitro tool to quantitatively predict drug-drug interactions when it is known that the perpetrator is also a substrate of drug transporters.

Primary cultures of cryopreserved human hepatocytes have become the preferred model system to determine the potential of compounds to induce hepatic enzymes and transporters via nuclear receptor (NR) activation. Recently, it has been shown that cryopreserved hepatocytes retain viability, morphological integrity, and a more stable phenotype, including metabolic capacity, when maintained in micropatterned cocultures (MPCCs) with 3T3-J2 cells. However, no reports have been published demonstrating its potential as a surrogate for testing the NR activation potential of new compounds. The purpose of this study was to examine the maintenance and fidelity of key xenobiotic receptor pathways in human MPCCs, namely AhR, CAR, PXR, and PPARα. MPCCs from two separate batches of cryopreserved hepatocytes were treated with prototype NR activators and 17 compounds from the ToxCast and JRC datasets that are known to be hepatotoxic and/or activators of different NRs. Points of departure for chemical-induced changes in gene expression were calculated for NR-specific target genes after 24- and 72-hour treatments. Good concordance with induction of relevant target genes by the prototypical inducers was observed. Human MPCCs recapitulated outcomes between known activators and their respective receptor/targets, thus representing a reliable, robust, and reproducible surrogate model for the measurement of changes in NR-mediated gene expression.

Long-term coculture models of hepatocytes are promising tools to study drug transport, clearance, and hepatoxicity. In this report we compare the basal expression of drug disposition genes and the inductive response of prototypical inducers (rifampin, phenobarbital, phenytoin) in hepatocyte two-dimensional monocultures and the long-term coculture model (HepatoPac). All the inducers used in the study increased the expression and activity of CYP3A4, CYP2B6 and CYP2C enzymes in the HepatoPac cultures. The coculture model showed a consistent and higher induction of CYP2C enzymes compared with the monocultures. The EC50 of rifampin for CYP3A4 and CYP2C9 was up to 10-fold lower in HepatoPac than the monocultures. The EC50 of rifampin calculated from the clinical drug interaction studies correlated well with the EC50 observed in the HepatoPac cultures. Owing to the long-term stability of the HepatoPac cultures, we were able to directly measure a half-life (t1/2) for both CYP3A4 and CYP2B6 using the depletion kinetics of mRNA and functional activity. The t1/2 for CYP3A4 mRNA was 26 hours and that for the functional protein was 49 hours. The t1/2 of CYP2B6 was 38 hours (mRNA) and 68 hours (activity), which is longer than CYP3A4 and shows the differential turnover of these two proteins. This is the first study to our knowledge to report the turnover rate of CYP2B6 in human hepatocytes. The data presented here demonstrate that the HepatoPac cultures have the potential to be used in long-term culture to mimic complex clinical scenarios.

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.

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 = 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

Generating accurate in vitro intrinsic clearance data is an important aspect of predicting in vivo human clearance. Primary hepatocytes in suspension are routinely used to predict in vivo clearance; however, incubation times have typically been limited to 4-6 hours, which is not long enough to accurately evaluate the metabolic stability of slowly metabolized compounds. HepatoPac is a micropatterened hepatocytefibroblast coculture system that can be used for continuous incubations of up to 7 days. This study evaluated the ability of human HepatoPac to predict the in vivo clearance (CL) of 17 commercially available compounds with low to intermediate clearance (<12 ml/min/kg). In vitro half-life for disappearance of each compound was converted to hepatic clearance using the well stirred model, with and without correction for plasma protein binding. Hepatic CL, using three individual donors, was accurately predicted for 11 of 17 compounds (59%; predicted clearance within 2-fold of observed human in vivo clearance values). The accuracy of prediction increased to 82% (14 of 17 compounds) with an acceptance criterion defined as within 3-fold. When considering only low clearance compounds (<5 ml/min per kg), which represented 10 of the 17 compounds, the accuracy of prediction was 70% within 2-fold and 100% within 3-fold. In addition, the turnover of three slowly metabolized compounds (alprazolam, meloxicam, and tolbutamide) in HepatoPac was directly compared with turnover in suspended hepatocytes. The turnover of alprazolam and tolbutamide was approximately 2-fold greater using HepatoPac compared with suspended hepatocytes, which was roughly in line with the extrapolated values (correcting for the longer incubation time and lower cell number with HepatoPac). HepatoPac, but not suspended hepatocytes, demonstrated significant turnover of meloxicam. These results demonstrate the utility of HepatoPac for prediction of in vivo hepatic clearance, particularly with low clearance compounds.

The current landscape of in vitro models used to identify drug- or chemical-induced hepatotoxicity relies heavily on cell culture models consisting of HepG2, induced pluripotent stem cell-derived, or primary hepatocytes. While these in vitro models offer powerful approaches for predicting toxicity, each system has challenges, including variable metabolic capacity, brief ex vivo life span in culture, and adoption with standard automated microscopy high-content screening (HCS) systems to measure reproducibility data at the single-cell level. In this report we introduce a novel primary hepatocyte coculture model, HepatoPac™, as an alternative to current model systems for evaluation of in vitro hepatotoxicity in 96-well microtiter plate format examined by HCS. The coculture model consists of primary hepatocytes that are micropatterned to form a discrete microarchitecture or "hepatocyte islands" that are surrounded by supporting fibroblasts resulting in long-term viability and metabolic function of primary hepatocytes. Using multiple HCS image capture and image analysis strategies, we established methods to interrogate various morphometric parameters, such as size, shape, and intensity, at the island or single-cell level. We applied these approaches to identify subpopulations of both fibroblasts and hepatocytes that exhibited alterations in nuclear parameters, cell permeability, mitochondria function, and apoptosis using known reference control compounds and an eight-point dose curve. Subpopulation analysis with additional bioprobe sets can provide a powerful means of addressing differential cell and tissue susceptibilities during compound profiling. Our data show that the HepatoPac is amendable for HCS imaging applications and provides a unique approach for studying hepatotoxicity over prolonged periods of time.

Generating accurate in vitro intrinsic clearance data is an important aspect of predicting in vivo human clearance. Primary hepatocytes in suspension are routinely used to predict in vivo clearance; however, incubation times have typically been limited to 4–6 hours, which is not long enough to accurately evaluate the metabolic stability of slowly metabolized compounds. HepatoPac is a micropatterened hepatocyte-fibroblast coculture system that can be used for continuous incubations of up to 7 days. This study evaluated the ability of human HepatoPac to predict the in vivo clearance (CL) of 17 commercially available compounds with low to intermediate clearance (<12 ml/min per kg). In vitro half-life for disappearance of each compound was converted to hepatic clearance using the well stirred model, with and without correction for plasma protein binding. Hepatic CL, using three individual donors, was accurately predicted for 10 of 17 compounds (59%; predicted clearance within 2-fold of observed human in vivo clearance values). The accuracy of prediction increased to 76% (13 of 17 compounds) with an acceptance criterion defined as within 3-fold. When considering only low clearance compounds (<5 ml/min per kg), which represented 10 of the 17 compounds, the accuracy of prediction was 60% within 2-fold and 90% within 3-fold. In addition, the turnover of three slowly metabolized compounds (alprazolam, meloxicam, and tolbutamide) in HepatoPac was directly compared with turnover in suspended hepatocytes. The turnover of alprazolam and tolbutamide was approximately 2-fold greater using HepatoPac compared with suspended hepatocytes, which was roughly in line with the extrapolated values (correcting for the longer incubation time and lower cell number with HepatoPac). HepatoPac, but not suspended hepatocytes, demonstrated significant turnover of meloxicam. These results demonstrate the utility of HepatoPac for prediction of in vivo hepatic clearance, particularly with low clearance compounds.

ABSTRACT Primary hepatocytes display functional and structural instability in standard monoculture systems. We have previously developed a model in which primary hepatocytes are organized in domains of empirically optimized dimensions and surrounded by murine embryonic fibroblasts (HepatoPac™). Here, we assess the long‐term phenotype of freshly isolated and cryopreserved rat hepatocytes in a 96‐well HepatoPac format. The viability, cell polarity (actin microfilaments, bile canaliculi), and functions (albumin, urea, Phase I/II enzymes, transporters) of fresh and cryopreserved rat hepatocytes were retained in HepatoPac at similar levels for at least 4 weeks as opposed to rapidly declining over 5 days in collagen/Matrigel™ sandwich cultures. Pulse or continuous exposure of rat HepatoPac to GW‐7647, a selective agonist of PPARα, caused reproducible induction of CYP4A1 and 3‐hydroxy‐3‐methylglutaryl‐CoA synthase over 4 weeks. In conclusion, rat HepatoPac in a 96‐well format can be used for chronic dosing of highly functional hepatocytes and assessment of perturbed hepatocellular pathways. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:204‐212, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21469

Because drug-induced liver injury (DILI) remains a major reason for late-stage drug attrition, predictive assays are needed that can be deployed throughout the drug discovery process. Clinical DILI can be predicted with a sensitivity of ~50% and a false positive (FP) rate of ~5% using 24-h cultures of sandwich-cultured primary human hepatocytes and imaging of four cell injury endpoints (Xu et al., 2008). We hypothesized that long-term drug dosing in a functionally stable model of primary hepatocytes (micropatterned cocultures [MPCCs]) could provide for increased predictivity over short-term dosing paradigms. We used MPCCs with either primary human or rat hepatocytes to understand possible species differences along with standard endpoints (glutathione levels, ATP levels, albumin, and urea secretion) to test 45 drugs either known or not known to cause clinical DILI. Human MPCCs correctly detected 23 of 35 compounds known to cause DILI (65.7% sensitivity), with a FP rate of 10% for the 10 negative compounds tested. Rat MPCCs correctly detected 17 of 35 DILI compounds (48.6% sensitivity) and had a higher FP rate than human MPCCs (20 vs. 10%). For an additional 19 drugs with the most DILI concern, human MPCCs displayed a sensitivity of 100% when at least two hepatocyte donors were used for testing. Furthermore, MPCCs were able to detect relative clinical toxicities of structural drug analogs. In conclusion, MPCCs showed superiority over conventional short-term cultures for predictions of clinical DILI, and human MPCCs were more predictive for human liabilities than their rat counterparts.