Use of Micropatterned Cocultures to Detect Compounds That Cause Drug-Induced Liver Injury in Humans

Abstract

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.

A testing strategy to predict risk for drug-induced liver injury in humans using high-content screen assays and the ‘rule-of-two’ model

“…Thus, a test was defined as positive when its harmless ratio was <100, otherwise it was considered as negative. Subsequently, the readout of the HCS assay was assigned as positive when at least one of the selected cellular parameters was positive; this so-called logical OR method has been widely adopted for in vitro testing Khetani et al 2013). …”

“…In vitro cytotoxicity assays are poorly predictive of human hepatotoxicity . Consequently, monitoring multiple and mechanistically relevant endpoints in HCS assays improves predictive performance (Khetani et al 2013). Unfortunately, the use of human hepatocyte cell cultures to screen multiple endpoints for prolonged periods of time proved to be difficult (i.e., limited viability of cryopreserved human hepatocytes and the cost of pluripotent stem cell derived liver cells) (Khetani et al 2013).…”

“…It is considered as a promising methodology in the drug discovery and early preclinical drug development phase. Several studies reported the utility of HCS for assessing the risk of liver liabilities caused by drugs or chemicals Xu et al 2008;Khetani et al 2013). For example, Xu et al (2008) used primary human hepatocytes and determined an approximately 50 % sensitivity and 95 % specificity for over 300 drugs/chemicals.…”

“…Notwithstanding, the reported sensitivities are relatively low and thus will inevitably increase the risk of drug failure in the subsequent development program. Some efforts such as extending the culture duration of hepatocytes were proposed (Khetani et al 2013); however, the throughput of drug testing decreased while the cost of in vitro screening increased.…”

Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells

“…[1][2][3][4] Several in vitro models comprising of microsomes, cell lines, primary hepatocytes, and liver slices have been developed to provide predictive information related to drug toxicity. [5][6][7][8][9][10] Primary hepatocytes are relatively easy to isolate and retain most of the important in vivo functions for several days in monolayer static culture, [11][12][13] and up to 2 weeks or longer when cocultured with stromal cell support, [14][15][16] under collagen gel or on micropatterned surfaces, [15][16][17] and continue to be the most studied for drug screening purposes in various formats. 9,10,18 A major advance in primary hepatocyte culture is the use of a collagen gel sandwich, wherein primary hepatocytes are cultured between two layers of type I collagen gel for extended time periods.…”