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First Annual Hepregen User Group Meeting

Presentations of Real-world HepatoPac™ Data & Round Table Discussions

Hepregen User Group Meeting
Friday, November 30, 2012
8:30am - 4:00pm
Hotel Marlowe
Cambridge, MA




"We would like to thank all our speakers and attendees for the huge success of Hepregen's First User Group Meeting. The meeting brought together users and prospective users of Hepregen's HepatoPac™ micro-liver platform and discussed practical applications in the fields of drug metabolism and toxicology. The quality of the presentations and round table discussions was fantastic. Due to the positive feedback we received, we anticipate holding regular User Group Meetings in the future."

— Mann Shoffner, Director of Marketing and Sales

» Request copies of User Group Presentations

User Group Agenda
8:30 – 9:00	Breakfast
9:00 – 9:15	Introductory Remarks
9:15-9:45	Keynote Address	Sangeeta Bhatia, Professor, MIT The History of HepatoPac Microliver Technology
9:45–12:00	DMPK Session
9:45-10:30		Scott Obach, Pfizer Use of Micropattern Human Hepatocyte Culture for Metabolite Identification and Clearance Predictions
10:30-10:45	Coffee Break
10:45-11:30		Diane Ramsden, Boehringer-Ingelheim Bridging In Vitro and In Vivo Metabolism: Applications of HepatoPac™
11:30-12:00	Roundtable Discussion
12:00-1:00	Lunch
1:00-3:15	Safety Session
1:00-1:45		Okey Ukairo, Hepregen Corporation A Micropatterned Culture with Primary Hepatocytes and Kupffer Macrophages for Studying Inflammation Mediated Drug-Drug Interactions
1:45-2:30		Salman Khetani, Colorado State University The Use of HepatoPac™ to Detect Compounds that Cause Drug Induced Liver Injury in Humans
2:30-2:45	Coffee Break
2:45-3:15	Roundtable Discussion
3:15-3:45	Hepregen Updates	New HepatoPac Species; Characterization Data and HepatoPac for In-house Use
3:45-4:00	Concluding Remarks

Presentation Abstracts

Sangeeta Bhatia
Title: The History of HepatoPac Microliver Technology

Scott Obach
Title: Use of Micro Patterned Cultured Human Hepatocytes for the Study of Low Clearance Compounds

Abstract: In modern drug discovery, we are frequently faced with candidate compounds that are slowly, but extensively metabolized. That is, the intrinsic clearance is low (i.e. low rate), but the percentage of clearance that is metabolic is high (i.e. high extent). It can be very challenging to predict the human CL for such compounds, because turnover is too slow to be reliably measured. Also, it can be challenging to generate metabolites for such compounds in vitro. We have used the HepatoPac micropatterned human hepatocyte culture system to address these challenges. This system was incubated for up to a week without a change in medium so that metabolism can be observable. For clearance prediction, we found that low CLint drugs had lability values that were scalable to in vivo clearance. Metabolite profiles observed were more reflective of in vivo human profiles than could be yielded by other in vitro systems (e.g. suspended hepatocytes).

Diane Ramsden
Title: Bridging In Vitro and In Vivo Metabolism: Applications of HepatoPac™

Abstract: HepatoPac™ was employed to evaluate the metabolic clearance of Faldaprevir, which is a hepatitis C serine protease inhibitor which effectively reduces viral load in patients. Preclinical studies indicated that Faldaprevir is a low clearance compound that did not exhibit any appreciable levels of circulating metabolites in animals or humans. As such, it was expected that metabolic clearance was a minor pathway. The human 14C-ADME study revealed that two mono-hydroxylated metabolites were the major excretory metabolites in feces constituting approximately 40% of the total administered drug. This unexpected finding prompted additional studies to determine why the minor change in structure (addition of 16 amu) produced chemical entities that were excreted rather than released into blood. HepatoPac™ expresses high activity for drug transporters and drug metabolizing enzymes which allowed sufficient incubation time to permit formation of these metabolites. Outcomes from these studies will be presented.

Investigations into the utility of HepatoPac™ for evaluating CYP450 protein-protein interactions were undertaken. By utilizing HepatoPac™, we were able to follow CYP2C9 activity in response to selective knockdown of CYP3A4 using siRNA and also following recovery of CYP3A4. CYP3A4 specific gene silencing resulted in a 70% decrease in CYP3A4 activity (midazolam hydroxylation) commensurate with a 67% increase in CYP2C9 activity (diclofenac hydroxylation). Conversely, once the siRNA was removed, both CYP3A4 and CYP2C9 activities returned to pre-knockdown levels. This work demonstrated that modulation of CYP3A4 levels can lead to appreciable increases in CYP2C9 activity in human hepatocytes, although the clinical consequence of these findings is not yet clear*. In conjunction with the siRNA knockdown of CYP3A4 activity and IL-6 mediated suppression of CYP3A4, linear regression models of log transformed percent activity remaining were obtained. Both models resulted in comparable slopes that were then used to calculate the kdeg value for CYP3A4. The overall theme of the talk will be to focus on how the unique attributes of HepatoPac® enabled these studies to be designed and performed.

*Funded in part by a grant from the National Institutes of Health (GM 086891) to T.S.T.

Okey Ukairo
Title: A Micropatterned Culture with Primary Hepatocytes and Kupffer Macrophages for Studying Inflammation Mediated Drug-Drug Interactions

Abstract: It is well established that inflammatory processes (Renton, 2001) affect the metabolism, distribution, and elimination of certain drugs potentially unfavorable pharmacological and toxicological consequences. This is especially important with biologics, which may induce inflammation responses. An in vitro model that mimics liver inflammation may provide better predictive data in preclinical testing. We have developed a micropatterned co-culture of primary hepatocytes and embryonic fibroblasts (HepatoPac™) that retains high levels of phenotypic functions such as drug metabolism enzymes for 4 weeks in vitro. Here, we supplement the HepatoPac platform with primary Kupffer macrophages in order to mimic one component of inflammation. Species-matched Kupffer cells were added to human and rat HepatoPac at multiple ratios (to mimic both the physiologically normal and inflamed states of the liver) to generate a tri-culture with primary hepatocytes and embryonic fibroblasts (HepatoPac- Kupffer cell co-culture). Recent evidence suggests that interaction between inflammatory stress and certain drugs may precipitate toxic responses. Here, we assess whether stimulation of HepatoPac-Kupffer cell co-cultures with LPS sensitizes the cultures to trovafloxacin (TVX) toxicity. Rat or human HepatoPac-Kupffer cell co-cultures were treated with increasing concentrations of TVX (+/- LPS) and assessed for changes in hepatic ATP content. TVX caused a concentration-dependent toxicity in the HepatoPac-Kupffer cell co-cultures which was potentiated by addition of 50ng/mL LPS to the cultures (TC50= 87.29 vs 27.77 Cmax for the rat platform and 68.24 vs 30.26 Cmax for the human platform). This effect was not observed with the non-toxic analog, levofloxacin. Treatment with pentoxifylline (an inhibitor of TNFa transcription) significantly decreased TVX/LPS- induced rat HepatoPac toxicity suggesting a synergistic effect between TNFa and trovafloxacin (TC50= 19.73 vs. 76.36 Cmax). In conclusion, rat or human HepatoPac- Kupffer cell co-cultures may be used to predict drug induced liver injury mediated by inflammatory stress.

Salman Khetani
Title: The Use of HepatoPac™ to Detect Compounds that Cause Drug Induced Liver Injury in Humans

Abstract: Since 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 rate of ~5% using 24 hour 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 (HepatoPac) could provide for increased predictivity over short-term dosing paradigms. We used HepatoPac 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 HepatoPac correctly detected 23 of 35 compounds known to cause DILI (65.7% sensitivity) with a false positive rate of 10% for the 10 negative compounds tested. Rat HepatoPac correctly detected 17 of 35 DILI compounds (48.6% sensitivity) and had a higher false positive rate than human HepatoPac (20% versus 10%). For an additional 19 drugs with the most DILI concern, human HepatoPac displayed a sensitivity of 100% when at least 2 hepatocyte donors were used for testing. Furthermore, HepatoPac were able to detect relative clinical toxicities of structural drug analogs. In conclusion, HepatoPac showed superiority over conventional short-term cultures for predictions of clinical DILI and human HepatoPac were more predictive for human liabilities than their rat counterparts.

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