Determination of Low Intrinsic Clearance Values using Primary Human Hepatocytes and the HepaRG ® Cell Line - A Comparison of Methods Petter Svanberg, Britta Bonn, Annika Janefeldt, Kajsa Kanebratt, Ia Hultman, Paul Courtney, Anshul Gupta, Ken Grime AstraZeneca R&D 20th International Symposium on Microsomes and Drug Oxidations, Stuttgart, Germany, 18-22 May, 2014 Background Oral drugs typically require effective half lives in the region of 10 – 20 h for once or twice daily dosing. For candidate drugs with low distribution volumes it is necessary to define intrinsic clearance (CLint) values of 0.1 - 1μL/min/million human hepatocytes (Grime et al., 2013). We wanted to compare in vitro systems that potentially can provide a solution to the problem of robustly defining low CLint values in human hepatocytes. Recently the HepatoPac™ Platform and a novel relay suspension method (Di et al., 2012) have shown promising results producing reliable low CLint values. XenoTech have made a well characterised platable pool of cryopreserved human hepatocytes (5 donors) commercially available, which makes plated heptocytes methods attractive to evaluate. Also of interest is the HepaRG ® human hepatoma cell line, since it offers stable expression of drug metabolising enzymes (DMEs) (Kanebratt et al., 2008, Aninat et al., 2006). We are evaluating all four methods but this poster focuses on HepaRG & plated primary hepatocytes since data from Hepatopac and Relay at present are inconclusive. Methods CLint for a set of DME substrates (Table 1) with known low turnover was determined in each in vitro system. Plated human hepatocytes The platable pooled human hepatocytes (lot 1310168) were purchased from XenoTech, thawed and plated according to vendors protocol for 4 hours. Culture media with 1μM substrate was added and repeated samples were withdrawn during incubations. Formation of 1OH-Midazolam and 4OH-Diclofenac and depletion of Naloxone were assessed to determine DME activity change over time. HepaRG ® Cryopreserved differentiated HepaRG ® cells were plated in 96-wells plates for 5 days prior to start of incubation for 24 hours. Substrate was added to the cells and media samples withdrawn from one well per timepoint. Substrates or metabolites were quantified using LCMSMS. Results and discussion Both HepaRG ® and plated hepatocytes produced CLints for low turnover substrates in the expected range (Table 1). The plated primary hepatocytes gave a linear decline for 2D6 and 2C19 substrates (Fig. 1). Moreover 80 minute metabolite formation and CLint-studies with specific DME- substrates (Diclofenac (2C9), Midazolam (3A4) and Naloxone (UGT2B7)) at different timepoints during culture shows sustained activity for 9 hours (Fig. 2). Diclofenac shows lower CLint in this evaluation than in literature (Table 1), we also couldn’t determine S-Warfarin CLint, which may indicate 2C9 is lower in activity in this batch or the ten hours incubation is not enough to produce a reliable CLint. The same holds true for Theophylline and CYP1A2. For in-house 3A4 substrates and Disopyramide we were able to determine Clints down to 0.2-0.4 uL/min/million cells (Table 1). Further plated experiments are needed to fully characterize DMEs in the plated system. CLint values correlate well between hepatocytes run in suspension and plated (Fig. 4) with the important difference that major DMEs seem to retain same activity up to 10 hours of plating giving possibilities to determine lower CLints. References Grime H. K, Barton P, McGinnity F D (2013). Mol. Pharmaceutics 10 1191-1206 Kanebratt K, Andersson B. T (2008). Drug metabolism and disposition 36:71444-1452 Aninat C, Piton A, Glaise D, Le Charpentier T, Langouet S, Morel F, Guguen-Guillouzo C, Guilouzo A (2006). Drug metabolism and disposition 34:1 75-83 Brown H, Griffin M, Houston J B (2007). Drug metabolism and disposition 35 293–301 McGinnity F D, Soars D M, Urbanowicz A R, Riley J R (2007). Drug metabolism and disposition 32 11 1247-1253 Di L, Trapa, P, Obach S, Atkinson K, Bi Y, Wolford A, Tan B, McDonald S T, Lai Y, Tremaine L M (2012). Drug metabolism and disposition 40:9 1860-1865 Lau Y Y, Sapidou E, Cui X, White E R, Cheng K C. (2002). Drug metabolism and disposition 30 12 1446-1454 0 5 10 15 20 25 30 35 40 45 0 3 9 18 25 42 Exp 1 Exp 2 Exp 3 Exp 4 Incubation time (h) Clint (uL/min/million cells) Figure 4. CLint for 7 of the compounds when tested in Suspension method (2 hr incubation) compared to CLints from the Plated method (10 hr incubation). CLints from Plated on the y-axis, CLints from suspension on the x-axis. . Intrinsic Clearance in Suspension versus Plated human hepatocytes 0 5 10 15 20 25 30 35 40 0 10 20 30 40 Clint (uL/min/million cells). Clint (uL/min/million cells). Diclofenac Midazolam Naloxone Verapamil Sildenafil AZ5 Diazepam Table 1. Clint values and compound information a Brown et al., 2007, b McGinnity et al., 2004, , c Di et al., 2012, d Lau et al., 2002. e uL/min/million cells n.v. = no value Compound Ion class Drug metabolising enzyme CLint e Literature data CLint e Plated hhepatocytes CLint e HepaRG Bufuralol Base 2D6 17 a 10.3 Diazepam Neutral 2C19>3A4 0.3 b , 3 c , 1.4 d 0.9 ± 0.2 0.2, 0.6 Diclofenac Acid 2C9 38 a , 47 b 5.8 Disopyramide Base 3A4 1 c 0.2 0.2 Metoprolol Base 2D6 7 b 2.1 ± 0.7 0.6 ± 0.3 Midazolam Neutral 3A4 7 d , 14 b 7.3 Naloxone Base UGT2B7 28 d , 216 b 27.0, 38.0 Sildenafil Base 3A4>2C9,2C19 5 d 9.7 S-Warfarin Neutral 2C9>3A4 1 a , 1 c , 1 d n.v. 0.1, 0.3 Theophylline Neutral 1A2 0.6 c , 1 d n.v. n.v. Verapamil Base 3A4 16 d , 18 b 14.8 AZ1 Neutral 3A4 0.9 ± 0.3 0.4, 0.4 AZ2 Neutral 1.2 ± 0.3 1.2 ± 0.2 AZ3 Neutral 3A4 0.4 ± 0.2 0.1, 0.2 AZ4 Base 0.9 ± 0.2 0.3 ± 0.2 AZ5 Acid 3A4 1.3 ± 0.2 0.5 ± 0.2 AZ8 Neutral 0.4, 0.5 0.4 Linear depletion of Metoprolol (2D6), Diazepam (2C19/3A4) and Naloxone in plated hepatocytes. Figure 1. Depletion profiles for CYP2D6-substrate Metoprolol (left), CYP2C19/3A4-substrate Diazepam ( middle) and Naloxone (right). 100000 150000 200000 250000 300000 350000 400000 0 2 4 6 8 10 12 Incubation time (h) Peak area 60000 70000 80000 90000 100000 110000 120000 0 2 4 6 8 10 12 Incubation time (h) Peak area 1 10 100 1000 10000 100000 1000000 0 2 4 6 8 10 12 Incubation time (h) Peak area Conclusions Major drug metabolising enzymes seem to have sustained activity in XenoTech´s platable pool of human hepatocytes up to 10 hours directly after plating. The results in this evaluation demonstrates the use of both plated hepatocytes and HepaRG® in order to determine CLint values below 1 uL/min/million cells. Figure 2. Formation of metabolites at different timepoints during culture period, 4OH-Diclofenac to the left, 1OH-Midazolam in the middle. Each bar represent metabolite-formation (peak-area) during 60 minutes incubation. Depletion of Naloxone as measured by CLint at different timepoints during culture, to the right. 0 100 200 300 400 500 600 700 0 3 9 18 25 42 Exp 1 Exp 2 Exp 3 Exp 4 Peak area Incubation time (h) 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 0 3 9 18 25 42 Exp 1 Exp 2 Exp 3 Exp 4 Peak area Incubation time (h) 0 10000 20000 30000 40000 50000 60000 0 2 4 6 8 10 12 1OH-Midazolam Incubation time (h) Peak area 0 1000 2000 3000 4000 5000 6000 0 2 4 6 8 10 12 4OH-Diclofenac Incubation time (h) Peak area Figure 3. Formation of metabolites during 10 hours. Left panel ;1-OH-Diclofenac, right panel; 1OH- Midazolam. Metabolite formation for Diclofenac (2C9) and Midazolam (3A4). Glucuronidation of Naloxone (UGT2B7) in plated hepatocytes. Culture time (h) Culture time (h) Culture time (h) Culture time (h) Culture time (h)