Michelle Schaefer, Gerhard Schänzle, Daniel Bischoff, and Roderich D. Süssmuth.

Transcription

1 Upcyte Human Hepatocytes: a Potent In Vitro Tool for the Prediction of Hepatic Clearance of Metabolically Stable Compounds. Michelle Schaefer, Gerhard Schänzle, Daniel Bischoff, and Roderich D. Süssmuth. Drug Metabolism and Disposition SUPPLEMENTAL DATA Supplemental Table 1. Demographics, Supplier and Culture Characteristics of Primary Human Hepatocytes used for Correlation Analysis of P450 and UGT Enzyme Activity with Upcyte Human Hepatocytes No. Donor Supplier Race Age Sex Post Thaw Viability N Seeding Density x10 6 viable cells/well Culture Medium 1 Hu C 55 Male 90-96% WME 2 Hu C 51 Male 88% WME C 36 Female 94-96% WME C 52 Female 96% WME C 64 Female 93%, 94% WME 6 HC C 42 Female 81-86% XTC*/WME 7 HC C 43 Female 72%, 76% XTC*/WME 8 HC C 21 Male 94% XTC* 9 YJM 4 C 47 Female 92% HI # 10 CDP 4 C 58 Male 84% HI # C: Caucasian; N: Number of batches, equivalent to the maximum number of individual experiments performed for in situ determination of CYP450 enzyme activity; 1) LifeTechnologies GmbH (Darmstadt, Germany); 2) Corning/BD Gentest (Bedford, MA, USA); 3) Tebu-Bio GmbH (Offenbach, Germany)/XenoTech (Lenexa, KS, USA); 4) BioreclamationIVT GmbH (Frankfurt/Main, Germany/Baltimore, MD, USA) 1

2 ) CHRM medium (LifeTechnologies GmbH, Darmstadt, Germany) was used for thawing; Williams Medium E (WME) supplemented with 5% fetal bovine serum, 15 mm HEPES (ph 7.4), 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenous acid, 5.35 µg/ml linoleic acid, 1.25 mg/ml bovine serum albumin, 1 µm dexamethasone (DEX), 100 U/mL penicillin, 100 µg/ml streptomycin and 2 mm L-glutamine for plating (WME complete with 1 µm DEX); WME complete with 0.1 µm DEX for overlay and long-term culturing. * ) Hepatocyte Thawing Kit and Hepatocyte Re-Suspension Medium (XenoTec, Lenexa, KS, USA) were used for plating and XenoTec Hepatocyte Culture Medium for overlay and long-term culturing. ) WME complete with 0.1 µm DEX instead of XenoTec Hepatocyte Culture Medium was used for overlay and long-term culturing. ) High Viability CryoHepatocyte Recovery Kit (Corning/BD Gentest, Bedford, MA, USA) was used for thawing and plating; WME complete with 0.1 µm DEX for overlay and long-term culturing. #) InVitro Grow CP Medium (BioreclamationIVT GmbH, Frankfurt, Germany) was used for thawing and plating; InVitro Grow HI Medium (BioreclamationIVT GmbH) for overlay and log-term culturing. Supplemental Table 2. Assay conditions for the in situ determination of P450 isoform specific enzyme activity CYP450 isoform Probe Substrate Metabolite Substrate Conc. (µm) Incubation Time (min) ISTD 3A4 Testosterone 6ßOH-Testosterone 200 0, 15,30,45 [D 3 ]6ßOH-Testosterone 3A4/3A5 Midazolam 1`OH-Midazolam 25 0,5,10,20 [D 4 ]1OH-Midazolam 2C9 Diclofenac 4`OH-Diclofenac 100 0, 15,30,45 [ 13 C 6 ]4`OH-Diclofenac 2C19 S-Mephenytoin 4OH-Mephenytoin 100 0,30,60,90 [D 3 ]4OH-Mephenytoin 2C8 Amodiaquine Desethylamodiaquine 100 0, 15,30,45 [D 5 ]Desethylamodiaquine 2D6 Dextromethorphan Dextrorphan 50 0, 15,30,45 [D 3 ]Dextrorphan 1A2 Phenacetin Acetaminophen 50 0, 15,30,45 [D 4 ]Acetaminophen 2B6 Bupropion 2OH-Bupropion 75 0,30,60,90 [D 8 ]2OH-Bupropion UGT 7OH-Coumarin 7OH-Coumarin glucuronide 75 0,30,60,90 α-naphthylglucuronid SULT 7OH-Coumarin 7OH-Coumarin sulfate 75 0,30,60,90 α-naphthylglucuronid 2

3 Supplemental Table 3. HPLC/MS-MS conditions and parameters for P450 enzyme activity assays CYP450 isoform Metabolite HPLC Column Flow Rate (µl/min) Mass transition (m/z) Mode 3A4 6ßOH-Testosterone Pos A4/3A5 1`OH-Midazolam Pos C9 4`OH-Diclofenac Pos C19 4OH-Mephenytoin Pos C8 Desethylamodiaquine Pos D6 Dextrorphan Pos A2 Acetaminophen Pos B6 2OH-Bupropion Pos UGT 7OH-Coumarin glucuronide Neg SULT 7OH-Coumarin sulfate Neg Positive (Pos.); Negative (Neg.); Declustering Potential (DP); Collision Energy (CE) 1) YMC-Triart C18, YMC Europe, Dinslaken, Germany, 2) X-Terra MS C18, Waters, Eschborn, Germany DP (V) CE (V) Supplemental Table 4. MRM transitions and MS/MS parameters for reference drugs used in this study for determination of CL int by parent depletion assays. Reference Drug Mass transition (m/z) Mode DP (V) Alprazolam Positive Diazepam Positive Glimepiride Positive Meloxicam Positive Oxazepam Positive Prednisolone Positive Riluzole Positive Theophylline Positive Tolbutamide Positive Voriconazole Positive Warfarin Positive Atazanavir Positive Atomoxetine Positive Diclofenac Positive Flecainide Positive Imipramine Positive Lidocaine Positive Metoprolol Positive Midazolam Positive Ondansetron Positive Risperidone Positive CE (V) BI1052 (ISTD) Positive

4 Supplemental Table 5. In Vivo PK Data for 21 Reference Drugs Drug CL total F renal R B Nonrenal CL b fu p References ml/min/kg % ml/min/kg Alprazolam (1) , 2, 3, 4 Diazepam (3) 0.38 < , 5 Glimepiride (8) 0.62 < , 4, 5 Meloxicam (6) , 5 Oxazepam (11) 1.60 < , 8, 9 Prednisolone (2) , 7 Riluzole (10) 5.50 < , 6, 4 Theophylline (9) , 5 Tolbutamide (5) 0.20 < , 12, 2, 5 Voriconazole (4) 3.8 <2 n.a , 13, 5 Warfarin (7) < , 2 Atazanavir *) (13) n.a , 4 Atomoxetine #) (18) , 16, 4, 5 Diclofenac (14) 4.22 < , 4, 2 Flecainide (17) , 4, 5 Imipramine (20) 13.3 < , 7, 9 Lidocaine (15) , 19 Metoprolol (19) , 6 Midazolam (12) , 4, 2 Ondansetron (21) , 19, 4 Risperidone (16) , 21, 6, 22, 9 #) Pharmacokinetic parameters for extensive metabolizers, CL estimated from CL/F *) Nonrenal blood clearance estimated from total CL p after oral administration (Piliero et al., 2004) R B : Blood-to-plasma Ratio; fu p : fraction unbound in plasma; CL b : blood clearance (1) Smith et al., 1984; (2) Obach, 1999; (3) Greenblatt and Wright, 1993; (4) Thummel et al., 2011; (5) Chan et al., 2013; (6) Paixão et al., 2010; (7) Brown et al., 2007; (8) Sonne et al., 1988; (9) Hallifax et al., 2010; (10) Wokke, 1996; (11) Scott and Poffenbarger, 1979; (12) Stringer et al., 2008; (13) Purkins et al., 2003; (14) Piliero, 2004; (15) Simpson and Plosker, 2004; (16) Sauer et al., 2003; (17) Willis et al., 1979; (18) Sallee and Pollock, 1990; (19) Paixão et al., 2009; (20) Heizmann et al., 1983; (21) Heykants et al., 1994; (22) Mannens et al., 1994 Supplemental references for in vivo PK data (not cited in main manuscript): Greenblatt DJ and Wright CE (1993) Clinical pharmacokinetics of alprazolam. Therapeutic implications. Clin. Pharmacokinet. 24: Heizmann P, Eckert M, and Ziegler WH (1983) Pharmacokinetics and bioavailability of midazolam in man. Br. J. Clin. Pharmacol. 16 Suppl 1: 43S-49S. Heykants J, Huang ML, Mannens G, Meuldermans W, Snoeck E, Van BL, Van PA, and Woestenborghs R (1994) The pharmacokinetics of risperidone in humans: a summary. J. Clin. Psychiatry 55 Suppl: Mannens G, Meuldermans W, Snoeck E, and Heykants J (1994) Plasma protein binding of risperidone and its distribution in blood. Psychopharmacology (Berl) 114: Paixão P, Gouveia LF, and Morais JA (2009) Prediction of drug distribution within blood. Eur. J. Pharm. Sci. 36:

5 Paixão P, Gouveia LF, and Morais JA (2010) Prediction of the in vitro intrinsic clearance determined in suspensions of human hepatocytes by using artificial neural networks. Eur. J. Pharm. Sci. 39: Piliero PJ (2004) Atazanavir: A novel once-daily protease inhibitor. Drugs Today (Barc. ) 40: Purkins L, Wood N, Greenhalgh K, Eve MD, Oliver SD, and Nichols D (2003) The pharmacokinetics and safety of intravenous voriconazole - a novel wide-spectrum antifungal agent. Br. J. Clin. Pharmacol. 56 Suppl 1: 2-9. Sallee FR and Pollock BG (1990) Clinical pharmacokinetics of imipramine and desipramine. Clin. Pharmacokinet. 18: Sauer JM, Ponsler GD, Mattiuz EL, Long AJ, Witcher JW, Thomasson HR, and Desante KA (2003) Disposition and metabolic fate of atomoxetine hydrochloride: the role of CYP2D6 in human disposition and metabolism. Drug Metab Dispos. 31: Scott J and Poffenbarger PL (1979) Pharmacogenetics of tolbutamide metabolism in humans. Diabetes 28: Simpson D and Plosker GL (2004) Atomoxetine: a review of its use in adults with attention deficit hyperactivity disorder. Drugs 64: Smith RB, Kroboth PD, Vanderlugt JT, Phillips JP, and Juhl RP (1984) Pharmacokinetics and pharmacodynamics of alprazolam after oral and IV administration. Psychopharmacology (Berl) 84: Sonne J, Loft S, Døssing M, Vollmer-Larsen A, Olesen KL, Victor M, Andreasen F, and Andreasen PB (1988) Bioavailability and pharmacokinetics of oxazepam. Eur. J. Clin. Pharmacol. 35: Stringer R, Nicklin PL, and Houston JB (2008) Reliability of human cryopreserved hepatocytes and liver microsomes as in vitro systems to predict metabolic clearance. Xenobiotica 38: Willis JV, Kendall MJ, Flinn RM, Thornhill DP, and Welling PG (1979) The pharmacokinetics of diclofenac sodium following intravenous and oral administration. Eur. J. Clin. Pharmacol. 16: Wokke J (1996) Riluzole. Lancet 348:

6 Supplemental Table 6. Demographics and Donor-specific Batch Characteristics of Upcyte Human Hepatocytes Initial seeding (P0) Sub-culture (P1) Final seeding (P2) Donor Race Sex N Cell yield (10 6 /vial) Viability (%) No. of T150 flasks Cell yield (10 6 cells) Viability (%) No. of T150 flasks Cell yield (10 6 cells) Viability (%) C F *) *) *) C F *) 42.8 #) *) *) C F #) #) 95.3 *) not all cells used for further plating on T150 culture flasks #) low cell yield due to strong cell attachment and insufficient trypsination /re-suspension N: Number of batches and cell cultures; C: Caucasian; F: Female Summary of demographics and batch-related culture characteristics of the different donors of cryopreserved upcyte human hepatocytes (UHH) used in this study. Yield of viable cells and viability is reported for initial seeding (passage 0), expansion (passage 1) and final seeding to assay format (passage 2). Not all cells were used for further cultivation during the expansion phase. 6

7 Donor Donor Supplemental Table 7. Summary of P450, UGT and SULT Enzyme Activity and mrna Expression in UHH at Different Time Points Donor * ) Day 5 Day 7 Day 10 Day 14 mrna Activity mrna Activity mrna Activity mrna Activity 10³ fold ß-actin pmol/min/mio.cells 10³ fold ß-actin pmol/min/mio.cells 10³ fold ß-actin pmol/min/mio.cells 10³ fold ß-actin pmol/min/mio.cells CYP3A4 298 ± ± ± ± ± ± ± ± 19.2 CYP3A4/ ) ± ) ± ) ± 6.32 CYP2D ± ± ± ± ± ± ± ± 0.47 CYP2C ± ± ± ± ± ± ± ± 2.67 CYP2C ± ± ± ± ± ± ± ± 4.85 CYP2C ± ± ± ± ± 2.86 CYP2B6 988 ± ± ± ± ± 70.6 CYP1A ± ± ± ± ± ± ± ± 1.59 UGT 319 ± ± ± ± 66.3 SULT 16.1 ± ± ± ± 3.04 CYP3A ) ± ± ± ± ± ± ) ± 3.10 CYP3A4/ ) ± 1.01 CYP2D ) ± ± ± ± ± ± ) ± 0.01 CYP2C ) ± ± ± ± ± ± ) ± 1.26 CYP2C ) ± ± ± ± ± ± ) ± 0.57 CYP2C ± ± ) ± ± 3.85 CYP2B6 172 ± ± ) ± ± 8.77 CYP1A ) ± ± ± ± ± ± ) ± 0.57 UGT 159 ) ± ± ± ) ± 27.2 SULT 39.3 ) ± ± ± ) ± 1.66 CYP3A ± ± 3.19 CYP3A4/5 CYP2D ± ± 0.06 CYP2C ± ± 0.46 CYP2C ± ± 0.02 CYP2C ± 0.83 CYP2B6 117 ± 0.28 CYP1A ± ± 0.06 UGT 311 ± 1.96 SULT 9.45 ±

8 Supplemental Table 7. Summary of P450, UGT and SULT Enzyme Activity and mrna Expression in UHH at Different Time Points Total RNA was isolated and analyzed via qrt-pcr after the determination of in situ enzyme activity from the same batch of UHH by LC-MS/MS. Monitored test reactions were testosterone-6β-hydroxylation (CYP3A4), dextromethorphan-o-demethylation (CYP2D6), diclofenac-4'-hydroxylation (CYP2C9), S- mephenytoin-4'-hydroxylation (CYP2C19), phenacetin-o-deethylation (CYP1A2), bupropion-2- hydroxylation (CYP2B6), amodiaquine-n-deethylation (CYP2C8), midazolam-1 -hydroxylation (CYP3A4/3A5), 7-hydroxycoumarin-glucuronidation (UGT) and 7-hydroxycoumarin-sulfatation (SULT). In situ enzyme activity data are depicted as mean ± S.D. for at least three separate experiments (n 3), except for data specified ( ) mean ± S.D. for two separate experiments (n=2), or *) mean ± S.D. of duplicate determinations from a single experiment for donor 10-03). Each experiment was performed in triplicate or duplicate incubations per substrate (n=2 or n=3). The mrna data are shown as mean ± S.D. of at least two separate experiments (n=2) for donor and as mean ± S.D. of triplicate amplifications of a single experiment for donor and Each sample for mrna expression analysis represents a pool of four wells per culture condition or time point (n=4). Amplifications were performed in triplicate for each sample (n=3). 8

9 Supplement Table 8. In Vitro Predicted CL H Values for 21 Low and Intermediate Cleared Drugs using UHH from Three Donors (< 5 ml/min/kg) Low nonrenal CLb (5-15 ml/min/kg) Intermediate nonrenal CLb Substrate Predominant CYP450 isoform Observed In vivo CL nonrenal In vitro predicted CL H (N=3) (N=1) (N=1) Folderror In vitro predicted CL H Folderror In vitro predicted CL H * (ml/min/kg) (ml/min/kg) (ml/min/kg) (ml/min/kg) Alprazolam (1) 3A ± ± ± Prednisolone (2) 3A ± ± ± Diazepam (3) 2C ± ± ± Voriconazole (4) 2C ± ± n.d. Tolbutamide (5) 2C ± ± ± Meloxicam (6) 2C ± * ± n.d. Warfarin (7) 2C ± ± n.d. Glimepiride (8) 2C ± ± ± Theophylline (9) 1A n.d n.d n.d. Riluzole (10) 1A ± ± ± Oxazepam (11) UGT2B ± ± ± Midazolam (12) 3A ± ± ± Atazanavir (13) 3A ± * ± n.d. Diclofenac (14) 2C ± ± ± Lidocaine (15) 1A ± ± ± Risperidone (16) 2D ± ± ± Flecainide (17) 2D ± ± n.d. Atomoxetine (18) 2D ± ± ± Metoprolol (19) 2D ± ± n.d Imipramine (20) 2D6/1A ± n.t. n.t. Ondansetron (21) 2D ± n.t. n.t. n.t: not tested; n.d.: not determinable; CL H : hepatic clearance; CL b : blood clearance; * ) n=2 replicates; ) R² < 0.7 Folderror 9

10 Supplement Table 8. In Vitro Predicted CL H Values for 21 Low and Intermediate Cleared Drugs using UHH from Three Donors In vivo CL H was predicted for up to 21 reference drugs using sandwich-cultured UHH from donors , and Experiments were performed at day 7 in triplicate (n=3) or duplicate (n=2; donor 10-03) incubations per compound. Predicted CL H data obtained from UHH of donor represent mean ± S.D. from three individual experiments (n=3) for 19 compounds and mean ± S.D. from two individual experiments (n=2) for the two compounds ondansetron and imipramine. Data for donor and donor are derived from a single experiment (n=1). Prediction fold errors were calculated from the mean values and used to assess accuracy and bias. Depletion profiles showing a linear regression coefficient of R 2 < 0.7 are highlighted. Supplemental Table 9: Inter-Assay Variability of P450, UGT and SULT Enzyme Activity Determined at Day 7 in Sandwich-Cultured Upcyte Hepatocytes (151-03) Phase I/II Enzyme Mean absolute metabolite formation rate (pmol/min/mio.cells) S.D. Number of experiments %CV CYP3A CYP2C CYP2C CYP2D CYP1A UGT SULT S.D. : standard deviation; %CV: coefficient of variation 10

11 Supplement Figure 1: Morphology of Upcyte Hepatocytes at different stages in culture Donor Donor Donor A) B) C) D) Phase-contrast micrographs of upcyte human hepatocytes (UHH) from donors , and are shown during proliferation 6 h (A), 24 h (B) and 48 h post seeding (C) and at high density (24-well format), exemplary for sandwich culture (SC) configuration 24 h after seeding (D). Magnification 100 X. 11

12 Supplement Figure 2: Morphology of UHH in comparison to PHH UHH Donor PHH Donor Hu1601 Phase-contrast micrographs of sandwich-cultured upcyte human hepatocytes (UHH) from donors are shown in comparison to plated primary human hepatocytes (donor Hu1601). Similarities in morphology e.g. bile canalicular-like structures (white arrow) were observed by microscopic inspection. Magnification 400 X. 12

13 F o r m a t io n o f 6 ß - O H - T e s t o s t e r o n e (p m o l/m in /m io.c e lls ) F o r m a t io n o f 4 - O H - M e p h e n y t o in (p m o l/m in /m io.c e lls ) Supplement Figure 3: Impact of Culture Format on P450 Enzyme Activities D a y s 0 Comparison of P450 enzyme activities determined for UHH (donor ) in sandwich culture (filled circles and triangles) and monolayer culture (open circles and triangles). Circles represent the formation of 6ß-OH-testosterone (CYP3A4), triangles the formation of 4-OHmephenytoin (CYP2C19). Data for each time point represent mean ± S.D. of at least two and up to 5 individual experiments (n=2-5), each with duplicate or triplicate determinations in separate wells of the culture plate (n=2 or n=3) and as mean ± S.D. of duplicate or triplicate determination from a single experiment (n=1; for day 1, 3, 4 and 18). 13

14 % p a re n t re m a in in g L o g 2 F o ld c h a n g e Supplemental Figure 4: Time-dependent Changes in P450 mrna Expression in Upcyte Human Hepatocytes D 6 3 A 4 2 C 9 2 C A 2 2 B 6 Expression levels of CYP450 mrna at day 14 relative to day 7 were determined by qrt- PCR for sandwich cultured UHH of donor in two individual experiments (n=2; filled bars) and for UHH of donor in a single experiment (n=1; open bars). Data are shown as mean of triplicate amplification for each experiment; errors are calculated from the S.D. by the efficiency-corrected comparative Ct-method. Elevated levels of gene expression are indicated by values > 0 and down-regulated genes by values < 0. Dashed lines represent a 2- fold change in expression relative to day 7. Supplemental Figure 5: Impact of Culture Format on Metabolic Turnover M C S C ** 0 A L P T L B D Z M Alprazolam (ALP), tolbutamide (TLB) and diazepam (DZM) Comparison of the percentage of parent compound remaining at the end of the incubation period (96h for ALP and TLB; 48h for DZM) in monolayer cultures (MC) and sandwich cultures (SC) of upcyte human hepatocytes (donor ). Values represent mean ± S.D. of duplicate incubations (n=2) from a single experiment. 14

15 (m l/m in /k g ) (m l/m in /k g ) % p a re n t re m a in in g % p a re n t re m a in in g Supplemental Figure 6: Effect of Culture Time on Metabolic Turnover and CL H Prediction Accuracy A B * ** ** *** A L P T L B O X A 0 n.t A L P T L B O X A. n.t. not tested C In v it r o p r e d ic t e d C L H D In v itro p r e d ic te d C L H In v iv o C L n o n r e n a l (m l/m in /k g ) In v iv o C L n o n r e n a l (m l/m in /k g ) Alprazolam (1), diazepam (3), voriconazole (4), tolbutamide (5), meloxicam (6), warfarin (7), glimepiride (8), oxazepam (11), lidocaine (15), atomoxetine (18), metoprolol (19) (A, B) Metabolic turnover of alprazolam (ALP), tolbutamide (TLB) and oxazepam (OXA), expressed as percentage of parent compound remaining at the end of the incubation period (96h for donor ; 120h for donor ) was determined at day 7 (white bars), day 10 (light grey bars) and day 14 (dark grey bars) in sandwich-cultured UHH of donor (A) and donor (B). Values represent mean ± S.D. of duplicate (n=2) or triplicate incubations (n=3) from a single experiment. (C, D) Correlation between in vitro predicted and in vivo CL nonrenal depending on pre-culture time in sandwich cultures of UHH from donors (C) and (D). Experiments were performed at day 5 (squares), day 7 (circles), day 10 (triangles) and day 14 (diamonds). Data are shown exemplarily for 5 (donor ) and 11 reference drugs (donor ), respectively, as mean ± S.D. of duplicate (n=2) or triplicate (n=3) incubations from a single experiment. Solid lines represent conformity; dashed lines 2- and 3-fold ranges, respectively. Significance was determined by applying an unpaired t-test (* p<0.05, ** p<0.01, *** p < 0.001). 15

16 Supplemental Figure 7: Parent Depletion over Time Profiles Parent depletion profiles in sandwich-cultured UHH (donor ) are depicted exemplarily for alprazolam (ALP), diazepam (DZM) and glimepiride (GLP) together with respective control incubations without cells (w/o). Data are shown as mean ± S.D. of triplicate incubations (n=3) or as single control incubations (w/o cells) from a single experiment. Supplemental Figure 8: Chemical Structure of Internal Standard BI1052 N N Chiral N N N N Cl O Cl 16