Abstract

Drug–drug interaction (DDI) assessment of therapeutic peptides is an evolving area. The industry generally follows DDI guidelines for small molecules, but the translation of data generated with commonly used in vitro systems to in vivo is sparse. In the current study, we investigated the ability of advanced human hepatocyte in vitro systems, namely HepatoPac, spheroids, and Liver-on-a-chip, to assess potential changes in regulation of CYP1A2, CYP2B6, CYP3A4, SLCO1B1, and ABCC2 in the presence of selected therapeutic peptides, proteins, and small molecules. The peptide NN1177, a glucagon and GLP-1 receptor co-agonist, did not suppress mRNA expression or activity of CYP1A2, CYP2B6, and CYP3A4 in HepatoPac, spheroids, or Liver-on-a-chip; these findings were in contrast to the data obtained in sandwich cultured hepatocytes. No effect of NN1177 on SLCO1B1 and ABCC2 mRNA was observed in any of the complex systems. The induction magnitude differed across the systems (e.g., rifampicin induction of CYP3A4 mRNA ranged from 2.8-fold in spheroids to 81.2-fold in Liver-on-a-chip). Small molecules, obeticholic acid and abemaciclib, showed varying responses in HepatoPac, spheroids, and Liver-on-a-chip, indicating a need for EC50 determinations to fully assess translatability data. HepatoPac, the most extensively investigated in this study (3 donors), showed high potential to investigate DDIs associated with CYP regulation by therapeutic peptides. Spheroids and Liver-on-a-chip were only assessed in one hepatocyte donor and further evaluations are required to confirm their potential. This study establishes an excellent foundation toward the establishment of more clinically-relevant in vitro tools for evaluation of potential DDIs with therapeutic peptides.

SIGNIFICANT STATEMENT At present, there are no guidelines for drug–drug interaction (DDI) assessment of therapeutic peptides. Existing in vitro methods recommended for assessing small molecule DDIs do not appear to translate well for peptide drugs, complicating drug development for these moieties. Here, we establish evidence that complex cellular systems have potential to be used as more clinically-relevant tools for the in vitro DDI evaluation of therapeutic peptides.

Introduction

In vitro studies needed to assess potential drug–drug interactions (DDIs) for therapeutic peptides during drug development remain unclear with no specific guidance currently available from health authorities. In contrast, in vitro to in vivo extrapolation (IVIVE) for small molecule DDI assessments is well-established (European Medicines Agency 2012; United States Food and Drug Administration 2020; International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use 2023). Although peptide DDI assessment is not part of the regulatory requirement, many companies in the pharmaceutical industry follow the same procedures used for small molecules (Säll et al., 2023).

Evaluating potential perpetrator effects of therapeutic peptides on cytochrome P450 (CYP) enzymes and transporters expression/activity is important as several biologics have been reported to alter CYP expression/activity in vitro and/or in vivo (Cheung et al., 1996; Liddle et al., 1998; Iber et al., 2001; Jürgens et al., 2002; Woodcroft et al., 2002; Song and Chiang, 2006). For example, the protein fibroblast growth factor (FGF)-21 suppressed CYP3A4 expression in vitro (Woolsey et al., 2016); glucagon suppressed Cyp2c11 expression in rat hepatocytes in a concentration-dependent manner, with similar trends seen for CYP7A1 in human hepatocytes (Iber et al., 2001; Song and Chiang, 2006); insulin suppressed Cyp2e1 in rat hepatocytes (Woodcroft et al., 2002); and growth hormone (GH) induced multiple CYP enzymes in vitro, with a 910% increase in CYP3A4 mRNA in plated human hepatocytes (Liddle et al., 1998). However, treatment with GH has clinically indicated induction of CYPs in GH-deficient adults but in healthy men, GH only resulted in induction of CYP1A2 and not CYP3A4 (Cheung et al., 1996; Jürgens et al., 2002). Other than these, several proinflammatory cytokines, such as interleukin-6 (IL-6), are known to suppress CYP expression with clinical implications (Coutant et al., 2023; Dunvald et al., 2022; Gatti and Pea, 2022).

Recently, we published in vitro and clinical DDI assessments for the peptide NN1177, a linear 29-amino acid synthetic peptide (∼4.57 kDa) co-agonist for the glucagon and the glucagon-like peptide-1 receptors (Säll et al., 2022). This co-agonist suppressed CYP2B6 and CYP3A4 in a dose-dependent manner in vitro using freshly isolated primary human hepatocytes from three donors in a sandwich culture (a standard method recommended in DDI guidelines for small molecule drugs). CYP1A2 was also suppressed in one donor. However, a follow up clinical DDI trial showed no effect on the pharmacokinetic profile of midazolam (CYP3A4 substrate), while the area under the curve (AUC) of caffeine (CYP1A2 substrate) was decreased after NN1177 treatment (Säll et al., 2022). These findings highlight the disconnect between the clinical outcome and in vitro results obtained while following standard DDI guidelines for small molecule drugs and challenges in translation of in vitro CYP suppression data to in vivo. Similarly, the small molecule abemaciclib suppressed the mRNA for CYP3A4, CYP1A2, and CYP2B6, but no clinically relevant effects were observed (Turner et al., 2020). Moreover, obeticholic acid (OCA) suppressed CYP1A2 and CYP3A4 in vitro, but a clinical DDI trial with OCA showed no effect on midazolam (CYP3A4 substrate) (Edwards et al., 2017; Ishida et al., 2019), and while caffeine AUC did increase, the AUC of the CYP1A2-mediated major metabolite of caffeine did not (Edwards et al., 2017; Ishida et al., 2019).

Current evidence of IVIVE for CYP suppression data are limited and molecular mechanisms behind suppression are often unclear (European Medicines Agency 2012; Hariparsad et al., 2017; United States Food and Drug Administration 2020). In a survey performed by the IQ consortium, 16/17 respondents had observed downregulation in routine CYP induction studies. However, only three companies performed follow-up clinical studies of in vitro downregulation, and none showed any clinical significance (Hariparsad et al., 2017). The current in vitro systems used for the evaluation of DDIs, such as plated human hepatocytes in a monolayer or in a sandwich culture, clearly do not translate well for CYP suppression (Hariparsad et al., 2017). A potential reason for this lack of translation could be related to low CYP baseline expression levels in sandwich cultured human hepatocytes. Accordingly, human hepatocytes rapidly de-differentiate in sandwich culture, which could affect their potential to correctly detect changes in CYP regulation caused by indirect mechanisms, which are the expected effect of peptides/proteins (Bell et al., 2016). Maintaining native expression profiles is important for in vitro systems to provide data for informative IVIVE (Hariparsad et al., 2017). This was shown by Hendriks et al., where the small molecule AZD1208 was only identified correctly as an CYP3A4 inducer through a complex pathway in 3D spheroids of hepatocytes, but not in 2D sandwich culture of human hepatocytes (SCHH) (Hendriks et al., 2020).

Novel culture formats of human hepatocytes have emerged in recent years. The current study investigated three such systems: (1) HepatoPac, in which human hepatocytes are cultured in islands and then co-cultured with stromal cells (Khetani and Bhatia, 2008); (2) 3D spheroids, in which hepatocytes in ultra-low adherent plates self-assemble into spheroids (Bell et al., 2016); and (3) a microphysiological Liver-on-a-chip system, in which human hepatocytes are seeded in a scaffold with continuous perfusion of the cells (Rubiano et al., 2021; Docci et al., 2022). All these are long-term culture systems that retain hepatocyte identity and more stable CYP expression compared with SCHH throughout culture time (Khetani and Bhatia, 2008; Bell et al., 2016; Rubiano et al., 2021). Similar results are expected for transporters, but the experimental evidence is, so far, sparse.

In the current study, nine drugs (two small molecules, five peptides, and two proteins) were initially screened as perpetrators for their potential to regulate the expression CYP enzymes (CYP3A4, CYP1A2, and CYP2B6) and the transporters SLCO1B1 (OATP1B1) and ABCC2 (MRP2) in SCHH. Subsequently, peptide NN1177, protein rFGF-19, as well as small molecules OCA and abemaciclib, were selected for further evaluation in HepatoPac, spheroids, and Liver-on-a-chip. This work aims to systematically evaluate different complex and more physiologically relevant in vitro systems for their ability to investigate CYP and transporter regulation and improve the in vitro prediction of potential clinical DDI risks associated with therapeutic peptides.

Materials and Methods

Cryopreserved primary human hepatocytes were purchased from BioIVT (Westbury, NY) (donor QNT, BGF, and IVL). Donor demographic information (ethnicity, gender, and age) is listed in Supplemental Table 1. Fetal bovine serum, Glycogen RNA grade, Insulin-Transferrin-Selenium (ITS -G), dexamethasone Nunclon Sphera 96-well plates, ATP solution, hepatocyte culture media (Williams E medium w/o phenol red), plating supplement A kit, maintenance supplement B kit, and cryopreserved hepatocyte recovery medium (CHRM Medium) were purchased from Thermo Fisher Scientific (Waltham, MA). Transferrin, hydrocortisone, linoleic acid, D-(+)-Glucose (45%), penicillin-streptomycin, 30% bovine serum albumin (BSA) solution, isopropanolol, fatty acid-free BSA, BSA, sodium selenite, and human recombinant FGF-19 were purchased from Sigma-Aldrich (St. Louis, MO). GlutaMAX supplements were purchased from Thermo Fisher Scientific or from Life Technologies (Bleiswijk, Netherlands). Phosphate-buffered saline, Williams E medium without phenol red and glucose, Hepes, TRIzol reagent, PenStrep, and fetal bovine serum (FBS) were acquired from Life technologies. Sterile filtered DMSO was purchased from R&D systems. Acetic acid was purchased from Biosolve Chimie (Dieuze, France). BD Matrigel basement membrane matrix was purchased from Corning B.V. (Glendale, AZ). Forty-eight-well plate were either coated in-house with rat tail collagen type I (Corning B.V.) or purchased pre-coated from Corning B.V. Phenacetin, acetaminophen, testosterone, 6β-hydroxytestosterone, acetaminophen-(ring-d4), omeprazole, phenobarbital, rifampicin, flumazenil, and mercaptoethanol were purchased from Merck (Kenilworth, NJ). (±) Hydroxybupropion, (±)-hydroxybupropion-D6, and 6β-hydroxytestosterone-D3 solutions were obtained from Cerilliant (Merck). Phenobarbital was provided by the hospital pharmacy at Odense University Hospital, Odense. Bupropion hydrochloride were from either Merck or Toronto Research Chemical. Recombinant human IL-6 was purchased from Peprotech EC, Ltd. (London, UK). Human native glucagon, GLP-1, and glucagon co-agonist analog (NN1177), gastric inhibitory polypeptide (GIP) analog (NN0194), amylin analog (NN1213), FGF-21 analog (NN0119), and human GH (Norditropin (somatropin)) were procured internally at Novo Nordisk. OCA and abemaciclib mesylate were purchased from Abcam PLC (Cambridge, UK) and SCBT (Dallas, TX), respectively. RNA isolation kits, RNeasy mini kit and RNeasy micro kit, Qiazol, and RNase-Free DNase kit were procured from Qiagen (Hilden, Germany), and NucleoSpin RNA mini kit was from Macherey-Nagel (Duren, Germany). The iScript cDNA synthesis kit was procured from Bio-Rad (Hercules, CA) and the cDNA synthesis kit from Thermo Fisher Scientific. TaqMan fast advanced master mix and TaqMan gene expression assays (Supplemental Table 2) were procured from ThermoFisher scientific.

Test Compounds and Controls

The compounds OCA (0.1 and 1.0 µM), abemaciclib (0.5 and 5.0 µM), native glucagon (30 nM), NN1177 (100 and 1000 nM), amylin analog (0.6 and 6.0 µM), GIP analog (0.72 and 7.2 µM), recombinant FGF-19 (80 and 800 ng/ml), FGF-21 analog (0.236 and 2.36 µM), and GH (0.88 and 8.8 µg/ml) were initially tested in SCHH and OCA, abemaciclib, NN1177, and FGF-19 was afterward tested using HepatoPac, Liver-on-a-chip liver chip, and spheroids of hepatocytes at the same concentrations. The concentrations of the compounds tested were based on the expected or the reported clinically relevant total human plasma steady state maximum concentrations concentrations and 10-fold maximum concentration steady state (Rossi et al., 2014; US Food and Drug Administration 2015; Patnaik et al., 2016; Säll et al., 2022).

As positive controls, omeprazole (50 µM) was used for aryl hydrocarbon receptor-mediated induction of CYP1A2, phenobarbital (750 µM) constitutively active receptor-mediated induction of CYP2B6, and rifampicin (20 µM) was the positive control for pregnane X receptor-mediated induction of the CYP3A4. IL-6 at 10 ng/ml was used as a potential positive control for suppression of CYP3A4, CYP1A2 and CYP2B6. Relevant vehicle controls (glucagon, flumazenil, rifampicin, omeprazole, phenobarbital, amylin analog, GIP analog, OCA, and abemaciclib were dissolved in DMSO, GH and FGF-21 were in formulation buffer, FGF-19 in PBS with 0.1% BSA, IL-6 in 10 mM acetic acid, and NN1177 were in incubation media with 0.1% DMSO), were used for each compound and the concentration DMSO in the culture media did not exceed 0.1%.

Sandwich-Cultured Human Hepatocyte Preparation and Treatment

Hepatocytes (donor IVL and QNT) were thawed quickly and resuspended in CHRM media. Cell suspension was centrifuged at 95g for 15 minutes. Supernatant was discarded carefully, and cell pellet was resuspended in warm (37°C) hepatocyte plating medium. Before seeding, cell viability was estimated using the Trypan blue dye exclusion test. The human hepatocyte cell density was adjusted to 0.7 to 1 million viable cells per ml of the hepatocyte platting medium (William’s E medium w/o phenol red, 5% FBS, cocktail A (1% PenStrep, 4 µg/ml Insulin, 2 mM GlutaMAX, and 15 mM HEPES), and 1 µM dexamethasone). The hepatocytes were then seeded onto collagen I-coated 48-well plates at ∼0.2 million viable cells per well 200 µl of the medium. Cells were cultured in a 37°C incubator with 5% CO2 and 95% relative humidity. Four to six hours after seeding, plating efficiency was evaluated by visual inspection under microscope and cells were washed once with platting medium to clear dead and unattached cells. Afterward, cells were overlaid with cold maintenance (William’s E medium without phenol red, cocktail B (0.5% PenStrep, 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 µg/ml selenous acid, 1.25 mg/ml BSA, 5.35 µg/ml linoleic acid, 2 mM GlutaMAX, and 15 mM HEPES), and 0.1 µM dexamethasone) supplemented with 0.25 mg/ml Matrigel and the plate was placed in the incubator immediately. After overnight settling, the cells were treated daily for 3 days (total 72 hours) with test compounds or control compounds. Cell morphology was evaluated visually at each media change. For assessment of viability, a lactate dehydrogenase release assay was used for all conditions, following the procedure of the manufacturer (Roche kit supplied by Merck). After 72 hours of treatment, the activity of CYP1A2, CYP2B6, and CYP3A4 were measured using phenacetin, bupropion, and testosterone, respectively. The cells were initially treated with 100 µM phenacetin for 1 hour and then washed with medium, and then treated with a cocktail of 500 µM bupropion and 200 µM testosterone for 1 hour. After activity assay, the cells were lysed using the Qiagen RNeasy kit with the addition of beta-mercaptoethanol in the lysis buffer and stored at −80°C. The RNA isolation followed the protocol of the manufacturer.

HepatoPac

HepatoPac was used in a 24-well format with 21,000 hepatocytes per well, and donor BGF, IVL, and QNT were used, as well as a stromal-only plates as control plates. The cells were seeded at BioIVT (Westbury) and shipped 5–6 days after seeding. The cells arrived 2–4 days after shipment and the medium was change immediately upon arrival with the supplied maintenance medium. The cells were then kept in an incubator at 37°C with 10% CO2 and 95% relative humidity. The medium was changed again 1–2 days after arrival with maintenance medium and the experiment was started at day 12 after seeding of hepatocytes. The experiment was initiated by changing the media with the supplied application medium supplemented with 1.25 mg/ml BSA with an equilibration period of 1–2 hours before treatment. After the equilibration period, the cells were treated daily for 3 days (total 72 hours) with test compounds and control compounds (total volume: 400 µl). The cells were visually inspected upon each media change. After treatment, the activity of CYP1A2, CYP2B6, and CYP3A4 were measured using phenacetin, bupropion, and testosterone, respectively, in application medium supplied with 1.25 mg/ml BSA. The cells were initially treated with 100 µM phenacetin for 1 hour and then washed with medium, and then treated with a cocktail of 500 µM bupropion and 200 µM testosterone for 1 hour. Before the experiments, the linearity of the metabolite formation was ensured. The rate of metabolite formation was measured using liquid crystallography tandem mass spectrometry. After the activity assay, cells were lysed with 350 µl RLT buffer containing 10 µl/ml mercaptoethanol. Lysed cells were immediately kept at −80°C. RNA isolation using Qiagen RNeasy kit or Macherey-Nagel NucleoSpin RNA purification, and the procedure was according to the manufacture’s protocol.

3D Spheroids of Hepatocytes

Donor BGF was used for formation of hepatocyte 3D spheroids. Hepatocytes were thawed quickly and resuspended in CHRM media. Cell suspension was centrifuged at 100 g for 10 minutes. Before seeding, cell viability was estimated using Trypan blue dye. The cells were diluted with plating media medium (William’s E medium w/o phenol red, 5% FBS, cocktail A (1% PenStrep, 4 µg/ml Insulin, 2mM GlutaMAX, and 15 mM HEPES), and 1 µM dexamethasone), and for each well 1.500 cells in 100 ul were transferred to an ultra-low attachment 96-well plate. The plates were afterward shortly centrifuged for 2 minutes at 200 g and placed in a 37°C incubator with 5% CO2 and 95% relative humidity for 5 days to let the hepatocyte self-assemble into spheroids. At days 5,6, and 7 the media was changed with maintenance media (William’s E medium without phenol red, 1.25 mg/ml BSA, 0.1 µM dexamethasone, 100 units of penicillin, 0.1 mg/ml streptomycin, 1.72 µM insulin, 68.75 nM transferrin, 3.87 nM sodium selenite, and 2 mM GlutaMAX). At day 8 after cell seeding, the cells were treated daily for 3 days (total 72 hours) with test compounds and controls (total volume: 100 µl). Triplicate samples of 16 spheroids were treated for each RNA isolation, and triplicate samples of single spheroids were treated for the activity of CYP1A2 and of CYP2B6/CYP3A4, respectively. After treatment, 16 spheroids per replicate were collected and lysed using Qiazol and stored for later RNA isolation at −80°C. Simultaneous spheroids of each treatment were added to an activity solution of either 100 µM phenacetin or of 500 µM bupropion and 200 µM testosterone. The activity assays were then terminated after either 2, 8, and 24 hours, where 8 hours was used for further analysis (Supplemental Fig. 10). RNA isolation from spheroids was performed with phenol-chloroform extraction method (Qiazol) that included glycogen as RNA co-precipitant. The viability of the 3D spheroids was assessed after thawing, during cultivation and after treatment based on the quantification of ATP using the CellTiter-Glo Cell viability assay following the protocol of the manufacturer (Promega).

Liver-on-a-chip

Donor BGF was used for the PhysioMimix Liver-on-a-chip MPS-LC12 system (CN Bio Innovations). The day before cell seeding, cells were primed with seeding media prepared with William’s E media without glucose and phenol red, supplemented with 15 mM HEPES, 2 mM GlutaMax, 5.5 mM D-(+)-glucose, 200 pM insulin, 100 nM hydrocortisone, 5% FBS, and 1% PenStrep, using the designated priming program in an incubator at 37°C and with 5% CO2 and 95% relative humidity. The day after priming, hepatocytes were thawed quickly and resuspended in CHRM media. Cell suspension was centrifuged at 100 g for 10 minutes. Before seeding, the cell viability was estimated using a NucleoCounter (Chemometec). Viable hepatocytes at a volume of 0.6 million were added to each well with a total volume of 1600 µl and the seeding program was run. One day after seeding, the media was changes to maintenance media prepared with William’s E media without glucose and phenol red, supplemented with 15 mM HEPES, 2 mM GlutaMax, 5.5 mM D-(+)-glucose, 200 pM insulin, 100 nM hydrocortisone. 1.25 mg/ml fatty-acid free BSA, 6.25 ng/ml sodium selenite, 6.25 µg/ml transferrin, 20 µM linoleic acid, and 0.5% PenStrep. The media was subsequently changed on days 4, 6, and 8 after seeding using the designated media change program. At day 11 after cell seeding, the cells were treated daily for 3 days (total 72 hours) with test compounds and controls (total volume: 1600 µl). The amount of secreted human albumin present in the media was used to assess the viability and activity of the hepatocytes. After treatment of compounds for 72 hours the activity of CYP1A2, CYP2B6, and CYP3A4 were measured using phenacetin, bupropion, and testosterone, respectively, in maintenance medium. The cells were initially treated with 100 µM phenacetin for 1 hour, washed with medium, and then treated with a cocktail of 500 µM bupropion and 200 µM testosterone for 1 hour. Before the experiment, the linearity of the metabolite formation was ensured. The rate of metabolite formation was measured using liquid crystallography tandem mass spectrometry. After the activity assay, the cell-containing scaffolds were transferred to tubes with Trizol and prefilled with 0.1 mm zirconium beads (Merck). The cells were then homogenized/detached with a Bed Bug microtube homogenizer and the supernatant transferred to −80°C. RNA isolation was performed using Direct-zol RNA Microprep kit (Zymo Research) following the protocol of the manufacturer.

During each media change, media was stored at −80°C. After the end of the experiment the albumin content in the spent media was analyses. The amount of albumin present was measured using the Human Serum Albumin DuoSet ELISA and DuoSet ELISA Ancillary Reagent Kit 2, following the protocol of the manufacture (R&D Systems).

Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and Analysis

Isolated RNA from all the in vitro systems was reverse transcripted, and the prepared cDNA was used for qPCR. TaqMan Fast Universal PCR Master Mix was used for the qPCR reaction together with the TaqMan assays CYP1A2, CYP2B6, CYP3A4, CYP7A1, SLCO1B1, GAPDH, RPLP0, ABCC2, and HPRT1 (Supplemental Table 2). The PCR reaction was done using the QuantStudio 12K Flex system (Applied biosystems), and the data were analyzed using R-studio using the delta-delta ct method. For Hepatopac, the stromal-only cells did not give a ct value with the used TaqMan assays.

Liquid Crystallography Tandem Mass Spectrometry

For each activity assay, after the incubation of phenacetin, bupropion, and testosterone, samples were withdrawn and mixed with 1:1 ice-cold acetonitrile containing the internal standards acetaminophen-(ring-d4) or hydroxybupropion-D6 and 6β-Hydroxytestosterone-D3, respectively. The samples were then stored at -20°C until analysis. The analysis was performed with an ACQUITY UPLC HHS T3 1.8 mm 2.1 × 100 mm column at a column temperature of 40°C. A gradient mobile phase system was used consisting of eluent A (0.1% formic acid in water) and eluent B (0.1% formic acid in acetonitrile). The flow rate was 0.4 ml/min. Analysis was performed on a SYNAPT G2 (Waters) hybrid quadrupole-time-of-flight mass spectrometer with positive electrospray ionization mode using multiple reaction monitoring (Supplemental Table 3). The response of the formed metabolites acetaminophen (CYP1A2), hydroxybupropion (CYP2B6), and 6β-hydroxytestosterone (CYP3A4) were compared with a standard curve of each respective metabolite, and the respective controls were subtracted. For the Hepatopac samples, 90% of the cells were stromal cells; therefore, 90% of the formation of the metabolites in stromal-only cells were subtracted for each test compound and each metabolite, respectively.

ResultsInitial Screening in Sandwich Cultured Human Hepatocytes

Initial screening of 5 peptides (NN1177, native glucagon, GH, amylin analog, GIP analog), two proteins (recombinant FGF-19 and FGF-21-analog), and two small molecules (abemaciclib and OCA) was performed in SCHH (two donors). The regulation of CYP1A2, CYP2B6, and CYP3A4 by different compounds is shown in Fig. 1. Lactate dehydrogenase release was measured after treatment with each compound (Supplemental Fig. 1) and indicated no cytotoxicity. Control inducers (e.g., rifampicin for CYP3A4) noted for all enzymes/donors behaved as expected (Supplemental Fig. 2).

Fig. 1.

In vitro assessment of mRNA expression of CYP1A2, CYP2B6, and CYP3A4 in sandwich culture human hepatocytes treated with nine different compounds in two donor (Blue = IVL and red = QNT). Each sample is relative to the respective control and the ct-values are normalized to RPLP0 (ribosomal protein, large, P0) expression. Data are presented as boxplots with individual dots represent individual experiments (technical replicate) (n = 2–9). The horizontal solid line represents no change compared with controls samples and the dotted lines are the cut-off of 2- and 0.5-fold change.

No relevant or concentration-dependent effects on CYP1A2 expression were observed in SCHH treated with NN1177, glucagon, FGF-21 analog, amylin analog, GIP analog, or GH. The exception was FGF-19, which induced CYP1A2 by 2.8-fold at 800 ng/ml (donor IVL), whereas abemaciclib suppressed CYP1A2 at the highest concentration with a decrease of 0.26-fold. OCA also showed slight CYP1A2 suppression in donor IVL at the highest concentration (1.0 µM: 0.41-fold). No relevant effects on CYP2B6 expression were observed for FGF-19, glucagon, OCA, FGF-21 analog, amylin analog, GIP analog, or GH. NN1177 increased the expression of CYP2B6 by 3.4-fold in donor IVL at both concentrations investigated. Abemaciclib slightly suppressed CYP2B6 with 0.46- and 0.51-fold in donor QNT and IVL, respectively. OCA, NN1177, FGF-19, and glucagon attenuated expression of CYP3A4 below 0.5-fold in a concentration-dependent manner in both donors. GH slightly upregulated CYP3A4 for both tested concentrations, whereas no relevant effect was observed for abemaciclib, amylin analog, GIP analog, or FGF-21 analog. No relevant effects were observed on SLCO1B1 (OATP1B1) expression by any proteins or small molecules investigated (Supplemental Fig. 3). However, CYP7A1 expression was attenuated by OCA, glucagon, NN1177, and FGF-19 in a concentration-dependent manner (Supplemental Fig. 3), and the GIP analog suppressed CYP7A1 at both concentrations in donor QNT. Based on these results and available clinical DDI data (Edwards et al., 2017; Turner et al., 2020; Säll et al., 2022), four compounds (NN1177, FGF-19, OCA, and abemaciclib) were selected for further testing in more advanced in vitro systems.

General Evaluation of CYP Regulation in Complex In Vitro Systems

Induction and suppression potential were investigated in HepatoPac, 3D spheroids, and Liver-on-a-chip using known inducers of CYP1A2, CYP2B6, and CYP3A4 (omeprazole, phenobarbital, and rifampicin, receptively). IL-6 was used as a potential suppression control. HepatoPac studies were performed using 3 donors (IVL, QNT, and BGF), whereas spheroids and Liver-on-a-chip were performed in donor BGF (Fig. 2). Due to specific requirements (QNT could not form spheroids) of these advanced systems, it was only possible to have one common hepatocyte donor (BGF) in all three systems.

Fig. 2.

In vitro fold change in mRNA (white boxplot, left column) and activity (gray boxplot, right column) of CYP1A2, CYP2B6, and CYP3A4 after treatment with induction and suppression control compounds in HepatoPac, Liver-on-a-chip and spheroids (Donors: blue = IVL, red = QNT, and green = BGF). Each sample is relative to the respective control and the ct-values for the mRNA levels are normalized to HPRT1 expression. Activity of CYP1A2, CYP2B6, and CYP3A4 was measuring by the conversion of phenacetin to acetaminophen, bupropion to hydroxybupropion and testosterone to 6β-hydroxytestosterone, respectively. For spheroids the activity after 8 hours is depicted. The boxplot represents the overall results for all donors with each dot representing an individual experiment (technical replicate) (n = 3 – 6). Omeprazole = 50 µM, phenobarbital = 750 µM, rifampicin = 20 µM, and IL-6 = 10 ng/ml. The horizontal solid line represents no change compared with control samples and the dotted lines are the cut-off of 2- and 0.5-fold change.

In all the in vitro systems, the induction controls responded with an increase in mRNA and activity of the CYP enzyme induced. However, the magnitude of the response differed substantially across the different systems (Fig. 2). The induction of CYP1A2 by omeprazole showed the following rank order Liver-on-a-chip (40.6-fold) > spheroids (13.5-fold) > HepatoPac (8.9-fold) at the mRNA level, whereas at the activity level, the order was spheroids (18.7-fold) > Liver-on-a-chip (12.1-fold) > HepatoPac (3.6-fold). CYP2B6 was induced by phenobarbital at the mRNA level in the order HepatoPac (11.2-fold) > spheroids (5.0-fold) > Liver-on-a-chip (4.0-fold) and for activity was HepatoPac (9.2-fold) > Liver-on-a-chip (3.5-fold) > spheroids (2.5-fold). Rifampicin induced CYP3A4 in the order Liver-on-a-chip (81.2-fold mRNA and 20.0-fold activity) > HepatoPac (4.1-fold mRNA and 6.5-fold activity) > spheroids (2.8-fold mRNA and 2.0-fold activity). IL-6 did not have a relevant suppression effect (>0.5-fold) on the expression of CYP1A2, CYP2B6, or CYP3A4 at the mRNA or activity level in spheroids. Similar observations were made in the Liver-on-a-chip system where only CYP2B6 activity was 0.47-fold suppressed and CYP1A2 mRNA was 2.9-fold induced. In HepatoPac, IL-6 did suppress mRNA and activity of CYP1A2 and CYP2B6 in donor QNT (0.20-fold mRNA and 0.20-fold activity, and 0.13-fold mRNA and 0.27-fold activity, respectively), but not below 0.5-fold in donors IVL or BGF. For CYP3A4, IL-6 suppressed the mRNA in all donors (QNT: 0.07-fold, IVL: 0.42-fold, and BGF: 0.19-fold), but the activity was suppressed below 0.5-fold cut-off only in donor QNT (0.23-fold).

The absolute rate of the metabolite formation in each system, donor and CYP enzyme is depicted in Table 1. The absolute activity is in a similar range for all donors in HepatoPac with one exception: the donor BGF showed approximately half the activity for CYP3A4 and CYP1A2 compared with the other donors. The order of activity is the same for all donors: CYP3A4 > CYP1A2 > CYP2B6. A similar order is observed for spheroids with approximately the same magnitude of difference in activity between the CYP enzymes. For Liver-on-a-chip, a different order of activity was observed: CYP1A2 > CYP3A4 > CYP2B6. Comparing the activity across systems and assuming no loss of cells after seeding, then the metabolite formation rate is in order: HepatoPac > Spheroids > Liver-on-a-chip. The number of cells in Liver-on-a-chip was not determined, and it has previously been shown that the seeding efficiency is around 50% (Docci et al., 2022). Considering seeding efficiency, Liver-on-a-chip still had far less activity per million hepatocytes of all investigated CYP enzymes compared with the other systems. Generally, the fold change in activity and mRNA in the presence of NN1177, FGF-19, OCA, and abemaciclib correlated well for all in vitro systems investigated (Supplemental Fig. 9)

TABLE 1

Absolute activity of metabolite formation of probe substrates of CYP1A2, CYP2B6, and CYP3A4. The activity is normalized by the number of seeded cells. Cells seeded: HepatoPac ≈ 21,000, Spheroids ≈ 1,500, and Liver-on-a-chip ≈ 600,000. The metabolite formation is depicted as mean ± S.D. n = 3–6 technical replicates

The Effect of NN1177, FGF-19, Obeticholic Acid, and Abemaciclib on CYP1A2, CYP2B6, and CYP3A4 in Complex In Vitro Systems

The fold change in mRNA and activity data obtained in HepatoPac, spheroids, and Liver-on-a-chip is shown for CYP1A2 (Fig. 3), CYP2B6 (Fig. 4), and CYP3A4 (Fig. 5). Changes in mRNA expression of CYP7A1, SLCO1B1, and ABCC2 are shown in Supplemental Figs. 4, 5, and 6, respectively. Furthermore, the normalized base mRNA expression of the investigated genes is shown in Supplemental Fig. 11. The ATP content in spheroids after each treatment was in the range of the media control for all samples except 5.0 µM abemaciclib (Supplemental Fig. 7). For 5.0 µM abemaciclib, there was no ATP content detected in the spheroids and visual inspection showed poor condition. CYP1A2, CYP2B6, and CYP3A4 activity/mRNA were low/not detected following treatment of 5.0 µM abemaciclib in the spheroids. In HepatoPac, 5.0 µM abemaciclib resulted in a high number of large vacuoles (data not shown). The human albumin secreted by hepatocytes to the media was used as an integrity control for Liver-on-a-chip and showed that all treatments except abemaciclib and FGF-19 had a similar albumin production of approximately 1 µg/d after the last day of treatment (Supplemental Fig. 8). In the case of abemaciclib and FGF-19, a decrease in albumin concentration in the media was observed compared with the respective control.

Fig. 3.

In vitro fold change of mRNA (white boxplot, left column) and activity (gray boxplot, right column) of CYP1A2 after treatment with NN1177, FGF-19, obeticholic acid, and abemaciclib in HepatoPac, Liver-on-a-chip and spheroids (Donors: blue = IVL, red = QNT, and green = BGF). Each sample is relative to the respective control and the ct-values for the mRNA levels are normalized to HPRT1 expression. Activity of CYP1A2 was measuring by the conversion of phenacetin to acetaminophen. For spheroids the activity after 8 hours is depicted. The boxplot represents the overall results for all donors with each dot representing an individual experiment (technical replicate) (n = 2–6). The horizontal solid line represents no change compared with control samples and the dotted lines are the cut-off of 2- and 0.5-fold change. FGF: Fibroblast growth factor.

Fig. 4.

In vitro fold change of mRNA (white boxplot, left column) and activity (gray boxplot, right column) of CYP2B6 after treatment with NN1177, FGF-19, obeticholic acid, and abemaciclib in HepatoPac, Liver-on-a-chip and spheroids (Donors: blue = IVL, red = QNT, and green = BGF). Each sample is relative to the respective control and the ct-values for the mRNA levels are normalized to HPRT1 expression. Activity of CYP2B6 was measuring by the conversion of bupropion to hydroxybupropion. For spheroids the activity after 8 hours is depicted. The boxplot represents the overall results for all donors with each dot representing an individual experiment (technical replicate) (n = 2–6). The horizontal solid line represents no change compared with control samples and the dotted lines are the cut-off of 2- and 0.5-fold change. FGF: Fibroblast growth factor.

Fig. 5.

In vitro fold change of mRNA (white boxplot, left column) and activity (gray boxplot, right column) of CYP3A4 after treatment with NN1177, FGF-19, obeticholic acid, and abemaciclib in HepatoPac, Liver-on-a-chip and spheroids (Donors: blue = IVL, red = QNT, and green = BGF). Each sample is relative to the respective control and the ct-values for the mRNA levels are normalized to HPRT1 expression. Activity of CYP3A4 was measuring by the conversion of testosterone to 6β-hydroxytestosterone. For spheroids the activity after 8 hours is depicted. The boxplot represents the overall results for all donors with each dot representing an individual experiment (technical replicate) (n = 2–6). The horizontal solid line represents no change compared with control samples and the dotted lines are the cut-off of 2- and 0.5-fold change. FGF: Fibroblast growth factor.

OCA suppressed in a concentration-dependent manner the mRNA expression and activity of CYP1A2, CYP2B6, and CYP3A4 in HepatoPac (1.0 µM, mRNA: 0.35, 0.30, and 0.17-fold, respectively) and in Liver-on-a-chip (1.0 µM, mRNA: 0.53, 0.38, and 0.46-fold, respectively), but not in spheroids. Notably, the expression of CYP7A1 was highly suppressed by OCA in HepatoPac (0.1 and 1.0 µM: 0.04 and 0.02-fold, respectively) and in Liver-on-a-chip (0.1 and 1.0 µM: 0.41 and 0.03-fold, respectively), in contrast to spheroids (Supplemental Fig. 4). OCA showed no relevant effect on ABCC2 (MRP2) expression, and only a minor suppression of SLCO1B1 was observed in HepatoPac at 1.0 µM (0.46-fold).

Overall, abemaciclib caused no relevant effects at 0.5 µM at either the mRNA or activity level for CYP1A2, CYP2B6, CYP3A4, CYP7A1, SLC01B1, and ABCC2 in HepatoPac, spheroids, or Liver-on-a-chip, except for the activity of CYP2B6 (0.39-fold of activity) and mRNA of CYP1A2 (2.4-fold) and CYP3A4 (3.7-fold) in Liver-on-a-chip. In individual donors, QNT in HepatoPac, abemaciclib did suppress mRNA/activity below 0.5-fold for CYP1A2 (0.35-fold mRNA and 0.37-fold activity), CYP2B6 (0.43-fold mRNA), CYP3A4 (0.33-fold mRNA), SLCO1B1 (0.37-fold mRNA), and CYP7A1 (0.26-fold mRNA) (also mRNA in donor BGF of 0.39-fold), and the activity in donor BGF was suppressed for CYP2B6 (0.35-fold) and CYP3A4 (0.37-fold). Five micromolars of abemaciclib caused more than 0.5-fold suppression of both mRNA and activity of all CYPs in HepatoPac, except in donor BGF in HepatoPac with 0.54-fold. In the case of CYP1A2 and CYP2B6, no activity was observed in the spheroids at this high concentration of abemaciclib. This finding agrees with the observed formation of vacuoles in HepatoPac and absence of ATP content in these spheroids. CYPs in Liver-on-a-chip were not suppressed by this high concentration of abemaciclib, except the activity of CYP2B6 (0.43-fold), and induction was found for CYP1A2 (3.0-fold) and CYP3A4 (4.74-fold) mRNA.

The mRNA expression and activity of CYP1A2, CYP2B6, and CYP3A4 were not affected by NN1177 in spheroids and HepatoPac, except for donor QNT in HepatoPac, where the expression of CYP2B6 mRNA at 1000 nM was changed 0.36-fold. No suppression was observed in Liver-on-a-chip, but CYP2B6 activity was induced 2.17-fold at 100 nM, and CYP3A4 was also induced at 100 nM (mRNA 2.82-fold) and at 1000 nM (mRNA = 3.80-fold and activity = 2.16-fold). CYP7A1 mRNA was slightly suppressed only in spheroids at 1000 nM (0.39-fold), but no relevant effects were seen in any of the in vitro systems on SLCO1B1 or ABCC2 mRNA expression.

FGF-19 did not have any relevant effect on the mRNA expression or activity of any CYP enzymes investigated in spheroids, except for CYP7A1 (0.40-fold at 800 ng/ml). In HepatoPac, 800 ng/ml FGF-19 suppressed the activity of CYP1A2 (0.28-fold) in donor QNT and the mRNA expression and activity in donor BGF (0.46 and 0.45-fold, respectively) and QNT (0.22 and 0.23-fold, respectively). The expression of CYP3A4 was also suppressed by 800 ng/ml in donor QNT (0.17-fold mRNA and 0.28-fold activity) and IVL (0.34-fold mRNA). in the case of Liver-on-a-chip, CYP1A2 and CYP3A4 mRNA expression was induced by FGF-19 by 2.58 and 2.37-fold, respectively. In contrast, CYP7A1 was suppressed by FGF-19 in a concentration-dependent manner in all in vitro systems with the largest effect seen HepatoPac (0.10-fold across all donors), followed by spheroids (0.40-fold), and Liver-on-a-chip (0.47-fold). No relevant effects of FGF-19 were observed on SLCO1B1 or ABCC2 in any of the advanced in vitro systems.

Discussion

In the current study, four in vitro systems were evaluated for their ability to investigate potential CYP/transporter regulation by therapeutic peptides. Nine compounds were initially investigated in SCHH; subsequently, one peptide (NN1177), one protein (FGF-19), and two small molecules (abemaciclib and OCA) were further investigated in HepatoPac, spheroid, and Liver-on-a-chip systems. HepatoPac, the most extensively investigated in this study (three donors), showed high potential to investigate DDIs associated with CYP regulation by therapeutic peptides. Spheroids and Liver-on-a-chip were only assessed in one hepatocyte donor and further evaluations are required to confirm their potential.

A recent study highlighted difficulties associated with predicting clinical DDIs for peptides such as NN1177. NN1177 suppressed multiple CYPs (CYP3A4, CYP2B6, CYP1A2) following clinically relevant exposure in freshly isolated human hepatocytes (Säll et al., 2022). However, pharmacokinetic profiles from a follow-up clinical DDI trial showed no indication of downregulation of CYP enzymes following NN1177 exposure (Säll et al., 2022). Here, we investigated four in vitro systems with a range of complexities and their ability to evaluate peptide DDI risks. NN117 suppressed CYP3A4 and CYP1A2 mRNA expression in SCHH, in line with data previously reported in freshly isolated hepatocytes (Säll et al., 2022), but CYP2B6 was not suppressed in this system. In more advanced cell systems (HepatoPac, spheroids, and Liver-on-a-chip), the mRNA expression and activity of CYP1A2, CYP2B6, and CYP3A4 were not suppressed by NN1177, with the exception that suppression of CYP2B6 was noted in one donor in HepatoPac. Less stable expression of enzymes in SCHH may rationalize these differences in findings between SCHH and the more complex systems (HepatoPac, spheroids, and Liver-on-a-chip). Given available clinical DDI data for NN1177, the absence of an effect on CYP3A4 would have been correctly predicted by HepatoPac and spheroids if performed before the clinical study, whereas SC