Establishment of a hepatocyte-kupffer cell coculture model for assessment of proinflammatory cytokine effects on metabolizing enzymes and drug transporters.
Elevated levels of proinflammatory cytokines associated with infection and inflammation can modulate cytochrome P450 enzymes, leading to potential disease-drug interactions and altered small-molecule drug disposition. We established a human-derived hepatocyte-Kupffer cell (Hep:KC) coculture model to assess the indirect cytokine impact on hepatocytes through stimulation of KC-mediated cytokine release and compared this model with hepatocytes alone. Characterization of Hep:KC cocultures showed an inflammation response after treatment with lipopolysaccharide and interleukin (IL)-6 (indicated by secretion of various cytokines). Additionally, IL-6 exposure upregulated acute-phase proteins (C-reactive protein, alpha-1-acid glycoprotein, and serum amyloid A2) and downregulated CYP3A4. Compared with hepatocytes alone, Hep:KC cocultures showed enhanced IL-1β-mediated effects but less impact from both IL-2 and IL-23. Hep:KC cocultures treated with IL-1β exhibited a higher release of proinflammatory cytokines, an increased upregulation of acute-phase proteins, and a larger extent of metabolic enzyme and transporter suppression. IC50 values for IL-1β-mediated CYP3A4 suppression were lower in Hep:KC cocultures (98.0-144 pg/ml) compared with hepatocytes alone (IC50 > 5000 pg/ml). Cytochrome suppression was preventable by blocking IL-1β interaction with IL-1R1 using an antagonist cytokine or an anti-IL-1β antibody. Unlike IL-1β, IL-6-mediated effects were comparable between hepatocyte monocultures and Hep:KC cocultures. IL-2 and IL-23 caused a negligible inflammation response and a minimal inhibition of CYP3A4. In both hepatocyte monocultures and Hep:KC cocultures, IL-2RB and IL-23R were undetectable, whereas IL-6R and IL-1R1 levels were higher in Hep:KC cocultures. In summary, compared with hepatocyte monocultures, the Hep:KC coculture system is a more robust in vitro model for studying the impact of proinflammatory cytokines on metabolic enzymes.
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A) theresa.nguyen@merck.com.
Theresa V
TV
Nguyen
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A).
Okechukwu
O
Ukairo
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A).
Salman R
SR
Khetani
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A).
Michael
M
McVay
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A).
Chitra
C
Kanchagar
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A).
Wolfgang
W
Seghezzi
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A).
Gulesi
G
Ayanoglu
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A).
Onyi
O
Irrechukwu
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A).
Raymond
R
Evers
D016475:3T3 Cells; D000328:Adult; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002097:C-Reactive Protein / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D018920:Coculture Techniques / Q000379:methods; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D006023:Glycoproteins / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D007378:Interleukins / Q000378:metabolism*; D007728:Kupffer Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D008875:Middle Aged; D000685:Serum Amyloid A Protein / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology
D002352:Carrier Proteins; D006023:Glycoproteins; D007378:Interleukins; D000685:Serum Amyloid A Protein; D002097:C-Reactive Protein; D051544:Cytochrome P-450 CYP3A