Abstract

Like most infections and certain inflammatory diseases, some therapeutic proteins cause a cytokine-mediated suppression of hepatic drug-metabolizing enzymes, which may lead to pharmacokinetic interactions with small-molecule drugs. We propose a new in vitro method to evaluate the whole blood–mediated effects of therapeutic proteins on drug-metabolizing enzymes in human hepatocytes cocultured with Kupffer cells. The traditional method involves treating hepatocyte cocultures with the therapeutic protein, which detects hepatocyte- and macrophage-mediated suppression of cytochrome P450 (P450). The new method involves treating whole human blood with a therapeutic protein to stimulate the release of cytokines from peripheral blood mononuclear cells (PBMCs), after which plasma is prepared and added to the hepatocyte coculture to evaluate P450 enzyme expression. In this study, human blood was treated for 24 hours at 37°C with bacterial lipopolysaccharide (LPS) or ANC28.1, an antibody against human T-cell receptor CD28. Cytokines were measured in plasma by sandwich immunoassay with electrochemiluminescense detection. Treatment of human hepatocyte cocultures with LPS or with plasma from LPS-treated blood markedly reduced the expression of CYP1A2, CYP2B6, and CYP3A4. However, treatment of hepatocyte cocultures with ANC28.1 did not suppress P450 expression, but treatment with plasma from ANC28.1-treated blood suppressed CYP1A2, CYP2B6, and CYP3A4 activity and mRNA levels. The results demonstrated that applying plasma from human blood treated with a therapeutic protein to hepatocytes cocultured with Kupffer cells is a suitable method to identify those therapeutic proteins that suppress P450 expression by an indirect mechanism—namely, the release of cytokines from PBMCs.