Anti-CD28 Monoclonal Antibody–Stimulated Cytokines Released from Blood Suppress CYP1A2, CYP2B6, and CYP3A4 in Human Hepatocytes In Vitro

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.

Direct and cytokine‐mediated effects of albumin‐fused growth hormone, TV‐1106, on CYP enzyme expression in human hepatocytes in vitro

“…Indirect, cytokine-mediated, suppression of mRNA and reduction in enzyme activity of CYP1A2 and CYP3A4 by plasma from blood treated with LPS were expected. 18,24 In our study the cytokinemediated effect of albumin-fused rhGH is attenuated, likely due to reduced cytokine response to TV-1106 as compared to rhGH.…”

“…We developed a method for evaluation of biologics as direct and cytokine‐mediated determinants of hepatic biotransformation of xenobiotics. The assay is based on a treatment of whole blood with the biologic drug, followed by application of the resulted stimulated plasma to cultured hepatocytes . The CYPs selected for this study dominate xenobiotic metabolism in human liver and are suppressed by IL‐6 in vitro.…”

“…The assay is based on a treatment of whole blood with the biologic drug, followed by application of the resulted stimulated plasma to cultured hepatocytes. 18 The CYPs selected for this study dominate xenobiotic metabolism in human liver and are suppressed by IL-6 in vitro. We examined the ability of TV-1106 or rhGH to initiate cytokine release in whole blood and the effect of the drugs themselves or the stimulated plasma on CYPs 1A2, 2C19, and 3A4 expression in human hepatocytes.…”

Effects of monocyte chemoattractant protein‐1, macrophage inflammatory protein‐1α, and interferon‐α2a on P450 enzymes in human hepatocytes in vitro

“…However, it is possible that the CYP3A4 mRNA response to INF‐α2a was attenuated by the addition of normal plasma (10% v/v) to the cell culture medium in our study. In a previous study, we observed that the addition of plasma (10%, v/v) from blood incubated with saline (2% v/v, 24 hour) to cell culture medium for 72 hour suppressed CYP3A4 mRNA in hepatocytes from three donors …”

Kupffer cell inactivation by carbon monoxide bound to red blood cells preserves hepatic cytochrome P450 via anti-oxidant and anti-inflammatory effects exerted through the HMGB1/TLR-4 pathway during resuscitation from hemorrhagic shock

“…Recent in vitro and in vivo studies have clearly shown that Kupffer cell activation is associated with a decrease in hepatic CYP expression and activity [6][7][8]. In addition, ROS and cytokines appear to cause a decrease in CYP expression resulting from an enhancement in CYP degradation and synthesis [9,10].…”