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

Abstract Some immunomodulatory agents stimulate the release of cytokines capable of suppressing P450 enzymes and potentially affecting pharmacokinetics of coadministered medications. Cytokines released in response to an immunomodulator in the blood ex vivo can be used to screen for the potential for drug‐drug interactions. Tilsotolimod, an investigational agonist of Toll‐like receptor 9, stimulated the release of macrophage chemoattractant protein‐1 (MCP‐1), macrophage inflammatory protein‐1α (MIP‐1α), and interferon‐α2a (INF‐α2a) in blood obtained from healthy donors. Although tilsotolimod did not directly affect CYP1A2, CYP2B6, or CYP3A4 expression or activity, the cytokines stimulated by the drug reduced CYP1A2 and CYP2B6 enzyme activities in cultured human hepatocytes. This study sought to identify which cytokines were responsible for tilsotolimod's indirect effects on P450 enzymes in vitro. A 72‐h treatment with recombinant human chemokines MCP‐1 and MIP‐1α did not alter CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP3A4, or signal transducer and activator of transcription 1 (STAT1) mRNA expression or CYP1A2, CYP2B6, or CYP3A4/5 enzyme activity in cocultures of human hepatocytes and Kupffer cells. INF‐α2a, at 2.5 ng/mL but not at the lower concentrations applied to the cells, increased CYP1A2 and STAT1 mRNA by 2.4‐ and 5.2‐fold, respectively, and reduced CYP2B6 enzyme activity to 46% of control. This study established that INF‐α2a, but not MCP‐1 or MIP‐1α, mediated tilsotolimod effects on CYP1A2 and CYP2B6 expression in human hepatocytes.

the cytokines stimulated by the drug reduced CYP1A2 and CYP2B6 enzyme activities in cultured human hepatocytes. This study sought to identify which cytokines were responsible for tilsotolimod's indirect effects on P450 enzymes in vitro. A 72-h treatment with recombinant human chemokines MCP-1 and MIP-1α did not alter CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP3A4, or signal transducer and activator of transcription 1 (STAT1) mRNA expression or CYP1A2, CYP2B6, or CYP3A4/5 enzyme activity in cocultures of human hepatocytes and Kupffer cells. INF-α2a, at 2.5 ng/mL but not at the lower concentrations applied to the cells, increased CYP1A2 and STAT1 mRNA by 2.4-and 5.2-fold, respectively, and reduced CYP2B6 enzyme activity to 46% of control. This study established that INF-α2a, but not MCP-1 or MIP-1α, mediated tilsotolimod effects on CYP1A2 and CYP2B6 expression in human hepatocytes.

K E Y W O R D S
chemokine, CYP enzymes, cytokine, interferon-alpha

| INTRODUC TI ON
Some immunomodulatory drugs stimulate the innate immune system to release the cytokines which may change the expression of drug metabolizing enzymes. In drug development, regulation of mutiple pro-and anti-inflammatory cytokines by Toll-like receptors (TLR) has gained attention in parallel to targetting the therpapeutic potential of these receptors. 1,2 In a previous study, tilsotolimod, an agonist of TLR9 designed to enhance T-cell responses to tumor antigens, stimulated the release of cytokines in human blood and the effects of these cytokines on P450 enzymes in human hepatocytes treated for 72 hours were measured in vitro. 3 Tarantino and co-authors (2018) demonstrated that the treatment of blood with tilsotolimod (10 µg/mL) resulted in significant elevation of interferon gamma-induced protein-10 (IP-10) and macrophage chemoattractant protein-1 (MCP-1), while a higher concentration (100 µg/ mL) significantly increased macrophage inflammatory protein-1α (MIP-1α), tumor necrosis factor-α (TNF-α), and interferon-α2a (INF-α2a) over control. The cytokines contained in plasma isolated from whole blood treated with tilsotolimod (100 µg/mL) reduced CYP1A2 and CYP2B6 enzyme activity to 47% and 13% of control, respectively, in human hepatocytes. In the same study, bacterial lipopolysaccharide (LPS), an agonist of TLR4, significantly elevated IP-10, MIP-1α, and pro-inflammatory IL-2, IL-6, IL-12p70, and TNF-α but not MCP-1 or INF-α2a. MCP-1 and IFN-α2a were increased 7.2-and 1500-fold, respectively, in the tilsotolimod-treated plasma over the LPS-treated plasma. The LPS-treated plasma suppressed the CYP1A2 and CYP2B6 enzymatic activity by 79% and 84%, respectively. 3 The aim of this study was to establish which of the tilsotolimod-stimulated cytokines were responsible for the suppres-

| Cell culture and treatments, mRNA and enzyme activity analysis
The cell culture procedures and treatments as well as the analysis of mRNA and enzyme activities were conducted as recently described. 5 Demographic data of liver donors, two men and one woman, are provided in Table 1. The modified Chee's medium was supplemented with 10% normal pooled human plasma in order to replicate conditions of the initial in vitro evaluation of tilsotolimod. Hepatocytes were treated with MCP-1 (2, 10 or 50 ng/mL), MIP-1α (0.4, 2 or 10 ng/mL), and INF-α2a (0.1, 0.5, or 2.5 ng/mL) with dosing solutions prepared fresh daily for each of 3 days of treatments. These concentrations were 0.2-, 1-, or 5-fold the concentration found in plasma stimulated with tilsotolimod (100 µg/mL). 3 The levels of STAT1 mRNA were evaluated only for treatments with IL-6, INF-α2a, and phenobarbital. Representative cultures were examined daily with light microscopy for signs of treatment toxicity such as cell membrane deterioration, swollen nuclei, or increased number of cytoplasmic vacuoles.
Differences between treatment and control groups were analyzed with one-tailed t test (SigmaPlot ™ 12.5, Systat Software, Inc).

| Effects of MCP-1, MIP-1α, or INF-α2a on P450 mRNA expression in human hepatocytes
For brevity, only the average reduction to less than 50% or the increase of at least 2-fold over the medium control in cultured cells from three donors were reported.

| D ISCUSS I ON
In this study, recombinant INF-α2a induced CYP1A2 mRNA 2.4fold in cultured hepatocytes, consistent with the 1.5-fold increase F I G U R E 1 Effects of IL-6 and INF-α2a on CYP1A2, CYP2B6, and CYP3A4 mRNA expression and enzyme activity in culture of primary human hepatocytes. Hepatocyte-Kupffer cell cocultures (N = 3, experiment in cells from each donor was conducted once) were incubated for 72 hour with medium alone, medium containing phenobarbital (750 µmol/L), IL-6 (2, 10 or 50 ng/mL), or INF-α2a (0.1, 0.5 or 2.5 ng/mL). The mRNA levels of each P450 were quantitated by qPCR with normalization to glyceraldehyde 3-phosphate dehydrogenase mRNA and to the mRNA levels of the P450 in the medium control cultures. The enzymatic activity of the P450 enzymes were determined as described in Materials and Methods. Data were normalized to medium control cultures values set to equal 1. Patterned bars indicate observations of mRNA or enzyme activity levels below 50% of the control or observations of enzyme activity above 2-fold of the control. Asterisk (*) indicates difference from the control with onetailed t test, P < .05 (SigmaPlot™ 12.5, Systat Software, Inc)  increase or a 50% reduction at concentration of the cytokine that was 5-fold higher than that found in the plasma from tilsotomodtreated blood.
The effects of INF-α2b, which is pharmacologically indistinguishable from its allelic variant INF-α2a, on P450 enzymes in cocultures of human primary hepatocytes and non-parenchymal liver cells were previously examined. 6 Chen and coauthors  The effects of MCP-1 or MIP-1α on P450 enzymes have not been reported, but since these chemokines are expected to be elevated by some drugs targeting TLRs, they will need to be considered from a drug safety perspective. 9 It is a limitation of this study that cytokine-stimulated differentiation of monocytes to macrophages could not be evaluated in vitro, as macrophages are a source of pro-inflammatory cytokines. 10 This study established that INF-α2a, in a dose-dependent manner, increased CYP1A2 mRNA and reduced CYP2B6 enzyme activity in human hepatocytes in vitro. We concluded that INF-α2a, but not chemokines MCP-1 or MIP-1α, was likely responsible for appreciable P450 changes observed in hepatocytes cultured with plasma from tilsotolimod-treated blood. It is possible that other cytokines modulated by tilsotolimod but not evaluated in this study, could also modify CYP enzymes mRNA expression or activity.

D I SCLOS U R E
All the authors are employees of Sekisui XenoTech LLC and do not have a conflict of interest related to preparation and publication of this research article.

AUTH O R CO NTR I B UTI O N S
Czerwiński participated in research design and performed data analysis. Green and Westland conducted the experiments. Czerwiński and Ogilvie wrote or contributed to the writing of the manuscript.