De-Risking Bio-therapeutics for Possible Drug Interactions Using Cryopreserved Human Hepatocytes

Abstract

Inflammatory diseases such as rheumatoid arthritis and psoriasis are characterized by increases in circulating cytokines, which play an important role in modulation of the disease state. Several marketed bio-therapeutics target cytokines and act as effective treatment strategies. Previous in-vitro and in-vivo studies have suggested that cytokines may have both direct and indirect effects on drug metabolizing enzyme levels in the liver. Few studies have characterized models to evaluate the risk of potential drug interactions that might be mediated by changes in cytokine levels. In the present studies the potential of three cytokines (IL-2, IL-6 and TNF-α) to modulate gene expression and activity of the major human cytochrome P450 (CYP) enzymes (CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4) in cryopreserved human hepatocytes (CHH) was investigated. Significant decreases in the activity of all 6 CYP isoforms occurred in hepatocytes incubated with TNF-α or IL-6 (17-85%; and 22-76% of untreated control values, respectively). TNF-α down-regulated the gene expression of CYP1A2, 2D6 and 3A4 only, whereas IL-6 down-regulated gene expression of all of the tested CYP isoforms except 2D6. IL-2 had only mild effects on CYP activity and mRNA levels of examined isoforms. In CHH exposed to TNF-α, changes in CYP activity were not always paralleled by gene expression alterations for three of the examined CYP isoforms. These studies highlight several potential pitfalls in using isolated human hepatocytes for determination of drug interactions by bio-therapeutics including lack of correlation of mRNA and activity measurements for some CYP isoforms when using single time point determinations, and appropriateness of the model for indirect acting cytokine and cytokine modulators.

Interleukins-12 and -23 Do Not Alter Expression or Activity of Multiple Cytochrome P450 Enzymes in Cryopreserved Human Hepatocytes

“…Cytotoxicity of the various treatments was assessed by visual inspection during the culture process and after experimentation, as well as by using an in vitro lactic dehydrogenase toxicology assay kit (TOX-7; Sigma-Aldrich) as described previously (Dallas et al, 2012). None of the cytokines used in the present study showed appreciable cytotoxicity at 10 ng/ml.…”

“…Six separate lots of cryopreserved human hepatocytes were purchased from commercial suppliers (donor characteristics are listed in Supplemental Table 1). Cell lots were chosen by first verifying the ability of the donors to regulate P450s generally by measuring P450 induction following incubations with well characterized nuclear receptor activators, i.e., an assay integrity test, as described previously (Dallas et al, 2012). Cells were thawed, prepared, and incubated according to Dallas et al (2012).…”

“…Cell lots were chosen by first verifying the ability of the donors to regulate P450s generally by measuring P450 induction following incubations with well characterized nuclear receptor activators, i.e., an assay integrity test, as described previously (Dallas et al, 2012). Cells were thawed, prepared, and incubated according to Dallas et al (2012). Briefly, at 48 hours postseeding the cells were dosed with freshly spiked William's E media containing 10 ng/ml IL-6, IL-12, IL-23, or IL-12+23 in combination.…”

“…The effect of the various cytokines on the activity of the P450 isoforms screened was determined by monitoring formation of the metabolite of each P450-specific probe substrate in the absence and presence of the cytokines as previously described (Dallas et al, 2012). A 12-point standard curve and five quality control levels in triplicate were prepared by spiking blank Kreb's Henseleit buffer with a serially diluted mixture of metabolite reference standards in methanol and included in the analytical plate with the pooled postincubation samples.…”

Therapeutic Protein Drug–Drug Interactions: Navigating the Knowledge Gaps–Highlights from the 2012 AAPS NBC Roundtable and IQ Consortium/FDA Workshop

“…For example, in isolated human hepatocytes, although the suppression of CYP3A4 and CYP1A2 by IL-6 is well established, recent publications have revealed some key limitations in the predictive value of the model (6)(7)(8). The human hepatocyte model is now being applied to new cytokine targets such as the pro-inflammatory cytokine, IL-23.…”