2014

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

Published:  17 October 2014

Maciej CzerwiƄski, Faraz Kazmi, Andrew Parkinson, and David B. Buckley

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.

Drugs as Victims and Perpetrators and the Pharmacokinetic Concept of Maximum Exposure

Published:  01 October 2014

Brian W. Ogilvie, Andrew Parkinson

This article describes the dramatic but predictable impact of two perpetrators on the disposition of a victim drug. For a single elimination pathway, victim potential can be quantified as fm (fractional metabolism by an enzyme) or fe (fractional elimination by a transporter) as follows:

mathml_0-1.gif

where AUCi and AUCui are the plasma AUC (area under the curve) values in the presence and absence of an inhibitor of the enzyme (or transporter) responsible for eliminating the victim drug, respectively, and where AUCEM and AUCPM are the plasma AUC values in genetically determined extensive metabolizers (EMs) and poor metabolizers, respectively. For a drug that is cleared by two parallel pathways (routes A and B), victim potential can be similarly quantified:

mathml_1-1.gif

If routes A and B both had an fm (or fe) value of 0.49 (i.e., fmA = 0.49 and fmB = 0.49), then loss of only route A or only route B would result in a 1.96-fold increase in systemic exposure to the victim drug (1.96 = 1/(1 − 0.49)). However, loss of both route A and route B would result in a dramatic 50-fold increase in systemic exposure (50 = 1/(1 − 0.98)). This article provides several examples of perpetrator–perpetrator–victim interactions that demonstrate (i) the clinical relevance of this phenomenon, which is often referred to as maximum exposure; (ii) the large difference in the impact of an enzyme/transporter inhibitor in genetically determined EMs and PMs; and (iii) the potentially large impact of a perpetrator drug that inhibits multiple-drug-metabolizing enzymes or transporters.

In vitro inhibition of human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes by rose bengal: system-dependent effects on inhibitory potential

Published:  01 July 2014

Faraz Kazmi, Lois J. Haupt, Jennifer R. Horkman, Brian D. Smith, David B. Buckley, Eric A. Wachter, and Jamie M. Singer

1. Rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) is being developed for the treatment of cutaneous melanoma and hepatocellular carcinoma. Interestingly, rose bengal can generate singlet oxygen species upon exposure to light. 2. We evaluated rose bengal as an in vitro inhibitor of cytochrome P450 (CYP) or UDP-glucuronosyltransferase (UGT) enzymes in both human liver microsomes (HLM) and cryopreserved human hepatocytes (CHHs) under both yellow light and dark conditions. 3. Rose bengal directly inhibited CYP3A4/5 and UGT1A6 in HLM under yellow light with inhibitor concentration that causes 50% inhibition (IC50) values of 0.072 and 0.035 μM, respectively; whereas much less inhibition was observed in the dark with the IC50 values increasing 43- and 120-fold, respectively. To determine if a more physiologically-relevant test system could be protected from such an effect, rose bengal was evaluated as an inhibitor of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4/5 and UGT enzymes in CHH. All IC50 values were similar (64 ± 8 μM) and little to no effect of light on inhibitory potential was observed. 4. Given the IC50 values in CHH increased an order of magnitude compared to HLM and the atypical pharmacokinetics of the drug, the risk of rose bengal to cause clinically relevant drug-drug interactions is likely low, particularly when administered to cancer patients on an intermittent schedule.