Role of Toll-like receptor 4 in drug-drug interaction between paclitaxel and irinotecan in vitro

Highlights

• New mechanism of paclitaxel and Irinotecan drug-drug interaction is proposed.

• Paclitaxel activates Toll-like receptor 4 to alter drug metabolizing enzymes/transporters in hepatocytes.

• Activation of Toll-like receptor 4 by paclitaxel alters irinotecan pharmacokinetics in hepatocytes.

• Potential role of Toll-like receptors as novel modulators of drug-drug interactions

Abstract

The bacterial receptor, Toll-like receptor (TLR) 4 mediates inflammatory responses and has been linked to a broad array of diseases. TLR4 agonists are being explored as potential treatments for cancer and other diseases. We have previously shown that activation of TLR4 by lipopolysaccharide (LPS) leads to down-regulation of drug metabolizing enzymes/transporters (DMETs), and altered pharmacokinetics/pharmacodynamics (PK/PD) of drugs. These changes can increase the risk of drug-drug interactions (DDIs) in patients on multiple medications. Clinically, DDI was observed for combination chemotherapy of paclitaxel (TLR4 ligand) and irinotecan. To determine the role of TLR4 in DDI between paclitaxel and irinotecan in vitro, primary hepatocytes from TLR4-wild-type (WT) and mutant mice were pre-treated with paclitaxel, followed by irinotecan. Gene expression of DMETs was determined. Paclitaxel treatment increased the levels of irinotecan metabolites, SN-38 and SN-38 glucuronide (SN-38G) in TLR4-dependent manner. Paclitaxel-mediated induction of genes involved in irinotecan metabolism such as Cyp3a11 and Ugt1a1 was TLR4-dependent, while induction of the transporter Mrp2 was TLR4-independent. These novel findings demonstrate that paclitaxel can affect irinotecan metabolism by a TLR4-dependent mechanism. This provides a new perspective towards evaluation of marketed drugs according to their potential to exert DDIs in TLR4-dependent manner.

Introduction

Toll-like receptor 4 (TLR4) is a transmembrane receptor that detects components of microbial pathogens and plays a critical role in innate immunity (Marshak-Rothstein, 2006). Emerging genetic data also support the association of TLR4 with several diseases (Bochud et al., 2009, de Oliveira and Silva, 2012). Thus, activating or suppressing TLR4 provides access to a new generation of therapeutics (Manthey et al., 1993, Thoelen et al., 2001, Mullarkey et al., 2003, Ziakas et al., 2013).

Currently, TLR4 agonists are being explored extensively as immunomodulators and vaccine adjuvants for allergic diseases, cancers and infectious diseases (Hawkins et al., 2002, Evans et al., 2003, Lee et al., 2003, Przetak et al., 2003, Stover et al., 2004, Cluff et al., 2005, Krieg, 2006, Kanzler et al., 2007, Casella and Mitchell, 2008). Additionally, medications including opioid agonists (Hutchinson et al., 2010a, Hutchinson et al., 2010b) and widely-used chemotherapy drug, Paclitaxel (Byrd-Leifer et al., 2001, Zimmer et al., 2008) have been reported to activate TLR4 signaling in macrophages. Studies show that TLR4 signaling enhances resistance to paclitaxel therapy in breast and ovarian cancers (Rajput et al., 2013, Wang et al., 2014), and contributes to paclitaxel-induced peripheral neuropathy (Li et al., 2014).

Marked antitumor activity of paclitaxel in ovarian and breast cancer has led to its extensive evaluation as combination therapy with other cytotoxic agents for various metastatic cancers (Bellmunt et al., 2000, Kondagunta et al., 2005, Grau et al., 2009, Li et al., 2011). However, pharmacokinetics (PK) drug-drug interactions (DDIs) pose major clinical problem with such combination therapy. In a Phase-I study, DDI was observed in patients with advanced small non-small cell lung cancer (NSCLC) (Hotta et al., 2004) treated with paclitaxel and the potent anticancer drug, irinotecan. In this study, pre-treatment of paclitaxel, followed by irinotecan, increased the area under curve (AUC) of the toxic metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38) (Kasai et al., 2002).

The plasma level of both paclitaxel and irinotecan is determined by drug metabolism and disposition. Paclitaxel is an auto inducer, and induces drug metabolizing enzymes (DMEs) and transporters involved in its disposition (Nallani et al., 2003). It undergoes metabolism via cytochrome P450 (CYP) 2C8 (Rahman et al., 1994, Dai et al., 2001) and 3A4 (Cresteil et al., 1994, Harris et al., 1994); with biliary excretion via MDR1B (also known as P-glycoprotein [Pgp]) (Monsarrat et al., 1993, Kang et al., 2001). Irinotecan, a topoisomerase I inhibitor, is metabolized by two pathways (Fig. 1) (i) bio activation by carboxylesterase (CES) to form active and toxic metabolite, SN-38 (Satoh et al., 1994, Slatter et al., 1997) which is detoxified to SN-38 glucuronide (SN-38G) by uridine diphosphate glucuronosyltransferases (UGT) 1A1; (ii) oxidation by CYP3A4/5 enzyme (Santos et al., 2000) to form APC (7-ethyl-10[4-N-(5-aminopentanoicacid)-1-piperidino] carbonyloxycamptothecin) and NPC (7-ethyl-10[4-amino-1-piperidino]carbonyloxycamptothecin). NPC is converted to SN-38 by CES. Irinotecan and its metabolites are predominantly eliminated in the bile. Accumulation of SN-38 in the intestine is primarily due to deconjugation of SN-38G to SN-38 by bacterial β-glucuronidase, which accounts for life-threatening diarrhea (Sparreboom et al., 1998, Buajordet et al., 2001).

We have previously shown that activation of TLR4 by lipopolysaccharide (LPS) downregulate DME/transporter (DMET) genes (Ghose et al., 2009) leading to altered PK/pharmacodynamics (PD) of drugs (Gandhi et al., 2012). We observed that downregulation of DMETs by LPS was associated with reduced expression of the xenobiotic nuclear receptor (NR), pregnane X receptor (PXR) (Ghose et al., 2004a). PXR forms a dimer with the central NR, retinoid X receptor (RXR)-alpha to regulate DMET genes. It is well-established that alterations in DMET expression/activity can cause DDIs (Kohler et al., 2000, Polasek et al., 2011). Since TLR4 is activated by paclitaxel, we hypothesized that paclitaxel will cause TLR4-mediated alterations in DMETs, leading to changes in irinotecan PK. We found that in TLR4-wild type (WT) hepatocytes, paclitaxel increased the levels of SN-38 and SN-38G, while no such induction was observed in TLR4-mutant hepatocytes. Gene expression of Cyp3a11 and Ugt1a1 was induced up to ~ 600 and ~ 4 fold respectively by paclitaxel in TLR4-WT hepatocytes. In the TLR4-mutant hepatocytes Cyp3a11 was induced ~ 300-fold by paclitaxel, while Ugt1a1 expression was not increased. Thus, our finding proposes a novel mechanism of regulation of DDIs by TLR4 and indicates two possible unwanted effects due to TLR4 involvement in regulation of DMEs, (i) SN-38 levels may be affected in individuals with TLR4 polymorphism, resulting in changes irinotecan efficacy and safety; and (ii) TLR4 agonists pose potential for DDIs when given as combination therapy with other drugs.