Roles of Toll-Like Receptors, Nuclear Receptors and Obesity in Drug Metabolism

Drug metabolism is affected in various diseases and altered physiological states such as cancer, cardiovascular diseases, diabetes, obesity, liver disorders, etc. The common factor underlying all these disease states is inflammation. Accumulating evidence has shown that inflammation can alter the gene expression and activity of several drug metabolizing enzymes and transporters (DMETs) however, the exact mechanism is unknown. Although well established studies have attributed the down-regulation of DMET activities and protein expression to the reduction in their gene expression levels, further research is needed to establish the clinical relevance of this down-regulation. Furthermore, clinical relevance of down-regulation of DMETs during inflammation or pathophysiological conditions needs to be ascertained. Our overall aim is to determine molecular mechanisms that regulate gene expression of DMETs during inflammation and ascertain the clinical relevance of this down-regulation in a mouse model of obesity. Inflammatory responses in the liver are primarily mediated by specific trans-membrane receptors known as Toll-like receptors (TLRs). These receptors are key components of the innate immune response and are present on immune cells as well as hepatocytes in the liver. Out of the 13 known TLRs, we selected TLR2, TLR3 and TLR4 (activated by gram-positive bacterial components, viral components and gram-negative bacterial components, respectively) as they play major roles in immune reactions elicited during various pathophysiological conditions. TLR signaling is mediated by adaptor proteins Toll/interleukin (IL)1 receptor (TIR) domain containing adaptor protein (TIRAP) and TIR domain containing adaptor inducing interferon (IFN)-β (TRIF). We have shown that activation of TLR2 by lipoteichoic acid (LTA) and TLR4 by lipopolysaccharide (LPS) caused down-regulation of gene expression and activity of key hepatic DMETs. This down-regulation was associated with reduced expression of the nuclear receptors, constitutive androstane receptor (CAR) and pregnane X receptor (PXR), which heterodimerize with the central nuclear receptor, retinoid x receptor alpha (RXRα). Our central hypothesis is that drug metabolism is altered by activation of innate immune components and in obesity. We propose to achieve our goals by pursuing the following Specific Aims: Aim 1: To determine the role of the adaptor protein, TRIF down-regulating the gene expression of DMEs and transporters. We hypothesize that the adaptor protein TRIF mediates the down-regulation of DMETs through TLR4 and TLR3. We observed that down-regulation of DMETs on activation of TLR4 and TLR3 is independent of the adaptor protein TRIF. We also found that the MAP kinase, c-Jun-N-terminal kinase (JNK) is involved in mediating the down-regulation of DMET through TLR3 & TLR4. Aim 2: To determine the role of nuclear receptor CAR in TLR2- & 4-mediated down-regulation of DMEs and transporters. We hypothesize that CAR mediates the down-regulation of DMEs and transporters through TLR2 and TLR4. CAR was shown to play a differential role in regulating gene expression of DMETs in TLR2 and TLR4 induced inflammation in mice. We found that CAR is involved in down-regulation of hepatic DMET genes through TLR2, but not through TLR4. Aim 3: We have shown that gene expression of uridine glucuronosyl transferase (Ugt) 1a1 enzyme is reduced during obesity. Our goal is to determine the role of obesity in Ugt-mediated glucuronidation of irinotecan metabolite. We hypothesize that obesity induced-alteration in Ugt enzymes leads to accumulation of the active and toxic metabolite of irinotecan i.e. SN-38. We found that the rate of formation of SN-38 Glucuronide (SN-38G) was 2-fold lower in the DIO mice compared to the lean controls. Plasma exposure of SN-38 increased by 2-folds and that of SN-38G decreased significantly in the DIO mice compared to the lean controls. Thus, reduction in Ugt1a expression and activity in DIO mice contributes to accumulation of SN-38 which could be the potential reason for irinotecan-induced liver toxicity during obesity. Since TLRs are activated in a variety of pathophysiological conditions, this study will have a significant impact on drug development. The outcomes of this proposed study will provide important information regarding the signaling pathways that govern drug metabolism and hepatotoxicity of clinically relevant drugs at therapeutic doses during pathophysiological conditions.