Irinotecan-mediated diarrhea is mainly correlated with intestinal exposure to SN-38: Critical role of gut Ugt.

BACKGROUND AND PURPOSE: Irinotecan-induced diarrhea (IID) results from intestinal damages by its active metabolite SN-38. Alleviation of these damages has focused on lowering luminal SN-38 concentrations. However, it is unclear if the enteric bioavailability of SN-38 is mostly dependent on luminal SN-38 concentrations. EXPERIMENTAL APPROACH: Irinotecan (50 mg/kg, i.p. once daily for 6 days) was administered to female wildtype FVB, Mdr1a (-/-), Mrp2 (-/-) and Bcrp1 (-/-) mice for pharmacokinetic (PK), toxicokinetic (TK) and biodistribution studies. Plasma PK/TK profiles and tissues drug distribution were determined after first or sixth daily doses, along with activities of blood and gut esterases and intestinal Ugts. Caco-2 cells and bile-cannulate mice were used to further investigate intestinal and biliary disposition of irinotecan and its metabolites. KEY RESULTS: Significant differences in IID severity were observed with the susceptible rank of Bcrp1(-/-) > wildtype FVB > Mdr1a(-/-) > Mrp2(-/-). This rank order did not correlate with biliary excretion rates of SN-38/SN-38G. Rather, the severity was best correlated (R = 0.805) with the intestinal ratio of Css SN-38/SN-38G, a measure of gut Ugt activity. On the contrary, IID was poorly correlated with plasma AUC ratio of SN-38/SN-38G (R = 0.227). Increased intestinal esterase activities due to repeated dosing and gut efflux transporter functionality are the other key factors that determine SN-38 enteric exposures. CONCLUSION AND IMPLICATIONS: Intestinal SN-38 exposure is mainly affected by intestinal Ugt activities and blood esterase activities, and strongly correlated with severity of IID. Modulating intestinal SN-38 concentration and gut Ugt expression should be the focus of future studies to alleviate IID.

Neratinib in HER2-Positive Breast Cancer Patients.

OBJECTIVE: To review the chemistry, pharmacology, pharmacokinetics, safety, and efficacy of neratinib in human epidermal growth factor receptor (HER2)+ breast cancer (BC). DATA SOURCES: A PubMed search was performed using the term neratinib between September 12, 2018, and November 21, 2018. References of published articles and reviews were also assessed for additional information. STUDY SELECTION AND DATA EXTRACTION: English-language preclinical and clinical studies on the chemistry, pharmacology, pharmacokinetics, safety, and efficacy of neratinib were evaluated. DATA SYNTHESIS: Neratinib, an irreversible inhibitor of HER1, HER2, and HER4, is Food and Drug Administration approved for the extended adjuvant treatment of stage I-III HER2+ BC to follow trastuzumab-based therapy. A phase III study has demonstrated statistically significant improvement in 5-year disease-free survival rate (90.2 vs 87.7; hazard ratio = 0.73, 95% CI = 0.57-0.92, P = 0.0083). Its most common adverse effect is diarrhea, observed in more than 90% of patients. The incidence of grade 3/4 diarrhea (~40%) is reduced by half with loperamide prophylaxis, which is recommended for the first 8 weeks of neratinib therapy. Other common adverse reactions are nausea and fatigue. The patients need to be monitored for liver function tests and drug interactions with acid-reducing agents, CYP3A4 inhibitors/inducers, and P-glycoprotein substrates with narrow therapeutic window. Relevance to Patient Care and Clinical Practice: American Society of Clinical Oncology and National Comprehensive Cancer Network clinical guidelines suggest the use of neratinib for extended adjuvant therapy following 1-year trastuzumab in stage I to III HER2+ BC. Diarrhea remains a clinically significant but manageable adverse event. CONCLUSION: Neratinib significantly improves treatment outcomes and has manageable toxicity in stage I to III HER2+ BC patients.

Role of c-Jun-N-Terminal Kinase in Pregnane X Receptor-Mediated Induction of Human Cytochrome P4503A4 In Vitro.

Cytochrome P450 CYP3A4 is the most abundant drug-metabolizing enzyme and is responsible for the metabolism of ∼50% of clinically available drugs. Induction of CYP3A4 impacts the disposition of its substrates and leads to harmful clinical consequences, such as failure of therapy. To prevent such undesirable consequences, the molecular mechanisms of regulation of CYP3A4 need to be fully understood. CYP3A4 induction is regulated primarily by the xenobiotic nuclear receptor pregnane-X receptor (PXR). After ligand binding, PXR is translocated to the nucleus, where it binds to the CYP3A4 promoter and induces its gene expression. PXR function is modulated by phosphorylation(s) by multiple kinases. In this study, we determined the role of the c-Jun N-terminal kinase (JNK) in PXR-mediated induction of CYP3A4 enzyme in vitro. Human liver carcinoma cells (HepG2) were transfected with CYP3A4 luciferase and PXR plasmids, followed by treatment with JNK inhibitor (SP600125; SP) and PXR activators rifampicin (RIF) or hyperforin. Our results indicate that SP treatment significantly attenuated PXR-mediated induction of CYP3A4 reporter activity, as well as gene expression and enzyme activity. JNK knockdown by siRNA (targeting both JNK 1 and 2) also attenuated CYP3A4 induction by RIF. Interestingly, SP treatment attenuated JNK activation by RIF. Furthermore, treatment with RIF increased PXR nuclear levels and binding to the CYP3A4 promoter; SP attenuated these effects. This study shows that JNK is a novel mechanistic regulator of CYP3A4 induction by PXR.

Role of Adaptor Protein Toll-Like Interleukin Domain Containing Adaptor Inducing Interferon ß in Toll-Like Receptor 3- and 4-Mediated Regulation of Hepatic Drug Metabolizing Enzyme and Transporter Genes.

The expressions and activities of hepatic drug-metabolizing enzymes and transporters (DMETs) are altered during infection and inflammation. Inflammatory responses in the liver are mediated primarily by Toll-like receptor (TLR)-signaling, which involves recruitment of Toll/interleukin (IL)-1 receptor (TIR) domain containing adaptor protein (TIRAP) and TIR domain containing adaptor inducing interferon (IFN)-ß (TRIF) that eventually leads to induction of proinflammatory cytokines and mitogen-activated protein kinases (MAPKs). Lipopolysaccharide (LPS) activates the Gram-negative bacterial receptor TLR4 and polyinosinic:polycytidylic acid (polyI:C) activates the viral receptor TLR3. TLR4 signaling involves TIRAP and TRIF, whereas TRIF is the only adaptor protein involved in the TLR3 pathway. We have shown previously that LPS-mediated downregulation of DMETs is independent of TIRAP. To determine the role of TRIF, we treated TRIF(+/+) and TRIF(-/-) mice with LPS or polyI:C. LPS downregulated (∼40%-60%) Cyp3a11, Cyp2a4, Ugt1a1, Mrp2 mRNA levels, whereas polyI:C downregulated (∼30%-60%) Cyp3a11, Cyp2a4, Cyp1a2, Cyp2b10, Ugt1a1, Mrp2, and Mrp3 mRNA levels in TRIF(+/+) mice. This downregulation was not attenuated in TRIF(-/-) mice. Induction of cytokines by LPS was observed in both TRIF(+/+) and TRIF(-/-) mice. Cytokine induction was delayed in polyI:C-treated TRIF(-/-) mice, indicating that multiple mechanisms mediating polyI:C signaling exist. To assess the role of MAPKs, primary hepatocytes were pretreated with specific inhibitors before treatment with LPS/polyI:C. We found that only the c-jun-N-terminal kinase (JNK) inhibitor attenuated the down-regulation of DMETs. These results show that TRIF-independent pathways can be involved in the downregulation of DMETs through TLR4 and 3. JNK-dependent mechanisms likely mediate this downregulation.

Role of constitutive androstane receptor in Toll-like receptor-mediated regulation of gene expression of hepatic drug-metabolizing enzymes and transporters.

Impairment of drug disposition in the liver during inflammation has been attributed to downregulation of gene expression of drug-metabolizing enzymes (DMEs) and drug transporters. Inflammatory responses in the liver are primarily mediated by Toll-like receptors (TLRs). We have recently shown that activation of TLR2 or TLR4 by lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, leads to the downregulation of gene expression of DMEs/transporters. However, the molecular mechanism underlying this downregulation is not fully understood. The xenobiotic nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), regulate the expression of DMEs/transporter genes. Downregulation of DMEs/transporters by LTA or LPS was associated with reduced expression of PXR and CAR genes. To determine the role of CAR, we injected CAR(+/+) and CAR(-/-) mice with LTA or LPS, which significantly downregulated (~40%-60%) RNA levels of the DMEs, cytochrome P450 (Cyp)3a11, Cyp2a4, Cyp2b10, uridine diphosphate glucuronosyltransferase 1a1, amine N-sulfotransferase, and the transporter, multidrug resistance-associated protein 2, in CAR(+/+) mice. Suppression of most of these genes was attenuated in LTA-treated CAR(-/-) mice. In contrast, LPS-mediated downregulation of these genes was not attenuated in CAR(-/-) mice. Induction of these genes by mouse CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene was sustained in LTA- but not in LPS-treated mice. Similar observations were obtained in humanized CAR mice. We have replicated these results in primary hepatocytes as well. Thus, LPS can downregulate DME/transporter genes in the absence of CAR, whereas the effect of LTA on these genes is attenuated in the absence of CAR, indicating the potential involvement of CAR in LTA-mediated downregulation of DME/transporter genes.

Chlorpromazine-induced hepatotoxicity during inflammation is mediated by TIRAP-dependent signaling pathway in mice.

Inflammation is a major component of idiosyncratic adverse drug reactions (IADRs). To understand the molecular mechanism of inflammation-mediated IADRs, we determined the role of the Toll-like receptor (TLR) signaling pathway in idiosyncratic hepatotoxicity of the anti-psychotic drug, chlorpromazine (CPZ). Activation of TLRs recruits the first adaptor protein, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) to the TIR domain of TLRs leading to the activation of the downstream kinase, c-Jun-N-terminal kinase (JNK). Prolonged activation of JNK leads to cell-death. We hypothesized that activation of TLR2 by lipoteichoic acid (LTA) or TLR4 by lipopolysaccharide (LPS) will augment the hepatotoxicity of CPZ by TIRAP-dependent mechanism involving prolonged activation of JNK. Adult male C57BL/6, TIRAP(+/+) and TIRAP(-/-) mice were pretreated with saline, LPS (2 mg/kg) or LTA (6 mg/kg) for 30 min or 16 h followed by CPZ (5 mg/kg) or saline (vehicle) up to 24h. We found that treatment of mice with CPZ in presence of LPS or LTA leads to ~3-4 fold increase in serum ALT levels, a marked reduction in hepatic glycogen content, significant induction of serum tumor necrosis factor (TNF) α and prolonged JNK activation, compared to LPS or LTA alone. Similar results were observed in TIRAP(+/+) mice, whereas the effects of LPS or LTA on CPZ-induced hepatotoxicity were attenuated in TIRAP(-/-) mice. For the first time, we show that inflammation-mediated hepatotoxicity of CPZ is dependent on TIRAP, and involves prolonged JNK activation in vivo. Thus, TIRAP-dependent pathways may be targeted to predict and prevent inflammation-mediated IADRs.

Impact of hepatic malignancy on CYP3A4 gene expression.

BACKGROUND: The aim of this study was to examine differences in a major enzyme system for hepatic metabolism of drugs, CYP3A4, by measuring RNA levels in the liver tissue of subjects with and without hepatic malignancy and with primary versus metastatic liver tumors. MATERIALS AND METHODS: We identified liver specimens from a hospital-wide tissue repository of patients having liver resection for a clinical indication. Total RNA isolation, complementary DNA synthesis, and real-time quantitative polymerase chain reaction were performed according to the standards. Demographic, clinical, and laboratory data were obtained from medical records. Standard statistical analyses were performed with significance set to α=0.05. RESULTS: Liver tissue from 27 subjects was available for analysis: 13 were without malignancy and 14 had either primary liver malignancies (n=7) or metastatic disease (n=7). Median age was 57 y, and half of the subjects were men. More than 80% of subjects were overweight or obese without differentiation between benign or malignant tumors. Fewer than 20% of subjects had diabetes or hypercholesterolemia. No preresection laboratory differences were noted between the groups (benign versus malignant or primary versus metastatic disease). Subjects with malignant liver tumors had significantly lower relative-fold CYP3A4 RNA content than those with benign liver tumors (P=0.009), but no difference in the CYP3A4 RNA content between primary and metastatic disease was seen. CONCLUSIONS: This study demonstrates differences in the expression of CYP3A4 in benign and malignant human liver tumors and contributes to understanding the possible impact of malignancy on hepatic metabolism.

Neratinib causes non-recoverable gut injury and reduces intestinal cytochrome P450 3A enzyme in mice.

Neratinib is a pan-HER tyrosine kinase inhibitor newly approved by FDA in 2017 to treat HER2-positive breast cancer, but the phase III trial of neratinib showed that 96% of the patients taking neratinib experienced diarrhea. So far very few mechanistic studies explore neratinib-induced gastrointestinal (GI) toxicity. Hereby, we performed toxicity studies in mice to characterize the potential mechanism underlying this adverse effect. C57BL/6 J mice were separated into three groups A, B, C. Group A received vehicle; group B was orally dosed with 100 mg/kg neratinib once daily for 18 days. Group C was dosed with 100 mg/kg neratinib for 12 days and switched to vehicle for 6 days. Intestine and liver were collected for further analysis. Human intestine-derived cells were treated with neratinib in vitro. Our results showed that 12 days treatment of neratinib caused persistent histological damage in mouse GI tract. Both gene expression and activity of Cyp3a11, the major enzyme metabolizing neratinib in mice was reduced in small intestine. The gene expression of proinflammatory cytokines increased throughout the GI tract. Such damages were not recovered after 6 days without neratinib treatment. In addition, in vitro data showed that neratinib was potent in killing human intestine-derived cell lines. Based on such findings, we hypothesized that neratinib downregulates intestinal CYP3A enzyme to cause excessive drug disposition, eventually leading to gut injury.

Development of a physiologically based pharmacokinetic model to predict irinotecan disposition during inflammation.

Irinotecan, a first-line chemotherapy for gastrointestinal (GI) cancers has been causing fatal toxicities like bloody diarrhea and steatohepatitis for years. Irinotecan goes through multiple-step drug metabolism after injection and one of its intermediates 7-ethyl-10-hydroxy-camptothecin (SN-38) is responsible for irinotecan side effect. However, it is unclear what is the disposition kinetics of SN-38 in the organs subjected to toxicity. No studies ever quantified the effect of each enzyme or transporter on SN-38 distribution. In current study, we established a new physiologically based pharmacokinetic (PBPK) model to predict the disposition kinetics of irinotecan. The PBPK model was calibrated with in-house mouse pharmacokinetic data and evaluated with external datasets from the literature. We separated the contribution of each parameters in irinotecan pharmacokinetics by calculating the normalized sensitivity coefficient (NSC). The model gave robust prediction of SN-38 distribution in GI tract, the site of injury. We identified that bile excretion and UDP-glucuronosyltransferases (UGT) played more important roles than fecal excretion and renal clearance in SN-38 pharmacokinetics. Our NSC showed that the impact of enzyme and transporter on irinotecan and SN-38 pharmacokinetics evolved when time continued. Additionally, we mapped out the effect of inflammation on irinotecan metabolic pathways with PBPK modelling. We discovered that inflammation significantly increased the blood and liver exposure of irinotecan and SN-38 in the mice receiving bacterial endotoxin. Inflammation suppressed UGT, microbial metabolism but increased fecal excretion. The present PBPK model can serve as an efficacious and versatile tool to quantitively assess the risk of irinotecan toxicity.

Irinotecan decreases intestinal UDP-glucuronosyltransferase (UGT) 1A1 via TLR4/MyD88 pathway prior to the onset of diarrhea.

Irinotecan is a first-line treatment for colorectal cancer and the prodrug of 7-ethyl-10-hydroxy-camptothecin (SN-38). However, its fatal gastrointestinal (GI) toxicity raises serious concern. In liver, irinotecan generates its inactive metabolite, SN-38G via UDP-glucuronosyltransferase (UGT)1A1. Subsequently, SN-38G is excreted into GI tract where it is reactivated by microbiome to yield the toxic metabolite, SN-38. Activation of toll-like receptor (TLR)/myeloid differentiation primary response 88 (MyD88) by bacterial endotoxin decreases drug-metabolizing enzymes. In this study, we treated C57BL6/J mice with 50 mg/kg irinotecan once daily until observing grade 4 diarrhea. Mice were sacrificed on day0, day2 and day8. Based on the finding in C57BL6/J mice, we repeated the treatment in Tlr2-/-, Tlr4-/- and Myd88-/- mice to determine the impact of inflammation on UGT metabolism. Our toxicity study in C57BL6/J mice showed that mice started bloody diarrhea after 6 days' injection of irinotecan. Ugt1a1 expression in GI tract started decreasing after 24h since first dose, before the onset of diarrhea. In Tlr4-/- and Myd88-/- mice, no Ugt1a1 reduction was observed in distal GI tract after irinotecan injection. In Tlr2-/- mice, intestinal Ugt1a1 expression was down-regulated. Our results indicate that after two doses of irinotecan, mice started losing capability of detoxifying SN-38. TLR4 plays more important role in Ugt1a1 reduction than TLR2, despite that TLR2 and TLR4 share MyD88 as common adaptor protein. We concluded that irinotecan reduced intestinal Ugt1a1 via TLR4/MyD88 pathway, which eventually triggers the onset of diarrhea. Our finding unveils a novel mechanism underlying irinotecan-induced diarrhea and provides a new direction to prevent chemotherapy side effect.

Differential role of Toll-interleukin 1 receptor domain-containing adaptor protein in Toll-like receptor 2-mediated regulation of gene expression of hepatic cytokines and drug-metabolizing enzymes.

Pharmacological activities of drugs are impaired during inflammation because of reduced expression of hepatic drug-metabolizing enzyme genes (DMEs) and their regulatory nuclear receptors (NRs): pregnane X receptor (PXR), constitutive androstane receptor (CAR), and retinoid X receptor (RXRα). We have shown that a component of Gram-positive bacteria, lipoteichoic acid (LTA) induces proinflammatory cytokines and reduces gene expression of hepatic DMEs and NRs. LTA is a Toll-like receptor 2 (TLR2) ligand, which initiates signaling by recruitment of Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) to the cytoplasmic TIR domain of TLR2. To determine the role of TIRAP in TLR2-mediated regulation of DME genes, TLR2(+/+), TLR2(-/-), TIRAP(+/+), and TIRAP(-/-) mice were given LTA injections. RNA levels of the DMEs (Cyp3a11, Cyp2b10, and sulfoaminotransferase), xenobiotic NRs (PXR and CAR), and nuclear protein levels of the central NR RXRα were reduced ∼ 50 to 60% in LTA-treated TLR2(+/+) but not in TLR2(-/-) mice. Induction of hepatic cytokines (interleukin-1ß, tumor necrosis factor-α, and interleukin-6), c-Jun NH(2)-terminal kinase, and nuclear factor-κΒ was blocked in TLR2(-/-) mice. As expected, expression of hepatic DMEs and NRs was reduced by LTA in TIRAP(+/+) but not in TIRAP(-/-) mice. Of interest, cytokine RNA levels were induced in the livers of both the TIRAP(+/+) and TIRAP(-/-) mice, whereas LTA-mediated induction of serum cytokines was attenuated in TIRAP(-/-) mice. LTA-mediated down-regulation of DME genes was attenuated in hepatocytes from TLR2(-/-) or TIRAP(-/-) mice and in small interfering RNA-treated hepatocytes. Thus, the effect of TLR2 on DME genes in hepatocytes was mediated by TIRAP, whereas TIRAP was not involved in mediating the effects of TLR2 on cytokine expression in the liver.

Immunization and Drug Metabolizing Enzymes: Focus on Hepatic Cytochrome P450 3A.

OBJECTIVE: Infectious disease emergencies like the 2013-2016 Ebola epidemic and the 2009 influenza and current SARS-CoV-2 pandemics illustrate that vaccines are now given to diverse populations with preexisting pathologies requiring pharmacological management. Many natural biomolecules (steroid hormones, fatty acids, vitamins) and ~60% of prescribed medications are processed by hepatic cytochrome P450 (CYP) 3A4. The objective of this work was to determine the impact of infection and vaccines on drug metabolism. METHODS: The impact of an adenovirus-based vaccine expressing Ebola glycoprotein (AdEBO) and H1N1 and H3N2 influenza viruses on hepatic CYP 3A4 and associated nuclear receptors was evaluated in human hepatocytes (HC-04 cells) and in mice. RESULTS: CYP3A activity was suppressed by 55% in mice 24 h after administration of mouse-adapted H1N1, while ˂10% activity remained in HC-04 cells after infection with H1N1 and H3N2 due to global suppression of cellular translation capacity, indicated by reduction (70%, H1N1, 56%, H3N2) of phosphorylated eukaryotic translation initiation factor 4e (eIF4E). AdEBO suppressed CYP3A activity in vivo (44%) and in vitro (26%) 24 hours after infection. CONCLUSION: As the clinical evaluation of vaccines for SARS-CoV-2 and other global pathogens rise, studies to evaluate the impact of new vaccines and emerging pathogens on CYP3A4 and other metabolic enzymes are warranted to avoid therapeutic failures that could further compromise the public health during infectious disease emergencies.

Potential role of drug metabolizing enzymes in chemotherapy-induced gastrointestinal toxicity and hepatotoxicity.

INTRODUCTION: Toxicity of chemotherapy drugs is the leading cause of poor therapeutic outcome in many cancer patients. Gastrointestinal (GI) toxicity and hepatotoxicity are among the most common side effects of current chemotherapies. Emerging studies indicate that many chemotherapy-induced toxicities are driven by drug metabolism, but very few reviews summarize the role of drug metabolism in chemotherapy-induced GI toxicity and hepatotoxicity. In this review, we highlighted the importance of drug metabolizing enzymes (DMEs) in chemotherapy toxicity. AREAS COVERED: Our review demonstrated that altered activity of DMEs play important role in chemotherapy-induced GI toxicity and hepatotoxicity. Besides direct changes in catalytic activities, the transcription of DMEs is also affected by inflammation, cell-signaling pathways, and/or by drugs in cancer patients due to the disease etiology. EXPERT OPINION: More studies should focus on how DMEs are altered during chemotherapy treatment, and how such changes affect the metabolism of chemotherapy drug itself. This mutual interaction between chemotherapies and DMEs can lead to excessive exposure of parent drug or toxic metabolites which ultimately cause GI adverse effect.

Effects of inflammation on irinotecan pharmacokinetics and development of a best-fit PK model.

Irinotecan is a chemotherapeutic drug used in the treatment of advanced colorectal cancer and elevated blood concentrations of its active metabolite, SN-38 leads to increased gastrointestinal (GI) toxicity and diarrhea in patients. In this study, we investigated the effects of inflammation on the pharmacokinetics (PK) of irinotecan (CPT-11) and its active metabolite, SN-38. Mice were i.p.-injected with either saline or lipopolysaccharide (LPS) to induce inflammation. After 16 h, irinotecan was administered orally. Blood was collected from the tail vein of mice from 0 to 24 h after dosing. Concentrations of irinotecan, SN-38 and SN-38G were analyzed using LC-MS/MS. The AUC, Cmax, and tmax were derived using WinNonlin® 5.2. A PK model was developed using Phoenix NLME® to describe the PK of irinotecan and SN-38 during inflammation. Results indicated a significant increase in the blood concentrations of irinotecan and SN-38 in mice during inflammation. The AUC of irinotecan and SN-38 in LPS group were 2.6 and 2-folds, respectively, of those in control saline-treated mice. The Cmax of irinotecan and SN-38 in LPS treated mice were 2.4 and 2.3-folds of those in saline-treated mice. The PK model was successfully developed and validated. The best-fit plots of individual PK analysis showed a good correlation between observed and predicted concentrations of irinotecan and SN-38. Together, this study reveals that SN-38 concentrations are elevated during inflammation, which may increase the GI toxicity and diarrhea in patients who receive irinotecan; and the developed PK model can quantitatively describe the PK of irinotecan and SN-38 during inflammation.

Regulation of gene expression of hepatic drug metabolizing enzymes and transporters by the Toll-like receptor 2 ligand, lipoteichoic acid.

Expression of hepatic drug metabolizing enzymes (DMEs) is altered in infection and inflammation. However, the role of Gram+ve bacterial components and their receptor, Toll-like receptor (TLR) 2 in regulation of hepatic DMEs is unknown. Gene expression of DMEs is regulated by members of the nuclear receptor superfamily (PXR, CAR and RXRalpha). The TLR2 ligand, lipoteichoic acid (LTA) reduced RNA levels of CAR and its target genes, Cyp2b10, Cyp2a4 and Sultn in mouse liver ( approximately 60-80% reduction). Hepatic genes regulated by PXR and CAR, Cyp3a11 and Mrp2 were moderately reduced by LTA, along with approximately 50% reduction of PXR RNA and nuclear protein levels of RXRalpha. The effects of LTA were significantly attenuated by pre-treatment with the Kupffer cell inhibitor, gadolinium chloride, indicating that Kupffer cells contribute to LTA-mediated down-regulation of hepatic genes. These results indicate that treatment with Gram+ve bacterial components preferentially down-regulate CAR and its target genes in the liver.

Transcriptomic profiling identifies novel mechanisms of transcriptional regulation of the cytochrome P450 (Cyp)3a11 gene.

Cytochrome P450 (CYP)3A is the most abundant CYP enzyme in the human liver, and a functional impairment of this enzyme leads to unanticipated adverse reactions and therapeutic failures; these reactions result in the early termination of drug development or the withdrawal of drugs from the market. The transcriptional regulation mechanism of the Cyp3a gene is not fully understood and requires a thorough investigation. We mapped the transcriptome of the Cyp3a gene in a mouse model. The Cyp3a gene was induced using the mPXR activator pregnenolone-16alpha-carbonitrile (PCN) and was subsequently downregulated using lipopolysaccharide (LPS). Our objective was to identify the transcription factors (TFs), epigenetic modulators and molecular pathways that are enriched or repressed by PCN and LPS based on a gene set enrichment analysis. Our analysis shows that 113 genes were significantly upregulated (by at least 1.5-fold) with PCN treatment, and that 834 genes were significantly downregulated (by at least 1.5-fold) with LPS treatment. Additionally, the targets of the 536 transcription factors were enriched by a combined treatment of PCN and LPS, and among these, 285 were found to have binding sites on Cyp3a11. Moreover, the repressed targets of the epigenetic markers HDAC1, HDAC3 and EZH2 were further suppressed by LPS treatment and were enhanced by PCN treatment. By identifying and contrasting the transcriptional regulators that are altered by PCN and LPS, our study provides novel insights into the transcriptional regulation of CYP3A in the liver.

Xiao-Chai-Hu-Tang (XCHT) Intervening Irinotecan's Disposition: The Potential of XCHT in Alleviating Irinotecan-Induced Diarrhea.

BACKGROUND: Diarrhea is a severe side effect of irinotecan, a pro-drug of SN-38 used for the treatment of many types of cancers. Pre-clinical and clinical studies showed that decreasing the colonic exposure of SN-38 can mitigate irinotecan-induced diarrhea. OBJECTIVE: The purpose of this study is to evaluate the anti-diarrhea potential of Xiao-Chai-Hu-Tang (XCHT), a traditional Chinese herbal formula, against irinotecan-induced diarrhea by determining if and how XCHT alters the disposition of SN-38. METHODS: LC-MS/MS was used to quantify the concentrations of irinotecan and its major metabolites (i.e., SN-38, SN-38G). An Intestinal perfusion model was used to determine the effect of XCHT on the biliary and intestinal secretions of irinotecan, SN-38, and SN-38G. Pharmacokinetic (PK) studies were performed to determine the impact of XCHT on the blood and fecal concentrations of irinotecan, SN-38, and SN-38G. RESULTS: The results showed that XCHT significantly inhibits both biliary and intestinal excretions of irinotecan, SN-38, and SN-38G (range: 35% to 95%). PK studies revealed that the fecal concentrations of irinotecan and SN-38 were significantly decreased from 818.35 ± 120.2 to 411.74 ± 138.83 µg/g or from 423.95 ± 76.44 to 245.63 ± 56.72 µg/g (p<0.05) by XCHT, respectively, suggesting the colonic exposure of SN-38 is significantly decreased by XCHT. PK studies also showed that the plasma concentrations of irinotecan, SN-38, and SN-38G were not affected by XCHT. CONCLUSION: In conclusion, XCHT significantly decreased the exposure of SN-38 in the gut without affecting its plasma level, thereby possessing the potential of alleviating irinotecan-induced diarrhea without negatively impacting its therapeutic efficacy.

Regulation of hepatic drug-metabolizing enzyme genes by Toll-like receptor 4 signaling is independent of Toll-interleukin 1 receptor domain-containing adaptor protein.

During inflammation, drug metabolism and clearance are altered due to suppression of hepatic drug-metabolizing enzyme (DME) genes and their regulatory nuclear receptors (NRs) [pregnane X receptor, constitutive androstane receptor, and retinoid X receptor alpha (RXRalpha)]. The bacterial endotoxin, lipopolysaccharide (LPS), induces expression of proinflammatory cytokines in the liver, which contribute to altered DME expression. LPS binds to the cell-surface receptor, Toll-like receptor 4 (TLR4), which initiates a signal transduction cascade, including recruitment of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP). However, the role of TLR4 and TIRAP in LPS-mediated regulation of hepatic DME gene expression is not known. Wild-type (C3HeB/FeJ), TLR4-mutant (C3H/HeJ), TIRAP(+/+), and TIRAP(-/-) mice were injected i.p. with LPs. RNA levels of the major hepatic DME, Cyp3a11 and Ugt1a1, and the NRs were suppressed approximately 60 to 70% by LPS in wild-type but not in the TLR4-mutant mice. The nuclear protein levels of RXRalpha were reduced by LPS in wild-type but not in TLR4-mutant mice. Induction of hepatic cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interleukin-6), c-Jun N-terminal kinase, and nuclear factor-kappaB was blocked in TLR4-mutant mice. Surprisingly, LPS had the same effect on cytokines, kinases, NRs, and DME genes in livers of both TIRAP(+/+) and TIRAP(-/-) mice, indicating that TIRAP is not essential for TLR4-mediated suppression of NRs and DMEs in liver. However, TIRAP(-/-) mice have reduced serum cytokine expression compared with TIRAP(+/+) mice in response to LPS. This shows that although TIRAP mediates inflammatory responses induced by LPS, it is not essential in regulating LPS-mediated alterations of gene expression in liver.

Impact of diet on irinotecan toxicity in mice.

Irinotecan is highly effective in the treatment of metastatic colorectal cancer as well as many other cancers. However, irinotecan is known to cause severe diarrhea, which pose significant problems in patients undergoing irinotecan based chemotherapy. Dietary and herbal components have shown promise in improving gastrointestinal health. Therefore, we compared the effect of grain-based chow diet containing phytoestrogens and corn/alfalfa as fat source to purified diets containing either animal-derived fat source (lard) or plant-derived fat source (soybean oil) on irinotecan-induced toxicities in mice. The concentration of the toxic metabolite, SN-38, was measured in the serum, and the activity of main enzyme, carboxylesterase (CEs) involved in biotransformation of irinotecan to SN-38 formation was measured in the liver. We found that the grain-based diet was protective against irinotecan-induced diarrhea. Interestingly, purified diet containing lard caused fatty liver in mice, while grain-based chow diet containing corn/alfa-alfa or purified diet with soybean oil did not cause fat deposition in the liver. Serum SN-38 concentration was significantly higher in the mice fed with purified diets compared to the chow-fed mice. Hepatic CEs activity was induced in the presence of irinotecan in mice on purified diets, but not chow diet. These results indicate that components of grain-based natural diet (presumably phytoestrogens and/or the macronutrients balance) compared to purified diets may have a beneficial effect by controlling the adverse effects of irinotecan in cancer patients.