Perspectives on the evaluation and adoption of complex in vitro models in drug development: Workshop with the FDA and the pharmaceutical industry (IQ MPS Affiliate).

Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualifi­cation plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharma­ceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.

3D cell culture models: Drug pharmacokinetics, safety assessment, and regulatory consideration.

Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two-dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species-specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell- and organ-based assays for more accurate representation of human susceptibility to drug response. Among others, the three-dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs-on-chips, and from single-cell type static 3D models to cell co-culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver-, intestine-, kidney-, and neuron-based 3D cellular models.

In vitro assessment of farnesoid X receptor antagonism to predict drug-induced liver injury risk.

Drug-induced liver injury (DILI) continues to be a major cause of drug attrition and restrictive labeling. Given the importance of farnesoid X receptor (FXR) in bile acid homeostasis, drug-related FXR antagonism may be an important mechanism of DILI. However, a comprehensive assessment of this phenomenon broadly in the context of DILI is lacking. As such, we used an orthogonal approach comprising a FXR target gene assay in primary human hepatocytes and a commercially available FXR reporter assay to investigate the potential FXR antagonistic effects of an extensive test set of 159 compounds with and without association with clinical DILI. Data were omitted from analysis based on the presence of cytotoxicity to minimize false positive assay signals and other complications in data interpretation. Based on the experimental approaches employed and corresponding data, the prevalence of FXR antagonism was relatively low across this broad DILI test set, with 16-24% prevalence based on individual assay results or combined signals in both assays. Moreover, FXR antagonism was not highly predictive for identifying clinically relevant hepatotoxicants retrospectively, where FXR antagonist classification alone had minimal to moderate predictive value as represented by positive and negative likelihood ratios of 2.24-3.84 and 0.72-0.85, respectively. The predictivity did not increase significantly when considering only compounds with high clinical exposure (maximal or efficacious plasma exposures > 1.0 µM). In contrast, modest gains in predictive value of FXR antagonism were observed considering compounds that also inhibit bile salt export pump. In addition, we have identified novel FXR antagonistic effects of well-studied hepatotoxic drugs, including bosentan, tolcapone and ritonavir. In conclusion, this work represents a comprehensive evaluation of FXR antagonism in the context of DILI, including its overall predictivity and challenges associated with detecting this phenomenon in vitro.

Introduction to a manuscript series on the characterization and use of microphysiological systems (MPS) in pharmaceutical safety and ADME applications.

Safety related drug failures continue to be a challenge for pharmaceutical companies despite the numerous complex and lengthy in vitro assays and in vivo studies that make up the typical safety screening funnel. A lack of complete translation of animal data to humans can explain some of those shortcomings. Differences in sensitivity and drug disposition between animals and humans may also play a role. Many gaps exist for potential target tissues of drugs that cannot be adequately modeled in vitro. Microphysiological systems (MPS) may help to better model these target tissues and provide an opportunity to better assess some aspects of human safety prior to clinical studies. There is hope that these systems can supplement current preclinical drug safety and disposition evaluations, filling gaps and enhancing our ability to predict and understand human relevant toxicities. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) MPS Affiliate is a group of pharmaceutical industry scientists who seek to expedite appropriate characterization and incorporation of MPS to potentially improve drug safety assessment and provide safer and more effective medicines to patients. In keeping with this mission, the IQ MPS Affiliate scientists have prepared a series of organotypic manuscripts for several key drug safety and disposition target tissues (lung, liver, kidney, skin, gastrointestinal, cardiovascular, and blood brain barrier/central nervous system). The goal of these manuscripts is to provide key information related to likely initial contexts of use (CoU) and key characterization data needed for incorporation of MPS in pharmaceutical safety screening including a list of characteristic functions, cell types, toxicities, and test agents (representing major mechanisms of toxicity) that can be used by MPS developers. Additional manuscripts focusing on testing biologically based therapeutics and ADME considerations have been prepared as part of this effort. These manuscripts focus on general needs for assessing biologics and ADME endpoints and include similar information to the tissue specific manuscripts where appropriate. The current manuscript is an introduction to several general concepts related to pharmaceutical industry needs with regard to MPS application and other MPS concepts that apply across the organ specific manuscripts.

Liver microphysiological systems development guidelines for safety risk assessment in the pharmaceutical industry.

The liver is critical to consider during drug development because of its central role in the handling of xenobiotics, a process which often leads to localized and/or downstream tissue injury. Our ability to predict human clinical safety outcomes with animal testing is limited due to species differences in drug metabolism and disposition, while traditional human in vitro liver models often lack the necessary in vivo physiological fidelity. To address this, increasing numbers of liver microphysiological systems (MPS) are being developed, however the inconsistency in their optimization and characterization often leads to models that do not possess critical levels of baseline performance that is required for many pharmaceutical industry applications. Herein we provide a guidance on best approaches to benchmark liver MPS based on 3 stages of characterization that includes key performance metrics and a 20 compound safety test set. Additionally, we give an overview of frequently used liver injury safety assays, describe the ideal MPS model, and provide a perspective on currently best suited MPS contexts of use. This pharmaceutical industry guidance has been written to help MPS developers and end users identify what could be the most valuable models for safety risk assessment.

Variance component analysis of circulating miR-122 in serum from healthy human volunteers.

Micro-RNA (miR)-122 is a promising exploratory biomarker for detecting liver injury in preclinical and clinical studies. Elevations in serum or plasma have been associated with viral and autoimmune hepatitis, non-alcoholic steatohepatitis (NASH), hepatocellular carcinoma, and drug-induced liver injury (DILI). However, these associations were primarily based upon population differences between the disease state and the controls. Thus, little is known about the variability and subsequent variance components of circulating miR-122 in healthy humans, which has implications for the practical use of the biomarker clinically. To address this, we set out to perform variance components analysis of miR-122 in a cohort of 40 healthy volunteers. Employing a quantitative real-time polymerase chain reaction (qRT-PCR) assay to detect miR-122 and other circulating miRNAs in human serum, the relative expression of miR-122 was determined using two different normalization approaches: to the mean expression of a panel of several endogenous miRNAs identified using an adaptive algorithm (miRA-Norm) and to the expression of an exogenous miRNA control (Caenorhabditis elegans miR-39). Results from a longitudinal study in healthy volunteers (N = 40) demonstrated high variability with 117- and 111-fold 95% confidence reference interval, respectively. This high variability of miR-122 in serum appeared to be due in part to ethnicity, as 95% confidence reference intervals were approximately three-fold lower in volunteers that identified as Caucasian relative to those that identified as Non-Caucasian. Variance analysis revealed equivalent contributions of intra- and inter-donor variability to miR-122. Surprisingly, miR-122 exhibited the highest variability compared to other 36 abundant miRNAs in circulation; the next variable miRNA, miR-133a, demonstrated a 45- to 62-fold reference interval depending on normalization approaches. In contrast, alanine aminotransferase (ALT) activity levels in this population exhibited a 5-fold total variance, with 80% of this variance due to inter-donor sources. In conclusion, miR-122 demonstrated higher than expected variability in serum from healthy volunteers, which has implications for its potential utility as a prospective biomarker of liver damage or injury.

Structure- and Ligand-Based Discovery of Chromane Arylsulfonamide Nav1.7 Inhibitors for the Treatment of Chronic Pain.

Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Nav1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response in a Nav1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.

Interrogation of transcriptomic changes associated with drug-induced hepatic sinusoidal dilatation in colorectal cancer.

Drug-related sinusoidal dilatation (SD) is a common form of hepatotoxicity associated with oxaliplatin-based chemotherapy used prior to resection of colorectal liver metastases (CRLM). Recently, hepatic SD has also been associated with anti-delta like 4 (DLL4) cancer therapies targeting the NOTCH pathway. To investigate the hypothesis that NOTCH signaling plays an important role in drug-induced SD, gene expression changes were examined in livers from anti-DLL4 and oxaliplatin-induced SD in non-human primate (NHP) and patients, respectively. Putative mechanistic biomarkers of bevacizumab (bev)-mediated protection against oxaliplatin-induced SD were also investigated. RNA was extracted from whole liver sections or centrilobular regions by laser-capture microdissection (LCM) obtained from NHP administered anti-DLL4 fragment antigen-binding (F(ab')2 or patients with CRLM receiving oxaliplatin-based chemotherapy with or without bev. mRNA expression was quantified using high-throughput real-time quantitative PCR. Significance analysis was used to identify genes with differential expression patterns (false discovery rate (FDR) < 0.05). Eleven (CCL2, CCND1, EFNB2, ERG, ICAM1, IL16, LFNG, NOTCH1, NOTCH4, PRDX1, and TGFB1) and six (CDH5, EFNB2, HES1, IL16, MIK67, HES1 and VWF) candidate genes were differentially expressed in the liver of anti-DLL4- and oxaliplatin-induced SD, respectively. Addition of bev to oxaliplatin-based chemotherapy resulted in differential changes in hepatic CDH5, HEY1, IL16, JAG1, MMP9, NOTCH4 and TIMP1 expression. This work implicates NOTCH and IL16 pathways in the pathogenesis of drug-induced SD and further explains the hepato-protective effect of bev in oxaliplatin-induced SD observed in CRLM patients.

Utility of spherical human liver microtissues for prediction of clinical drug-induced liver injury.

Drug-induced liver injury (DILI) continues to be a major source of clinical attrition, precautionary warnings, and post-market withdrawal of drugs. Accordingly, there is a need for more predictive tools to assess hepatotoxicity risk in drug discovery. Three-dimensional (3D) spheroid hepatic cultures have emerged as promising tools to assess mechanisms of hepatotoxicity, as they demonstrate enhanced liver phenotype, metabolic activity, and stability in culture not attainable with conventional two-dimensional hepatic models. Increased sensitivity of these models to drug-induced cytotoxicity has been demonstrated with relatively small panels of hepatotoxicants. However, a comprehensive evaluation of these models is lacking. Here, the predictive value of 3D human liver microtissues (hLiMT) to identify known hepatotoxicants using a panel of 110 drugs with and without clinical DILI has been assessed in comparison to plated two-dimensional primary human hepatocytes (PHH). Compounds were treated long-term (14 days) in hLiMT and acutely (2 days) in PHH to assess drug-induced cytotoxicity over an 8-point concentration range to generate IC50 values. Regardless of comparing IC50 values or exposure-corrected margin of safety values, hLiMT demonstrated increased sensitivity in identifying known hepatotoxicants than PHH, while specificity was consistent across both assays. In addition, hLiMT out performed PHH in correctly classifying hepatotoxicants from different pharmacological classes of molecules. The hLiMT demonstrated sufficient capability to warrant exploratory liver injury biomarker investigation (miR-122, HMGB1, α-GST) in the cell-culture media. Taken together, this study represents the most comprehensive evaluation of 3D spheroid hepatic cultures up to now and supports their utility for hepatotoxicity risk assessment in drug discovery.

Histone demethylases UTX and JMJD3 are required for NKT cell development in mice.

BACKGROUND: Natural killer (NK)T cells and conventional T cells share phenotypic characteristic however they differ in transcription factor requirements and functional properties. The role of histone modifying enzymes in conventional T cell development has been extensively studied, little is known about the function of enzymes regulating histone methylation in NKT cells. RESULTS: We show that conditional deletion of histone demethylases UTX and JMJD3 by CD4-Cre leads to near complete loss of liver NKT cells, while conventional T cells are less affected. Loss of NKT cells is cell intrinsic and not due to an insufficient selection environment. The absence of NKT cells in UTX/JMJD3-deficient mice protects mice from concanavalin A-induced liver injury, a model of NKT-mediated hepatitis. GO-analysis of RNA-seq data indicates that cell cycle genes are downregulated in UTX/JMJD3-deleted NKT progenitors, and suggest that failed expansion may account for some of the cellular deficiency. The phenotype appears to be demethylase-dependent, because UTY, a homolog of UTX that lacks catalytic function, is not sufficient to restore their development and removal of H3K27me3 by deletion of EZH2 partially rescues the defect. CONCLUSIONS: NKT cell development and gene expression is sensitive to proper regulation of H3K27 methylation. The H3K27me3 demethylase enzymes, in particular UTX, promote NKT cell development, and are required for effective NKT function.

Why Does the Intestine Lack Basolateral Efflux Transporters for Cationic Compounds? A Provocative Hypothesis.

Transport proteins in intestinal epithelial cells facilitate absorption of nutrients/compounds that are organic anions, cations, and zwitterions. For two decades, we have studied intestinal absorption and transport of hydrophilic ionic compounds, with specific focus on transport properties of organic cations and their interactions with intestinal transporters and tight junction proteins. Our data reveal how complex interactions between a compound and transporters in intestinal apical/basolateral (BL) membranes and tight junction proteins define oral absorption, and that the BL membrane lacks an efflux transporter that can transport positively charged compounds. Based on our investigations of transport mechanisms of zwitterionic, anionic, and cationic compounds, we postulate that physicochemical properties of these ionic species, in relation to the intestinal micro pH environment, have exerted evolutionary pressure for development of transporters that can handle apical uptake/efflux of all 3 ionic species and BL efflux of anions and zwitterions, but such evolutionary pressure is lacking for development of a BL efflux transporter for cationic compounds. This review provides an overview of intestinal uptake/efflux transporters and describes our studies on intestinal transport of cationic, anionic, and zwitterionic drugs that led to hypothesize that there are no cation-selective BL efflux transporters in the intestine.

Drug-induced allergic hepatitis develops in mice when myeloid-derived suppressor cells are depleted prior to halothane treatment.

UNLABELLED: Clinical evidence suggests that many cases of serious idiosyncratic drug-induced liver injury are mediated by the adaptive immune system in response to hepatic drug-protein adducts, also referred to as "drug-induced allergic hepatitis"; but detailed mechanistic proof has remained elusive due to the lack of animal models. We have hypothesized that drug-induced allergic hepatitis is as rare in animals as it is in humans due at least in part to the tolerogenic nature of the liver. We provide evidence that immune tolerance can be overcome in a murine model of halothane-induced liver injury initiated by trifluoroacetylated protein adducts of halothane formed in the liver. Twenty-four hours after female Balb/cJ mice were initially treated with halothane, perivenous necrosis and an infiltration of CD11b(+) Gr-1(high) cells were observed in the liver. Further study revealed a subpopulation of myeloid-derived suppressor cells within the CD11b(+) Gr-1(high) cell fraction that inhibited the proliferation of both CD4(+) and CD8(+) T cells. When CD11b(+) Gr-1(high) cells were depleted from the liver with Gr-1 antibody treatment, enhanced liver injury was observed at 9 days after halothane rechallenge. Toxicity was associated with increased serum levels of interleukin-4 and immunoglobulins G1 and E directed against hepatic trifluoroacetylated protein adducts, as well as increased hepatic infiltration of eosinophils and CD4(+) T cells, all features of an allergic reaction. When hepatic CD4(+) T cells were depleted 5 days after halothane rechallenge, trifluoroacetylated protein adduct-specific serum immunoglobulin and hepatotoxicity were reduced. CONCLUSION: Our data provide a rational approach for developing animal models of drug-induced allergic hepatitis mediated by the adaptive immune system and suggest that impaired liver tolerance may predispose patients to this disease.

Four cation-selective transporters contribute to apical uptake and accumulation of metformin in Caco-2 cell monolayers.

Metformin is the frontline therapy for type II diabetes mellitus. The oral bioavailability of metformin is unexpectedly high, between 40 and 60%, given its hydrophilicity and positive charge at all physiologic pH values. Previous studies in Caco-2 cell monolayers, a cellular model of the human intestinal epithelium, showed that during absorptive transport metformin is taken up into the cells via transporters in the apical (AP) membrane; however, predominant transport to the basolateral (BL) side occurs via the paracellular route because intracellular metformin cannot egress across the BL membrane. Furthermore, these studies have suggested that the AP transporters can contribute to intestinal accumulation and absorption of metformin. Transporter-specific inhibitors as well as a novel approach involving a cocktail of transporter inhibitors with overlapping selectivity were used to identify the AP transporters that mediate metformin uptake in Caco-2 cell monolayers; furthermore, the relative contributions of these transporters in metformin AP uptake were also determined. The organic cation transporter 1, plasma membrane monoamine transporter (PMAT), serotonin reuptake transporter, and choline high-affinity transporter contributed to approximately 25%, 20%, 20%, and 15%, respectively, of the AP uptake of metformin. PMAT-knockdown Caco-2 cells were constructed to confirm the contribution of PMAT in metformin AP uptake because a PMAT-selective inhibitor is not available. The identification of four intestinal transporters that contribute to AP uptake and potentially intestinal absorption of metformin is a significant novel finding that can influence our understanding of metformin pharmacology and intestinal drug-drug interactions involving this highly prescribed drug.

Thymic stromal lymphopoietin and interleukin-4 mediate the pathogenesis of halothane-induced liver injury in mice.

UNLABELLED: Liver eosinophilia has been associated with incidences of drug-induced liver injury (DILI) for more than 50 years, although its role in this disease has remained largely unknown. In this regard, it was recently shown that eosinophils played a pathogenic role in a mouse model of halothane-induced liver injury (HILI). However, the signaling events that drove hepatic expression of eosinophil-associated chemokines, eotaxins, eosinophil infiltration, and subsequent HILI were unclear. We now provide evidence implicating hepatic epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) and type 2 immunity, in particular, interleukin-4 (IL-4) production, in mediating hepatic eosinophilia and injury during HILI. TSLP was constitutively expressed by mouse hepatocytes and increased during HILI. Moreover, the severity of HILI was reduced in mice deficient in either the TSLP receptor (TSLPR) or IL-4 and was accompanied by decreases in serum levels of eotaxins and hepatic eosinophilia. Similarly, concanavalin A-induced liver injury, where type 2 cytokines and eosinophils play a significant role in its pathogenesis, was also reduced in TSLPR-deficient mice. Studies in vitro revealed that mouse and human hepatocytes produce TSLP and eotaxins in response to treatment with combinations of IL-4 and proinflammatory cytokines IL-1ß and tumor necrosis factor alpha. CONCLUSION: This report provides the first evidence implicating roles for hepatic TSLP signaling, type 2 immunity, and eosinophilia in mediating liver injury caused by a drug.

Organic cation transporter 1 (OCT1/mOct1) is localized in the apical membrane of Caco-2 cell monolayers and enterocytes.

Organic cation transporters (OCTs) are members of the solute carrier 22 family of transporter proteins that are involved in absorption, distribution, and excretion of organic cations. OCT3 is localized in the apical (AP) membrane of enterocytes, but the literature is ambiguous about OCT1 (mOct1) localization, with some evidence suggesting a basolateral (BL) localization in human and mouse enterocytes. This is contrary to our preliminary findings showing AP localization of OCT1 in Caco-2 cell monolayers, an established model of human intestinal epithelium. Therefore, this study aims at determining the localization of OCT1 (mOct1) in Caco-2 cells, and human and mouse enterocytes. Functional studies using OCT1-specific substrate pentamidine showed transporter-mediated AP but not BL uptake in Caco-2 cells and human and mouse intestinal tissues. OCT1 inhibition decreased AP uptake of pentamidine by ∼50% in all three systems with no effect on BL uptake. A short hairpin RNA-mediated OCT1 knockdown in Caco-2 cells decreased AP uptake of pentamidine by ∼50% but did not alter BL uptake. Immunostaining and confocal microscopy in all three systems confirmed AP localization of OCT1 (mOct1). Our studies unequivocally show AP membrane localization of OCT1 (mOct1) in Caco-2 cells and human and mouse intestine. These results are highly significant as they will require reinterpretation of previous drug disposition and drug-drug interaction studies where conclusions were drawn assuming BL localization of OCT1 in enterocytes. Most importantly, these results will require revision of the regulatory guidance for industry in the United States and elsewhere because it has stated that OCT1 is basolaterally localized in enterocytes.

Sorafenib hepatobiliary disposition: mechanisms of hepatic uptake and disposition of generated metabolites.

Sorafenib is an orally active tyrosine kinase inhibitor used in the treatment of renal and hepatocellular carcinoma. This study was designed to establish whether transport proteins are involved in the hepatic uptake of sorafenib and to determine the extent of biliary excretion of sorafenib and its metabolites in human hepatocytes. Initial uptake was assessed in freshly isolated, suspended human hepatocytes in the presence of inhibitors and modulators. [(14)C]Sorafenib (1 µM) uptake at 4°C was reduced by about 61-63% of the uptake at 37°C, suggesting a high degree of passive diffusion. Hepatocyte uptake of [(14)C]sorafenib was not Na(+) dependent or influenced by the organic anion transporter 2 inhibitor ketoprofen. However, initial [(14)C]sorafenib hepatocyte uptake was reduced by 46 and 30% compared with control values in the presence of the organic anion transporting polypeptide inhibitor rifamycin SV and the organic cation transporter (OCT) inhibitor decynium 22, respectively. [(14)C]Sorafenib (0.5-5 µM) uptake was significantly higher in hOCT1-transfected Chinese hamster ovary cells compared with mock cells, and inhibited by the general OCT inhibitor, 1-methyl-4-phenylpryidinium. OCT1-mediated uptake was saturable with a Michaelis-Menten constant of 3.80 ± 2.53 µM and a V(max) of 116 ± 42 pmol/mg/min. The biliary excretion index and in vitro biliary clearance of sorafenib (1 µM) in sandwich-cultured human hepatocytes were low (∼11% and 11 ml/min/kg, respectively). Results suggest that sorafenib uptake in human hepatocytes occurs via passive diffusion, by OCT1, and by organic anion transporting polypeptide(s). Sorafenib undergoes modest biliary excretion, predominantly as a glucuronide conjugate(s).

Cigarette smoke exposure greatly increases alcohol consumption in adolescent C57BL/6 mice.

BACKGROUND: Alcohol and tobacco are often used together, and alcoholism is much more common among smokers compared with nonsmokers. Studies in humans suggest that nicotine (an active ingredient in cigarette smoke) can increase the consumption of alcohol. Research on rats and mice demonstrated mixed results; some studies report that nicotine increases alcohol consumption, while others show a decrease in drinking. Because cigarette smoke includes many other chemicals, these also may play a significant role in alcohol consumption. For example, 2 of these other constituents, monoamine oxidase inhibitors and acetaldehyde, increase alcohol tolerance and/or alcohol consumption in rodents. This study was designed to investigate how cigarette smoke from tobacco may modify self-administration of alcohol in adolescent C57BL/6 mice, a critical time when adolescent humans begin abusing drugs. METHODS: C57BL/6 male mice (4 to 5 weeks old) were acclimated for 3 weeks to consume a 10% (w/v) alcohol solution during a 2-hour daily access in the dark. Subsequently, half the animals were exposed to cigarette smoke for 6 h/d for 16 days. The remaining animals (control) were placed in a smoke-free adjacent chamber. Immediately following the 6-hour period in the chambers, the control and smoke-exposed mice were given access to the 10% alcohol solution for 2 hours. RESULTS: Animals exposed to cigarette smoke for 6 h/d consumed approximately 3- to 5-fold more alcohol than the mice in the control group throughout the 16-day study. The mice in the smoke group had a blood alcohol concentration that was nearly 4-fold that of the control mice. CONCLUSIONS: Cigarette smoke increases alcohol consumption several fold higher than reported studies using nicotine treatment alone in adolescent rodents. Thus, this model should be useful to determine the roles of other bioactive components in cigarette smoke that may be important in the high co-abuse of smoking and alcohol consumption.

Eosinophils mediate the pathogenesis of halothane-induced liver injury in mice.

UNLABELLED: Drug-induced liver injury (DILI) is a major health issue, as it remains difficult to predict which new drugs will cause injury and who will be susceptible to this disease. This is due in part to the lack of animal models and knowledge of susceptibility factors that predispose individuals to DILI. In this regard, liver eosinophilia has often been associated with DILI, although its role remains unclear. We decided to investigate this problem in a murine model of halothane-induced liver injury (HILI). When female Balb/cJ mice were administered halothane, eosinophils were detected by flow cytometry in the liver within 12 hours and increased thereafter proportionally to liver damage. Chemokines, eotaxin-1 (CCL11) and eotaxin-2 (CCL24), which are known to attract eosinophils, increased in response to halothane treatment. The severity of HILI was decreased significantly when the study was repeated in wildtype mice made deficient in eosinophils with a depleting antibody and in eosinophil lineage-ablated ΔdblGata(-/-) mice. Moreover, depletion of neutrophils by pretreating animals with Gr-1 antibody prior to halothane administration failed to reduce the severity of HILI at antibody concentrations that did not affect hepatic eosinophils. Immunohistochemical staining for the granule protein, major basic protein, revealed that eosinophils accumulated exclusively around areas of hepatocellular necrosis. CONCLUSION: Our findings indicate that eosinophils have a pathologic role in HILI in mice and suggest that they may contribute similarly in many clinical cases of DILI.

Endogenous interleukin-4 regulates glutathione synthesis following acetaminophen-induced liver injury in mice.

In a recent study, we reported that interleukin (IL)-4 had a protective role against acetaminophen (APAP)-induced liver injury (AILI), although the mechanism of protection was unclear. Here, we carried out more detailed investigations and have shown that one way IL-4 may control the severity of AILI is by regulating glutathione (GSH) synthesis. In the present studies, the protective role of IL-4 in AILI was established definitively by showing that C57BL/6J mice made deficient in IL-4 genetically (IL-4(-/-)) or by depletion with an antibody, were more susceptible to AILI than mice not depleted of IL-4. The increased susceptibility of IL-4(-/-) mice was not due to elevated levels of hepatic APAP-protein adducts but was associated with a prolonged reduction in hepatic GSH that was attributed to decreased gene expression of γ-glutamylcysteine ligase (γ-GCL). Moreover, administration of recombinant IL-4 to IL-4(-/-) mice postacetaminophen treatment diminished the severity of liver injury and increased γ-GCL and GSH levels. We also report that the prolonged reduction of GSH in APAP-treated IL-4(-/-) mice appeared to contribute toward increased liver injury by causing a sustained activation of c-Jun-N-terminal kinase (JNK) since levels of phosphorylated JNK remained significantly higher in the IL-4(-/-) mice up to 24 h after APAP treatment. Overall, these results show for the first time that IL-4 has a role in regulating the synthesis of GSH in the liver under conditions of cellular stress. This mechanism appears to be responsible at least in part for the protective role of IL-4 against AILI in mice and may have a similar role not only in AILI in humans but also in pathologies of the liver caused by other drugs and etiologies.

Functional adaptation of the N-methyl-D-aspartate receptor to inhibition by ethanol is modulated by striatal-enriched protein tyrosine phosphatase and p38 mitogen-activated protein kinase.

The hippocampal N-methyl-D-aspartate receptor (NMDAR) activity plays important roles in cognition and is a major substrate for ethanol-induced memory dysfunction. This receptor is a glutamate-gated ion channel, which is composed of NR1 and NR2 subunits in various brain areas. Although homomeric NR1 subunits form an active ion channel that conducts Na⁺ and Ca²âº currents, the incorporation of NR2 subunits allows this channel to be modulated by the Src family of kinases, phosphatases, and by simple molecules such as ethanol. We have found that short-term ethanol application inhibits the NMDAR activity via striatal enriched protein tyrosine phosphatase (STEP)-regulated mechanisms. The genetic deletion of the active form of STEP, STEP61, leads to marked attenuation of ethanol inhibition of NMDAR currents. In addition, STEP61 negatively regulates Fyn and p38 mitogen-activated protein kinase (MAPK), and these proteins are members of the NMDAR super molecular complex. Here we demonstrate, using whole-cell electrophysiological recording, Western blot analysis, and pharmacological manipulations, that neurons exposed to a 3-h, 45 mM ethanol treatment develop an adaptive attenuation of short-term ethanol inhibition of NMDAR currents in brain slices. Our results suggest that this adaptation of NMDAR responses is associated with a partial inactivation of STEP61, an activation of p38 MAPK, and a requirement for NR2B activity. Together, these data indicate that altered STEP61 and p38 MAPK signaling contribute to the modulation of ethanol inhibition of NMDARs in brain neurons.