Timothy P. Reilly

Role of neutrophils in a mouse model of halothane-induced liver injury

Drug‐induced liver injury (DILI) is a major safety concern in drug development. Its prediction and prevention have been hindered by limited knowledge of the underlying mechanisms, in part the result of a lack of animal models. We developed a mouse model of halothane‐induced liver injury and characterized the mechanisms accounting for tissue damage. Female and male Balb/c, DBA/1, and C57BL/6J mice were injected intraperitoneally with halothane. Serum levels of alanine aminotransferase and histology were evaluated to determine liver injury. Balb/c mice were found to be the most susceptible strain, followed by DBA/1, with no significant hepatotoxicity observed in C57BL/6J mice. Female Balb/c and DBA/1 mice developed more severe liver damage compared with their male counterparts. Bioactivation of halothane occurred similarly in all three strains based on detection of liver proteins adducted by the reactive metabolite. Mechanistic investigations revealed that hepatic message levels of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β); IL‐6, and IL‐8 were significantly higher in halothane‐treated Balb/c mice compared to DBA/1 and C57BL/6J mice. Moreover, a higher number of neutrophils were recruited into the liver of Balb/c mice upon halothane treatment compared with DBA/1, with no obvious neutrophil infiltration detected in C57BL/6J mice. Neutrophil depletion experiments demonstrated a crucial role for these cells in the development of halothane‐induced liver injury. The halothane‐initiated hepatotoxicity and innate immune response‐mediated escalation of tissue damage are consistent with events that occur in many cases of DILI. In conclusion, our model provides a platform for elucidating strain‐based and gender‐based susceptibility factors in DILI development. (HEPATOLOGY 2006;44:1421–1431.)

Role of immune reactions in drug-induced liver injury (DILI)

Although some drugs cause drug-induced liver injury (DILI) through direct damage to hepatocytes or intereference with bile secretion, others cause delayed, often idiosyncratic, DILI with clinical features, such as mild lymphocytic infiltrate, that are reminiscent of allergic reactions involving activation of the adaptive immune system. Even in cases of direct drug-induced hepatotoxicity, infiltration of inflammatory cells into the liver is often observed, suggesting a role for the innate immune system (e.g., neutrophils, macrophages, and so on). Therefore, a variety of hypotheses for the pathogenesis of DILI center around a pathogenic role of drug- (or drug-metabolite–) specific adaptive immune cells, as well as hepatic-injury–induced innate immune responses in the development, progression, and/or resolution of DILI.

Impact of Compliance with a Care Bundle on Acute Kidney Injury Outcomes: A Prospective Observational Study.

Background A recent report has highlighted suboptimal standards of care for acute kidney injury (AKI) patients in England. The objective of this study was to ascertain if improvement in basic standard of care by implementing a care bundle (CB) with interruptive alert improved outcomes in patients with AKI. Methods An AKI CB linked to electronic recognition of AKI, coupled with an interruptive alert, was introduced to improve basic care delivered to patients with AKI. Outcomes were compared in patients who had the CB completed within 24 hours (early CB group) versus those who didn’t have the CB completed or had it completed after 24 hours. Results In the 11-month period, 2297 patients had 2500 AKI episodes, with 1209 and 1291 episodes occurring before and after implementation of the AKI CB with interruptive alert, respectively. The CB was completed within 24 hours in 306 (12.2%) of AKI episodes. In-hospital case-fatality was significantly lower in the early CB group (18% versus 23.1%, p 0.046). Progression to higher AKI stages was lower in the early CB group (3.9% vs. 8.1%, p 0.01). In multivariate analysis, patients in the early CB group had lower odds of death at discharge (0.641; 95% CI 0.46, 0.891), 30 days (0.707; 95% CI 0.527, 0.950), 60 days (0.704; 95% CI 0.526, 0.941) and after a median of 134 days (0.771; 95% CI 0.62, 0.958). Conclusions Compliance with AKI CB was associated with a decrease in case-fatality and reduced progression to higher AKI stage. Further interventions are required to improve utilization of the CB.

Targeting the kidney and glucose excretion with dapagliflozin: preclinical and clinical evidence for SGLT2 inhibition as a new option for treatment of type 2 diabetes mellitus

Sodium-glucose cotransporter-2 (SGLT2) inhibitors are a novel class of glucuretic, antihyperglycemic drugs that target the process of renal glucose reabsorption and induce glucuresis independently of insulin secretion or action. In patients with type 2 diabetes mellitus, SGLT2 inhibitors have been found to consistently reduce measures of hyperglycemia, including hemoglobin A1c, fasting plasma glucose, and postprandial glucose, throughout the continuum of disease. By inducing the renal excretion of glucose and its associated calories, SGLT2 inhibitors reduce weight and have the potential to be disease modifying by addressing the caloric excess that is believed to be one of the root causes of type 2 diabetes mellitus. Additional benefits, including the possibility for combination with insulin-dependent antihyperglycemic drugs, a low potential for hypoglycemia, and the ability to reduce blood pressure, were anticipated from the novel mechanism of action and have been demonstrated in clinical studies. Mechanism-related risks include an increased incidence of urinary tract and genital infections and the possibility of over-diuresis in volume-sensitive patients. Taken together, the results of Phase III clinical studies generally point to a positive benefit-risk ratio across the continuum of diabetes patients. To date, data on dapagliflozin, a selective SGLT2 inhibitor in development, demonstrate that the kidney is an efficacious and safe target for therapy, and that SGLT2 inhibition may have benefits for patients with type 2 diabetes mellitus beyond glycemic control.

Mice Lacking Natural Killer T Cells Are More Susceptible to Metabolic Alterations following High Fat Diet Feeding

Current estimates suggest that over one-third of the adult population has metabolic syndrome and three-fourths of the obese population has non-alcoholic fatty liver disease (NAFLD). Inflammation in metabolic tissues has emerged as a universal feature of obesity and its co-morbidities, including NAFLD. Natural Killer T (NKT) cells are a subset of innate immune cells that abundantly reside within the liver and are readily activated by lipid antigens. There is general consensus that NKT cells are pivotal regulators of inflammation; however, disagreement exists as to whether NKT cells exert pathogenic or suppressive functions in obesity. Here we demonstrate that CD1d−/− mice, which lack NKT cells, were more susceptible to weight gain and fatty liver following high fat diet (HFD) feeding. Compared with their WT counterparts, CD1d−/− mice displayed increased adiposity and greater induction of inflammatory genes in the liver suggestive of the precursors of NAFLD. Calorimetry studies revealed a significant increase in food intake and trends toward decreased metabolic rate and activity in CD1d−/− mice compared with WT mice. Based on these findings, our results suggest that NKT cells play a regulatory role that helps to prevent diet-induced obesity and metabolic dysfunction and may play an important role in mechanisms governing cross-talk between metabolism and the immune system to regulate energy balance and liver health.

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

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‐1high cells were observed in the liver. Further study revealed a subpopulation of myeloid‐derived suppressor cells within the CD11b+Gr‐1high cell fraction that inhibited the proliferation of both CD4+ and CD8+ T cells. When CD11b+Gr‐1high 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. (Hepatology 2015;62:546–557

Occurrence of Spontaneous Pancreatic Lesions in Normal and Diabetic Rats: A Potential Confounding Factor in the Nonclinical Assessment of GLP-1–Based Therapies

Glucagon-like peptide 1–based therapies, collectively described as incretins, produce glycemic benefits in the treatment of type 2 diabetes. Recent publications raised concern for a potential increased risk of pancreatitis and pancreatic cancer with incretins based in part on findings from a small number of rodents. However, extensive toxicology assessments in a substantial number of animals dosed up to 2 years at high multiples of human exposure do not support these concerns. We hypothesized that the lesions being attributed to incretins are commonly observed background findings and endeavored to characterize the incidence of spontaneous pancreatic lesions in three rat strains (Sprague-Dawley [S-D] rats, Zucker diabetic fatty [ZDF] rats, and rats expressing human islet amyloid polypeptide [HIP]; n = 36/group) on a normal or high-fat diet over 4 months. Pancreatic findings in all groups included focal exocrine degeneration, atrophy, inflammation, ductular cell proliferation, and/or observations in large pancreatic ducts similar to those described in the literature, with an incidence of exocrine atrophy/inflammation seen in S-D (42–72%), HIP (39%), and ZDF (6%) rats. These data indicate that the pancreatic findings attributed to incretins are common background findings, observed without drug treatment and independent of diet or glycemic status, suggesting a need to exercise caution when interpreting the relevance of some recent reports regarding human safety.

Nonclinical Toxicology Assessments Support the Chronic Safety of Dapagliflozin, a First-in-Class Sodium-Glucose Cotransporter 2 Inhibitor

Dapagliflozin, a first-in-class, selective inhibitor of sodium-glucose cotransporter 2 (SGLT2), promotes urinary glucose excretion to reduce hyperglycemia for the treatment of type 2 diabetes. A series of nonclinical studies were undertaken to evaluate dapagliflozin in species where it was shown to have pharmacologic activity comparable with that in humans at doses that resulted in supratherapeutic exposures. In vitro screening (>300 targets; 10 μmol/L) indicated no significant off-target activities for dapagliflozin or its primary human metabolite. Once daily, orally administered dapagliflozin was evaluated in Sprague-Dawley rats (≤6 months) and in beagle dogs (≤1 year) at exposures >5000-fold those observed at the maximum recommended human clinical dose (MRHD; 10 mg). Anticipated, pharmacologically mediated effects of glucosuria, osmotic diuresis, and mild electrolyte loss were observed, but there were no adverse effects at clinically relevant exposures, including in the kidneys or urogenital tract. The SGLT2−/− mice, which show chronic glucosuria, and dapagliflozin-treated, wild-type mice exhibited similar safety profiles. In rats but not dogs, dapagliflozin at >2000-fold MRHD exposures resulted in tissue mineralization and trabecular bone accretion. Investigative studies suggested that the effect was not relevant to human safety, since it was partially related to off-target inhibition of SGLT1, which was observed only at high doses of dapagliflozin and resulted in intestinal glucose malabsorption and increased intestinal calcium absorption. The rigorous assessment of supra- and off-target dapagliflozin pharmacology in nonclinical species allowed for a thorough evaluation of potential toxicity, providing us with confidence in its safety in patients with diabetes.

Coagulation-Dependent Gene Expression and Liver Injury in Rats Given Lipopolysaccharide with Ranitidine but Not with Famotidine

In an animal model of drug idiosyncrasy, rats cotreated with nonhepatotoxic doses of lipopolysaccharide (LPS) and ranitidine (RAN) develop hepatocellular injury, whereas rats treated with LPS and famotidine (FAM) do not. The coagulation system and neutrophils (PMNs) are requisite mediators of LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression in LPS/RAN-treated rats requires coagulation system activation and that these changes are absent in rats given LPS and FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 × 106 endotoxin units/kg i.v.) or its vehicle, and then 1 h later, they were treated with heparin (3000 U/kg) or its vehicle. One hour thereafter, they were given RAN (30 mg/kg), FAM (6 mg/kg, a pharmacologically equiefficacious dose, or 28.8 mg/kg, an equimolar dose), or vehicle (i.v.). They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity, coagulation system activation, and liver gene expression (2 h only). Statistical filtering of gene array results and real-time polymerase chain reaction identified groups of genes expressed in LPS/RAN-treated rats but not LPS/FAM-treated rats that were either changed or unchanged by heparin administration. For example, LPS/RAN-induced mRNA expression of the inflammatory mediators interleukin-6, cyclooxygenase-2, and macrophage inflammatory protein-2 (MIP-2) was reduced by anticoagulation. Enhancement of serum MIP-2 and plasminogen activator inhibitor-1 concentrations in LPS/RAN-treated rats was prevented by anticoagulation. The results suggest cross-talk between hemostasis-induced gene expression and inflammation (e.g., PMN function) in the genesis of hepatocellular injury in LPS/RAN-treated rats. In contrast, neither the expression of such genes nor hepatocellular necrosis occurred in rats treated with LPS/FAM.

Targeting Renal Glucose Reabsorption for the Treatment of Type 2 Diabetes Mellitus Using the SGLT2 Inhibitor Dapagliflozin

Abstract Sodium–glucose co–transporter 2 (SGLT2) plays a key role in glucose homeostasis as the key transporter responsible for most renal glucose reabsorption in the proximal tubules of the kidney. Dapagliflozin is a potent, selective, and reversible inhibitor of SGLT2 that lowers blood glucose levels in an insulin–independent fashion. This novel agent has been studied extensively in patients with type 2 diabetes mellitus (T2DM). In these clinical trials, dapagliflozin significantly decreased glycated hemoglobin and fasting plasma glucose levels when administered alone or as add–on treatment in patients who were already receiving metformin, a sulfonylurea (glimepiride), pioglitazone, or insulin. Moreover, dapagliflozin decreased body weight when taken as monotherapy or in combination with metformin, a sulfonylurea, or insulin, and mitigated weight gain in patients receiving pioglitazone. Consistent with preclinical toxicology studies, dapagliflozin has a manageable adverse event profile that is largely predictable from its mechanism of action. While there are no clinically significant negative effects on renal function or electrolytes, dapagliflozin treatment is associated with increased frequencies of urinary tract infections and vulvovaginitis/balanitis. With a mechanism of action that is distinct from and complementary to that of existing antihyperglycemic therapies, dapagliflozin is an effective antihyperglycemic agent that is well tolerated and may enhance weight loss. As such, dapagliflozin promises to become an important adjunctive therapy for comprehensive treatment of T2DM.