Duloxetine (DLX) is a dual serotonin and norepinephrine reuptake inhibitor, widely used for the treatment of major depressive disorder. Although DLX has shown good efficacy and safety, serious adverse effects (e.g., liver injury) have been reported. The mechanisms associated with DLX-induced toxicity remain elusive. Drug metabolism plays critical roles in drug safety and efficacy. However, the metabolic profile of DLX in mice is not available, although mice serve as commonly used animal models for mechanistic studies of drug-induced adverse effects. Our study revealed 39 DLX metabolites in human/mouse liver microsomes and mice. Of note, 13 metabolites are novel, including five N -acetyl cysteine adducts and one reduced glutathione (GSH) adduct associated with DLX. Additionally, the species differences of certain metabolites were observed between human and mouse liver microsomes. CYP1A2 and CYP2D6 are primary enzymes responsible for the formation of DLX metabolites in liver microsomes, including DLX-GSH adducts. In summary, a total of 39 DLX metabolites were identified, and species differences were noticed in vitro. The roles of CYP450s in DLX metabolite formation were also verified using human recombinant cytochrome P450 (P450) enzymes and corresponding chemical inhibitors. Further studies are warranted to address the exact role of DLX metabolism in its adverse effects in vitro (e.g., human primary hepatocytes) and in vivo (e.g., Cyp1a2-null mice). SIGNIFICANCE STATEMENT This current study systematically investigated Duloxetine (DLX) metabolism and bioactivation in liver microsomes and mice. This study provided a global view of DLX metabolism and bioactivation in liver microsomes and mice, which are very valuable to further elucidate the mechanistic study of DLX-related adverse effects and drug-drug interaction from metabolic aspects.

No abstract

Sepsis is a multifaceted host response to infection that dramatically affects patient outcomes and the cost of health care. Animal models are necessary to replicate the complexity and heterogeneity of clinical sepsis. However, these models entail a high risk of pain and distress due to tissue trauma, inflammation, endotoxin-mediated hyperalgesia, and other mechanisms. Several recent studies and initiatives address the need to improve the welfare of animals through analgesics and standardize the models used in preclinical sepsis research. Ultimately, the goal is to provide high-fidelity, humane animal models that better replicate the clinical course of sepsis, to provide more effective translation and advance therapeutic discovery. The purpose of this review is to discuss the current understanding of the roles of pain and analgesia in rodent models of sepsis. The current definitions of sepsis along with an overview of pain in human sepsis are described. Finally, welfare concerns associated with animal models of sepsis and the most recent considerations for relief of pain and distress are reviewed.

Regulatory guidelines mandate housing for laboratory mice at temperatures below their thermoneutral zone, creating chronic cold stress. However, increases in housing temperature could alter immune responses. We hypothesized housing mice at temperatures within their thermoneutral zone would improve sepsis survival and alter immune responses. Male C57BL/6 mice were housed at 22°C or 30°C after cecal ligation and puncture (CLP) for 10 days. Survival of mice housed at 30°C (78%) after CLP was significantly increased compared with mice housed at 22°C (40%). Experimental groups were repeated with mice euthanized at 0, 12, 24, and 48 h post-surgery to examine select immune parameters. Raising housing temperature minimally altered systemic, peritoneal, or splenic cell counts. However, IL-6 levels in plasma and peritoneal lavage fluid were significantly lower at 12 h post-surgery in mice housed at 30°C compared with 22°C. Bacterial colony counts from peritoneal lavage fluid were significantly lower in mice housed at 30°C and in vivo studies suggested this was the result of increased phagocytosis by neutrophils. As previously demonstrated, adoptive transfer of fibrocytes significantly increased sepsis survival compared with saline at 22°C. However, there was no additive effect when adoptive transfer was performed at 30°C. Overall, the results demonstrated that thermoneutral housing improves survival after CLP by increasing local phagocytic activity and technical revisions may be necessary to standardize the severity of the model across different housing temperatures. These findings stress the pronounced impact housing temperature has on the CLP model and the importance of reporting housing temperature.

No abstract

No abstract

ID 24646 Poster Board 20Duloxetine (DLX), a dual serotonin and norepinephrine reuptake inhibitor for the treatment of major depressive disorder, is mainly metabolized by CYP1A2 and CYP2D6 in human. Despite DLX has a good tolerance for most patients, the cases with serious liver injury have been reported. Here, we investigated the roles of P450-mediated metabolism in the DLX hepatotoxicity in vitro and in vivo. We found that DLX is less toxic to CYP1A2-or CYP2D6-overexpressed HepG2 cells compared to their counterpart HepG2 cells transduced with empty vector, indicating that P450-mediated metabolism attenuates DLX cytotoxicity to HepG2. The CYP1A2 (a-naphthoflavone and fluvoxamine) or CYP2D6 inhibitors (paroxetine, fluoxetine, and quinidine) significantly enhanced the toxicity of DLX to human primary hepatocytes. In Cyp1a2-knockout (Cyp1a2-KO) and counterpart wild-type (WT) mice, 50 mg/ kg or 100 mg/kg (p.o.) of DLX for consecutive 5 days did not significantly increase the plasma ALT and AST levels compared with the vehicle group in both WT and Cyp1a2-KO mice, suggesting that Cyp1a2 is not a fundamental contributor to DLX detoxification at least in mice. However, DLX and a mouse Cyp2d inhibitor propranolol in both WT and Cyp1a2-KO mice lead to severe liver injury. The ALT levels increased for 2.9-and 8.7-fold in 50 mg/kg and 100 mg/kg groups, respectively in WT mice co-treated with propranolol. Similar increased folds of ALT were also observed in Cyp1a2-KO mice. H&E staining of mouse livers also confirmed the liver toxicity, presented as cell necrosis, in 100 mg/kg group in both WT and Cyp1a2-KO mice with the co-treatment of Cyp2d inhibitor.In summary, our findings suggests that impairing P450-mediated metabolism exacerbates DLX hepatoxicity in vitro and in vivo. Further studies are warranted to verify the roles of CYP1A2 and CYP2D6 in DLX liver toxicity using liver humanized mice and their inhibitors.

Pexidartinib (PLX, TURALIOTM) is a novel small molecule tyrosine‐kinase inhibitor with highly selective activity against colony‐stimulating factor‐1 receptor. PLX was developed for the treatment of symptomatic tenosynovial giant cell tumor in adult patients and approved by the US FDA in 2019. Despite its effectivity, frequently reported clinical cases with severe hepatotoxicity led to a black‐box warning issued to PLX by the FDA. However, the mechanisms of PLX‐induced hepatotoxicity remain largely unknown and the roles of PLX metabolism in its toxicity have not been investigated. Using liquid chromatography/mass spectrometry‐based metabolomic approaches, this study revealed 36 PLX metabolites in human liver microsome (HLM) and mouse liver microsome (MLM), including characterized 11 glutathione (GSH) ‐PLX adducts and 3 methoxyamine‐trapped aldehydes (hydrazones). Using recombinant cytochrome P450 enzymes, CYP3A4 and CYP3A5 were identified as the primary enzymes contributing to the formation of major metabolites, GSH‐PLX adducts, and methoxyamine‐trapped hydrazones. Their formation was significantly suppressed by the CYP3A inhibitor ketoconazole and in the liver S9 fraction of Cyp3a‐null mice, further confirming the role of CYP3A in PLX Phase I metabolism. More interestingly, we found that PLX is less toxic to CYP3A4‐ or CYP3A5‐overexpressed HepG2 cells compared to their counterpart HepG2 cells transduced with empty vector, indicating that CYP3A‐mediated metabolism attenuates PLX cytotoxicity to HepG2. In summary, 36 PLX metabolites including 11 GSH adducts and 3 hydrazones were identified in HLM and MLM. CYP3A4 and CYP3A5 are responsible for the Phase I biotransformation of PLX and alleviate its cytotoxicity in HepG2. Further studies are warranted to elucidate the role of metabolism in PLX hepatotoxicity and the mechanism associated with PLX‐induced liver injury.