Metabolism and Effects on Endogenous Metabolism of Paracetamol (Acetaminophen) in a Porcine Model of Liver Failure.
ABSTRACT: The metabolic fate, toxicity, and effects on endogenous metabolism of paracetamol (acetaminophen, APAP) in 22 female Landrace cross large white pigs were evaluated in a model of acute liver failure (ALF). Anesthetized pigs were initially dosed at 250?mg/kg via an oroduodenal tube with APAP serum concentrations maintained above 300?mg/l using maintenance doses of 0.5-4?g/h until ALF. Studies were undertaken to determine both the metabolic fate of APAP and its effects on the endogenous metabolic phenotype of ALF in using 1H NMR spectroscopy. Increased concentrations of citrate combined with pre-ALF increases in circulating lactate, pyruvate, and alanine in plasma suggest mitochondrial dysfunction and a switch in hepatic energy metabolism to glycolysis in response to APAP treatment. A specific liquid chromatography-tandem mass spectrometry assay was used to quantify APAP and metabolites. The major circulating and urinary metabolite of APAP was the phenolic glucuronide (APAP-G), followed by p-aminophenol glucuronide (PAP-G) formed from N-deacetylated APAP. The PAP produced by N-deacetylation was the likely cause of the methemoglobinemia and kidney toxicity observed in this, and previous, studies in the pig. The phenolic sulfate of APAP, and the glutathione-derived metabolites of the drug were only found as minor components (with the cysteinyl conjugate detected but not the mercapturate). Given its low sulfation, combined with significant capacity for N-deacetylation the pig may represent a poor translational model for toxicology studies for compounds undergoing significant metabolism by sulfation, or which contain amide bonds which when hydrolyzed to unmask an aniline lead to toxicity. However, the pig may provide a useful model where extensive amide hydrolysis is seen for drugs or environmental chemicals in humans, but not in, eg, the rat and dog which are the preclinical species normally employed for safety assessment.
Project description:Acetaminophen (APAP)-induced liver injury is the most common cause of acute liver failure (ALF) in the Western world. APAP toxicity progresses to multiorgan dysfunction and thus has broader whole-body implications. Importantly, greater 30-day mortality has been observed in liver transplant recipients following ALF due to APAP-related versus non-APAP-related causes. Reasons for this discrepancy have yet to be determined. Extrahepatic toxicities of APAP overdose may represent underappreciated and unaddressed comorbidities within this patient population. In the present study, rapid induction of apoptosis following APAP overdose was observed in the intestine, an organ that greatly influences the physiology of the liver. Strikingly, apoptotic cells appeared to be strictly restricted to the intestinal crypts. The use of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) reporter mice confirmed that the LGR5-positive (+) crypt base stem cells were disproportionately affected by APAP-induced cell death. Although the apoptotic cells were cleared within 24 hours after APAP treatment, potentially long-lived consequences on the intestine due to APAP exposure were indicated by prolonged deficits in gut barrier function. Moreover, small intestinal cell death was found to be independent of tumor necrosis factor receptor signaling and may represent a direct toxic insult to the intestine by exposure to high concentrations of APAP. Conclusion: APAP induces intestinal injury through a regulated process of apoptotic cell death that disproportionately affects LGR5+ stem cells. This work advances our understanding of the consequences of APAP toxicity in a novel organ that was not previously considered as a significant site of injury and thus presents potential new considerations for patient management.
Project description:Analyses of outcomes after acute liver failure (ALF) have typically included all ALF patients regardless of whether they were listed for liver transplantation (LT). We hypothesized that limiting analysis to listed patients might provide novel insights into factors associated with outcome, focusing attention on disease evolution after listing. Listed adult ALF patients enrolled in the US Acute Liver Failure Study Group registry between 2000 and 2013 were analyzed to determine baseline factors associated with 21-day outcomes after listing. We classified 617 patients (36% of overall ALF group) by 3-week outcome after study admission: 117 were spontaneous survivors (SSs; survival without LT), 108 died without LT, and 392 underwent LT. Only 22% of N-acetyl-p-aminophenol (APAP) ALF patients were listed; however, this group of 173 patients demonstrated greater illness severity: higher coma grades and more patients requiring ventilator, vasopressor, or renal replacement therapy support. Only 62/173 (36%) of APAP patients received a graft versus 66% for drug-induced liver injury patients, 86% for autoimmune-related ALF, and 71% for hepatitis B-related ALF. APAP patients were more likely to die than non-APAP patients (24% versus 17%), and the median time to death was sooner (2 versus 4.5 days). Despite greater severity of illness, the listed APAP group still had a SS rate of 40% versus 11% for non-APAP causes (P?<?0.001). APAP outcomes evolve rapidly, mainly to SS or death. Patients with APAP ALF listed for LT had the highest death rate of any etiology, whereas more slowly evolving etiologies yielded higher LT rates and, consequently, fewer deaths. Decisions to list and transplant must be made early in all ALF patients, particularly in those with APAP ALF.
Project description:INTRODUCTION:Acetaminophen (paracetamol, APAP) is widely used as an analgesic and antipyretic drug in children and neonates. A number of enzymes contribute to the metabolism of acetaminophen, and genetic factors might be important to explain variability in acetaminophen metabolism among individuals. METHODS:The current investigation utilized a previously published parent-metabolite population pharmacokinetic model describing acetaminophen glucuronidation, sulfation, and oxidation to examine the potential role of genetic variability on the relevant metabolic pathways. Neonates were administered 30-min intravenous infusions of acetaminophen 15 mg/kg every 12 h (< 28 weeks' gestational age [GA]) or every 8 h (? 28 weeks GA) for 48 h. A total of 18 sequence variations (SVs) in UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), and cytochrome P450 (CYP) genes from 33 neonates (aged 1-26 days) were examined in a stepwise manner for an effect on the metabolic formation clearance of acetaminophen by glucuronidation (UGT), sulfation (SULT), and oxidation (CYP). The stepwise covariate modeling procedure was performed using NONMEM® version 7.3. RESULTS:Incorporation of genotype as a covariate for one SV located in the UGT1A9 gene promoter region (rs3832043, - 118 > insT, T9 > T10) significantly improved model fit (likelihood ratio test, p < 0.001) and reduced between-subject variability in glucuronide formation clearance. Individuals with the UGT1A9 T10 polymorphism, indicating insertion of an additional thymidine nucleotide, had a 42% reduction in clearance to APAP-glucuronide as compared to their wild-type counterparts. CONCLUSION:This study shows a pharmacogenetic effect of an SV in the UGT1A9 promoter region on the metabolism of acetaminophen in neonates.
Project description:Overdose of acetaminophen (APAP), the active ingredient of Tylenol, is the leading cause of drug-induced acute liver failure in the United States. As such, it is necessary to develop novel strategies to prevent or manage APAP toxicity. In this report, we reveal a novel function of the liver X receptor (LXR) in preventing APAP-induced hepatotoxicity. Activation of LXR in transgenic (Tg) mice or by an LXR agonist conferred resistance to the hepatotoxicity of APAP, whereas the effect of LXR agonist on APAP toxicity was abolished in LXR-deficient mice. The increased APAP resistance in LXR Tg mice was associated with increased APAP clearance, increased APAP sulfation, and decreased formation of toxic APAP metabolites. The hepatoprotective effect of LXR may have resulted from the induction of antitoxic phase II conjugating enzymes, such as Gst and Sult2a1, as well as the suppression of protoxic phase I P450 enzymes, such as Cyp3a11 and Cyp2e1. Promoter analysis suggested the mouse Gst isoforms as novel transcriptional targets of LXR. The suppression of Cyp3a11 may be accounted for by the inhibitory effect of LXR on the PXR-responsive transactivation of Cyp3a11. The protective effect of LXR in preventing APAP toxicity is opposite to the sensitizing effect of pregnane X receptor, constitutive androstane receptor, and retinoid X receptor alpha.We conclude that LXR represents a potential therapeutic target for the prevention and treatment of Tylenol toxicity.
Project description:Acetaminophen (APAP) is one of the most popular and safe pain medications worldwide. However, due to its wide availability, it is frequently implicated in intentional or unintentional overdoses where it can cause severe liver injury and even acute liver failure (ALF). In fact, APAP toxicity is responsible for 46% of all ALF cases in the United States. Early mechanistic studies in mice demonstrated the formation of a reactive metabolite, which is responsible for hepatic glutathione depletion and initiation of the toxicity. This insight led to the rapid introduction of N-acetylcysteine as a clinical antidote. However, more recently, substantial progress was made in further elucidating the detailed mechanisms of APAP-induced cell death. Mitochondrial protein adducts trigger a mitochondrial oxidant stress, which requires amplification through a MAPK cascade that ultimately results in activation of c-jun N-terminal kinase (JNK) in the cytosol and translocation of phospho-JNK to the mitochondria. The enhanced oxidant stress is responsible for the membrane permeability transition pore opening and the membrane potential breakdown. The ensuing matrix swelling causes the release of intermembrane proteins such as endonuclease G, which translocate to the nucleus and induce DNA fragmentation. These pathophysiological signaling mechanisms can be additionally modulated by removing damaged mitochondria by autophagy and replacing them by mitochondrial biogenesis. Importantly, most of the mechanisms have been confirmed in human hepatocytes and indirectly through biomarkers in plasma of APAP overdose patients. The extensive necrosis caused by APAP overdose leads to a sterile inflammatory response. Although recruitment of inflammatory cells is necessary for removal of cell debris in preparation for regeneration, these cells have the potential to aggravate the injury. This review touches on the newest insight into the intracellular mechanisms of APAP-induced cells death and the resulting inflammatory response. Furthermore, it discusses the translation of these findings to humans and the emergence of new therapeutic interventions.
Project description:CYP2E1 is recognized as the most important enzyme for initiation of acetaminophen (APAP)-induced toxicity. In this study, the resistance of Cyp2e1-null mice to APAP treatment was confirmed by comparing serum aminotransferase activities and blood urea nitrogen levels in wild-type and Cyp2e1-null mice. However, unexpectedly, profiling of major known APAP metabolites in urine and serum revealed that the contribution of CYP2E1 to APAP metabolism decreased with increasing APAP doses administered. Measurement of hepatic glutathione and hydrogen peroxide levels exposed the importance of oxidative stress in determining the consequence of APAP overdose. Subsequent metabolomic analysis was capable of constructing a principal components analysis (PCA) model that delineated a relationship between urinary metabolomes and the responses to APAP treatment. Urinary ions high in wild-type mice treated with 400 mg/kg APAP were elucidated as 3-methoxy-APAP glucuronide (VII) and three novel APAP metabolites, including S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid (VI, formed by a Cys-APAP transamination reaction in kidney), 3,3'-biacetaminophen (VIII, an APAP dimer), and a benzothiazine compound (IX, originated from deacetylated APAP), through mass isotopomer analysis, accurate mass measurement, tandem mass spectrometry fragmentation, in vitro reactions, and chemical treatments. Dose-, time-, and genotype-dependent appearance of these minor APAP metabolites implied their association with the APAP-induced toxicity and potential biomarker application. Overall, the oxidative stress elicited by CYP2E1-mediated APAP metabolism might significantly contribute to APAP-induced toxicity. The combination of genetically modified animal models, mass isotopomer analysis, and metabolomics provides a powerful and efficient technical platform to characterize APAP-induced toxicity through identifying novel biomarkers and unraveling novel mechanisms.
Project description:The benefit of therapeutic hypothermia (TH) in acute liver failure (ALF) has not been previously demonstrated in a controlled fashion. This study sought to determine the impact of TH on 21-day survival and complications in ALF patients at high risk for cerebral edema. This was a retrospective cohort study of ALF patients in the US Acute Liver Failure Study Group with grade III or IV hepatic encephalopathy. TH (32°C-35°C) was used in 97 patients (8%); 1135 (92%) who were not cooled were controls. Intracranial pressure was monitored in 38 TH ALF patients (39.2% versus 22% of controls, P?<?0.001). Rates of bleeding (12% for both) and bloodstream (17% versus 18%) and tracheal infections (21% versus 23%, P?>?0.5 for all) were similar. Unadjusted 21-day overall (62% versus 60%) and transplant-free survival rates (45% versus 39%, P?>?0.4 for both) were similar. Multivariate models were created for acetaminophen (APAP) patients (n?=?582) and non-APAP patients (n?=?613). For APAP patients, the Model for End-Stage Liver Disease [MELD; odds ratio (OR)?=?0.91 per increment, 95% confidence interval (CI)?=?0.89-0.94, P?<?0.001] and vasopressors (OR?=?0.16, 95% CI?=?0.11-0.24, P?<?0.001) were associated with decreased 21-day spontaneous survival. Survival was improved with TH in APAP patients who were <25 years old (age of 25 years: OR?=?2.735, 95% CI?=?1.001-7.467) but worsened for APAP patients who were 64 years old or older (age of 64 years: OR?=?0.167, 95% CI?=?0.028-0.999). For non-APAP patients, MELD (OR?=?0.93 per increment, 95% CI?=?0.91-0.95, P?<?0.001) and vasopressors (OR?=?0.60, 95% CI?=?0.40-0.90, P?=?0.01) were associated with worse outcomes, whereas TH had no impact (P?=?0.93). In conclusion, TH in ALF was not associated with increased bleeding or infections. Although young APAP ALF patients may benefit, TH did not consistently affect 21-day survival. A prospective trial is required to clarify the utility of TH in ALF patients.
Project description:To identify novel factors or mechanisms that are important for the resistance of tissues to chemical toxicity, we have determined the mechanisms underlying the previously observed increases in resistance to acetaminophen (APAP) toxicity in the lateral nasal gland (LNG) of the male Cyp2g1-null/Cyp2a5-low mouse. Initial studies established that Cyp2a5-null mice, but not a newly generated strain of Cyp2g1-null mice, were resistant to APAP toxicity in the LNG; therefore, subsequent studies were focused on the Cyp2a5-null mice. Compared with the wild-type (WT) male mouse, the Cyp2a5-null male mouse had intact capability to metabolize APAP to reactive intermediates in the LNG, as well as unaltered circulating levels of APAP, APAP-GSH, APAP-glucuronide, and APAP-sulfate. However, it displayed reduced tissue levels of APAP and APAP-GSH and increased tissue levels of testosterone and salivary androgen-binding protein (ABP) in the LNG. Furthermore, we found that ABP was able to compete with GSH and cellular proteins for adduction with reactive metabolites of APAP in vitro. The amounts of APAP-ABP adducts formed in vivo were greater, whereas the amounts of APAP adducts formed with other cellular proteins were substantially lower, in the LNG of APAP-treated male Cyp2a5-null mice compared with the LNG of APAP-treated male WT mice. We propose that through its critical role in testosterone metabolism, CYP2A5 regulates 1) the bioavailability of APAP and APAP-GSH (presumably through modulation of the rates of xenobiotic excretion from the LNG) and 2) the expression of ABP, which can quench reactive APAP metabolites and thereby spare critical cellular proteins from inactivation.
Project description:Introduction:For patients with obstructive sleep apnoea (OSA) who are initially non-compliant with continuous (automatic) positive airway pressure (CPAP/APAP) therapy, a bilevel PAP (Spont/VAuto) therapy transition pathway is available to improve therapy adherence. The aim of this retrospective study was to compare PAP therapy usage data of patients with non-compliant OSA (ncOSA) on CPAP/APAP who were switched to bilevel PAP. Methods:A PAP telemonitoring database was queried between 1 January 2015 and 31 July 2016 for eligible patients started on CPAP/APAP and non-CMS (United States Center for Medicare and Medicaid Services) compliant and switched to bilevel PAP within 90 days of starting CPAP/APAP therapy. PAP therapy data on all patients were compared before switch (CPAP/APAP) and after switch (VAuto/Spont). Results:Of the 1496 patients with ncOSA identified, 30.3% used CPAP, 62.3% APAP, and 7.4% both APAP and CPAP before switching to a bilevel mode. 47.8% patients switched to Spont mode and 52.2% to VAuto mode. PAP usage significantly improved by 0.9 h/day (p<0.001) and all other device metrics (residual apnoea-hypopnoea index and unintentional mask leak) also improved after the switch. No patients had achieved US CMS criteria for compliance before the switch, and 56.8% did after. Conclusion:This shows for the first time that there may be potential benefit from switching from CPAP/APAP to bilevel PAP for patients struggling with PAP adherence.
Project description:Acetaminophen (APAP) is the most common cause of acute liver failure (ALF) in the west. It is unknown if APAP overdose in combination with diphenhydramine or opioids confers a different clinical presentation or prognosis. Study objectives were to compare (1) baseline patient characteristics; (2) initial clinical presentation; and (3) clinical outcomes among patients with ALF due to APAP alone or in combination with diphenhydramine or opioids.We analyzed 666 cases of APAP-related liver failure using the Acute Liver Failure Study Group database from 1998 to 2012. The database contains detailed demographic, laboratory, and clinical outcome data, including hemodialysis, transplantation, and death and in-hospital complications such as arrhythmia and infection.The final sample included 666 patients with APAP liver injury. A total 30.3% of patients were overdosed with APAP alone, 14.1% with APAP/diphenhydramine, and 56.6% with APAP/opioids. Patients taking APAP with opioids were older, had more comorbidities, and were more likely to have unintentional overdose (all P<0.0001). On presentation, 58% in the APAP/opioid group had advanced encephalopathy as compared with 43% with APAP alone (P=0.001) The APAP/diphenhydramine group presented with the highest serum aminotransferase levels, no differences in laboratory values were noted at 3 days postenrollment. No significant differences were observed in clinical outcomes among the groups.Most patients with APAP-induced ALF were taking APAP combination products. There were significant differences in patient characteristics and clinical presentation based on the type of product ingested, however, there were no differences noted in delayed hepatotoxicity or clinical outcomes.