The thrombopoietin mimetic JNJ-26366821 reduces the late injury and accelerates the onset of liver recovery after acetaminophen-induced liver injury in mice.

Acetaminophen (APAP)-induced hepatotoxicity is comprised of an injury and recovery phase. While pharmacological interventions, such as N-acetylcysteine (NAC) and 4-methylpyrazole (4-MP), prevent injury there are no therapeutics that promote recovery. JNJ-26366821 (TPOm) is a novel thrombopoietin mimetic peptide with no sequence homology to endogenous thrombopoietin (TPO). Endogenous thrombopoietin is produced by hepatocytes and the TPO receptor is present on liver sinusoidal endothelial cells in addition to megakaryocytes and platelets, and we hypothesize that TPOm activity at the TPO receptor in the liver provides a beneficial effect following liver injury. Therefore, we evaluated the extent to which TPOm, NAC or 4-MP can provide a protective and regenerative effect in the liver when administered 2 h after an APAP overdose of 300 mg/kg in fasted male C57BL/6J mice. TPOm did not affect protein adducts, oxidant stress, DNA fragmentation and hepatic necrosis up to 12 h after APAP. In contrast, TPOm treatment was beneficial at 24 h, i.e., all injury parameters were reduced by 42-48%. Importantly, TPOm enhanced proliferation by 100% as indicated by PCNA-positive hepatocytes around the area of necrosis. When TPOm treatment was delayed by 6 h, there was no effect on the injury, but a proliferative effect was still evident. In contrast, 4MP and NAC treated at 2 h after APAP significantly attenuated all injury parameters at 24 h but failed to enhance hepatocyte proliferation. Thus, TPOm arrests the progression of liver injury by 24 h after APAP and accelerates the onset of the proliferative response which is essential for liver recovery.

Clinically relevant therapeutic approaches against acetaminophen hepatotoxicity and acute liver failure.

Liver injury and acute liver failure caused by an acetaminophen (APAP) overdose is a significant clinical problem in western countries. With the introduction of the mouse model of APAP hepatotoxicity in the 1970 s, fundamental mechanisms of cell death were discovered. This included the recognition that part of the APAP dose is metabolized by cytochrome P450 generating a reactive metabolite that is detoxified by glutathione. After the partial depletion of glutathione, the reactive metabolite will covalently bind to sulfhydryl groups of proteins, which is the initiating event of the toxicity. This insight led to the introduction of N-acetyl-L-cysteine, a glutathione precursor, as antidote against APAP overdose in the clinic. Despite substantial progress in our understanding of the pathomechanisms over the last decades viable new antidotes only emerged recently. This review will discuss the background, mechanisms of action, and the clinical prospects of the existing FDA-approved antidote N-acetylcysteine, of several new drug candidates under clinical development [4-methylpyrazole (fomepizole), calmangafodipir] and examples of additional therapeutic targets (Nrf2 activators) and regeneration promoting agents (thrombopoietin mimetics, adenosine A2B receptor agonists, Wharton's Jelly mesenchymal stem cells). Although there are clear limitations of certain therapeutic approaches, there is reason to be optimistic. The substantial progress in the understanding of the pathophysiology of APAP hepatotoxicity led to the consideration of several drugs for development as clinical antidotes against APAP overdose in recent years. Based on the currently available information, it is likely that this will result in additional drugs that could be used as adjunct treatment for N-acetylcysteine.

High-Dose Acetaminophen with Concurrent CYP2E1 Inhibition Has Profound Anticancer Activity without Liver Toxicity.

Acetaminophen (AAP) is metabolized by a variety of pathways such as sulfation, glucuronidation, and fatty acid amide hydrolase-mediated conversion to the active analgesic metabolite AM404. CYP2E1-mediated metabolism to the hepatotoxic reactive metabolite NAPQI (N-acetyl-p-benzoquinone imine) is a minor metabolic pathway that has not been linked to AAP therapeutic benefits yet clearly leads to AAP liver toxicity. N-acetylcysteine (NAC) (an antioxidant) and fomepizole (a CYP2E1 inhibitor) are clinically used for the treatment of AAP toxicity. Mice treated with AAP in combination with fomepizole (plus or minus NAC) were assessed for liver toxicity by histology and serum chemistry. The anticancer activity of AAP with NAC and fomepizole rescue was assessed in vitro and in vivo. Fomepizole with or without NAC completely prevented AAP-induced liver toxicity. In vivo, high-dose AAP with NAC/fomepizole rescue had profound antitumor activity against commonly used 4T1 breast tumor and lewis lung carcinoma lung tumor models, and no liver toxicity was detected. The antitumor efficacy was reduced in immune-compromised NOD-scid IL2Rgammanull mice, suggesting an immune-mediated mechanism of action. In conclusion, using fomepizole-based rescue, we were able to treat mice with 100-fold higher than standard dosing of AAP (650 mg/kg) without any detected liver toxicity and substantial antitumor activity. SIGNIFICANCE STATEMENT: High-dose acetaminophen can be given concurrently with CYP2E1 inhibition to allow for safe dose escalation to levels needed for anticancer activity without detected evidence of toxicity.

Lack of mitochondrial Cyp2E1 drives acetaminophen-induced ER stress-mediated apoptosis in mouse and human kidneys: Inhibition by 4-methylpyrazole but not N-acetylcysteine.

Acetaminophen (APAP) overdose causes liver injury and acute liver failure, as well as acute kidney injury, which is not prevented by the clinical antidote N-acetyl-L-cysteine (NAC). The absence of therapeutics targeting APAP-induced nephrotoxicity is due to gaps in understanding the mechanisms of renal injury. APAP metabolism through Cyp2E1 drives cell death in both the liver and kidney. We demonstrate that Cyp2E1 is localized to the proximal tubular cells in mouse and human kidneys. Virtually all the Cyp2E1 in kidney cells is in the endoplasmic reticulum (ER), not in mitochondria. By contrast, hepatic Cyp2E1 is in both the ER and mitochondria of hepatocytes. Consistent with this subcellular localization, a dose of 600 mg/kg APAP in fasted C57BL/6J mice induced the formation of APAP protein adducts predominantly in mitochondria of hepatocytes, but the ER of the proximal tubular cells of the kidney. We found that reactive metabolite formation triggered ER stress-mediated activation of caspase-12 and apoptotic cell death in the kidney. While co-treatment with 4-methylpyrazole (4MP; fomepizole) or the caspase inhibitor Ac-DEVD-CHO prevented APAP-induced cleavage of procaspase-12 and apoptosis in the kidney, treatment with NAC had no effect. These mechanisms are clinically relevant because 4MP but not NAC also significantly attenuated APAP-induced apoptotic cell death in primary human kidney cells. We conclude that reactive metabolite formation by Cyp2E1 in the ER results in sustained ER stress that causes activation of procaspase-12, triggering apoptosis of proximal tubular cells, and that 4MP but not NAC may be an effective antidote against APAP-induced kidney injury.

Hepatocyte-specific deletion of small heterodimer partner protects mice against acetaminophen-induced hepatotoxicity via activation of Nrf2.

Acetaminophen (APAP) overdose stands as the primary cause of acute liver failure in the United States. APAP hepatotoxicity involves hepatic glutathione (GSH) depletion and mitochondrial damage. To counteract the toxicity of APAP, the nuclear factor erythroid 2 like 2 (Nrf2) activates the expression of genes responsible for drug detoxification and GSH synthesis. In this study, we present evidence that the elimination of hepatocyte small heterodimer partner, a critical transcriptional repressor for liver metabolism, results in Nrf2 activation and protects mice from APAP-induced acute liver injury. Initial investigations conducted on wildtype (WT) mice revealed a swift downregulation of Shp mRNA within the first 24 h after APAP administration. Subsequent treatment of hepatocyte-specific Shp knockout (ShpHep-/-) mice with 300 mg/kg APAP for 2 h exhibited comparable bioactivation of APAP with that observed in the WT controls. However, a significant reduction in liver injury was observed in ShpHep-/- after APAP treatment for 6 and 24 h. The decreased liver injury correlated with a faster recovery of GSH, attributable to heightened expression of Nrf2 target genes involved in APAP detoxification and GSH synthesis. Moreover, in vitro studies revealed that SHP protein interacted with NRF2 protein, inhibiting the transcription of Nrf2 target genes. These findings hold relevance for humans, as overexpression of SHP hindered APAP-induced NRF2 activation in primary human hepatocytes. In conclusion, our studies have unveiled a novel regulatory axis involving SHP and NRF2 in APAP-induced acute liver injury, emphasizing SHP as a promising therapeutic target in APAP overdose-induced hepatotoxicity.

Nrf2 as a therapeutic target in acetaminophen hepatotoxicity: A case study with sulforaphane.

Acetaminophen (APAP) overdose can cause severe liver injury and acute liver failure. The only clinically approved antidote, N-acetylcysteine (NAC), is highly effective but has a narrow therapeutic window. In the last 2 decades, activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates acute phase proteins and antioxidant defense genes, has emerged as a putative new therapeutic target against APAP hepatotoxicity. However, virtually all studies that propose Nrf2 activation as mechanism of protection used prolonged pretreatment, which is not a clinically feasible approach to treat a drug overdose. Therefore, the objective of this study was to assess if therapeutic activation of Nrf2 is a viable approach to treat liver injury after APAP overdose. We used the water-soluble Nrf2 activator sulforaphane (SFN; 5 mg/kg) in a murine model of APAP hepatotoxicity (300 mg/kg). Our results indicate that short-term treatment (≤3 h) with SFN alone did not activate Nrf2 or its target genes. However, posttreatment with SFN after APAP partially protected at 6 h likely due to more rapid activation of the Nrf2-target gene heme oxygenase-1. A direct comparison of SFN with NAC given at 1 h after APAP showed a superior protection with NAC, which was maintained at 24 h unlike with SFN. Thus, Nrf2 activators have inherent problems like the need to create a cellular stress to activate Nrf2 and delayed adaptive responses which may hamper sustained protection against APAP hepatotoxicity. Thus, compared to the more direct acting antidote NAC, Nrf2 activators are less suitable for this indication.

Pre- and Post-Sexual Maturity Liver-specific ERα Knockout Does Not Impact Hepatic Mitochondrial Function.

Compared with males, premenopausal women and female rodents are protected against hepatic steatosis and present with higher functioning mitochondria (greater hepatic mitochondrial respiration and reduced H2O2 emission). Despite evidence that estrogen action mediates female protection against steatosis, mechanisms remain unknown. Here we validated a mouse model with inducible reduction of liver estrogen receptor alpha (ERα) (LERKO) via adeno-associated virus (AAV) Cre. We phenotyped the liver health and mitochondrial function of LERKO mice (n = 10-12 per group) on a short-term high-fat diet (HFD), and then tested whether timing of LERKO induction at 2 timepoints (sexually immature: 4 weeks old [n = 11 per group] vs sexually mature: 8-10 weeks old [n = 8 per group]) would impact HFD-induced outcomes. We opted for an inducible LERKO model due to known estrogen-mediated developmental programming, and we reported both receptor and tissue specificity with our model. Control mice were ERαfl/fl receiving AAV with green fluorescent protein (GFP) only. Results show that there were no differences in body weight/composition or hepatic steatosis in LERKO mice with either short-term (4-week) or chronic (8-week) high-fat feeding. Similarly, LERKO genotype nor timing of LERKO induction (pre vs post sexual maturity) did not alter hepatic mitochondrial O2 and H2O2 flux, coupling, or OXPHOS protein. Transcriptomic analysis showed that hepatic gene expression in LERKO was significantly influenced by developmental stage. Together, these studies suggest that hepatic ERα is not required in female protection against HFD-induced hepatic steatosis nor does it mediate sexual dimorphism in liver mitochondria function.

Interruption of bile acid uptake by hepatocytes after acetaminophen overdose ameliorates hepatotoxicity.

BACKGROUND & AIMS: Acetaminophen (APAP) overdose remains a frequent cause of acute liver failure, which is generally accompanied by increased levels of serum bile acids (BAs). However, the pathophysiological role of BAs remains elusive. Herein, we investigated the role of BAs in APAP-induced hepatotoxicity. METHODS: We performed intravital imaging to investigate BA transport in mice, quantified endogenous BA concentrations in the serum of mice and patients with APAP overdose, analyzed liver tissue and bile by mass spectrometry and MALDI-mass spectrometry imaging, assessed the integrity of the blood-bile barrier and the role of oxidative stress by immunostaining of tight junction proteins and intravital imaging of fluorescent markers, identified the intracellular cytotoxic concentrations of BAs, and performed interventions to block BA uptake from blood into hepatocytes. RESULTS: Prior to the onset of cell death, APAP overdose causes massive oxidative stress in the pericentral lobular zone, which coincided with a breach of the blood-bile barrier. Consequently, BAs leak from the bile canaliculi into the sinusoidal blood, which is then followed by their uptake into hepatocytes via the basolateral membrane, their secretion into canaliculi and repeated cycling. This, what we termed 'futile cycling' of BAs, led to increased intracellular BA concentrations that were high enough to cause hepatocyte death. Importantly, however, the interruption of BA re-uptake by pharmacological NTCP blockage using Myrcludex B and Oatp knockout strongly reduced APAP-induced hepatotoxicity. CONCLUSIONS: APAP overdose induces a breach of the blood-bile barrier which leads to futile BA cycling that causes hepatocyte death. Prevention of BA cycling may represent a therapeutic option after APAP intoxication. LAY SUMMARY: Only one drug, N-acetylcysteine, is approved for the treatment of acetaminophen overdose and it is only effective when given within ∼8 hours after ingestion. We identified a mechanism by which acetaminophen overdose causes an increase in bile acid concentrations (to above toxic thresholds) in hepatocytes. Blocking this mechanism prevented acetaminophen-induced hepatotoxicity in mice and evidence from patients suggests that this therapy may be effective for longer periods after ingestion compared to N-acetylcysteine.

Comparing N-acetylcysteine and 4-methylpyrazole as antidotes for acetaminophen overdose.

Acetaminophen (APAP) overdose can cause hepatotoxicity and even liver failure. N-acetylcysteine (NAC) is still the only FDA-approved antidote against APAP overdose 40 years after its introduction. The standard oral or intravenous dosing regimen of NAC is highly effective for patients with moderate overdoses who present within 8 h of APAP ingestion. However, for late-presenting patients or after ingestion of very large overdoses, the efficacy of NAC is diminished. Thus, additional antidotes with an extended therapeutic window may be needed for these patients. Fomepizole (4-methylpyrazole), a clinically approved antidote against methanol and ethylene glycol poisoning, recently emerged as a promising candidate. In animal studies, fomepizole effectively prevented APAP-induced liver injury by inhibiting Cyp2E1 when treated early, and by inhibiting c-jun N-terminal kinase (JNK) and oxidant stress when treated after the metabolism phase. In addition, fomepizole treatment, unlike NAC, prevented APAP-induced kidney damage and promoted hepatic regeneration in mice. These mechanisms of protection (inhibition of Cyp2E1 and JNK) and an extended efficacy compared to NAC could be verified in primary human hepatocytes. Furthermore, the formation of oxidative metabolites was eliminated in healthy volunteers using the established treatment protocol for fomepizole in toxic alcohol and ethylene glycol poisoning. These mechanistic findings, together with the excellent safety profile after methanol and ethylene glycol poisoning and after an APAP overdose, suggest that fomepizole may be a promising antidote against APAP overdose that could be useful as adjunct treatment to NAC. Clinical trials to support this hypothesis are warranted.

Liver-specific deletion of mechanistic target of rapamycin does not protect against acetaminophen-induced liver injury in mice.

BACKGROUND: Acetaminophen (APAP) overdose can cause liver injury and liver failure, which is one of the most common causes of drug-induced liver injury in the United States. Pharmacological activation of autophagy by inhibiting mechanistic target of rapamycin (mTOR) protects against APAP-induced liver injury likely via autophagic removal of APAP-adducts and damaged mitochondria. In the present study, we aimed to investigate the role of genetic ablation of mTOR pathways in mouse liver in APAP-induced liver injury and liver repair/regeneration. METHODS: Albumin-Cre (Alb-Cre) mice, mTORf/f and Raptorf/f mice (C57BL/6J background) were crossbred to produce liver-specific mTOR knockout (L-mTOR KO, Alb Cre+/-, mTORf/f) and liver-specific Raptor KO (L-Raptor, Alb Cre+/-, Raptor f/f) mice. Alb-Cre littermates were used as wild-type (WT) mice. These mice were treated with APAP for various time points for up to 48 h. Liver injury, cell proliferation, autophagy and mTOR activation were determined. RESULTS: We found that genetic deletion of neither Raptor, an important adaptor protein in mTOR complex 1, nor mTOR, in the mouse liver significantly protected against APAP-induced liver injury despite increased hepatic autophagic flux. Genetic deletion of Raptor or mTOR in mouse livers did not affect APAP metabolism and APAP-induced c-Jun N-terminal kinase (JNK) activation, but slightly improved mouse survival likely due to increased hepatocyte proliferation. CONCLUSIONS: Our results indicate that genetic ablation of mTOR in mouse livers does not protect against APAP-induced liver injury but may slightly improve liver regeneration and mouse survival after APAP overdose.

Delayed administration of N-acetylcysteine blunts recovery after an acetaminophen overdose unlike 4-methylpyrazole.

N-acetylcysteine (NAC) is the only clinically approved antidote against acetaminophen (APAP) hepatotoxicity. Despite its efficacy in patients treated early after APAP overdose, NAC has been implicated in impairing liver recovery in mice. More recently, 4-methylpyrazole (4MP, Fomepizole) emerged as a potential antidote in the mouse APAP hepatotoxicity model. The objective of this manuscript was to verify the detrimental effect of NAC and its potential mechanism and assess whether 4MP has the same liability. C57BL/6J mice were treated with 300 mg/kg APAP; 9 h after APAP and every 12 h after that, the animals received either 100 mg/kg NAC or 184.5 mg/kg 4MP. At 24 or 48 h after APAP, parameters of liver injury, mitochondrial biogenesis and cell proliferation were evaluated. Delayed NAC treatment had no effect on APAP-induced liver injury at 24 h but reduced the decline of plasma ALT activities and prevented the shrinkage of the areas of necrosis at 48 h. This effect correlated with down-regulation of key activators of mitochondrial biogenesis (AMPK, PGC-1α, Nrf1/2, TFAM) and reduced expression of Tom 20 (mitochondrial mass) and PCNA (cell proliferation). In contrast, 4MP attenuated liver injury at 24 h and promoted recovery at 48 h, which correlated with enhanced mitochondrial biogenesis and hepatocyte proliferation. In human hepatocytes, 4MP demonstrated higher efficacy in preventing cell death compared to NAC when treated at 18 h after APAP. Thus, due to the wider treatment window and lack of detrimental effects on recovery, it appears that at least in preclinical models, 4MP is superior to NAC as an antidote against APAP overdose.

Fructose Protects Against Acetaminophen-Induced Hepatotoxicity Mainly by Activating the Carbohydrate-Response Element-Binding Protein α-Fibroblast Growth Factor 21 Axis in Mice.

Acetaminophen (N-acetyl-para-aminophenol [APAP]) overdose is the most common cause of drug-induced liver injury in the Western world and has limited therapeutic options. As an important dietary component intake, fructose is mainly metabolized in liver, but its impact on APAP-induced liver injury is not well established. We aimed to examine whether fructose supplementation could protect against APAP-induced hepatotoxicity and to determine potential fructose-sensitive intracellular mediators. We found that both high-fructose diet feeding before APAP injection and fructose gavage after APAP injection reduced APAP-induced liver injury with a concomitant induction of the hepatic carbohydrate-response element-binding protein α (ChREBPα)-fibroblast growth factor 21 (FGF21) pathway. In contrast, Chrebpα liver-specific-knockout (Chrebpα-LKO) mice failed to respond to fructose following APAP overdose, suggesting that ChREBPα is the essential intracellular mediator of fructose-induced hepatoprotective action. Primary mouse hepatocytes with deletion of Fgf21 also failed to show fructose protection against APAP hepatotoxicity. Furthermore, overexpression of FGF21 in the liver was sufficient to reverse liver toxicity in APAP-injected Chrebpα-LKO mice. Conclusion: Fructose protects against APAP-induced hepatotoxicity likely through its ability to activate the hepatocyte ChREBPα-FGF21 axis.

Impaired protein adduct removal following repeat administration of subtoxic doses of acetaminophen enhances liver injury in fed mice.

Acetaminophen (APAP) is a widely used analgesic and is safe at therapeutic doses. However, an overdose of APAP is hepatotoxic and accidental overdoses are increasingly common due to the presence of APAP in several combination medications. Formation of protein adducts (APAP-CYS) is central to APAP-induced liver injury and their removal by autophagy is an essential adaptive response after an acute overdose. Since the typical treatment for conditions such as chronic pain involves multiple doses of APAP over time, this study investigated APAP-induced liver injury after multiple subtoxic doses and examined the role of autophagy in responding to this regimen. Fed male C57BL/6J mice were administered repeated doses (75 mg/kg and 150 mg/kg) of APAP, followed by measurement of adducts within the liver, mitochondria, and in plasma, activation of the MAP kinase JNK, and markers of liver injury. The role of autophagy was investigated by treatment of mice with the autophagy inhibitor, leupeptin. Our data show that multiple treatments at the 150 mg/kg dose of APAP resulted in protein adduct formation in the liver and mitochondria, activation of JNK, and hepatocyte cell death, which was significantly exacerbated by inhibition of autophagy. While repeated dosing with the milder 75 mg/kg dose did not cause mitochondrial protein adduct formation, JNK activation, or liver injury, autophagy inhibition resulted in hepatocyte death even at this lower dose. These data illustrate the importance of adaptive responses such as autophagy in removing protein adducts and preventing liver injury, especially in clinically relevant situations involving repeated dosing with APAP.

Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.

Dual roles of p62/SQSTM1 in the injury and recovery phases of acetaminophen-induced liver injury in mice.

Acetaminophen (APAP) overdose can induce liver injury and is the most frequent cause of acute liver failure in the United States. We investigated the role of p62/SQSTM1 (referred to as p62) in APAP-induced liver injury (AILI) in mice. We found that the hepatic protein levels of p62 dramatically increased at 24 h after APAP treatment, which was inversely correlated with the hepatic levels of APAP-adducts. APAP also activated mTOR at 24 h, which is associated with increased cell proliferation. In contrast, p62 knockout (KO) mice showed increased hepatic levels of APAP-adducts detected by a specific antibody using Western blot analysis but decreased mTOR activation and cell proliferation with aggravated liver injury at 24 h after APAP treatment. Surprisingly, p62 KO mice recovered from AILI whereas the wild-type mice still sustained liver injury at 48 h. We found increased number of infiltrated macrophages in p62 KO mice that were accompanied with decreased hepatic von Willebrand factor (VWF) and platelet aggregation, which are associated with increased cell proliferation and improved liver injury at 48 h after APAP treatment. Our data indicate that p62 inhibits the late injury phase of AILI by increasing autophagic selective removal of APAP-adducts and mitochondria but impairs the recovery phase of AILI likely by enhancing hepatic blood coagulation.

Mitochondrial protein adduct and superoxide generation are prerequisites for early activation of c-jun N-terminal kinase within the cytosol after an acetaminophen overdose in mice.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States and formation of APAP-protein adducts, mitochondrial oxidant stress and activation of the mitogen activated protein (MAP) kinase c-jun N-terminal kinase (JNK) are critical for APAP-induced cell death. However, direct evidence linking these mechanistic features are lacking and were investigated by examining the early temporal course of these changes in mice after 300 mg/kg APAP. Protein adducts were detectable in the liver (0.05-0.1 nmol/mg protein) by 15 and 30 min after APAP, which increased (>500 %) selectively in mitochondria by 60 min. Cytosolic JNK activation was only evident at 60 min, and was significantly attenuated by scavenging superoxide specifically in the cytosol by TEMPO treatment. Treatment of mouse hepatocytes with APAP revealed mitochondrial superoxide generation within 15 min, accompanied by hydrogen peroxide production without change in mitochondrial respiratory function. The oxidant stress preceded JNK activation and its mitochondrial translocation. Inhibitor studies identified the putative source of mitochondrial superoxide as complex III, which released superoxide towards the intermembrane space after APAP resulting in activation of JNK in the cytosol. Our studies provide direct evidence of mechanisms involved in mitochondrial superoxide generation after NAPQI-adduct formation and its activation of the MAP kinase cascade in the cytosol, which are critical features of APAP hepatotoxicity.

4-methylpyrazole protects against acetaminophen-induced acute kidney injury.

Acetaminophen (APAP) hepatotoxicity is the most common cause of acute liver failure in the United States, and while a significant percentage of APAP overdose patients develop kidney injury, molecular mechanisms involved in APAP-induced nephrotoxicity are relatively unknown. We have shown that 4-methylpyrazole (4MP, Fomepizole) protects against APAP-induced liver injury by inhibiting reactive metabolite formation through Cyp2E1, and analysis of data from APAP overdose patients indicated that kidney dysfunction strongly correlated with severe liver injury. Since Cyp2E1 is also expressed in the kidney, this study explored protection by 4MP against APAP-induced nephrotoxicity. Male C57BL/6 J mice were treated with either 300 or 600 mg/kg APAP with or without 4MP for 2, 6 or 24 h, followed by measurement of APAP metabolism and tissue injury. Interestingly, levels of APAP and its non-oxidative metabolites were significantly higher in kidneys when compared to the liver. APAP-protein adducts were present in both tissues within 2 h, but were absent in kidney mitochondria, unlike in the liver. While GSH depletion was seen in both tissues, activation of c-jun N-terminal kinase and its translocation to the mitochondria, which is a critical feature of APAP-induced liver injury, was not detected in the kidney. Treatment with 4MP attenuated APAP oxidative metabolite generation, GSH depletion as well as kidney injury indicating its potential use in protection against APAP-induced nephrotoxicity. In conclusion, since reactive metabolite formation seems to be common in both liver and kidney, 4MP mediated inhibition of Cyp2E1 protects against APAP-induced nephrotoxicity. However, downstream mechanisms of APAP-induced nephrotoxicity seem distinct from the liver.

Novel strategies for the treatment of acetaminophen hepatotoxicity.

INTRODUCTION: Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the western world. Despite extensive investigations into the mechanisms of cell death, only a single antidote, N-acetylcysteine, is in clinical use. However, there have recently been more efforts made to translate mechanistic insight into identification of therapeutic targets and potential new drugs for this indication. AREAS COVERED: After a short review of the key events in the pathophysiology of APAP-induced liver injury and recovery, the pros and cons of targeting individual steps in the pathophysiology as therapeutic targets are discussed. While the re-purposed drug fomepizole (4-methylpyrazole) and the new entity calmangafodipir are most advanced based on the understanding of their mechanism of action, several herbal medicine extracts and their individual components are also considered. EXPERT OPINION: Fomepizole (4-methylpyrazole) is safe and has shown efficacy in preclinical models, human hepatocytes and in volunteers against APAP overdose. The safety of calmangafodipir in APAP overdose patients was shown but it lacks solid preclinical efficacy studies. Both drugs require a controlled phase III trial to achieve regulatory approval. All studies of herbal medicine extracts and components suffer from poor experimental design, which questions their clinical utility at this point.

Novel Therapeutic Approaches Against Acetaminophen-induced Liver Injury and Acute Liver Failure.

Liver injury and acute liver failure caused by acetaminophen (APAP, N-acetyl-p-aminophenol, paracetamol) overdose is a significant clinical problem in most western countries. The only clinically approved antidote is N-acetylcysteine (NAC), which promotes the recovery of hepatic GSH. If administered during the metabolism phase, GSH scavenges the reactive metabolite N-acetyl-p-benzoquinone imine. More recently, it was shown that NAC can also reconstitute mitochondrial GSH levels and scavenge reactive oxygen/peroxynitrite and can support mitochondrial bioenergetics. However, NAC has side effects and may not be efficacious after high overdoses. Repurposing of additional drugs based on their alternate mechanisms of action could be a promising approach. 4-Methylpyrazole (4MP) was shown to be highly effective against APAP toxicity by inhibiting cytochrome P450 enzymes in mice and humans. In addition, 4MP is a potent c-Jun N-terminal kinase inhibitor expanding its therapeutic window. Calmangafodipir (CMFP) is a SOD mimetic, which is well tolerated in patients and has the potential to be effective after severe overdoses. Other drugs approved for humans such as metformin and methylene blue were shown to be protective in mice at high doses or at human therapeutic doses, respectively. Additional protective strategies such as enhancing antioxidant activities, Nrf2-dependent gene induction and autophagy activation by herbal medicine components are being evaluated. However, at this point, their mechanistic insight is limited, and the doses used are high. More rigorous mechanistic studies are needed to advance these herbal compounds. Nevertheless, based on recent studies, 4-methylpyrazole and calmangafodipir have realistic prospects to become complimentary or even alternative antidotes to NAC for APAP overdose.

The Effect of 4-Methylpyrazole on Oxidative Metabolism of Acetaminophen in Human Volunteers.

INTRODUCTION: Acetaminophen (APAP) is commonly ingested in both accidental and suicidal overdose. Oxidative metabolism by cytochrome P450 2E1 (CYP2E1) produces the hepatotoxic metabolite, N-acetyl-p-benzoquinone imine. CYP2E1 inhibition using 4-methylpyrazole (4-MP) has been shown to prevent APAP-induced liver injury in mice and human hepatocytes. This study was conducted to assess the effect of 4-MP on APAP metabolism in humans. METHODS: This crossover trial examined the ability of 4-MP to inhibit CYP2E1 metabolism of APAP in five human volunteers. Participants received a single oral dose of APAP 80 mg/kg, both with and without intravenous 4-MP, after which urinary and plasma oxidative APAP metabolites were measured. The primary outcome was the fraction of ingested APAP excreted as total oxidative metabolites (APAP-CYS, APAP-NAC, APAP-GSH). RESULTS: Compared with APAP alone, co-treatment with 4-MP decreased the percentage of ingested APAP recovered as oxidative metabolites in 24-hour urine from 4.48 to 0.51% (95% CI = 2.31-5.63%, p = 0.003). Plasma concentrations of these oxidative metabolites also decreased. CONCLUSIONS: These results show 4-MP effectively reduced oxidative metabolism of APAP in human volunteers ingesting a supratherapeutic APAP dose. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03878693.