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.

The Roles of Antidotes in Emergency Situations.

Management of the acutely poisoned patient requires supportive care and timely administration of antidotes to minimize ongoing toxicity and mortality. New applications for old antidotes include utilization of methylene blue and hydroxocobalamin in vasoplegia. Fomepizole is also being evaluated as a potential adjunct in acetaminophen toxicity. Other advancements include individualized acetylcysteine dosing regimens for acetaminophen toxicity and carnitine supplementation in valproic acid toxicity. Additional antidote considerations include administration of lipid emulsion in lipophilic xenobiotic exposure not responsive to standard resuscitative modalities. These expert recommendations provide guidance for providers caring for the acutely poisoned patient.

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.

Metabolic and mitochondrial treatments for severe paracetamol poisoning: a systematic review.

BACKGROUND: Paracetamol (acetaminophen) remains a leading cause of poisoning in Europe, North America, and Australia. For over four decades, acetylcysteine has been the antidote of choice. However, despite the use of acetylcysteine, some patients who ingest very large doses of paracetamol or who reach hospital late in the course of their poisoning, develop acute liver failure. Some will develop metabolic acidosis indicating mitochondrial toxicity. OBJECTIVE: We review the experimental and clinical data reported with the use of cimetidine, fomepizole, and calmangafodipir in the treatment of paracetamol toxicity to determine if these treatments alone or in combination with acetylcysteine might be of benefit. METHODS: We searched Ovid Medline 1946-2020, Embase 1947-2020, Scopus 2004-2020, Cochrane Databases of Systematic Reviews (CDSR), Cochrane Central Register of Controlled Trials (CENTRAL), and clinicaltrials.gov 1997-2020 for records including the concepts of paracetamol poisoning and cimetidine, fomepizole, calmangafodipir, and acetylcysteine. We included basic science studies in animals and all available study types in humans. We reviewed the reference lists of included articles to search for references missed in the original search. We registered the protocol in PROSPERO. RESULTS: We completed all search strategies on 20 August 2019, 27 January 2020, and 15 June 2020. These produced 6,826 citations. We identified and deleted 2,843 duplicate resulting in a total of 3,856 unique citations. After applying inclusion and exclusion criteria, 89 studies remained. The largest numbers of studies described the past use of cimetidine, and the more recent use of fomepizole.Cimetidine: There is good animal evidence that cimetidine blocks CYP 2E1 with the potential to inhibit the toxic metabolism of paracetamol. Early case reports were inconclusive regarding the benefit to humans in paracetamol poisoning. Two comparative trials found no benefit of cimetidine in paracetamol poisoning, but few patients had severe poisoning.Fomepizole: There is good animal evidence that fomepizole blocks CYP 2E1 with the potential to inhibit the toxic metabolism of paracetamol. There are no comparative trials of fomepizole for acute paracetamol poisoning. Case reports are inconclusive due to multiple other interventions including the use of acetylcysteine in all cases. The benefit of fomepizole as adjunct treatment has not been demonstrated.Calmangafodipir: Calmangafodipir, a drug mimicking superoxide dismutase, has emerged as a potential treatment for severe paracetamol toxicity because the formation of superoxide free radicals appears to explain part of the mitochondrial toxicity of extremely large paracetamol overdoses. Calmangafodipir has reached Phase I/II trial of safety in humans with acute paracetamol overdose. Planning for a Phase III study of efficacy is currently underway. CONCLUSIONS: The vast majority of patients with acute paracetamol overdose enjoy excellent outcomes with acetylcysteine alone. Although cimetidine and fomepizole inhibit CYP 2E1 in animals, there is insufficient evidence to recommend their use either as a primary treatment or adjunct therapy in paracetamol poisoning. Calmangafodipir remains investigational.

Antidotes for alcohol and glycol toxicity: translating mechanisms into treatments.

Translational toxicology can be defined as the movement of potential antidotes for the treatment of poisonings from basic mechanistic research to the marketplace. Because poisonings are infrequent, the clinical development of antidotes is fraught with trials and tribulations. Academic scientists often conduct basic mechanistic work with antidotes but are infrequently involved in further drug development. This article presents the development of 4-methylpyrazole (4MP) (fomepizole) as an antidote against toxic alcohol poisonings, particularly by methanol and ethylene glycol (EG).

Effect of lycopene on caspase-3 enzyme activation in liver of methanol-intoxicated rats: comparison with fomepizole.

Lycopene is one of the major carotenoids and is found almost exclusively in tomatoes and tomato products. This study was performed to evaluate the effect of lycopene on methanol-induced liver injury and to compare the results with those after fomepizole, which is used in treatment of methanol intoxication. Experiments were carried out with 30 female Wistar rats weighting 180-200 g. Rats were injected with a intraperitoneally dose of 3 g/kg methanol as a 50% solution in isotonic saline once for intoxication. Rats were pretreated with fomepizole (50 mg/kg) and/or lycopene (10 mg/kg) before methanol. After 24 hours all the drug-treated and intoxicated rats were sacrificed under anesthesia. Malondialdehyde (MDA) levels were determined in order to assess lipid peroxidation, and caspase-3 activity was determined by immunostaining of liver tissues to evaluate apoptosis. Methanol administration significantly increased the MDA level and caspase-3 activity in liver. Pretreatment with lycopene and/or fomepizole decreased the MDA levels significantly. Similarly, lycopene and fomepizole decreased methanol-induced caspase-3 activity. The findings of the present study demonstrate that methanol intoxication causes hepatic toxicity in rats and that this is likely a result of reactive oxygen species and apoptosis induction. Lycopene has protective effects against methanol-induced hepatic injury similar to fomepizole. It was demonstrated for the first time that both lycopene and fomepizole prevent methanol-induced hepatic injury by reducing the increase of lipid oxidation and caspase-3 activation.

Increase in cellular pool of low-molecular-weight iron during ethanol metabolism in rat hepatocyte cultures. Relationship with lipid peroxidation.

Ethanol-induced lipid peroxidation was studied in primary rat hepatocyte cultures supplemented with ethanol at the concentration of 50 mM. Lipid peroxidation was assessed by two indices: (1) conjugated dienes by second-derivative UV spectroscopy in lipid extract of hepatocytes (intracellular content), and (2) free malondialdehyde (MDA) by HPLC-UV detection and quantitation for the incubation medium (extracellular content). In cultures supplemented with ethanol, free MDA increased significantly in culture media, whereas no elevation of conjugated diene level was observed in the corresponding hepatocytes. The cellular pool of low-mol-wt (LMW) iron was also evaluated in the hepatocytes using an electron spin resonance procedure. An early increase of intracellular LMW iron (< or = 1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas alpha-tocopherol, which prevented lipid peroxidation, did not inhibit the increase of LMW iron. Therefore, the LMW iron elevation was the result of ethanol metabolism and was not secondarily induced by lipid hydroperoxides. Thus, ethanol caused lipid peroxidation in rat hepatocytes as shown by the increase of free MDA, although no conjugated diene elevation was detected. During ethanol metabolism, an increase in cellular LMW iron was observed that could enhance conjugated diene degradation.

Effects of ethanol on urinary acidification and on gluconeogenesis by isolated renal tubules.

Class I alcohol dehydrogenase (ADH) is present in the kidney of rats. Rats fed an alcohol-containing diet long-term had higher urinary pH and reduced titratable acidity compared with pair-fed controls; rates of ammonium excretion were unchanged. The effects of ethanol on the metabolism of isolated renal tubules were then studied. Gluconeogenesis from lactate, pyruvate, or glutamine was not inhibited by 10 mmol/L ethanol during 30- or 60-minute incubations, although there was a trend toward increased lactate/pyruvate ratios at 30 minutes in the presence of ethanol. When the medium was also supplemented with oleate, glucose synthesis from most substrates was decreased, and the addition of ethanol inhibited glucose synthesis dramatically. This interaction between oleate and ethanol was not abolished by 4-methylpyrazole, an inhibitor of ADH. This effect of ethanol was highly dependent on the concentration of oleate present in the medium and was not observed with palmitate or decanoate; the inhibition was reversed by increasing the medium concentration of albumin. We conclude that ethanol may mildly perturb the redox state of isolated kidney tubules without inhibiting glucose synthesis, and that ethanol and oleate interact to inhibit renal gluconeogenesis by a mechanism highly dependent on the fatty acid concentration. The mechanism by which ethanol in the diet reduces renal acid excretion remains unknown.

4-Methylpyrazole partially ameliorated the teratogenicity of retinol and reduced the metabolic formation of all-trans-retinoic acid in the mouse.

Oral administration of retinol (50 mg/kg) to NMRI mice on day 11 of gestation (vaginal plug = day 0) led to the metabolic formation of high quantities of all-trans retinoic acid and all-trans-4-oxoretinoic acid, both known as potent teratogenic agents in the mouse. A 96% reduction of the area under the concentration-versus-time-curve (AUC) of metabolically generated all-trans retinoic acid in maternal plasma, and an 84% decrease in the embryonic AUC were observed when mice had been pretreated with the alcohol dehydrogenase inhibitor 4-methylpyrazole. A similar reduction was observed for the major metabolite of all-trans retinoic acid in the mouse, all-trans-4-oxoretinoic acid. However, 4-methylpyrazole pretreatment decreased the AUC of retinol by 10% in maternal plasma and 15% in embryo. Treatment with retinol alone resulted in 55.6%, 43.9% and 56.0% skeletal anomalies of the forelimbs, hindlimbs and craniofacial structures, respectively. Pretreatment with 4-methylpyrazole lowered the retinol induced skeletal defects to 31.3%, 24.0% and 31.3%, respectively, in the forelimb, hindlimb and craniofacial region. Typical retinoid-induced malformations for gestational day 11, e.g. bent or reduced zeugopod or stylopod elements, or cleft palate, were significantly reduced by 4-methylpyrazole pretreatment but were still detected in significantly higher prevalence than in control mice. These data suggest that the teratogenic activity of a single high dose of vitamin A in mouse is partially but not exclusively dependent on the metabolic activation of retinol to all-trans retinoic acid. Thus it could be hypothesized that retinol is either a proximate teratogen or a coteratogen with all-trans retinoic acid.

Methanol-induced monoamine changes in hypothalamus and striatum of albino rats.

This paper suggests that like ethanol, methanol also produces certain changes in the steady state level of monoamines in hypothalamus and striatum of albino rats. Though, the toxic manifestations of methanol are attributed to the metabolic end product of methanol viz. formic acid by several workers, we report here that the methanol-induced brain monoamine changes, at least, could be attributed to the direct action of methanol rather than to its metabolic end products like formaldehyde or formate. Studies in the steady state level of rat brain monoamines have shown that after methanol administration (3 g/kg), there is severe depletion of dopamine level in striatum but a significant increase in the level of dopamine, serotonin and 5-hydroxy indole acetic acid in hypothalamus. At the same time, norepinephrine and epinephrine levels are reduced in hypothalamus as well as in striatum. These effects do not seem to be induced by metabolic acidosis. The changes in monoamine levels are very well correlated with the blood and brain level of methanol as evidenced by maintaining a higher methanol level either by simultaneous administration of ethanol or by blocking methanol metabolism by pretreatment with 4-methyl pyrazole and 3-amino-1,2,4-triazole. It is thus postulated that monoamine changes induced by methanol appear to be the direct effect of methanol per se on the monoaminergic neuronal membranes.

High pressure liquid chromatographic assay of 4-methylpyrazole. Measurements of plasma and urine levels.

4-Methylpyrazole (4-MP), a potent competitive inhibitor of alcohol dehydrogenase activity, has potential usefulness as a treatment means for methanol and ethylene glycol poisoning as well as severe disulfiram-ethanol interactions. Further study of the safety and metabolism of 4-MP in human subjects is needed before it can be used in such therapies. An HPLC assay has been developed to measure 4-MP levels in plasma and urine samples. The method was sensitive enough to quantitate 4-MP in an amount as low as 0.1 nmol. Recovery of 4-MP from spiked urine and plasma samples was greater than 90%. 4-MP levels in the plasma and urine of rats injected with an oral dose of 50 mg/kg of body weight were determined; the detectability limit in these samples was about 3 microM. The method is easy to perform and thus has practical application for research laboratories dealing with ethanol metabolism and clinical laboratories desiring to monitor 4-MP levels.