N-acetyl-p-benzoquinone Imine Formation Research Articles

The influence of acetaminophen on human health

In pain management, acetaminophen, a widely used analgesic and antipyretic, holds a central position. The metabolic pathways can lead to acetaminophen-induced hepatotoxicity, elucidating from glucuronidation to the cytochrome P450 system. It meticulously details the complexities of acetaminophen metabolism, culminating in the formation of the toxic metabolite, n-acetyl-p-benzoquinone imine (NAPQI). Providing a holistic understanding guiding clinical practice, it explores risk factors, clinical implications, and management strategies. Transitioning to respiratory risks, potential associations between acetaminophen use and asthma, rhinoconjunctivitis, and eczema in adolescents is investigated. The study offers nuanced insights, urging caution in acetaminophen use during adolescence. In addition, the role of drug metabolism in acetaminophen-induced hepatic necrosis is also analyzed. Despite its temporal origins, the studys identification of cytochrome P450 as a catalyst for NAPQI formation remains foundational, contributing mechanistic insights to drug-induced liver injury knowledge. Synthesizing these studies reveals a delicate balance between acetaminophen metabolism and detoxification, crucial in determining its hepatotoxic potential. This nuanced understanding informs clinical practice and beckons researchers to bridge gaps, embarking on further explorations. In essence, this synthesis becomes a compass guiding the scientific community toward a profound comprehension of acetaminophen, epitomizing the standards expected in high-quality, peer-reviewed literature.

Chlorzoxazone reduced the paracetamol-induced toxicity via competitive inhibition of CYP2E1-mediated metabolism

BackgroundDrug metabolism is crucial to attaining the therapeutic index of any drug. The metabolism and elimination of the drugs are governed mainly by P-glycoprotein (P-gp) and Cytochrome P450 (CYP). Paracetamol is mostly used as analgesic and antipyretic agent. The metabolism of paracetamol is primarily via Glucuronidation and sulphation at therapeutic doses. About 5–10% of paracetamol is metabolized via CYP mediated pathway. Cytochrome P450 2E1 (CYP2E1) is primarily responsible for forming a toxic metabolite of paracetamol called N-acetyl-p-benzoquinoneimine (NAPQI). Even at therapeutic doses, long-term usage of paracetamol leads to the hepatic and nephrotoxicity because of NAPQI. Several in-vitro and in-vivo studies conducted by different research groups and reported that chlorzoxazone is a substrate and inhibitor of CYP2E1. However, the effect of chlorzoxazone on the paracetamol (CYP2E1 substrate) metabolism via the CYP2E1 has not yet been reported. This study investigated the effect of chlorzoxazone on the CYP2E1-mediated metabolism of Paracetamol and NAPQI formation in Wistar rats.ResultsFor 15 days, animals were orally administered with Paracetamol (300 mg/kg) with and without Silymarin (100 mg/kg) (standard CYP2E1 inhibitor) and Chlorzoxazone (50 and 100 mg/kg). Analysis was performed using RP-HPLC on the 15th day to determine paracetamol and NAPQI concentration in the plasma. Paracetamol combination with chlorzoxazone (50 and 100 mg/kg) showed a dose-dependent increase in the AUC0–∞ and the peak plasma concentration (Cmax) of Paracetamol and a dose-dependent decrease of AUC0–∞ and Cmax of NAPQI compared to paracetamol control (p < 0.001). Chlorzoxazone significantly decreased the elevated liver and renal markers compared to paracetamol control. Simultaneously, Hepatic and nephrotic tissue studies showed that compared to the paracetamol control group, the combination of chlorzoxazone significantly ameliorated paracetamol-induced hepatotoxicity and nephrotoxicity.ConclusionFinally, this study revealed that paracetamol in combination with chlorzoxazone led to a significant decrease in the plasma levels of NAPQI and enhanced absorption of paracetamol in rats via the inhibition of CYP2E1- mediated metabolism. In addition, chlorzoxazone significantly ameliorated paracetamol-induced hepatotoxicity and nephrotoxicity.

Mitochondrial versus microsomal bioactivation of paracetamol by human liver and kidney tissues

Mitochondria appeared to be a major target for paracetamol (PAR)-induced hepatotoxicity. Studies suggested that microsomal CYPs catalyse bioactivation of PAR to N-acetyl-p-benzoquinone imine (NAPQI), which alkylates mitochondrial proteins, and causes transmission of death signal from mitochondria to nucleus. We hypothesised that local formation of NAPQI within mitochondria seems more likely compared to the translocation of NAPQI. We therefore tested whether the formation of NAPQI may be catalysed by mitochondrial CYPs. Cellular fractions were isolated from human liver and kidney to compare the metabolic capacities. Liver and kidney mitochondria are capable to generate NAPQI. Mitochondrial CYP2E1 and CYP3A4 activities were comparable to the microsomal counterparts in both organs. Previously reported higher kidney microsomal CYP2E1 activity in men compared women were observed in mitochondrial CYP2E1 as well in the present study. On the other hand, no correlation between kidney CYP2E1 activity and quantity of NAPQI formation, as well as no induction on mitochondrial permeability transition pore (mPTP) opening by PAR in kidney mitochondria strongly suggested a different toxicity mechanism in this organ.

Determining site occupancy of acetaminophen covalent binding to target proteins in vitro.

Acetaminophen (APAP)-related toxicity is caused by the formation of N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite able to covalently bind to protein thiols. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, using multiple reaction monitoring (MRM), was developed to measure APAP binding on selected target proteins, including glutathione S-transferases (GSTs). In vitro incubations with CYP3A4 were performed to form APAP in the presence of different proteins, including four purified GST isozymes. A custom alkylation agent was used to prepare heavy labeled modified protein containing a structural isomer of APAP on all cysteine residues for isotope dilution. APAP incubations were spiked with heavy labeled protein, digested with either trypsin or pepsin, followed by peptide fractionation by HPLC prior to LC-MRM analysis. Relative site occupancy on the protein-level was used for comparing levels of modification of different sites in target proteins, after validation of protein and peptide-level relative quantitation using human serum albumin as a model system. In total, seven modification sites were quantified, namely Cys115 and 174 in GSTM2, Cys15, 48 and 170 in GSTP1, and Cys50 in human MGST1 and rat MGST1. In addition, APAP site occupancies of three proteins from liver microsomes were also quantified by using heavily labeled microsomes spiked into APAP microsomal incubations. A novel approach employing an isotope-labeled alkylation reagent was used to determine site occupancies on multiple protein thiols.

Assessment of the biochemical pathways for acetaminophen toxicity: Implications for its carcinogenic hazard potential

In 2019 California's Office of Environmental Health Hazard Assessment (OEHHA) initiated a review of the carcinogenic hazard potential of acetaminophen. In parallel with this review, herein we evaluated the mechanistic data related to the steps and timing of cellular events following therapeutic recommended (≤4 g/day) and higher doses of acetaminophen that may cause hepatotoxicity to evaluate whether these changes indicate that acetaminophen is a carcinogenic hazard. At therapeutic recommended doses, acetaminophen forms limited amounts of N-acetyl-p-benzoquinone-imine (NAPQI) without adverse cellular effects. Following overdoses of acetaminophen, there is potential for more extensive formation of NAPQI and depletion of glutathione, which may result in mitochondrial dysfunction and DNA damage, but only at doses that result in cell death – thus making it implausible for acetaminophen to induce the kind of stable, genetic damage in the nucleus indicative of a genotoxic or carcinogenic hazard in humans. The collective data demonstrate a lack of a plausible mechanism related to carcinogenicity and are consistent with rodent cancer bioassays, epidemiological results reviewed in companion manuscripts in this issue, as well as conclusions of multiple international health authorities.

A novel pipeline of 2-(benzenesulfonamide)-N-(4-hydroxyphenyl) acetamide analgesics that lack hepatotoxicity and retain antipyresis

Although acetaminophen (ApAP) is one of the most commonly used medicines worldwide, hepatotoxicity is a risk with overdose or in patients with compromised liver function. ApAP overdose is the most common cause of acute fulminant hepatic failure. Oxidation of ApAP to N-acetyl-p-benzoquinone imine (NAPQI) is the mechanism for hepatotoxicity. 1 is a non-hepatotoxic, metabolically unstable lipophilic ApAP analog that is not antipyretic. The newly synthesized 3 is a non-hepatotoxic ApAP analog that is stable, lipophilic, and retains analgesia and antipyresis. Intraperitoneal or po administration of the new chemical entities (NCEs), 3b and 3r, in concentrations equal to a toxic dose of ApAP did not result in the formation of NAPQI. Unlike livers from NCE-treated mice, the livers from ApAP-treated mice demonstrated large amounts of nitrotyrosine, a marker of mitochondrial free radical formation, and loss of hepatic tight junction integrity. Given the widespread use of ApAP, hepatotoxicity risk with overuse, and the ongoing opioid epidemic, these NCEs represent a novel, non-narcotic therapeutic pipeline.

Quercetin reduced the formation of N-acetyl-p-benzoquinoneimine, a toxic metabolite of paracetamol in rats and isolated rat hepatocytes.

N-acetyl-p-benzoquinoneimine (NAPQI) is toxic metabolite of paracetamol formed primarily by cytochrome P4502E1 (CYP2E1) metabolic pathway when administered at therapeutic doses or overdose. The influence of quercetin (flavonoid) on the bioactivation of paracetamol to NAPQI was investigated using rat liver microsomes and rats in vivo. Paracetamol (80mg/kg) was administered orally without or with silymarin (100mg/kg), a known inhibitor of CYP2E1, CYP3A4 and quercetin (10 and 20mg/kg) to rats for 15 consecutive days. Area under the plasma concentration-time curve (AUC0-∞ ) and the peakplasma concentration (Cmax ) of paracetamol were dose-dependently increased with quercetin (10 and 20mg/kg) compared to paracetamol control group (p<0.001). On the other hand, the AUC0-∞ and Cmax of NAPQI were decreased significantly with quercetin. The same results were observed with silymarin also. The elevated liver and kidney functional enzymes/compounds were significantly reduced by quercetin and silymarin compared to paracetamol control group. The formation of NAPQI was reduced in the incubation samples in presence of quercetin in experiment using isolated rat hepatocytes. The presentstudy results revealed that quercetin might be inhibited the CYP2E1-mediated metabolism of paracetamol; thereby decreased the formation of NAPQI and protected the liver and kidney.

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Although acetaminophen (ApAP) is one of the most commonly used medicines worldwide, hepatotoxicity is a risk with overdose or in patients with compromised liver function. ApAP overdose is the most common cause of acute fulminant hepatic failure. Oxidation of ApAP to N-acetyl-p-benzoquinone imine (NAPQI) is the mechanism for hepatotoxicity. 1 is a non-hepatotoxic, metabolically unstable lipophilic ApAP analog that is not antipyretic. The newly synthesized 3 is a non-hepatotoxic ApAP analog that is stable, lipophilic, and retains analgesia and antipyresis. Intraperitoneal or po administration of the new chemical entities (NCEs), 3b and 3r, in concentrations equal to a toxic dose of ApAP did not result in the formation of NAPQI. Unlike livers from NCE-treated mice, the livers from ApAP-treated mice demonstrated large amounts of nitrotyrosine, a marker of mitochondrial free radical formation, and loss of hepatic tight junction integrity. Given the widespread use of ApAP, hepatotoxicity risk with overuse, and the ongoing opioid epidemic, these NCEs represent a novel, non-narcotic therapeutic pipeline.

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Although acetaminophen (ApAP) is one of the most commonly used medicines worldwide, hepatotoxicity is a risk with overdose or in patients with compromised liver function. ApAP overdose is the most common cause of acute fulminant hepatic failure. Oxidation of ApAP to N-acetyl-p-benzoquinone imine (NAPQI) is the mechanism for hepatotoxicity. 1 is a non-hepatotoxic, metabolically unstable lipophilic ApAP analog that is not antipyretic. The newly synthesized 3 is a non-hepatotoxic ApAP analog that is stable, lipophilic, and retains analgesia and antipyresis. Intraperitoneal or po administration of the new chemical entities (NCEs), 3b and 3r, in concentrations equal to a toxic dose of ApAP did not result in the formation of NAPQI. Unlike livers from NCE-treated mice, the livers from ApAP-treated mice demonstrated large amounts of nitrotyrosine, a marker of mitochondrial free radical formation, and loss of hepatic tight junction integrity. Given the widespread use of ApAP, hepatotoxicity risk with overuse, and the ongoing opioid epidemic, these NCEs represent a novel, non-narcotic therapeutic pipeline.

Effect of chrysin on the formation of N-acetyl-p-benzoquinoneimine, a toxic metabolite of paracetamol in rats and isolated rat hepatocytes

Paracetamol (N-acetyl-para amino phenol) is the most commonly used analgesic and antipyretic around the world. Its causes hepatotoxicity and nephrotoxicity at overdose or even at therapeutic doses. It is primarily metabolized by glucuronidation and sulfate conjugation. It is also metabolized by cytochrome-P450 system (CYP2E1, CYP1A2 and CYP 3A4), leading to the formation of N-acetyl-p-benzoquinoneimine (NAPQI). The present study was planned to investigate the influence of chrysin (known CYP2E1 and CYP3A4 inhibitor) on the bioactivation of paracetamol to NAPQI using rat liver microsomes in vitro and rats in vivo. Paracetamol (80 mg/kg) was administered orally without or with silymarin (100 mg/kg), a known CYP2E1 inhibitor and chrysin (100 and 200 mg/kg) to rats for 15 consecutive days. The area under the plasma concentration–time curve (AUC0-∞) and the peak plasma concentration (Cmax) of paracetamol were dose-dependently increased with chrysin (100 and 200 mg/kg) compared to paracetamol control group. On the other hand, the AUC0-∞ and Cmax of NAPQI were decreased significantly with chrysin (100 and 200 mg/kg). The elevated liver and kidney function markers were significantly reduced by chrysin and silymarin compared to paracetamol control group (P < 0.01). Histopathological studies of liver and kidney also well correlated with liver and kidney function tests. Chrysin also reduced the formation of NAPQI in the incubation samples of rat hepatocytes. The present study (both in vivo and in vitro) results revealed that chrysin might be inhibited the CYP2E1, CYP1A2 and CYP3A4-mediated metabolism of paracetamol; thereby decreased the formation of NAPQI and protected the liver and kidney.

Investigation of acetaminophen toxicity in HepG2/C3a microscale cultures using a system biology model of glutathione depletion.

We have integrated in vitro and in silico information to investigate acetaminophen (APAP) and its metabolite N-acetyl-p-benzoquinone imine (NAPQI) toxicity in liver biochip. In previous works, we observed higher cytotoxicity of HepG2/C3a cultivated in biochips when exposed to 1 mM of APAP for 72 h as compared to Petri cultures. We complete our investigation with the present in silico approach to extend the mechanistic interpretation of the intracellular kinetics of the toxicity process. For that purpose, we propose a mathematical model based on the coupling of a drug pharmacokinetic model (PK) with a systemic biology model (SB) describing the reactive oxygen species (ROS) production by NAPQI and the subsequent glutathione (GSH) depletion. The SB model was parameterized using (i) transcriptomic data, (ii) qualitative results of time lapses ROS fluorescent curves for both control and 1-mM APAP-treated experiments, and (iii) additional GSH literature data. The PK model was parameterized (i) using the in vitro kinetic data (at 160 μM, 1 mM, 10 mM), (ii) using the parameters resulting from a physiologically based pharmacokinetic (PBPK) literature model for APAP, and (iii) by literature data describing NAPQI formation. The PK-SB model predicted a ROS increase and GSH depletion due to the NAPQI formation. The transition from a detoxification phase and NAPQI and ROS accumulation was predicted for a NAPQI concentration ranging between 0.025 and 0.25 μM in the cytosol. In parallel, we performed a dose response analysis in biochips that shows a reduction of the final hepatic cell number appeared in agreement with the time and doses associated with the switch of the NAPQI detoxification/accumulation. As a result, we were able to correlate in vitro extracellular APAP exposures with an intracellular in silico ROS accumulation using an integration of a coupled mathematical and experimental liver on chip approach.

Fennel and Raspberry Leaf as Possible Inhibitors of Acetaminophen Oxidation

In addition to CYP2E1, several CYP isoenzymes, notably CYP1A2, 2D6, and 3A4, are suggested to contribute in acetaminophen oxidation and formation of the hepatotoxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). The in vitro CYP2E1 inhibitory potentials of fennel and raspberry leaf, herbs previously found to inhibit CYP1A2, 2D6, and 3A4 activities in vitro, were investigated. Extracts from commercially available herbal products were incubated with recombinant cDNA-expressed human CYP2E1. A validated LC/MS/MS methodology was applied for determination of 6-hydroxychlorzoxazone formation with disulfiram used as a positive inhibitory control. CYP2E1 IC50 inhibition constants were found to be 23 ± 4 and 27 ± 5 µg/ml for fennel and raspberry leaf, respectively, constants significantly lower than those presented in the literature for other herbal extracts. Together with previous findings, the presented in vitro data for CYP2E1 inhibition suggest that fennel and raspberry leaf have a significant potential of inhibiting all the major metabolic pathways for acetaminophen oxidation and NAPQI formation. Both herbs should be further investigated for their in vivo ability of inhibiting acetaminophen oxidation and NAPQI formation.

An Indole Derivative Protects Against Acetaminophen-Induced Liver Injury by Directly Binding to N-Acetyl-p-Benzoquinone Imine in Mice

Acetaminophen (APAP)-induced liver injury is mainly due to the excessive formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) through the formation of a reactive intermediate, N-acetyl-p-benzoquinone imine (NAPQI), in both humans and rodents. Here, we show that the indole-derived synthetic compound has a protective effect against APAP-induced liver injury in C57Bl/6 mice model. NecroX-7 decreased tert-butylhydroperoxide (t-BHP)- and APAP-induced cell death and ROS/RNS formation in HepG2 human hepatocarcinoma and primary mouse hepatocytes. In mice, NecroX-7 decreased APAP-induced phosphorylation of c-Jun N-terminal kinase (JNK) and 3-nitrotyrosine (3-NT) formation, and also protected mice from APAP-induced liver injury and lethality by binding directly to NAPQI. The binding of NecroX-7 to NAPQI did not require any of cofactors or proteins. NecroX-7 could only scavenge NAPQI when hepatocellular GSH levels were very low. NecroX-7 is an indole-derived potent antioxidant molecule, which can be bound to some types of radicals and especially NAPQI. It is well known that the NAPQI is a major intermediate of APAP, which causes necrosis of hepatocytes in rodents and humans. Thus, blocking NAPQI formation or eliminating NAPQI are novel strategies for the treatment or prevention of APAP-induced liver injury instead of GSH replenishment. Our data suggest that the indole-derivative, NecroX-7, directly binds to NAPQI when hepatic GSH levels are very low and the NAPQI-NecroX-7 complex is secreted to the blood from the liver. NecroX-7 shows more preventive and similar therapeutic effects against APAP-induced liver injury when compared to the effect of N-acetylcysteine in C57Bl/6 mice.

Paracetamol and Simvastatin: A Potential Interaction Resulting in Hepatotoxicity

The safety profile of paracetamol and simvastatin is sufficiently well known, although no interactions between these two medicinal products have been described in the scientific literature so far. A 66-year-old female patient who experienced myocardial infarction and underwent coronary artery bypass grafting 9 years ago was taking simvastatin at a daily dose of 10 mg. Liver enzyme tests were carried out regularly, and their results were always normal. Later on, the patient took 6 tablets of fixed combination medicinal product Gripex(TM) (paracetamol, pseudoephedrine, and dextromethorphan) per day due to a fever. The daily dose of paracetamol taken by the patient totaled 1.95 g. The patient developed severe jaundice, nausea, vomiting; blood bilirubin levels increased more than 3 times; alanine transaminase, more than 10 times; and asparagine transaminase, more than 5 times. Paracetamol is metabolized by CYP enzymes (CYP2E1, 1A2, 2A6, 3A4) to a reactive metabolite, N-acetyl-p-benzoquinone-imine (NAPQI). Under conditions of excessive NAPQI formation or reduction in glutathione stores by approximately 70%, NAPQI covalently binds to the cysteinyl sulfhydryl groups of cellular proteins, forming NAPQI-protein adducts. Simvastatin is a substrate of CYP3A4 enzyme. Clinical and pharmacological data, available in the published literature, allow the assumption that simvastatin may induce CYP3A4 and result in increased hepatoxicity of paracetamol.

In-source formation of N-acetyl- p-benzoquinone imine (NAPQI), the putatively toxic acetaminophen (paracetamol) metabolite, after derivatization with pentafluorobenzyl bromide and GC–ECNICI-MS analysis

Pentafluorobenzyl (PFB) bromide (PFB-Br) is a versatile derivatization reagent for numerous classes of compounds. Under electron-capture negative-ion chemical ionization (ECNICI) conditions PFB derivatives of acidic compounds readily and abundantly ionize to produce intense anions due to [M−PFB] −. In the present article we investigated the PFB-Br derivatization of unlabelled acetaminophen ( N-acetyl- p-aminophenol, NAPAP-d 0; paracetamol; MW 151) and tetradeuterated acetaminophen (NAPAP-d 4; MW 155) in anhydrous acetonitrile and their GC–ECNICI-MS behavior using methane as the buffer gas. In addition to the expected anions [M−PFB] − at m/ z 150 from NAPAP-d 0 and m/ z 154 from NAPAP-d 4, we observed highly reproducibly almost equally intense anions at m/ z 149 and m/ z 153, respectively. Selected ion monitoring of these ions is suitable for specific and sensitive quantification of acetaminophen in human plasma and urine. Detailed investigations suggest in-source formation of N-acetyl- p-benzoquinone imine (NAPQI; MW 149), the putatively toxic acetaminophen metabolite, from the PFB ether derivative of NAPAP. GC–ECNICI-MS of non-derivatized NAPAP did not produce NAPQI. The peak area ratio of m/ z 149 to m/ z 150 and of m/ z 153 to m/ z 154 decreased with increasing ion-source temperature in the range 100–250 °C. Most likely, NAPQI formed in the ion-source captures secondary electrons to become negatively charged (i.e., [NAPQI] −) and thus detectable. Formation of NAPQI was not observed under electron ionization (EI) conditions, i.e., by GC–EI-MS, from derivatized and non-derivatized NAPAP. NAPQI was not detectable in flow injection analysis LC–MS of native NAPAP in positive electrospray ionization (ESI) mode, whereas in negative ESI mode low extent NAPQI formation was observed (<5%). Our results suggest that oxidation of drug derivatives in the ion-sources of mass spectrometers may form intermediates that are produced from activated drugs in enzyme-catalyzed reactions.

PXII-13 Demonstration of chlamydia pneumoniae, mycoplasma pneumoniae, cytomegalovirus, and Epstein-Barr virus in atherosclerotic coronary arteries, in nonrheumatic calcific aortic valves and in rheumatic stenotic mitral valves by polymerase chain reaction

Acetaminophen (APAP) is safe at therapeutic levels but causes liver injury via N-acetyl-p-benzoquinone imine (NAPQI)-induced oxidative stress when overdose. Recent studies indicated that mitochondrial permeability transition (mPT) plays a key role in APAP-induced toxicity and leflunomide (LEF) protects against the toxicity through inhibition of c-jun NH2-terminal protein kinase (JNK)-mediated pathway of mPT. It is not clearly understood if LEF also exerts its protective effect through inhibition of APAP bioactivation to the toxic NAPQI. The present work was undertaken to study the effect of LEF on the bioactivation of APAP to NAPQI. Mechanism-based inhibition incubations performed in mouse and human liver microsomes (MLM and HLM) indicated that inhibition of APAP bioactivation to NAPQI was observed in MLM but not in HLM. Furthermore, LEF but not its active metabolite, A77–1726, was shown to be the main inhibitor. When APAP and LEF were incubated with human recombinant P450 enzymes, CYP1A2 was found to be the isozyme responsible for the inhibition of APAP bioactivation. Species variation in CYP1A2 enzymes probably accounted for the different observations in our MLM and HLM studies. We concluded that inhibition of NAPQI formation is not a probable pathway that LEF protects APAP-induced hepatotoxicity in human.

Intravenous neonatal paracetamol dosing: the magic of 10 days

Intravenous (i.v.) paracetamol (acetaminophen) is gaining increasing use in neonates (1–3), despite an off-label status in some countries. Product information for i.v. paracetamol does not consider the clearance maturation with age in neonates in its dosing recommendations. In Australasia the same dose of 15 mgAEkg 6 h is suggested for neonates over 10 days of age and for infants and children up to 33 kg. A reduced dose of 7.5 mgAEkg 6 h is proposed in term neonates <10 days of age. In Europe there is no labeled use for neonates <10 days of age. The registered dose is 7.5 mgAEkg 6 h for neonates after 10 days of age through to infants of 10 kg. A dose of 15 mgAEkg 6 h (max daily dose 60 mgAEkg) is recommended between 10 and 40 kg. These dosing regimens suggest something magic about 10 days. There are data concerning the pharmacokinetics of this formulation in neonates. Population pharmacokinetics of the i.v. prodrug (propacetamol) have been studied in neonates following single (4,5) and repeat dose administration (6). Parameter estimates from neonates given repeat doses of i.v. paracetamol over a median of 4 days duration are consistent with those estimated using propacetamol (7). Dosing regimens used in some institutions for neonates have been published in Pediatric Anesthesia (2,8), but there are few data validating the safety or effectiveness of these regimens. The idea that a target effect and associated target concentration can be used to make rational individual dose decisions has acceptance (9). A knowledge of neonatal pharmacokinetics assists with dose prediction to achieve the target concentration. There are, however, flaws and constraints of current knowledge that limit implementation of this approach to dosing.

Altered Disposition of Acetaminophen in Nrf2-null and Keap1-knockdown Mice

Acetaminophen (AA) is a widely used antipyretic drug that causes hepatotoxicity at high doses. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that mitigates electrophilic stress from AA by inducing genes, such as NAD(P)H:quinone oxidoreductase 1 (Nqo1), multidrug resistance-associated proteins (Mrps), and glutathione (GSH) synthesis enzymes. To determine whether Nrf2 activation alters the biotransformation and excretion of AA, male wild-type, Nrf2-null, and Keap1 (Kelch-like ECH-associated protein 1)-knockdown (Keap1-kd) mice (which have increased activation of Nrf2) were administered a single subtoxic dose of AA (50 mg/kg, iv), after which, AA and its metabolites (AA-glucuronide [AA-GLUC]; AA-sulfate [AA-SULF]; AA-glutathione [AA-GSH]) were quantified in plasma, bile, and liver. AA-GLUC concentrations were reduced in plasma and elevated in livers of Nrf2-null mice due to decreased glucuronidation activity and lower expression of the basolateral efflux transporter Mrp3. In contrast, Keap1-kd mice had higher plasma and lower hepatic AA-GLUC concentrations, due to higher Mrp3 expression. Lower glucuronidation activity of Nrf2-null mice increased the proportion of AA available for sulfation, resulting in elevated AA-SULF concentrations in plasma, bile, and liver. Decreased AA-sulfation activity in Keap1-kd mice resulted in lower AA-SULF concentrations. AA-GSH conjugates were increased in Nrf2-null mice and tended to be lower in Keap1-kd mice. Furthermore, Nqo1, an enzyme capable of detoxifying the reactive intermediate of AA metabolism, N-acetyl-p-benzoquinone imine (NAPQI), had 85% lower activity in Nrf2-null mice and 415% higher activity in Keap1-kd mice relative to wild-type. In conclusion, lack of Nrf2 results in decreased AA glucuronidation, leading to increased AA available for NAPQI formation and decreased efflux of AA-GLUC via Mrp3; however, activation of Nrf2, as in Keap1-kd mice, results in decreased sulfotransferase activity, decreased AA-SULF formation, and enhanced elimination of AA-GLUC due to increased expression of Mrp3.

Acetaminophen bioactivation by human cytochrome P450 enzymes and animal microsomes

Acetaminophen is a widely used analgesic antipyretic agent. When used at low doses, it is a safe drug, but at higher doses it can cause acute hepatic necrosis in humans and experimental animals. The key mechanism in the hepatotoxicity is cytochrome P450 (CYP)-catalysed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) that is capable of binding to cellular macromolecules and in that way an LC/MS liquid chromatography/mass spectrometry (LC/MS) method was developed to measure NAPQI formation by trapping it to reduced glutathione. This method was used to determine the bioactivation of acetaminophen at two concentrations: 50 μM therapeutic and 1 mM toxic by using nine human recombinant CYP enzymes: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4; and with different microsomes from experimental animals. At the toxic concentration the formation of NAPQI–glutathione was highest with CYP3A4 followed by CYP2E1, CYP1A2, and CYP2D6. At the therapeutic concentration, CYP3A4 had also the highest bioactivation capacity. In a comparison of the enzyme kinetics, CYP3A4 was the most efficient CYP with the lowest Km value 130 μM (95% confidence interval = 63–210 μM). Dexamethasone-induced rat liver microsomes had the most effective bioactivation capacity at therapeutic and toxic acetaminophen concentrations. This study suggests that CYP3A4 is the major CYP enzyme form catalysing acetaminophen oxidation to NAPQI in human liver.

Prevention of acetaminophen (APAP)-induced hepatotoxicity by leflunomide via inhibition of APAP biotransformation to N-acetyl- p-benzoquinone imine

Acetaminophen (APAP) is safe at therapeutic levels but causes liver injury via N-acetyl- p-benzoquinone imine (NAPQI)-induced oxidative stress when overdose. Recent studies indicated that mitochondrial permeability transition (mPT) plays a key role in APAP-induced toxicity and leflunomide (LEF) protects against the toxicity through inhibition of c-jun NH 2-terminal protein kinase (JNK)-mediated pathway of mPT. It is not clearly understood if LEF also exerts its protective effect through inhibition of APAP bioactivation to the toxic NAPQI. The present work was undertaken to study the effect of LEF on the bioactivation of APAP to NAPQI. Mechanism-based inhibition incubations performed in mouse and human liver microsomes (MLM and HLM) indicated that inhibition of APAP bioactivation to NAPQI was observed in MLM but not in HLM. Furthermore, LEF but not its active metabolite, A77–1726, was shown to be the main inhibitor. When APAP and LEF were incubated with human recombinant P450 enzymes, CYP1A2 was found to be the isozyme responsible for the inhibition of APAP bioactivation. Species variation in CYP1A2 enzymes probably accounted for the different observations in our MLM and HLM studies. We concluded that inhibition of NAPQI formation is not a probable pathway that LEF protects APAP-induced hepatotoxicity in human.