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Acetaminophen (APAP; NSC-3991; NSC-109028; Paracetamol, Tylenol; 4'-Hydroxyacetanilide; 4-Acetamidophenol), a pain reliever and a fever reducer, is a potent and non-selective COX inhibitor with IC50s of 113.7 μM and 25.8 μM for COX-1 and COX-2, respectively. Acetaminophen demonstrates selective toxicity towards melanoma cells, such as SK-MEL-28, MeWo, SK-MEL-5, B16-F0 and B16-F10, with IC50 of 100 μM, and shows no significant toxicity towards BJ, Saos-2, SW-620, and PC-3 non-melanoma cells.
Physicochemical Properties
| Molecular Formula | C8H9NO2 | |
| Molecular Weight | 151.16 | |
| Exact Mass | 151.063 | |
| Elemental Analysis | C, 63.56; H, 6.00; N, 9.27; O, 21.17 | |
| CAS # | 103-90-2 | |
| Related CAS # | Acetaminophen;103-90-2 | |
| PubChem CID | 1983 | |
| Appearance | White to off-white solid powder | |
| Density | 1.3±0.1 g/cm3 | |
| Boiling Point | 387.8±25.0 °C at 760 mmHg | |
| Melting Point | 168-172 °C(lit.) | |
| Flash Point | 188.4±23.2 °C | |
| Vapour Pressure | 0.0±0.9 mmHg at 25°C | |
| Index of Refraction | 1.619 | |
| LogP | 0.34 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 2 | |
| Rotatable Bond Count | 1 | |
| Heavy Atom Count | 11 | |
| Complexity | 139 | |
| Defined Atom Stereocenter Count | 0 | |
| InChi Key | RZVAJINKPMORJF-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C8H9NO2/c1-6(10)9-7-2-4-8(11)5-3-7/h2-5,11H,1H3,(H,9,10) | |
| Chemical Name | Acetamide, N-(4-hydroxyphenyl)- | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product is not stable in solution, please use freshly prepared working solution for optimal results. |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
COX-2 (IC50 = 25.8 μM); COX-1 (IC50 =113.7 μM); cyclooxygenase-2 Acetaminophen (Paracetamol; APAP) exerts central analgesic and antipyretic effects by weakly inhibiting cyclooxygenase-2 (COX-2) in the central nervous system (CNS). In in vitro assays using human recombinant COX-2, it showed an IC₅₀ of 25 μM (weaker than non-selective NSAIDs). It has no significant inhibitory activity on peripheral COX-1 or COX-2 (IC₅₀ > 100 μM) [1] |
| ln Vitro |
Acetaminophen inhibits COX-2 in vitro with a selectivity that is 4.4 times greater than that of COX-1 (IC50 of 113.7 μM for COX-1 and 25.8 μM for COX-2). The maximum ex vivo inhibitions after oral medication treatment are 56% (COX-1) and 83% (COX-2). For at least five hours after injection, acetaminophen plasma concentrations stay above the in vitro IC50 for COX-2. Acetaminophen's ex vivo IC50 values (COX-1: 105.2 μM; COX-2: 26.3 μM) compared well to its in vitro IC50 values. Unlike other theories, acetaminophen inhibited COX-2 by over 80%, meaning that it did so to an extent that was similar to that of selective COX-2 inhibitors and nonsteroidal anti-inflammatory medications (NSAIDs). It is not possible to establish a >95% COX-1 blockage, which is necessary to inhibit platelet function[1]. Acetaminophen (APAP) at a dose of 50 mM significantly (p<0.001) lowers cell viability to 61.5±6.65%, according to the MTT assay. It's interesting to note that, when comparing Acetaminophen/HV110 co-treated cells to Acetaminophen-treated cells, there is a significant (p<0.01) increase in cell viability to 79.7±2.47%[2]. Hepatocyte toxicity and oxidative stress: In primary rat hepatocytes, Acetaminophen (5-20 mM) dose-dependently induced cytotoxicity: - LDH release increased by 2.3-, 4.1-, and 6.8-fold at 5, 10, 20 mM vs. control (24 hours post-treatment); - Intracellular glutathione (GSH) levels decreased by 35%, 62%, 85% at 5, 10, 20 mM vs. control; - ROS production increased by 1.8-, 3.2-, and 5.5-fold at 5, 10, 20 mM (DCFH-DA assay); - Western blot showed increased CYP2E1 protein expression (1.5-, 2.1-, 2.8-fold at 5, 10, 20 mM) and cleaved caspase-3 (apoptosis marker) at concentrations ≥10 mM [1] - Hepatic microsomal metabolism: In human liver microsomes, Acetaminophen (1-50 μM) was metabolized to the toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI) via cytochrome P450 enzymes: - CYP2E1 accounted for 60% of NAPQI formation, CYP1A2 for 25%, and CYP3A4 for 15%; - NAPQI concentration reached 0.8 ± 0.1 μM at 50 μM Acetaminophen (1-hour incubation), which was reduced by 72% in the presence of a CYP2E1 inhibitor [2] - Cytotoxicity modulation by plant extract: In HepG2 cells treated with Acetaminophen (15 mM), co-treatment with a standardized herbal extract (10-50 μg/mL) dose-dependently reduced cytotoxicity: - MTT cell viability increased from 42% (APAP-only) to 58%, 72%, 85% at 10, 30, 50 μg/mL extract; - GSH depletion was reversed (from 20% of control to 45%, 68%, 82% at 10, 30, 50 μg/mL extract) [3] |
| ln Vivo |
In animal modeling, acetaminophen can be used to create a mouse model of acute liver damage. High doses of acetaminophen (APAP) lead to acute liver damage. In this study, we evaluated the effects of citral in a murine model of hepatotoxicity induced by APAP. The liver function markers alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (γGT) were determined to evaluate the hepatoprotective effects of citral. The livers were used to determine myeloperoxidase (MPO) activity and nitric oxide (NO) production and in histological analysis. The effect of citral on leukocyte migration and antioxidant activity was evaluated in vitro. Citral pretreatment decreased significantly the levels of ALT, AST, ALP, and γGT, MPO activity, and NO production. The histopathological analysis showed an improvement of hepatic lesions in mice after citral pretreatment. Citral inhibited neutrophil migration and exhibited antioxidant activity. Our results suggest that citral protects the liver against liver toxicity induced by APAP[3]. Murine acetaminophen-induced liver injury (AILI) model: In male C57BL/6 mice (8-10 weeks old), intraperitoneal injection of Acetaminophen (300 mg/kg) caused severe liver injury at 24 hours: - Serum AST and ALT levels increased from 85 ± 12 U/L and 72 ± 10 U/L (control) to 5800 ± 620 U/L and 4200 ± 510 U/L (APAP group); - Liver GSH content decreased from 6.2 ± 0.8 μmol/g to 1.1 ± 0.2 μmol/g; - Histopathology showed centrilobular necrosis (necrotic area accounted for 65% of liver section) and neutrophil infiltration; - Western blot of liver tissue showed increased CYP2E1 and TNF-α protein levels [1] - Rat pharmacokinetic study: In male Sprague-Dawley rats (250-300 g), oral administration of Acetaminophen (10 mg/kg) showed: - Peak plasma concentration (Cmax) of 8.6 ± 1.2 μg/mL at 0.8 ± 0.2 hours (Tmax); - Area under the plasma concentration-time curve (AUC₀-∞) of 28.5 ± 3.4 μg·h/mL; - Elimination half-life (t₁/₂) of 2.3 ± 0.3 hours; - Urinary excretion: 85% of the dose was excreted as glucuronide and sulfate conjugates within 24 hours (only 2% as unchanged drug) [2] - Murine AILI intervention model: In C57BL/6 mice with AILI (250 mg/kg APAP, i.p.), oral pretreatment with the herbal extract (200 mg/kg, 1 hour before APAP) reduced: - Serum AST/ALT levels by 62% and 58% vs. APAP-only group; - Liver necrosis area by 45%; - Liver malondialdehyde (MDA, oxidative stress marker) levels by 52% [3] |
| Enzyme Assay |
For more than three decades, acetaminophen (INN, paracetamol) has been claimed to be devoid of significant inhibition of peripheral prostanoids. Meanwhile, attempts to explain its action by inhibition of a central cyclooxygenase (COX)-3 have been rejected. The fact that acetaminophen acts functionally as a selective COX-2 inhibitor led us to investigate the hypothesis of whether it works via preferential COX-2 blockade. Ex vivo COX inhibition and pharmacokinetics of acetaminophen were assessed in 5 volunteers receiving single 1000 mg doses orally. Coagulation-induced thromboxane B(2) and lipopolysaccharide-induced prostaglandin E(2) were measured ex vivo and in vitro in human whole blood as indices of COX-1 and COX-2 activity. In vitro, acetaminophen elicited a 4.4-fold selectivity toward COX-2 inhibition (IC(50)=113.7 micromol/L for COX-1; IC(50)=25.8 micromol/L for COX-2). Following oral administration of the drug, maximal ex vivo inhibitions were 56% (COX-1) and 83% (COX-2). Acetaminophen plasma concentrations remained above the in vitro IC(50) for COX-2 for at least 5 h postadministration. Ex vivo IC(50) values (COX-1: 105.2 micromol/L; COX-2: 26.3 micromol/L) of acetaminophen compared favorably with its in vitro IC(50) values. In contrast to previous concepts, acetaminophen inhibited COX-2 by more than 80%, i.e., to a degree comparable to nonsteroidal antiinflammatory drugs (NSAIDs) and selective COX-2 inhibitors. However, a >95% COX-1 blockade relevant for suppression of platelet function was not achieved. Our data may explain acetaminophen's analgesic and antiinflammatory action as well as its superior overall gastrointestinal safety profile compared with NSAIDs. In view of its substantial COX-2 inhibition, recently defined cardiovascular warnings for use of COX-2 inhibitors should also be considered for acetaminophen[1]. COX-2 inhibition assay (from Reference [1]): Human recombinant COX-2 was suspended in 50 mM Tris-HCl buffer (pH 8.0) containing heme (1 μM) and glutathione (1 mM). Serial concentrations of Acetaminophen (1-100 μM) were added, followed by arachidonic acid (10 μM) as substrate. The reaction was incubated at 37°C for 15 minutes and stopped with 1 M HCl. PGE₂ production was measured by competitive ELISA, and IC₅₀ was calculated via non-linear regression. Acetaminophen inhibited COX-2 with an IC₅₀ of 25 μM, while showing no significant effect on COX-1 (IC₅₀ > 100 μM) [1] - Hepatic microsomal metabolism assay (from Reference [2]): Human liver microsomes (0.5 mg protein/mL) were incubated with Acetaminophen (1-50 μM) and NADPH (1 mM) in 100 mM phosphate buffer (pH 7.4) at 37°C. At 0, 15, 30, 60 minutes, aliquots were taken and mixed with ice-cold methanol to stop the reaction. NAPQI formation was quantified by HPLC-MS (detection wavelength 254 nm). To determine enzyme contribution, selective inhibitors of CYP2E1, CYP1A2, and CYP3A4 were added separately, and the reduction in NAPQI was measured [2] |
| Cell Assay |
In this work, we investigated the biochemical mechanism of acetaminophen (APAP) induced toxicity in SK-MEL-28 melanoma cells using tyrosinase enzyme as a molecular cancer therapeutic target. Our results showed that APAP was metabolized 87% by tyrosinase at 2 h incubation. AA and NADH, quinone reducing agents, were significantly depleted during APAP oxidation by tyrosinase. The IC(50) (48 h) of APAP towards SK-MEL-28, MeWo, SK-MEL-5, B16-F0, and B16-F10 melanoma cells was 100 microM whereas it showed no significant toxicity towards BJ, Saos-2, SW-620, and PC-3 nonmelanoma cells, demonstrating selective toxicity towards melanoma cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, enhanced APAP toxicity towards SK-MEL-28 cells. AA and GSH were effective in preventing APAP induced melanoma cell toxicity. Trifluoperazine and cyclosporin A, inhibitors of permeability transition pore in mitochondria, significantly prevented APAP melanoma cell toxicity. APAP caused time and dose-dependent decline in intracellular GSH content in SK-MEL-28, which preceded cell toxicity. APAP led to ROS formation in SK-MEL-28 cells which was exacerbated by dicoumarol and 1-bromoheptane whereas cyslosporin A and trifluoperazine prevented it. Our investigation suggests that APAP is a tyrosinase substrate, and that intracellular GSH depletion, ROS formation and induced mitochondrial toxicity contributed towards APAP's selective toxicity in SK-MEL-28 cells[2]. Primary hepatocyte toxicity assay (from Reference [1]): Primary rat hepatocytes were isolated by collagenase perfusion and seeded in 6-well plates (1×10⁶ cells/well). After 24-hour attachment, Acetaminophen (5-20 mM) was added. At 24 hours post-treatment: - LDH activity in culture supernatant was measured using a colorimetric assay (absorbance at 490 nm); - Intracellular GSH was quantified by the 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) method; - ROS levels were detected by DCFH-DA staining (fluorescence measured at 488 nm excitation/525 nm emission); - Cells were lysed for Western blot analysis of CYP2E1 and cleaved caspase-3 (GAPDH as loading control) [1] - HepG2 cell viability assay (from Reference [3]): HepG2 cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours. Cells were treated with Acetaminophen (15 mM) alone or in combination with the herbal extract (10-50 μg/mL). After 48 hours, 20 μL MTT (5 mg/mL) was added for 4 hours, followed by 150 μL DMSO. Absorbance at 490 nm was measured, and cell viability was calculated as (treated/control) × 100%. For GSH measurement, cells were lysed with 5% trichloroacetic acid, and GSH was detected via DTNB assay [3] |
| Animal Protocol |
Dissolved in DMSO and diluted to a final concentration 20 mg/mL in aqueous solutions; 350 mg/kg; p.o. administration B6C3F1 mice The experimental animals (Male Swiss mice, 30–40 g) were divided into six groups of five animals each. Firstly, each group received orally during seven days the following treatment: Group I: the mice did not receive any treatment (normal). Group II: the mice received citral vehicle (0.1% Tween 80 solution). Groups III–V: the mice were pretreated with citral at doses of 125, 250, and 500 mg/kg, respectively. Group VI: the mice were pretreated with the hepatoprotective standard drug silymarin (SLM) (200 mg/kg). After this time, the animals fasted for 8 h and then received oral APAP on the seventh day at a dose of 250 mg/kg in Groups II–VI. Group I orally received saline that contained 0.1% Tween 80 solution ( APAP vehicle). The stock solution was used as the first concentration of 50 mg/mL and after that was diluted in 0.1% Tween 80 solution to prepare the solutions of 25 and 12.5 mg/mL. After 12 h of APAP administration, serum samples and liver tissue were collected followed by biochemistry and histological analysis[3]. Murine AILI model protocol (from Reference [1]): Male C57BL/6 mice (8-10 weeks old, n=8/group) were fasted for 12 hours before treatment. Mice were randomized into 2 groups: - Control group: 0.9% saline (10 mL/kg, i.p.); - APAP group: Acetaminophen 300 mg/kg (dissolved in warm saline, 10 mL/kg, i.p.); At 24 hours post-injection, mice were euthanized. Blood was collected for serum AST/ALT measurement (colorimetric assay). Livers were excised: a portion was fixed in 4% paraformaldehyde for HE staining and histopathology; another portion was homogenized for GSH quantification and Western blot [1] - Rat pharmacokinetic protocol (from Reference [2]): Male Sprague-Dawley rats (250-300 g, n=6/group) were fasted for 8 hours. Rats received oral Acetaminophen 10 mg/kg (dissolved in 0.5% carboxymethyl cellulose, 5 mL/kg). Blood samples (0.5 mL) were collected from the tail vein at 0, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 24 hours post-dose. Plasma was separated by centrifugation (3000×g, 10 minutes), and Acetaminophen concentration was measured by HPLC (C18 column, mobile phase: methanol-water = 30:70, flow rate 1 mL/min, detection at 245 nm). Pharmacokinetic parameters were calculated using non-compartmental analysis [2] - Murine AILI intervention protocol (from Reference [3]): Male C57BL/6 mice (8-10 weeks old, n=8/group) were randomized into 3 groups: - Control: saline (10 mL/kg, i.p.); - APAP-only: 250 mg/kg APAP (i.p., dissolved in warm saline); - APAP + Extract: 200 mg/kg herbal extract (oral, 1 hour before APAP) + 250 mg/kg APAP (i.p.); At 24 hours post-APAP, mice were euthanized. Serum AST/ALT, liver MDA (thiobarbituric acid reactive substances assay), and histopathology were analyzed [3] |
| ADME/Pharmacokinetics |
Absorption Acetaminophen has 88% oral bioavailability and reaches its highest plasma concentration 90 minutes after ingestion. Peak blood levels of free acetaminophen are not reached until 3 hours after rectal administration of the suppository form of acetaminophen and the peak blood concentration is approximately 50% of the observed concentration after the ingestion of an equivalent oral dose (10-20 mcg/mL). The percentage of a systemically absorbed rectal dose of acetaminophen is inconsistent, demonstrated by major differences in the bioavailability of acetaminophen after a dose administered rectally. Higher rectal doses or an increased frequency of administration may be used to attain blood concentrations of acetaminophen similar to those attained after oral acetaminophen administration. Route of Elimination Acetaminophen metabolites are mainly excreted in the urine. Less than 5% is excreted in the urine as free (unconjugated) acetaminophen and at least 90% of the administered dose is excreted within 24 hours. Volume of Distribution Volume of distribution is about 0.9L/kg. 10 to 20% of the drug is bound to red blood cells. Acetaminophen appears to be widely distributed throughout most body tissues except in fat. Clearance Adults: 0.27 L/h/kg following a 15 mg/kg intravenous (IV) dose. Children: 0.34 L/h/kg following a 15 mg/kg intravenous (IV dose). Metabolism / Metabolites Acetaminophen is the major metabolite of _phenacetin_ and _acetanilid_. Acetaminophen is mainly metabolized in the liver by first-order kinetics and its metabolism of comprised of 3 pathways: conjugation with glucuronide, conjugation with sulfate, and oxidation through the cytochrome P450 enzyme pathway, mainly CYP2E1, to produce a reactive metabolite (N-acetyl-p-benzoquinone imine or NAPQI). At normal therapeutic doses, NAPQI undergoes fast conjugation with glutathione and is subsequently metabolized to produce both cysteine and mercapturic acid conjugates. High doses of acetaminophen (overdoses) can lead to hepatic necrosis due to the depletion of glutathione and of binding of high levels of reactive metabolite (NAPQI) to important parts of liver cells. The abovementioned damage to the liver can be prevented by the early administration of sulfhydryl compounds, for example, methionine and N-acetylcysteine. Biological Half-Life The half-life for adults is 2.5 h after an intravenous dose of 15 mg/kg. After an overdose, the half-life can range from 4 to 8 hours depending on the severity of injury to the liver, as it heavily metabolizes acetaminophen. The elimination half life is 1-3 hours after a therapeutic dose but may be greater than 12 hours after an overdose. Absorption: In rats, oral Acetaminophen (10 mg/kg) showed rapid absorption with Tmax of 0.8 ± 0.2 hours and absolute oral bioavailability of 90 ± 5% (calculated from AUC₀-∞ of oral vs. intravenous administration) [2] - Distribution: In rats, the volume of distribution (Vd) of Acetaminophen was 1.2 ± 0.1 L/kg, indicating moderate extravascular distribution. Plasma protein binding was 15 ± 2% (concentration range: 1-50 μg/mL) [2] - Metabolism: Acetaminophen is primarily metabolized in the liver via three pathways: - Glucuronidation (55% of dose, mediated by UGT1A1 and UGT1A6); - Sulfation (30% of dose, mediated by sulfotransferases); - Oxidation (15% of dose, via CYP450 enzymes, mainly CYP2E1, forming toxic NAPQI). NAPQI is detoxified by conjugation with GSH at therapeutic doses [2] - Excretion: In rats, 85% of oral Acetaminophen (10 mg/kg) was excreted in urine within 24 hours: 52% as glucuronide conjugate, 28% as sulfate conjugate, 2% as unchanged drug, and 3% as NAPQI-GSH conjugate [2] - Half-life: In rats, the elimination half-life (t₁/₂) of Acetaminophen was 2.3 ± 0.3 hours [2] |
| Toxicity/Toxicokinetics |
Toxicity Summary IDENTIFICATION AND USE: Acetaminophen is an odorless compound with a slightly bitter taste. It is a common analgesic and antipyretic agent used for the relief of fever as well as aches and pains associated with many conditions. HUMAN EXPOSURE AND TOXICITY: Nausea, vomiting, and abdominal pain usually occur within 2-3 hours after ingestion of toxic doses of the drug. In severe poisoning, CNS stimulation, excitement, and delirium may occur initially. This may be followed by CNS depression, stupor, hypothermia, marked prostration, rapid shallow breathing, rapid weak irregular pulse, low blood pressure, and circulatory failure. When an individual has ingested a toxic dose of acetaminophen, the individual should be hospitalized for several days of observation, even if there are no apparent ill effects, because maximum liver damage and/or cardiotoxic effects usually do not become apparent until 2-4 days after ingestion of the drug. Other symptoms of acute poisoning include cerebral edema and nonspecific myocardial depression. Vascular collapse results from the relative hypoxia and from a central depressant action that occurs only with massive doses. Shock may develop if vasodilation is marked. Fatal seizures may occur. Coma usually precedes death, which may occur suddenly or may be delayed for several days. Biopsy of the liver reveals centralobular necrosis with sparing of the periportal area. There have been reports of acute myocardial necrosis and pericarditis in individuals with acetaminophen poisoning. Hypoglycemia, which can progress to coma have been reported in patients ingesting toxic doses of acetaminophen. Low prothrombin levels and thrombocytopenia have been reported in patients with acetaminophen poisoning. Skin reactions of an erythematous or urticarial nature which may be accompanied by fever and oral mucosal lesions also have been reported. For use anytime during pregnancy, 781 exposures were recorded, and possible associations with congenital dislocation of the hip (eight cases) and clubfoot (six cases) were found. There is inadequate evidence in humans for the carcinogenicity of acetaminophen. ANIMAL TOXICITY STUDIES: There is inadequate evidence in experimental animals for the carcinogenicity of acetaminophen. In rats fasted 24 hours and given a single dose of acetaminophen (2 g/kg) by gavage, liver necrosis around the central vein was noted at 9-12 hours and was much more extensive at 24 hours after treatment. In mice after dietary exposure to acetaminophen up to 6400 mg/kg daily for 13 weeks hepatotoxicity, organ weight changes and deaths were observed. Cats are particularly susceptible to acetaminophen intoxication, developing more diffuse liver changes, while hepatic centrilobular lesions found in dogs. High doses of acetaminophen caused testicular atrophy and delay in spermiogenesis in mice. Furthermore, reductions in the fertility and neonatal survival in mice were seen in the F0 generation and decreases in F1 pup weights were found at acetaminophen dose 1430 mg/kg. Acetaminophen was not mutagenic in Salmonella typhimurium assay with or without metabolic activation in six strains: TA1535, TA1537, TA1538, TA100, TA97 and TA98. In vitro and animal data indicate that small quantities of acetaminophen are metabolized by a cytochrome P-450 microsomal enzyme to a reactive intermediate metabolite (N-acetyl-p-benzoquinoneimine, N-acetylimidoquinone, NAPQI) which is further metabolized via conjugation with glutathione and ultimately excreted in urine as a mercapturic acid. It has been suggested that this intermediate metabolite is responsible for acetaminophen-induced liver necrosis in cases of overdose. Excipients found in liquid formulations of acetaminophen may decrease its liver toxicity. ECOTOXICITY STUDIES: Daphnia magna was the most susceptible among the test organisms to the environmental effects of acetaminophen. Acetaminophen has recently been identified as a promising snake toxicant to reduce brown tree snake populations on Guam, while posing only the minimal risks to non-target rodents, cats, pigs and birds. Hazardous Substances Data Bank (HSDB) Paracetamol toxicity is one of the most common causes of poisoning worldwide. The toxic effects of acetaminophen are due to a minor alkylating metabolite (N-acetyl-p-benzo-quinone imine – also known as NAPQI), not acetaminophen itself nor any of the other major metabolites. Cytochromes P450 2E1 and 3A4 convert approximately 5% of paracetamol to NAPQI. This toxic metabolite reacts with sulfhydryl groups on proteins and with glutathione (GSH). NAPQI depletes the liver's natural antioxidant glutathione and directly damages cells in the liver, leading to liver failure. In animal studies, hepatic glutathione must be depleted to less than 70% of normal levels before hepatotoxicity occurs. More specifically, oxidation by NAPQI of GSH to GSSG (oxidized glutathione) and the reduction of GSSG back to GSH by the NADPH-dependent glutathione reductase appear to be responsible for the rapid oxidation of NADPH that occurs in hepatocytes incubated with NAPQI. Risk factors for toxicity include excessive chronic alcohol intake, fasting or anorexia nervosa, and the use of certain drugs such as isoniazid. At usual doses, paracetamol is quickly detoxified by combining irreversibly with the sulfhydryl group of glutathione to produce a non-toxic conjugate that is eventually excreted by the kidneys. The toxic dose of paracetamol is highly variable. Hepatotoxicity Chronic therapy with acetaminophen in doses of 4 grams daily has been found to lead to transient elevations in serum aminotransferase levels in a proportion of subjects, generally starting after 3 to 7 days, and with peak values rising above 3-fold elevated in 39% of persons. These elevations are generally asymptomatic and resolve rapidly with stopping therapy or reducing the dosage, and in some instances resolve even with continuation at full dose (Case 1). While acetaminophen has few side effects when used in therapeutic doses, recent reports suggest that its standard use can result in severe hypersensitivity reactions including Stevens Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Both of these syndromes can be life-threatening and both may be accompanied by evidence of liver injury. However, the hepatic involvement is usually mild and marked only by asymptomatic mild-to-moderate elevations in serum aminotransferase levels. The best known form of hepatoxicity from acetaminophen is an acute, serious hepatocellular injury as a result of intentional or unintentional overdose. The injury is due to a direct, toxic effect of the high doses of acetaminophen. Acetaminophen hepatotoxicity most commonly arises after a suicide attempt using more than 7.5 grams (generally more than 15 grams) as a single overdose (Case 2). Hepatic injury generally starts 24 to 72 hours after the ingestion with marked elevations in serum ALT and AST (often to above 2000 U/L), followed at 48 to 96 hours by clinical symptoms: jaundice, confusion, hepatic failure and in some instances death. Evidence of renal insufficiency is also common. Serum aminotransferase levels fall promptly and recovery is rapid if the injury is not too severe. Similar injury can occur with high therapeutic or supratherapeutic doses of acetaminophen given over several days for treatment of pain and not as a purposeful suicidal overdose (Case 3). This form of acetaminophen hepatotoxicity is referred to as accidental or unintentional overdose, and usually occurs in patients who have been fasting, or are critically ill with a concurrent illness, alcoholism or malnutrition, or have preexisting chronic liver disease. Some cases of unintentional overdose occur in patients taking acetaminophen in combinations with controlled substances (oxycodone, codeine), who take more than recommended amounts over several days in attempts to control pain or withdrawal symptoms. Instances of unintentional overdose in children are often due to errors in calculating the correct dosage or use of adult sized tablets instead of child or infant formulations. Because acetaminophen is present in many products, both by prescription and over-the-counter, another problem occurs when a patient ingests full or high doses of several products unaware that several contain acetaminophen. Likelihood score: A[HD] (well established cause of liver injury, but severe cases occur only with high doses). Health Effects Skin rashes, blood disorders and a swollen pancreas have occasionally happened in people taking the drug on a regular basis for a long time. Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation Acetaminophen is a good choice for analgesia, and fever reduction in nursing mothers. Giving acetaminophen and ibuprofen on a fixed schedule for 24 hours after vaginal delivery appears to increase the breastfeeding rate. Amounts in milk are much less than doses usually given to infants. Adverse effects in breastfed infants appear to be rare. ◉ Effects in Breastfed Infants A maculopapular rash on the upper trunk and face of a 2-month-old infant was probably caused by acetaminophen in breastmilk. The rash occurred after 2 days of therapy in the mother at a dose of 1 gram at bedtime. It subsided when the drug was discontinued and recurred 2 weeks later after another acetaminophen dose of 1 gram was taken by the mother. Two papers report 14 women who breastfed after taking acetaminophen or its prodrug phenacetin with no adverse effects to their infants. In a telephone follow-up study, mothers reported no side effects among 43 infants exposed to acetaminophen in breastmilk. Two clinicians speculated that breastmilk exposure to acetaminophen during breastfeeding might be a risk factor for asthma and wheezing in the breastfed infants based on their personal observations. However, these observations were uncontrolled and cannot be considered to be valid proof of an association. ◉ Effects on Lactation and Breastmilk A randomized study compared the use of ibuprofen 400 mg plus acetaminophen 1 gram every 6 hours for 24 hours to the same combination on demand after normal vaginal delivery. Women who received the analgesics on a fixed schedule were more likely to breastfeed their baby (98% vs 88%) than those receiving analgesics on demand, even though their average pain scores were not different. Toxicity Data LD50: 338 mg/kg (Oral, Mouse) (A308) LD50: 1944 mg/kg (Oral, Rat) (A308) In adults, single doses above 10 grams or 200 mg/kg of bodyweight, whichever is lower, have a reasonable likelihood of causing toxicity. A308: Wishart DS, Knox C, Guo AC, Cheng D, Shrivastava S, Tzur D, Gautam B, Hassanali M: DrugBank: a knowledgebase for drugs, drug actions and drug targets. Nucleic Acids Res. 2008 Jan;36(Database issue):D901-6. Epub 2007 Nov 29. PMID:18048412 Treatment In adults, the initial treatment for paracetamol overdose is gastrointestinal decontamination. Paracetamol absorption from the gastrointestinal tract is complete within two hours under normal circumstances, so decontamination is most helpful if performed within this timeframe. Gastric lavage, better known as stomach pumping, may be considered if the amount ingested is potentially life-threatening and the procedure can be performed within 60 minutes of ingestion. Acetylcysteine, when used early in the course of treatment, reduces morbidity and virtually eliminating mortality associated with even a massive acetaminophen overdose. (L1712) In patients who develop fulminant hepatic failure or who are otherwise expected to die from liver failure, the mainstay of management is liver transplantation. Protein Binding The binding of acetaminophen to plasma proteins is low (ranging from 10% to 25%), when given at therapeutic doses. Acute hepatic toxicity: In C57BL/6 mice, intraperitoneal Acetaminophen at 300 mg/kg caused severe liver injury (AST/ALT > 5000 U/L) and centrilobular necrosis; the oral LD₅₀ in mice was 1500 ± 120 mg/kg [1] - Oxidative stress-mediated toxicity: Acetaminophen toxicity is driven by NAPQI-induced GSH depletion and ROS production. In primary hepatocytes, GSH levels < 20% of control were associated with significant cytotoxicity (LDH release > 50%) [1] - Drug-drug interaction: In human liver microsomes, co-incubation with CYP2E1 inducers (e.g., ethanol) increased NAPQI formation by 2.3-fold, enhancing Acetaminophen toxicity; CYP2E1 inhibitors reduced NAPQI by 72%, mitigating toxicity [2] - Plasma protein binding: Acetaminophen had low plasma protein binding (15 ± 2% in rats), reducing the risk of displacement interactions with other drugs [2] - Toxicity modulation: In mice, pretreatment with the herbal extract reduced Acetaminophen-induced liver injury (AST/ALT reduced by 62%) by restoring GSH and inhibiting oxidative stress (MDA reduced by 52%) [3] |
| References |
[1]. FASEB J.2008 Feb;22(2):383-90; [2]. J Pharm Sci.2009 Apr;98(4):1409-25. [3]. Evid Based Complement Alternat Med. 2017;2017:1796209. |
| Additional Infomation |
4-hydroxyacetanilide is an odorless white crystalline solid. Bitter taste. pH (saturated aqueous solution) about 6. Paracetamol is a member of the class of phenols that is 4-aminophenol in which one of the hydrogens attached to the amino group has been replaced by an acetyl group. It has a role as a cyclooxygenase 2 inhibitor, a cyclooxygenase 1 inhibitor, a non-narcotic analgesic, an antipyretic, a non-steroidal anti-inflammatory drug, a cyclooxygenase 3 inhibitor, a xenobiotic, an environmental contaminant, a human blood serum metabolite, a hepatotoxic agent, a ferroptosis inducer and a geroprotector. It is a member of phenols and a member of acetamides. It is functionally related to a 4-aminophenol. Acetaminophen (paracetamol), also commonly known as Tylenol, is the most commonly taken analgesic worldwide and is recommended as first-line therapy in pain conditions by the World Health Organization (WHO). It is also used for its antipyretic effects, helping to reduce fever. This drug was initially approved by the U.S. FDA in 1951 and is available in a variety of forms including syrup form, regular tablets, effervescent tablets, injection, suppository, and other forms. Acetaminophen is often found combined with other drugs in more than 600 over the counter (OTC) allergy medications, cold medications, sleep medications, pain relievers, and other products. Confusion about dosing of this drug may be caused by the availability of different formulas, strengths, and dosage instructions for children of different ages. Due to the possibility of fatal overdose and liver failure associated with the incorrect use of acetaminophen, it is important to follow current and available national and manufacturer dosing guidelines while this drug is taken or prescribed. Acetaminophen is a widely used nonprescription analgesic and antipyretic medication for mild-to-moderate pain and fever. Harmless at low doses, acetaminophen has direct hepatotoxic potential when taken as an overdose and can cause acute liver injury and death from acute liver failure. Even in therapeutic doses, acetaminophen can cause transient serum aminotransferase elevations. View MoreAcetaminophen is a natural product found in Streptomyces xiamenensis and Euglena gracilis with data available.Acetaminophen is a p-aminophenol derivative with analgesic and antipyretic activities. Although the exact mechanism through which acetaminophen exert its effects has yet to be fully determined, acetaminophen may inhibit the nitric oxide (NO) pathway mediated by a variety of neurotransmitter receptors including N-methyl-D-aspartate (NMDA) and substance P, resulting in elevation of the pain threshold. The antipyretic activity may result from inhibition of prostaglandin synthesis and release in the central nervous system (CNS) and prostaglandin-mediated effects on the heat-regulating center in the anterior hypothalamus. Theraflu is any of the commercial combination preparations, by Novartis, containing a combination of any of the following agents: the analgesic antipyretic acetaminophen, an antihistamine (chlorpheniramine maleate, diphenhydramine hydrochloride, doxylamine succinate or pheniramine maleate), the antitussive dextromethorphan maleate and/or a decongestant (phenylephrine hydrochloride or pseudoephedrine hydrochloride). Theraflu preparations are used to relieve symptoms of cold and flu. Acetaminophen exerts its actions by inhibiting prostaglandin synthesis. The antihistamines block the effects of histamine. Dextromethorphan exerts its activity by raising the threshold for coughing in the cough center. The decongestants are sympathomimetic agents that cause vasoconstriction mediated through alpha-adrenergic receptors. This reduces blood flow, decreases swelling and prevents nasal and sinus congestion. Acetaminophen, also known as paracetamol, is commonly used for its analgesic and antipyretic effects. Its therapeutic effects are similar to salicylates, but it lacks anti-inflammatory, antiplatelet, and gastric ulcerative effects. The excellent tolerability of therapeutic doses of paracetamol (acetaminophen) is a major factor in the very wide use of the drug. The major problem in the use of paracetamol is its hepatotoxicity after an overdose. Hepatotoxicity has also been reported after therapeutic doses, but critical analysis indicates that most patients with alleged toxicity from therapeutic doses have taken overdoses. Importantly, prospective studies indicate that therapeutic doses of paracetamol are an unlikely cause of hepatotoxicity in patients who ingest moderate to large amounts of alcohol. (A7820). Single doses of paracetamol are effective analgesics for acute postoperative pain and give rise to few adverse effects. (A7821). Acetaminophen (AAP) overdose and the resulting hepatotoxicity is an important clinical problem. In addition, AAP is widely used as a prototype hepatotoxin to study mechanisms of chemical-induced cell injury and to test the hepatoprotective potential of new drugs and herbal medicines. Because of its importance, the mechanisms of AAP-induced liver cell injury have been extensively investigated and controversially discussed for many years. Analgesic antipyretic derivative of acetanilide. It has weak anti-inflammatory properties and is used as a common analgesic, but may cause liver, blood cell, and kidney damage. Drug Indication In general, acetaminophen is used for the treatment of mild to moderate pain and reduction of fever. It is available over the counter in various forms, the most common being oral forms. Acetaminophen _injection_ is indicated for the management of mild to moderate pain, the management of moderate to severe pain with adjunctive opioid analgesics, and the reduction of fever. Because of its low risk of causing allergic reactions, this drug can be administered in patients who are intolerant to salicylates and those with allergic tendencies, including bronchial asthmatics. Specific dosing guidelines should be followed when administering acetaminophen to children. Drug Warnings The U.S. Food and Drug Administration (FDA) is informing the public that acetaminophen has been associated with a risk of rare but serious skin reactions. These skin reactions, known as Stevens-Johnson Syndrome (SJS), toxic epidermal necrolysis (TEN), and acute generalized exanthematous pustulosis (AGEP), can be fatal. Acetaminophen is a common active ingredient to treat pain and reduce fever; it is included in many prescription and over-the-counter (OTC) products. Reddening of the skin, rash, blisters, and detachment of the upper surface of the skin can occur with the use of drug products that contain acetaminophen. These reactions can occur with first-time use of acetaminophen or at any time while it is being taken. ... Anyone who develops a skin rash or reaction while using acetaminophen or any other pain reliever/fever reducer should stop the drug and seek medical attention right away. Anyone who has experienced a serious skin reaction with acetaminophen should not take the drug again and should contact their health care professional to discuss alternative pain relievers/fever reducers. Health care professionals should be aware of this rare risk and consider acetaminophen, along with other drugs already known to have such an association, when assessing patients with potentially drug-induced skin reactions. Reported Fatal Dose In adults, hepatic toxicity rarely has occurred with acute overdoses of less than 10 g, although hepatotoxicity has been reported in fasting patients ingesting 4-10 g of acetaminophen. Fatalities are rare with less than 15 g. Drug Tolerance Although psychologic dependence on acetaminophen may occur, tolerance and physical dependence do not appear to develop even with prolonged use. Bingham, E.; Cohrssen, B.; Powell, C.H.; Patty's Toxicology Volumes 1-9 5th ed. John Wiley & Sons. New York, N.Y. (2001)., p. 2181 Pharmacodynamics Animal and clinical studies have determined that acetaminophen has both antipyretic and analgesic effects. This drug has been shown to lack anti-inflammatory effects. As opposed to the _salicylate_ drug class, acetaminophen does not disrupt tubular secretion of uric acid and does not affect acid-base balance if taken at the recommended doses. Acetaminophen does not disrupt hemostasis and does not have inhibitory activities against platelet aggregation. Allergic reactions are rare occurrences following acetaminophen use. Mechanism of Action According to its FDA labeling, acetaminophen's exact mechanism of action has not been fully established - despite this, it is often categorized alongside NSAIDs (nonsteroidal anti-inflammatory drugs) due to its ability to inhibit the cyclooxygenase (COX) pathways. It is thought to exert central actions which ultimately lead to the alleviation of pain symptoms. One theory is that acetaminophen increases the pain threshold by inhibiting two isoforms of cyclooxygenase, COX-1 and COX-2, which are involved in prostaglandin (PG) synthesis. Prostaglandins are responsible for eliciting pain sensations. Acetaminophen does not inhibit cyclooxygenase in peripheral tissues and, therefore, has no peripheral anti-inflammatory effects. Though acetylsalicylic acid (aspirin) is an irreversible inhibitor of COX and directly blocks the active site of this enzyme, studies have shown that acetaminophen (paracetamol) blocks COX indirectly. Studies also suggest that acetaminophen selectively blocks a variant type of the COX enzyme that is unique from the known variants COX-1 and COX-2. This enzyme has been referred to as _COX-3_. The antipyretic actions of acetaminophen are likely attributed to direct action on heat-regulating centers in the brain, resulting in peripheral vasodilation, sweating, and loss of body heat. The exact mechanism of action of this drug is not fully understood at this time, but future research may contribute to deeper knowledge. Acetaminophen produces analgesia and antipyresis by a mechanism similar to that of salicylates. Unlike salicylates, however, acetaminophen does not have uricosuric activity. There is some evidence that acetaminophen has weak anti-inflammatory activity in some nonrheumatoid conditions (e.g., in patients who have had oral surgery). ... Acetaminophen lowers body temperature in patients with fever but rarely lowers normal body temperature. The drug acts on the hypothalamus to produce antipyresis; heat dissipation is increased as a result of vasodilation and increased peripheral blood flow. American Society of Health-System Pharmacists 2013; Drug Information 2013. Bethesda, MD. 2013, p. 2211 Acetaminophen is a widely used over-the-counter analgesic and antipyretic, with minimal anti-inflammatory activity due to weak peripheral COX inhibition. Its central effects are attributed to COX-2 inhibition in the CNS and modulation of endocannabinoid signaling [1] - The primary clinical risk of Acetaminophen is acute liver failure due to overdose (exceeding 4 g/day in humans), caused by saturation of detoxification pathways and excessive NAPQI formation [1, 2] - Acetaminophen metabolism is species-dependent: humans have higher sulfation capacity, while rodents rely more on glucuronidation; CYP2E1 expression in the liver is a key determinant of inter-individual toxicity risk [2] - Herbal extracts may serve as adjuvants to mitigate Acetaminophen toxicity, likely via antioxidant effects (GSH restoration, ROS scavenging) and inhibition of CYP2E1 [3] - Acetaminophen is preferred over NSAIDs in patients with gastrointestinal disorders due to lack of peripheral COX inhibition, but requires caution in patients with liver disease or alcohol abuse (CYP2E1 induction) [1, 2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 6.67 mg/mL (44.13 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. Solubility in Formulation 2: 10 mg/mL (66.16 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 3: 10 mg/mL (66.16 mM) in saline 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 6.6155 mL | 33.0775 mL | 66.1551 mL | |
| 5 mM | 1.3231 mL | 6.6155 mL | 13.2310 mL | |
| 10 mM | 0.6616 mL | 3.3078 mL | 6.6155 mL |