Standard to Meet
| Compound Assessment
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Human Toxicity
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Compound causes one or more of the following:- cholestasis
- steatosis
- phospholipidosis
- fibrosis
- cell death
in humans; or exemplifies an in vitro mechanism underlying one of these toxicities.
| High doses can cause acute hepatic necrosis due to production of toxic quinone imine metabolite (NAPQI). From 1998 to 2003, acetaminophen was the leading cause of acute liver failure in the United States, with 48% of acetaminophen-related cases (131 of 275) associated with accidental overdose.
References:
- | Larson AM, Polson J, Fontana RJ, Davern TJ, Lalani E, Lee WM et al. Acute Liver Failure Study Group (ALFSG). Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study. Hepatology, 2005 Dec; 42(6):1364-72. |
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Toxicity occurs via one or more of the following mechanisms:- mitochondrial disruption
- inhibition of lipid transport or metabolism
- nuclear receptor modulation
- complexation of phospholipids
| Metabolism via CYP2E1 produces a toxic metabolite, N-acetyl-p-benzoquinoneimine (NAPQI). The toxic effects of acetaminophen are due to NAPQI, not acetaminophen itself nor any of the major metabolites. NAPQI is a quinone imine that has both redox cycling activity and alkylating activity. The imine depletes the mitochondrial membrane potential and glutathione to a comparable extent at toxic concentrations. Although glutathione depletion is most readily measured, it is assumed that NAPQI oxidizes/alkylates reactive protein thiols to an extent comparable to glutathione. It is not clear which reactivity, redox or alkylating, dominates the production of toxicity. At high doses high enough to significantly deplete glutathione, cell death is observed.
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Therapeutic target.
| Acetaminophen is thought to act primarily in the CNS, increasing the pain threshold by inhibiting both isoforms of cyclooxygenase, COX-1, COX-2, and COX-3 enzymes involved in prostaglandin (PG) synthesis. Unlike NSAIDs, acetaminophen does not inhibit cyclooxygenase in peripheral tissues and, thus, has no peripheral anti-inflammatory affects.
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Biochemical mechanism of toxicity.
| Cytotoxicity following production of toxic metabolite by CYP450 and GSH depletion.
Metabolism via CYP2E1 produces a toxic metabolite, N-acetyl-p-benzoquinoneimine (NAPQI). The toxic effects of acetaminophen are due to NAPQI, not acetaminophen itself nor any of the major metabolites. At therapeutic doses, NAPQI reacts with the sulfhydryl group of glutathione to produce a non-toxic conjugate that is excreted by the kidneys. High doses of acetaminophen may cause glutathione depletion, accumulation of NAPQI and hepatic necrosis. The maximum daily dose of acetaminophen is 4 g. Liver failure has been observed at doses as low as 6 g per day (drugbank)
Oral, mouse: LD50 = 338 mg/kg; Oral, rat: LD50 = 1944 mg/kg
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PK-ADME
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PK parameters 1:
- recommended dose
- Cmax
- Vd
- half-life
| Cmax 325 mg dose = 4.2 micro-g/ml. Half life 2.5 hours. (drugs.com)
Cmax 1000 mg dose = 17.98-20.55 micro-g/ml (formulation dependent). Half life 2.65-2.81 hours. (
Sevilla-Tirado Methods Find Exp Clin Pharmacol 2003, 25(7):531-535)
Vd = 0.9 L/kg.
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Therapeutic window.1
| The recommended daily dose is 4 g/day (60 mg/kg/day). Toxicity has been observed at the recommended dose and (rarely) at doses of 2.5 g/day. Although the therapeutic window is low, a specific toxicity threshold in humans has not been defined. Reference: Gerald Dal Pan, Bob Rappaport, Background Package for June 29-30, 2009 Meeting of the Drug Safety and Risk Management Committee, Anesthetic and Life Support Drugs Advisory Committee and Nonprescription Drugs Advisory Committee.
Species
| Route
| LD50 (mg/kg)
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Mouse
| Oral
| 340
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Rat
| Oral
| 1900
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Reference: Lee W. Drug-induced hepatotoxicity, New England Journal of Medicine, July 31, 2003; 349:474-485.
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Metabolically activated (optional), active metabolite known and available for testing.2
| Approximately 90 to 95% of a dose is conjugated in the liver with glucuronic acid and sulfuric acid. A small percentage of acetaminophen is oxidized by CYP2E1 to form N-acetyl-p-benzo-quinone imine (NAPQI), a toxic metabolite which is then conjugated to glutathione and excreted renally. Accumulation of NAPQI may occur if primary metabolic pathways are saturated.
Enzyme
| Metabolite
| Km
| Vmax
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Cytochrome P450 2D6
| NAPQI
| 1760
| 54.12
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Cytochrome P450 1A2
| NAPQI
| 950
| 0.12
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Cytochrome P450 2E1
| NAPQI
| 1290
| 120.84
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Omics and IC50 Data
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Gene expression profiles known.3
| E-TOXM-31 Transcription profiling of human hepatocyte and neuron cell cultures following exposure to various chemical compounds
Homo sapiens
Open TG-GATEs Human Liver
Status: Public on Feb 25, 2011 Title Genomics Assisted Toxicity Evaluation system study - Human Hepatocytes Organism(s) Homo Sapiens Citation(s) Takeki Uehara, Atsushi Ono, Toshiyuki Maruyama, Ikuo Kato, Hiroshi Yamada, Yasuo Ohno, Tetsuro Urushidani. The Japanese toxicogenomics project: application of toxicogenomics. Molecular nutrition & food research. 2010 Feb;54(2): 218-27 pmid:20041446
Series GSE5595
Status: Public on Sep. 19, 2007
Title: Acetaminophen (APAP) Rat Liver Test Gene Ex
Organism(s) Rattus norvegicus
Summary Gene expression test data set from rat liver samples exposed to either 150, 1500 or 2000 mg/kg of APAP for 3, 6 or 24 hours.
Bushel PR, Heinloth AN, Li J, Huang L et al. Blood gene expression signatures predict exposure levels. Proc Natl Acad Sci U S A 2007 Nov 13;104(46):18211-6. pmid:17984051
Series GSE8858
Study Title Liver Pharmacology and Xenobiotic Response Repertoire
Species Rattus norvegicus
Publications
Natsoulis G, Pearson CI, Gollub J, P Eynon B et al. The liver pharmacological and xenobiotic gene response repertoire. Mol Syst Biol 2008;4:175. pmid:18364709
E-MEXP-82
Title: Transcription profiling time course toxicogenomic profiles in CD-1 mice after nontoxic and nonlethal hepatotoxic paracetamol administration
Species: Mus musculus
Citation(s)
Williams DP, Garcia-Allan C, Hanton G, LeNet JL, Provost JP, Brain P, Walsh R, Johnston GI, Smith DA, Park BK. Time course toxicogenomic profiles in CD-1 mice after nontoxic and nonlethal hepatotoxic paracetamol administration. Chem Res Toxicol. 2004 Dec;17(12):1551-61.
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Proteomics profiles known.3
| Proteomic Analysis of Acetaminophen-Induced Changes in Mitochondrial Protein Expression Using Spectral Counting Chem. Res. Toxicol., 2011, 24 (4), pp 549-558
Alterations in the rat serum proteome during liver injury from acetaminophen exposure J Pharmacol Exp Ther (2006) 318: 792-802
Genomics and Proteomics Analysis of Acetaminophen Toxicity in Mouse Liver TOXICOLOGICAL SCIENCES 65, 135-150 (2002)
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Metabonomics profiles known.3
| Acetaminophen dosing of humans results in blood transcriptome and metabolome changes consistent with impaired oxidative phosphorylation Hepatology. 2010 Jan;51(1):227-36
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Fluxomics profiles known.3
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Epigenomics profiles known.3
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Observed IC50 for in vitro cellular efficacy.4
| IC50 values ranging from 4 to 200 uM dependant on cell type and stimulant. 7.2 and 4.2 M for PGE2 and PGF2 respectively for rheumatoid synoviocytes (Graham et al. Inflammopharmacology, Vol. 9, No. 1,2, pp. 131-142 (2001)
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Observed IC50 for in vitro cellular toxicity studies. 4
| Paracetamol IC50: 5-8 mM (mouse/human) VIOLLON et al Cell Biology and Toxicology Volume 11, Numbers 3-4, 195-227 (1991) 3D HepaTox Chip rat IC50 16.90mM (Toh et al. Lab Chip, 2009, 9, 2026-2035)
IC50 rat hepatocyte 14014 uM, 3T3 cells 80344 uM, Hela 31283 uM HepG2 29755 uM cell viability Wang et al. J of Toxicological Sciences Vol. 27 (2002) , No. 3 August 229-237
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Physical Properties
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Accepted and listed within the ToxCast/Tox21 program.5
| Included in phase II ToxCast List
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Defined and confirmed structure and isomeric form(s).
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Substance stability.
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Soluble in buffer solution at 30 times the in vitro IC50 for toxicity.6
| Water solubility 14 mg/ml (25°C) YALKOWSKY,SH & HE,Y (2003) from SRC PhysProp Database
Water solubility 16.7 mg/ml (25°C) Mota FL, Carneiro AP, Queimada AJ, Pinho SP, Macedo EA. Temperature and solvent effects in the solubility of some pharmaceutical compounds: measurements and modeling. Eur J Pharm Sci 2009; 37: 499-507.
estimated intrinsic solubility : 17.8 mg/ml
estimated solubility in pure water at pH 5.70: 17.8 mg/ml
estimated solubility in water at pH 7.4: 17.9 mg/ml
Calculations performed using ACD/PhysChem v 9.14
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Solubility in DMSO 100 times buffer solubility.
| 5 M Sigma Aldrich A7085 Product details
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Vessel binding properties.7
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Commercial availability at > 95% (> 99% is preferred).
| Sigma Aldrich (A7085) 100g/54.10€ purity >99% Sigma Aldrich A7085 Product details
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Vapor pressure. (Non-volatile)
| estimated vapor pressure: 1.94E-06 mmHg (Calculation performed using EPI Suite v4.10)
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Criteria Notes
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1. | The in vivo therapeutic window is used to estimate an appropriate concentration for in vitro toxicity assays. This in vitro concentration should also be consistent with the exposure implied by pharmacokinetics parameters. |
2. | We prefer compounds that require metabolic activation, although standards that are active in themselves will be accepted if they have otherwise valuable properties. We require knowing the active metabolite, and we prefer compounds where the metabolite is stable and can be independently tested in order to verify the mechanism of toxicity as well as of metabolic activation in the test cell line. |
3. | Literature data for at least one, but not necessarily all, of the ‘omics datasets is desired. This requirement can be waived in special cases. |
4. | The IC50’s for in vitro efficacy and toxicity should be consistent with the therapeutic ratio observed in the clinic. These parameters will be dependent on specific cell type and culture conditions, but differences of more than 30-fold in the in vitro vs. in vivo therapeutic ratios should be considered suspect and carefully justified. |
5. | This is not a requirement, but compounds utilized in the EPA testing program can be assumed to have physical properties verified to be suitable for in vitro cellular assays. |
6. | Sparing soluble compounds may be assayed for solubility in serum and the percent serum used specified here. |
7. | This property will be measured when a sample of compound becomes available. |
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