| Standard to Meet
|| Compound Assessment
| Human Toxicity
| Compound causes cholestasis, lipid-accumulating disorders (steatosis/phopholipidosis) or cell death in humans.
|| Hepatocellular and cholestatic injury has been seen with dicolfenac use (Banks et al HEPATOLOGY 95;22:820-827)
Total amount of patients in all trials was 1121. 583 patients took diclofenac and 538 ones took placebo. Meta-analysis was performed in StatsDirect software. We estimated 95% confidence interval, Q and 12 criteria, Mantel-Haenszel and DerSimonian-Laird statistics and relative risk of adverse reactions. Relative risk of hepatitis in diclofenac group did not differ from placebo. Hereby the fact of diclofenac hepatotoxicity needs more detailed study and genetic factors of risk should be taken into account.
Gomez-Lechon, M.J., Ponsoda, X., O’Connor, E., Donato, T., Jover, R., Castell, J.V., 2003. Diclofenac induces apoptosis in hepatocytes. Toxicol. in Vitro 17, 675–680
Patients using diclofenac showed no higher incidence of serious liver disease than did patients using other NSAIDs. The risk of clinically apparent liver injury was ˜ 1 case per 10,000 patient-years of NSAID use.
A.M. Walker, Quantitative studies of the risk of serious hepatic injury in persons using nonsteroidal antiinflammatory drugs. Arthritis Rheum. 40 (1997), pp. 201–208
Acute liver injury secondary to high exposure has not been observed clinically (with the exception of the dramatic onset of immunoallergic liver failure following rechallenge with the drug, but this is a totally different mechanism).
Diclofenac-induced liver injury is a paradigm for drug-related hepatotoxicity. Recent studies suggest that genetic factors favoring the formation and accumulation of the reactive acylglucuronide metabolite of diclofenac and an enhanced immune response to the metabolite-protein adducts are associated with increased susceptibility to hepatotoxicity
Clin Liver Dis. 2007 Aug;11(3):563-75, Nonsteroidal anti-inflammatory drug-induced hepatotoxicity. Aithal GP, Day CP.
| Toxicity is concentration dependent (non-idiosyncratic).
|| Banks et al report that low incidence of diclofenac toxicity is likely to be the result of metabolic idiosyncrasy, therefore within scope.
However, according to this publication (accessed 28 June, 2011).
For several reasons, diclofenac hepatotoxicity is an archetype of idiosyncratic DILI. About 15% of those patients regularly taking diclofenac develop elevated levels of liver enzymes, and a threefold rise in transaminase levels has been reported in 5%. A population-based study estimated that clinically relevant hepatotoxicity leading to hospital referral occurs in 6.3 (95% CI 3-12) per 100,000 diclofenac users. This finding implies that multiple steps are involved in the development of serious idiosyncratic DILI.
The majority (85%) of patients present with liver injury within 6 months of starting diclofenac therapy, and presentation after 1 year of treatment is rare (3%). Diclofenac is associated with a predominantly hepatocellular pattern of liver injury, but a cholestatic pattern of liver injury and cases resembling autoimmune hepatitis have also been described.[7,11] Therefore, it is likely that several factors—related to metabolism, consequences of formation of reactive metabolites and the response of the host to these initial insults—determine the degree of liver injury and its manifestations
The pathogenesis of low-incidence/high-severity diclofenac hepatotoxicity is largely unknown. One reason for this is the apparent lack of animal models available to date that mimic the human situation and with which one could investigate the mechanisms of the toxic response. Clearly, because liver injury from diclofenac is not a reproducible effect and lacks a simple dose–response relationship, it is generally accepted that individual patient-specific susceptibility factors eventually determine whether a patient will tolerate the drug (as in the vast majority of cases) or whether an individual in rare cases will develop a toxic response. Thus, diclofenac-induced liver injury is a typical example of idiosyncratic drug toxicity.
Ref. Aithal,G.P. "Diclofenac-induced liver injury: a paradigm of idiosyncratic drug toxicity" Expert Opin. Drug Saf. 3, 519-523 (2004)
| Therapeutic target.
|| NSAID (COX inhibitor)
The antiinflammatory effects of diclofenac are believed to be due to inhibition of both leukocyte migration and the enzyme cylooxygenase (COX-1 and COX-2), leading to the peripheral inhibition of prostaglandin synthesis. As prostaglandins sensitize pain receptors, inhibition of their synthesis is responsible for the analgesic effects of ketoprofen. Antipyretic effects may be due to action on the hypothalamus, resulting in peripheral dilation, increased cutaneous blood flow, and subsequent heat dissipation
| Biochemical mechanism of toxicity.
|| Impairment of ATP synthesis by mitochondria, and production of active metabolites, particularly n,5-dihydroxydiclofenac, which causes direct cytotoxicity. Mitochondrial permeability transition (MPT) has also been shown to be important in diclofenac-induced liver injury, resulting in generation of reactive oxygen species, mitochondrial swelling and oxidation of NADP and protein thiols. (O’Connor et al QJM. 2003 Nov;96(11):787-91)
Diclofenac-induced apoptosis, via the mitochondrial pathway which was related to CYP-mediated metabolism of diclofenac, with the highest apoptotic effect produced by the metabolite 5OH-diclofenac.
Oxidative stress at the mitochondrial level is in the root of MPT induction and caspase cascade activation
MJ Gómez-Lechón et al Biochem Pharmacol (2003) 66: 2155-67.
1)distinct reactive metabolites (quinone imines, arene oxides, acyl glucuronide, and iso-glucuronides) are involved in producing covalent protein modification, mostly in the canalicular plasma membrane and affecting the proteins' functions;
(2)oxidative stress is generated (by a putative cation radical or a nitroxide or by quinone imine cycling or thiol oxidation);
(3)mitochondria are a major subcellular target (uncoupling, MPT pore opening, oxidative stress, and apoptosis).
Urs A. Boelsterli Toxicology and Applied Pharmacology Volume 192, Issue 3, 1 November 2003, Pages 307-322
| PK parameters: recommended dose, Cmax, Vd, and half-life.
|| 99.7 % of diclofenac binds to serum proteins, mainly to albumin (99.4%).
Apparent volume of distribution calculated is 0.12-0.17 L/kg 1.3 L/kg (Drugbank)
Cmax in 12 healthy volunteers provided with 100mg sodium diclofenac from 2 preparations indicate 453-511ng/ml SD 217-272ng/ml Med J Malaysia Vol 59 No 3 August 2004 352-356
| Therapeutic window.
| Metabolically activated (optional), active metabolite known and available for testing.
Diclofenac undergoes ring hydroxylation, catalyzed by hCYP2C9, resulting in the formation of the major oxidative metabolite, 4′-hydroxydiclofenac [Stierlin and Faigle, 1979] Other, quantitatively minor products of oxidative metabolism include 5-hydroxydiclofenac, catalyzed by hCYP3A4 (featuring a Km that is 20× higher than that for CYP2C9).
Both the 4′-OH- and the 5-OH-diclofenac are structural alerts because they are potential protoxicants as they can be further oxidized to quinone imines at either ring system
| Omics and IC50 Data
| Gene expression profiles known.
|| Gene expression profiles in livers from diclofenac-treated rats reveal intestinal bacteria-dependent and -independent pathways associated with liver injury.
Deng X, Liguori MJ, Sparkenbaugh EM, Waring JF, Blomme EA, Ganey PE, Roth RA. J Pharmacol Exp Ther. 2008 Dec;327(3):634-44. Epub 2008 Sep 18.
In vitro gene expression analysis of hepatotoxic drugs in rat primary hepatocytes.
Suzuki H, Inoue T, Matsushita T, Kobayashi K, Horii I, Hirabayashi Y, Inoue T. J Appl Toxicol. 2008 Mar;28(2):227-36.
Public on Mar 28, 2008
Title Liver Pharmacology and Xenobiotic Response Repertoire
Organism Rattus norvegicus
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
Status Public on Mar 26, 2011
Title Non-steroidal anti-inflammatory drugs induce apoptosis and differentiation through an AP-1 and GADD45α dependent pathway in human acute myeloid leukemia cells
Organism Homo sapiens
Singh R, Koch A
Open TG-GATEs Human Liver
Status Public on Feb 25, 2011
Title Genomics Assisted Toxicity Evaluation system study - Human Hepatocytes
Organism(s) Homo Sapiens
Type Expression profiling by array
Summary TGP (Toxicogenomics project) is engaged by the Pharmaceutical Institute for Fundamental Research, National Institute of Health, and pharmaceutical companies (3) the public-private collaborative project. From FY 2002 through FY 2006 a large and high-quality Toxicogenomics database was built studying gene expression and toxicity information for 150 compounds in populations of rats and human hepatocytes exposed to drug.
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 Template:Pmid:20041446
Study Name Broad Connectivity Map (CMAP 2.0) compound database
(CMAP 1.0 i.e GSE5258 has been updated with this CMAP 2.0 dataset which is not available through GEO in its entirety)
Species Homo sapiens
Summary: A reference collection of genome-wide transcriptional expression data from cultured human cells (HL60 human promyelocytic leukemia, MCF-7 human breast carcinoma, and PC-3 human prostate cancer cell lines) treated with bioactive small molecules that enable the discovery of functional connections between drugs, genes and diseases through the transitory feature of common gene-expression changes.
Lamb J, Crawford ED, Peck D, Modell JW et al. The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science 2006 Sep 29;313(5795):1929-35. pmid:17008526
| Proteomics profiles known.
|| J Proteomics. 2010 Feb 10;73(4):721-32. Epub 2009 Oct 20.
Proteomic analysis in NSAIDs-treated primary cardiomyocytes.
Baek SM, Ahn JS, Noh HS, Park J, Kang SS, Kim DR.
BMC Med Genomics. 2010 Feb 23;3:5.
Insight in modulation of inflammation in response to diclofenac intervention: a human intervention study.
van Erk MJ, et al.
| Metabonomics profiles known.
| Fluxomics profiles known.
| Epigenomics profiles known.
| Observed IC50 for in vitro cellular efficacy.
|| Inhibits both COX-1(IC50 = 76 nM) and COX-2 (IC50 = 26 nM) activities. Also inhibits liver phenol sulfotransferase activity (IC50 approx. 9.5 nM) Calbiochem data sheet 2003
It inhibits human COX-1 and -2 with IC50 values of 0.9-2.7 and 1.5-20 µM. Laneuville, O., Breuer, D.K., DeWitt, D.L., et al.. J Pharmacol Exp Ther 271 927-934 (1994)
| Observed IC50 for in vitro cellular toxicity studies.
|| Rat primary hepatocytes 392+/-34 uM (n=16) and human primary hepatocytes 331+/-7 uM (n= 6), HepG2 763+/- 61 uM, (n=4) and FaO 682+/-64 uM,( n= 3), MDCK (908+/-56 uM, (n =2)
Ref: Bort et al., JPET 288:65–72, 1998
Apoptosis observed at 150 uM.
Ref: Francesca Cecere, Annarita Iuliano, Francesco Albano, Claudia Zappelli, Immacolata Castellano, Pasquale Grimaldi, Mariorosario Masullo, Emmanuele De Vendittis, and Maria Rosaria Ruocco1, "Diclofenac-Induced Apoptosis in the Neuroblastoma Cell Line SH-SY5Y: Possible Involvement of the Mitochondrial Superoxide Dismutase", J Biomed Biotechnol. 2010; 2010: 801726.
| Physical Properties
| Accepted and listed within the ToxCast/Tox21 program.
|| Included in phase II ToxCast List U.S EPA/ORD/NCCT. ToxCast Phase I and II Chemicals. December 14, 2010. Accessed May 25 2011.
| Defined and confirmed structure and isomeric form(s).
| Substance stability.
|| Phototransformation under natural sunlight
Aguera A, Perez Estrada LA, Ferrer I, Thurman EM, Malato S and Fernandez-Alba AR. Application of time-of-ﬂight mass spectrometry to the analysis of phototransformation products of diclofenac in water under natural sunlight. J. Mass Spectrom. 2005; 40: 908–915
| Soluble in buffer solution at 30 times the in vitro IC50 for toxicity.
|| The aqueous solubility values reported in literature for diclofenac are spread over a large range, with a factor of 100 separating the largest and the smallest, ranging from 0.02 to 1.8 mg/ml. The measured intrinsic solubility within this study is 0.001 mg/ml (25°C)
Llinas A, Burley JC, Box kJ, Glen RC and Goodman JM. Diclofenac solubility: independent determination of the intrinsic solubility of the three crystal forms. J Med Chem 2006; 50: 979-983
Diclofenac estimated intrinsic solubility : 9.08e-3mg/ml
Diclofenac estimated solubility in pure water at pH 4.35: 2.2512e-2 mg/ml
Diclofenac estimated solubility in water at pH 7.4: 11.72 mg/ml (Calculations performed using ACD/PhysChem v 9.14)
Solubility as a function of pH and other parameters available on the wiki
Diclofenac sodium salt water solubility: 50 mg/mL Sigma D6899 Product Details
Diclofenac sodium salt water solubility: 20.4 mg/g
Fele Zilnik L, Jazbinsek A,Hvala A, Vrecer F, Klamt A. Solubility of sodium diclofenac in different solvents. Fluid Phase Equilibria 261 (2007) 140–145
| Solubility in DMSO 100 times buffer solubility.
|| Diclofenac sodium salt DMSO solubility: 135 mg/g
Fele Zilnik L, Jazbinsek A,Hvala A, Vrecer F, Klamt A. Solubility of sodium diclofenac in different solvents. Fluid Phase Equilibria 261 (2007) 140–145
| Vessel binding properties.
| Commercial availability at > 95% (> 99% is preferred).
|| Diclofenac sodium salt Sigma Aldrich (D6899) 10g/79.00€ purity: >98%
Sigma D6899 Product Details
| Vapor pressure. (Non-volatile)
|| Estimated vapor pressure 6.14E-8 (Calculation performed using EPI Suite v4.10)
| Criteria Notes
|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.
|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.
|Literature data for at least one, but not necessarily all, of the ‘omics datasets is desired. This requirement can be waived in special cases.
|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.
|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.
|Sparing soluble compounds may be assayed for solubility in serum and the percent serum used specified here.
|This property will be measured when a sample of compound becomes available.