Toxicological evaluation of acyl glucuronides utilizing half-lives, peptide adducts, and immunostimulation assays
Introduction
Acyl glucuronidation is one of the major metabolic routes of carboxylic acid-containing drugs. Glucuronidation is an important phase II metabolic pathway for endogenous and exogenous substrates and is generally considered as a detoxification pathway. However, acyl glucuronides (AGs) are unstable under physiological conditions and consequently undergo hydrolysis or intramolecular rearrangement through the migration of the drug moiety from the 1-O-position to the 2-, 3-, or 4-positions on the glucuronic acid ring (Bailey and Dickinson, 2003, Benet et al., 1993, Smith et al., 1990). Because of their electrophilic nature and capacity to cause substitution reactions with nucleophilic groups in proteins or other macromolecules, AGs can covalently modify endogenous proteins leading to the adverse toxicity associated with carboxylic acid-containing drugs (Boelsterli, 2002, Faed, 1984). Till date, both direct and immune- and inflammation-mediated toxic pathways have been suggested as possible toxic mechanisms of AGs. Previous studies (Nakayama et al., 2009, Usui et al., 2009) have reported that zomepirac and bromfenac, which are carboxylic acid-containing drugs that have been withdrawn from the market, showed low covalent binding to proteins in human hepatocytes. In addition, the AGs of the widely used drugs naproxen, diclofenac, ketoprofen, and ibuprofen did not lead to cytotoxicity or genotoxicity in uridine 5′-diphosphate (UDP)-glucuronosyltransferase (UGT)–transfected human embryonic kidney 293 (HEK/UGT) cells and human hepatocytes (Koga et al., 2011). In contrast, it has been reported that mycophenolic acid AG induced tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6), and diclofenac AG induced IL-8 and monocyte chemoattractant protein 1 (MCP-1) in leukocytes (Miyashita et al., 2014, Wieland et al., 2000), suggesting that the induction of immune modulators could lead to immune- and/or inflammation-related adverse drug reactions.
Several in vitro assay methods to assess the toxicity of AGs have been proposed. The first is an evaluation of the half-lives of AGs in potassium phosphate buffer (KPB). The half-lives of AGs in withdrawn drugs were shorter than those in safe drugs. Therefore, this assay is useful to predict the risk of toxicity (Jinno et al., 2013, Sawamura et al., 2010). The second method is a Lys-Phe adducts assay, wherein Lys-Phe is used as a novel trapping agent that forms glycation adducts via a Schiff base. In this assay, a correlation was observed between the formation of a peptide adduct and the rearrangement rate of the primary AG of 7 drugs (Wang et al., 2004). The third method is an immunostimulation assay using human peripheral blood mononuclear cells (hPBMCs), wherein cytokines and chemokines, such as IL-6 and IL-8, were induced in hPBMCs by treatment with AGs (Miyashita et al., 2014, Wieland et al., 2000). Although the predictability of the assay of half-lives for drug toxicity has been evaluated (Jinno et al., 2013, Sawamura et al., 2010), the relationship between the results of the other two assays and drug toxicity remains to be investigated. The purpose of the present study was to evaluate the relationship of the results of assays of half-lives, peptide adducts, and immunostimulation to the toxic category of carboxylic acid-containing drugs defined by description in drug package inserts, and then to compare the usability of the three assays to assess the risk of toxicity of AGs in preclinical drug discovery. We modified the assay of peptide adducts by using dansylated Lys-Phe (dKF), peptide-AG adducts of which were easily detectable by fluorescence.
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Chemicals and reagents
Oxaprozin, pranoprofen, etodolac, and dKF were prepared in-house by chemosynthesis. Probenecid was obtained from Wako Pure Chemical Industries (Osaka, Japan). Diclofenac sodium salt, tolmetin sodium salt dihydrate, zomepirac sodium salt, mefenamic acid, bumetanide, furosemide, flufenamic acid, meclofenamic acid sodium salt, ibuprofen, and repaglinide were obtained from Sigma-Aldrich (St. Louis, MO). Montelukast sodium and telmisartan were obtained from Kemprotec (Middlesborough, UK).
Half-lives of AGs in KPB
In the present study, the 21 carboxylic acid-containing drugs shown in Table 1 were categorized on the basis of severe adverse drug reactions as follows. The 4 drugs categorized as “withdrawn” included drugs withdrawn from the market because of their toxicity, with potential to cause hepatotoxicity and anaphylaxis. The 3 drugs with a warning about fulminant hepatitis in drug package inserts in Japan or the United States or in the summary of product characteristics in Europe were categorized as
Discussion
The AGs of drugs are generally unstable and are believed to be involved in drug-induced toxicity via the formation of covalent adducts to endogenous proteins. In the process of drug discovery, it is important to predict and avoid the toxicity of AGs of new chemical entities, to reduce the need to withdraw drugs from the market, and to increase safety in clinical trials. Although there is increasing evidence that AGs form drug–protein adducts via their chemical reactivity (Grubb et al., 1993,
Conflict of interest
The authors declare no conflicts of interest.
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References (34)
- et al.
Acyl glucuronide reactivity in perspective: biological consequences
Chem. Biol. Interact.
(2003) - et al.
Predictability of the covalent binding of acidic drugs in man
Life Sci.
(1993) - et al.
A simple in vitro model to study the stability of acylglucuronides
J. Pharmacol. Toxicol. Methods
(2007) - et al.
The interleukin-1 family: back to the future
Immunity
(2013) - et al.
Studies on the in vitro reactivity of clofibryl and fenofibryl glucuronides. Evidence for protein binding via a Schiff's base mechanism
Biochem. Pharmacol.
(1993) - et al.
Metallothionein 2a gene expression is increased in subcutaneous adipose tissue of type 2 diabetic patients
Mol. Genet. Metab.
(2013) - et al.
p38 mitogen-activated protein kinase mediates IL-8 induction by the ribotoxin deoxynivalenol in human monocytes
Toxicol. Appl. Pharmacol.
(2006) - et al.
Blocking effect of anti-mouse interleukin-6 monoclonal antibody and glucocorticoid receptor antagonist, RU38486, on metallothionein-inducing activity of serum from lipopolysaccharide-treated mice
Toxicology
(1996) - et al.
Neutrophil recruitment by intradermally injected neutrophil attractant/activation protein-1
J. Investig. Dermatol.
(1991) - et al.
Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency in vitro
Toxicol. in Vitro
(2010)
Evaluation and mechanistic analysis of the cytotoxicity of the acyl glucuronide of nonsteroidal anti-inflammatory drugs
Drug Metab. Dispos.
Dipeptidyl peptidase IV is a target for covalent adduct formation with the acyl glucuronide metabolite of the anti-inflammatory drug zomepirac
Life Sci.
Induction of cytokine release by the acyl glucuronide of mycophenolic acid: a link to side effects?
Clin. Biochem.
Development of a cell-based assay system considering drug metabolism and immune- and inflammatory-related factors for the risk assessment of drug-induced liver injury
Toxicol. Lett.
Interleukin-8 and related chemotactic cytokines—CXC and CC chemokines
Adv. Immunol.
Xenobiotic acyl glucuronides and acyl CoA thioesters as protein-reactive metabolites with the potential to cause idiosyncratic drug reactions
Curr. Drug Metab.
Properties of acyl glucuronides: implications for studies of the pharmacokinetics and metabolism of acidic drugs
Drug Metab. Rev.
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