Elsevier

Toxicology in Vitro

Volume 30, Issue 1, Part B, 25 December 2015, Pages 241-249
Toxicology in Vitro

Toxicological evaluation of acyl glucuronides utilizing half-lives, peptide adducts, and immunostimulation assays

https://doi.org/10.1016/j.tiv.2015.10.013Get rights and content

Highlights

  • Acyl glucuronides (AGs) is suggested to be involved in the toxicity.

  • Half-lives and peptide adducts assay exhibited some false-positive.

  • We found 5 genes induced by AGs using DNA microarray analysis.

  • Immunostimulation assay using 5 genes as biomarkers showed high accuracy.

  • The risk assessment of AGs can contribute to improve drug safety.

Abstract

Chemical reactivity of acyl glucuronides (AGs) is believed to be involved in the toxicity of carboxylic acid-containing drugs. Both direct and immune-mediated toxicity have been suggested as possible mechanisms of toxicity; however, it remains unclear. In the present study, we performed assays of half-lives, peptide adducts, and immunostimulation to evaluate the potential risk of AGs of 21 drugs and analyzed the relationship to the toxic category. AGs of all withdrawn drugs tested in this study showed short half-lives and peptide adducts formation, but so did those of several safe drugs. In contrast, only AGs of withdrawn and warning drugs induced interleukin-8 (IL-8) in human peripheral blood mononuclear cells (hPBMCs). Using a DNA microarray assay, we found that zomepirac AG induced the mRNAs of 5 genes, including IL-8 in hPBMCs. In addition, withdrawn and warning drugs were distinguished from safe drugs by an integrated score of relative mRNA expression levels of 5 genes. The immunostimulation assay showed higher sensitivity, specificity, and accuracy compared with other methods. In preclinical drug development, the evaluation of the reactivity of AGs using half-lives and peptide adducts assays followed by the evaluation of immunostimulation by highly reactive AGs using hPBMCs can contribute to improved drug safety.

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.

Section snippets

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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