Risk Of Idiosyncratic Drug Reactions Research Articles

Involvement of Extracellular Vesicles in the Proinflammatory Response to Clozapine: Implications for Clozapine-Induced Agranulocytosis

Most idiosyncratic drug reactions (IDRs) appear to be immune-mediated, but mechanistic events preceding severe reaction onset remain poorly defined. Damage-associated molecular patterns (DAMPs) may contribute to both innate and adaptive immune phases of IDRs, and changes in extracellular vesicle (EV) cargo have been detected post-exposure to several IDR-associated drugs. To explore the hypothesis that EVs are also a source of DAMPs in the induction of the immune response preceding drug-induced agranulocytosis, the proteome and immunogenicity of clozapine- (agranulocytosis-associated drug) and olanzapine- (non-agranulocytosis-associated drug) exposed EVs were compared in two preclinical models: THP-1 macrophages and Sprague-Dawley rats. Compared with olanzapine, clozapine induced a greater increase in the concentration of EVs enriched from both cell culture media and rat serum. Moreover, treatment of drug-naïve THP-1 cells with clozapine-exposed EVs induced an inflammasome-dependent response, supporting a potential role for EVs in immune activation. Proteomic and bioinformatic analyses demonstrated an increased number of differentially expressed proteins with clozapine that were enriched in pathways related to inflammation, myeloid cell chemotaxis, wounding, transforming growth factor-β signaling, and negative regulation of stimuli response. These data indicate that, although clozapine and olanzapine exposure both alter the protein cargo of EVs, clozapine-exposed EVs carry mediators that exhibit significantly greater immunogenicity. Ultimately, this supports the working hypothesis that drugs associated with a risk of IDRs induce cell stress, release of proinflammatory mediators, and early immune activation that precedes severe reaction onset. Further studies characterizing EVs may elucidate biomarkers that predict IDR risk during development of drug candidates. SIGNIFICANCE STATEMENT: This work demonstrates that clozapine, an idiosyncratic drug-induced agranulocytosis (IDIAG)-associated drug, but not olanzapine, a safer structural analogue, induces an acute proinflammatory response and increases extracellular vesicle (EV) release in two preclinical models. Moreover, clozapine-exposed EVs are more immunogenic, as measured by their ability to activate inflammasomes, and contain more differentially expressed proteins, highlighting a novel role for EVs during the early immune response to clozapine and enhancing our mechanistic understanding of IDIAG and other idiosyncratic reactions.

Idiosyncratic Drug Reactions: A 35-Year Chemical Research in Toxicology Perspective.

When Larry Marnett founded Chemical Research in Toxicology, the study of idiosyncratic drug reactions (IDRs) was in its infancy. There was evidence that IDRs involve chemically reactive metabolites, and many of the papers in Chemical Research in Toxicology investigated the bioactivation of drugs. However, it became clear that not all drugs that form reactive metabolites are associated with a high risk of IDRs, and some drugs that do not appear to form reactive metabolites do cause IDRs. Some of the early Chemical Research in Toxicology papers investigated involvement of the adaptive immune system in the mechanism of IDRs, and HLA associations provided strong evidence for an immune mechanism of IDRs. This led to the question of how reactive metabolites might induce an immune response. The classic hapten hypothesis provided an obvious explanation, but a new hypothesis the danger hypothesis, added another dimension. Although there are common features to IDRs, it is becoming increasingly clear that there are also many differences in the mechanisms caused by different drugs. Other pharmacological effects of drugs may also play a role in the mechanism, and that is obviously true of IDRs caused by biological agents. The requirement for specific HLA and T-cell receptors is presumably the major factor that makes IDRs idiosyncratic. However, an innate immune response is required to prime the adaptive immune response. In contrast to the adaptive immune response, the innate immune response is unlikely to be idiosyncratic, and studies of the innate immune response to drugs may provide a much more accurate way to screen drugs for their potential to cause IDRs. For essential drugs that are known to cause IDRs, it may be possible to markedly decrease risk by a slow dose titration to induce immune tolerance. Significant progress has been made in the study of IDRs, but there is still a long way to go.

Are drugs containing a carboxylic acid functional group associated with a significant risk of idiosyncratic drug reactions

Are drugs containing a carboxylic acid functional group associated with a significant risk of idiosyncratic drug reactions

Afatinib-loaded immunoliposomes functionalized with cetuximab: A novel strategy targeting the epidermal growth factor receptor for treatment of non-small-cell lung cancer

Afatinib, a selective and irreversible inhibitor of tyrosine kinase, was approved for the treatment of advanced non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) overexpression in 2013. Cetuximab (CTX), an anti-EGFR monoclonal antibody, is co-administered with afatinib to improve efficacy. Unfortunately, dose-related adverse reactions caused by combination therapy have affected patient compliance, and have resulted in treatment discontinuation in severe cases. In the present study, afatinib was encapsulated in “liposomes” (LPs) to achieve longer circulation in the blood and an enhanced permeability-and-retention effect in tumors. Concomitantly, CTX was designed to bind to drug-loaded LPs to form “immuno-LPs” for tumor-cell selectivity and therapeutic activity. In vitro, the cellular internalization rate of immuno-LPs was significantly higher than that of LPs (p < 0.05). In vivo, a markedly increased area under the curve and prolonged terminal half-life were detected in rats injected with the two LP formulations, indicating that LP encapsulation protected afatinib from binding to hemoglobin to control the risk of idiosyncratic drug reactions. Compared with free afatinib and LPs, immuno-LPs exhibited strongly enhanced drug delivery and antitumor efficacy in an NSCLC xenograft model, with stronger tumor selectivity and potentially fewer side-effects. Hence, EGFR-targeting immuno-LPs appear to be promising for NSCLC treatment.

Determination of tyrosine kinase inhibitor afatinib in rat plasma using LC–MS/MS and its application to in vivo pharmacokinetic studies of afatinib liposomes

A sensitive quantitative liquid chromatography-tandem mass spectrometry assay for afatinib in rat plasma was developed and validated using afatinib dimaleate as a standard. The analyte was determined to be ionized using the positive ion multiple reaction monitoring mode through electrospraying on an AB Sciex Triple Quad™ 4500 system. Protein precipitation was used for sample preparation, and an Agilent Eclipse XDB-CN column (100 × 2.1 mm, 3.5 μm) was applied for chromatographic separation. The mobile phase was water and methanol (15:85, v/v) containing 0.1% formic acid with a flow rate of 0.5 mL/min. The linear range was 0.5–200 ng/mL, with r2 = 0.9994 ± 0.0004 calculated from linear regression, with 1/x2 as the weighting factor for the calibration. The average recovery of the plasma samples was stable and reproducible. The analyte is sufficiently stable for handling and analysis. The pharmacokinetic study and comparison were performed by analyzing plasma concentrations in rats administered afatinib solution or prepared afatinib liposomes using the developed determination method. The Cmax of the afatinib liposomes was nearly 400-fold higher than that of the afatinib solution. These results indicate that liposome-encapsulation protected afatinib from endogenous protein binding, thereby reducing the risk of idiosyncratic drug reactions by protein adducts. Thus, Afatinib liposomes seem to be a promising strategy for the treatment of non-small cell lung cancer.

Characterization of a highly sensitive and selective novel trapping reagent, stable isotope labeled glutathione ethyl ester, for the detection of reactive metabolites

IntroductionGlutathione (GSH) trapping assays are widely used to predict the post-marketing risk for idiosyncratic drug reactions (IDRs) in the pharmaceutical industry. Although several GSH derivatives have been introduced as trapping reagents for reactive intermediates, more sensitive and selective reagents are desired to prevent the generation of erroneous results. In this study, stable isotope labeled GSH ethyl ester (GSHEE-d5) was designed and its detection capability was evaluated. MethodsGSHEE-d5 was synthesized and its detection potential was compared with stable isotope labeled GSH ([13C2,15N]GSH) as a reference trapping reagent. The trapping reagents were added to human liver microsomes as a 1:1 mixture with GSHEE or GSH, respectively, and incubated with seven IDR positive drugs and three IDR negative drugs. The adducts formed between the reagents and reactive metabolites were analyzed by unit resolution mass spectrometer (MS) using isotope pattern-dependent scan with neutral loss filtering. ResultsA single-step reaction of GSH and ethanol-d6 produced GSHEE-d5 with a yield of 85%. The GSHEE-d5 assay detected adducts with all seven IDR positive drugs, and no adducts were detected with the three IDR negative drugs. In contrast, the [13C2,15N]GSH assay failed to detect adducts with three of the IDR positive drugs. In the case of diclofenac, the GSHEE-d5 assay showed a 4-times greater signal intensity than the [13C2,15N]GSH assay. DiscussionGSHEE-d5 enabled the detection of reactive metabolites with greater sensitivity and selectivity than [13C2,15N]GSH. These results demonstrate that GSHEE-d5 would be a useful trapping reagent for evaluating the risk of IDRs with unit resolution MS.