Chong Lor Huai, Ng Celine, Li Huan, Tian Edmund Feng, Ananthanarayanan Abhishek, McMillian Michael, Toh Yi-Chin
Department of Biomedical Engineering, National University of Singapore, 4 Engineering Drive 3, #04-08, Singapore 117583.
School of Applied Science, Temasek Polytechnic, Tampines Avenue 1, Singapore 529765.
ACS Omega. 2019 Aug 12;4(9):13902-13912. doi: 10.1021/acsomega.9b01551. eCollection 2019 Aug 27.
The clinical use of some drugs, such as carbamazepine, phenytoin, and allopurinol, is often associated with adverse cutaneous reactions. The bioactivation of drugs into immunologically reactive metabolites by the liver is postulated to be the first step in initiating a downstream cascade of pathological immune responses. Current mechanistic understanding and the ability to predict such adverse drug cutaneous responses have been partly limited by the lack of appropriate cutaneous drug bioactivation experimental models. Although in vitro human liver models have been extensively investigated for predicting hepatotoxicity and drug-drug interactions, their ability to model the generation of antigenic reactive drug metabolites that are capable of eliciting immunological reactions is not well understood. Here, we employed a human progenitor cell (HepaRG)-derived hepatocyte model and established highly sensitive liquid chromatography-mass spectrometry analytical assays to generate and quantify different reactive metabolite species of three paradigm skin sensitizers, namely, carbamazepine, phenytoin, and allopurinol. We found that the generation of reactive drug metabolites by the HepaRG-hepatocytes was sensitive to the medium composition. In addition, a functional assay based on the activation of U937 myeloid cells into the antigen-presenting cell (APC) phenotype was established to evaluate the immunogenicity potential of the reactive drug metabolites produced by HepaRG-derived hepatocytes. We showed that the reactive drug metabolites of known skin sensitizers could significantly upregulate 8, 1β, and 86 expressions in U937 cells compared to the metabolites from a nonskin sensitizer (i.e., acetaminophen). Thus, the extent of APC activation by HepaRG-hepatocytes conditioned medium containing reactive drug metabolites can potentially be used to predict their skin sensitization potential.
某些药物的临床应用，如卡马西平、苯妥英和别嘌醇，常常与皮肤不良反应相关。药物经肝脏生物活化形成具有免疫反应性的代谢产物，被认为是引发下游一系列病理性免疫反应的第一步。目前，由于缺乏合适的皮肤药物生物活化实验模型，对其作用机制的理解以及预测此类药物皮肤不良反应的能力受到了一定限制。尽管体外人肝脏模型已被广泛研究用于预测肝毒性和药物相互作用，但其模拟能够引发免疫反应的抗原性反应性药物代谢产物生成的能力尚不清楚。在此，我们采用了人祖细胞（HepaRG）来源的肝细胞模型，并建立了高灵敏度的液相色谱 - 质谱分析方法，以生成和定量三种典型皮肤致敏剂（即卡马西平、苯妥英和别嘌醇）的不同反应性代谢产物种类。我们发现，HepaRG - 肝细胞生成反应性药物代谢产物对培养基成分敏感。此外，还建立了一种基于将U937髓样细胞激活为抗原呈递细胞（APC）表型的功能测定方法，以评估HepaRG来源的肝细胞产生的反应性药物代谢产物的免疫原性潜力。我们发现，与非皮肤致敏剂（即对乙酰氨基酚）的代谢产物相比，已知皮肤致敏剂的反应性药物代谢产物能显著上调U937细胞中8、1β和86的表达。因此，含有反应性药物代谢产物的HepaRG - 肝细胞条件培养基激活APC的程度有可能用于预测其皮肤致敏潜力。
