Department of Pharmaceutical Analysis, Institute of Pharmacy, Nirma University, Sarkhej-Gandhinagar Highway, Post: Chandlodia, Via: Gota, Ahmedabad 382 481, Gujarat, India.
Chem Res Toxicol. 2022 Jul 18;35(7):1206-1219. doi: 10.1021/acs.chemrestox.1c00222. Epub 2022 Jun 22.
Pranlukast hydrate (PRN), a cysteinyl leukotriene receptor antagonist (CysLT), is used to treat bronchial asthma. The objective of this study is to perform the isolation, characterization, and toxicity analysis of stress degradation products of PRN. In high-performance liquid chromatography (HPLC), the separation was achieved using a Phenomenex Gemini C18 (250 × 4.6 mm, 5 μ) column; the ammonium format buffer (50 mM), pH 4, with formic acid: acetonitrile (50:50, v/v) was used as a mobile phase at a flow rate of 1.25 mL/min; and the photodiode array detector was used for detection at 230 nm. The drug was subjected to stress degradation as per ICH Q1A (R2) and ICH Q1B guidelines. The drug was found to be labile in alkaline (62.48% degradation) and photolytic (liquid state) (7.67% degradation) conditions, whereas the drug was found to be stable in acidic, peroxide, photolytic (solid state), and thermal conditions. The characterization of the drug and its degradation products was achieved using liquid chromatography-electrospray ionization-quadrupole time of flight tandem mass spectrometry (LC-ESI-QTOF-MS/MS), and the degradation mechanism was proposed. There were two degradation products observed in alkaline conditions (DP6 and DP9), whereas six novel degradation products were observed in photolytic degradation products (DP1, DP3, DP4, DP5, DP7, and DP10). The developed method was successfully validated as per the ICH Q2 (R1) guideline. The isolation of the alkaline degradation product DP9 was performed using preparative HPLC, and it was found to be 96.8% pure degradation product. The characterizations of the isolated degradation product (DP9) and procured impurity were performed using MS/MS, NMR, and FTIR. The mass of the procured impurity and DP9 were observed to be 404 and 500 Da, respectively. The in vitro cytotoxicity study of the procured impurity and DP9 was conducted using a 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay using an A549 cell line, and they were found to be cytotoxic at concentrations above 62.5 and 250 μg/mL, respectively. Furthermore, an in silico toxicity study was performed to predict the toxicity of all the major characterized degradation products of PRN using admetSAR software version 2.0. DP1, DP2, DP6, and DP10 were found to be hepatotoxic, mutagenic according to the micronucleus test, and aquatic toxic. We can conclude that the drug should be kept away from the direct exposure of light and the toxicity levels of DP1, DP2, DP6, and DP10 should be reduced below 0.1% to avoid their toxic effect.
普拉鲁司特水合物（PRN）是一种半胱氨酰白三烯受体拮抗剂（CysLT），用于治疗支气管哮喘。本研究的目的是对 PRN 的应激降解产物进行分离、表征和毒性分析。在高效液相色谱（HPLC）中，使用 Phenomenex Gemini C18（250×4.6mm，5μm）柱进行分离；以 50mM 氨格式缓冲液（pH4），含甲酸：乙腈（50：50，v/v）为流动相，流速为 1.25mL/min；采用光电二极管阵列检测器在 230nm 处进行检测。根据 ICH Q1A（R2）和 ICH Q1B 指南，对药物进行了应激降解。结果表明，药物在碱性（62.48%降解）和光解（液体状态）（7.67%降解）条件下不稳定，而在酸性、过氧化物、光解（固体状态）和热条件下稳定。采用液相色谱-电喷雾电离-四极杆飞行时间串联质谱（LC-ESI-QTOF-MS/MS）对药物及其降解产物进行了表征，并提出了降解机制。在碱性条件下观察到两种降解产物（DP6 和 DP9），而在光解降解产物中观察到六种新的降解产物（DP1、DP3、DP4、DP5、DP7 和 DP10）。根据 ICH Q2（R1）指南，对所开发的方法进行了成功验证。使用制备型 HPLC 分离碱性降解产物 DP9，发现其为 96.8%纯度的降解产物。采用 MS/MS、NMR 和 FTIR 对分离得到的降解产物（DP9）和获得的杂质进行了表征。获得的杂质和 DP9 的质量分别为 404 和 500Da。采用 MTT 法对获得的杂质和 DP9 进行了体外细胞毒性研究，使用 A549 细胞系，发现它们在浓度高于 62.5 和 250μg/mL 时具有细胞毒性。此外，使用 admetSAR 软件版本 2.0 对 PRN 的所有主要特征性降解产物进行了计算机毒性预测。DP1、DP2、DP6 和 DP10 被认为具有肝毒性，根据微核试验具有致突变性，并且对水生生物有毒。我们可以得出结论，药物应避免直接暴露在光线下，并且 DP1、DP2、DP6 和 DP10 的毒性水平应降低到 0.1%以下，以避免其毒性作用。
