P Johnsirani, Ch Vishnuvardhan, A Lingesh, V G M Naidu, Ch Naveen, N Satheeshkumar
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER-Hyderabad), Balanagar, Hyderabad, 500 0037 Telangana, India.
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER-Hyderabad), Balanagar, Hyderabad, 500 0037 Telangana, India; Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education and Research (NIPER-Hyderabad), Balanagar, Hyderabad 500 0037, Telangana, India.
J Pharm Biomed Anal. 2017 Mar 20;136:148-155. doi: 10.1016/j.jpba.2017.01.001. Epub 2017 Jan 3.
The present study describes the isolation, characterization and in vitro cytotoxic effect of all forced degradation products of niclosamide (NCM) an anthelmintic class of drug used specifically to treat tapeworms. NCM was subjected to forced degradation involving hydrolysis (acidic, alkaline and neutral), oxidative, photolysis and thermal stress, as per ICH (Q1A (R2)) suggested conditions. The drug under hydrolytic (acidic and basic) conditions showed extensive degradation, while it was stable under neutral hydrolytic, oxidative, photolytic and thermal stress conditions. A total of four degradation products (DPs) were observed and chromatographic separation of drug and its degradation products were achieved on a reverse phase Fortis diphenyl column (150×4.6mm, 5μm) using 0.1% formic acid and acetonitrile as mobile phase in gradient mode. All the four degradation products were isolated by semi preparative LC and its structures were characterized and confirmed by high resolution MS and H NMR spectroscopic techniques. In view of safety aspects, cytotoxicity assay were carried out for NCM and its four degradation products on human mononuclear cells and cell lines depicting the major organelle: neuronal (Neuro 2a), hepatic (HepG 2) and alveolar (A549). NCM was found to be non toxic on human mononuclear cells and cell lines at tested concentrations. However DP-1, DP-2, DP-3 and DP-4 showed significant increase in LDH release as compared to control at a concentration of 100μM. DP-1 and DP-3 exhibited toxicity on A549 cells with an IC of 92.18±4.93μM and 65.42±6.29μM respectively. DP-2, DP-3 and DP-4 were cytotoxic to Neuro 2a cells with an IC of 63.62±3.85μM, 86.09±6.19μM and 42.81±8.10μM respectively. The degradation products were found to be nontoxic on HepG 2 cells.
本研究描述了氯硝柳胺（NCM）所有强制降解产物的分离、表征及体外细胞毒性作用。氯硝柳胺是一种专门用于治疗绦虫的驱虫类药物。按照国际人用药品注册技术协调会（ICH）（Q1A (R2)）建议的条件，使NCM经历强制降解，包括水解（酸性、碱性和中性）、氧化、光解和热应激。该药物在水解（酸性和碱性）条件下显示出广泛降解，而在中性水解、氧化、光解和热应激条件下稳定。共观察到四种降解产物（DPs），并使用0.1%甲酸和乙腈作为流动相，在反相Fortis二苯基柱（150×4.6mm，5μm）上以梯度模式实现了药物及其降解产物的色谱分离。通过半制备液相色谱法分离出所有四种降解产物，并通过高分辨率质谱和氢核磁共振光谱技术对其结构进行了表征和确认。出于安全性考虑，对NCM及其四种降解产物在人单核细胞和代表主要细胞器的细胞系：神经元（Neuro 2a）、肝脏（HepG 2）和肺泡（A549）上进行了细胞毒性测定。在所测试的浓度下，发现NCM对人单核细胞和细胞系无毒。然而，与对照组相比，DP-1、DP-2、DP-3和DP-4在浓度为100μM时显示出乳酸脱氢酶（LDH）释放显著增加。DP-1和DP-3对A549细胞表现出毒性，其半数抑制浓度（IC）分别为92.18±4.93μM和65.42±6.29μM。DP-2、DP-3和DP-4对Neuro 2a细胞具有细胞毒性，其IC分别为63.62±3.85μM、86.09±6.19μM和42.81±8.10μM。发现这些降解产物对HepG 2细胞无毒。
