Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt.
Analyst. 2018 Sep 10;143(18):4366-4378. doi: 10.1039/c8an00772a.
The present study was designed to evaluate the potential protective effects of the cysteinyl leukotriene receptor blocker montelukast (MNK) and the xanthine oxidase inhibitor febuxostat (FBX) and their combination on a model of acute gouty arthritis induced by intraarticular injection of monourate sodium crystal (MUC) injection in rats. Additionally, we established an HPTLC method for the quantitative determination of both drugs simultaneously and applied this method to detect this combination in rat plasma. In this study, the treated male Wistar rats were grouped as follows: a negative control group injected with phosphate buffer saline (PBS) and a positive control group injected with MUC in their tibiotarsal joint. Four groups were treated orally with diclofenac (4 mg kg-1), MNK (10 mg kg-1), FBX (5 mg kg-1) and MNK plus FBX before MUC injection in their tibiotarsal joints. MUC injection in joints without pretreatment led to a progressive significant increase in joint diameter and heavy leucocytic infiltration compared to the PBS-injected joints. Treatment with diclofenac or a combination of MNK and FBX significantly decreased both joint diameters and leucocytic infiltration compared to the MUC group. MNK or FBX treatment attenuated leucocytic infiltration and significantly decreased joint diameters compared to the MUC group. Thus, MNK and FBX can prevent the development of MUC-induced acute gouty arthritis in rats. Also, MNK can be an alternative preventive treatment, particularly in elderly patients who cannot tolerate NSAIDs or corticosteroid. Furthermore, a new thin-layer chromatographic method combined with fluorescence detection was developed and validated for the simultaneous estimation of a mixture of FBX and MNK in spiked human plasma. In this method, we employed TLC aluminium plates precoated with silica gel G 60F254 as the stationary phase and chloroform : methanol (9 : 1, v/v) as the mobile phase. The optimized mobile phase selected for development gives compact bands (Rf values are 0.28 and 0.63 for FBX and MNK, respectively). The emission intensities were measured using a K400 optical filter after excitation at 322 nm. The linear regression data for the calibration plots showed excellent linear relationship (r = 0.9990 and 0.9996 for FBX and MNK, respectively), and the linearity range was 10.0-800.0 ng per band for both drugs. According to ICH-guidelines, this method was validated for precision, accuracy, robustness and selectivity. Also, the limits of detection and quantitation were calculated. In addition, stability studies on the studied mixture were performed. Statistical analysis proved that this method is reproducible and selective for the estimation of a mixture of FBX and MNK.
本研究旨在评估半胱氨酰白三烯受体拮抗剂孟鲁司特（MNK）和黄嘌呤氧化酶抑制剂非布司他（FBX）及其联合应用对尿酸单钠晶体（MUC）关节内注射诱导的急性痛风性关节炎模型的潜在保护作用。此外，我们建立了一种同时定量测定这两种药物的 HPTLC 方法，并应用该方法检测大鼠血浆中的这种联合用药。在这项研究中，将雄性 Wistar 大鼠分为以下几组：阴性对照组注射磷酸盐缓冲盐水（PBS），阳性对照组注射 MUC 于其胫跗关节。四组大鼠在 MUC 注射前分别口服双氯芬酸（4mg/kg-1）、MNK（10mg/kg-1）、FBX（5mg/kg-1）和 MNK+FBX。与 PBS 注射关节相比，未经预处理的 MUC 注射导致关节直径逐渐显著增加和白细胞浸润加重。与 MUC 组相比，双氯芬酸或 MNK 和 FBX 的联合治疗显著降低了关节直径和白细胞浸润。MNK 或 FBX 治疗减轻了白细胞浸润，并显著降低了与 MUC 组相比的关节直径。因此，MNK 和 FBX 可预防尿酸单钠晶体诱导的大鼠急性痛风性关节炎的发生。此外，MNK 可以作为一种替代的预防治疗方法，特别是在不能耐受 NSAIDs 或皮质类固醇的老年患者中。此外，我们还开发并验证了一种新的薄层色谱法结合荧光检测法，用于同时估计人血浆中 FBX 和 MNK 的混合物。在这种方法中，我们使用涂有硅胶 G 60F254 的 TLC 铝盘作为固定相，氯仿：甲醇（9：1，v/v）作为流动相。为了开发，选择了最佳的流动相，得到了紧凑的条带（FBX 和 MNK 的 Rf 值分别为 0.28 和 0.63）。用 K400 滤光片在 322nm 激发后测量发射强度。校准图的线性回归数据表明具有极好的线性关系（FBX 和 MNK 的 r 值分别为 0.9990 和 0.9996），线性范围分别为 10.0-800.0ng/条。根据 ICH 指南，对该方法的精密度、准确度、稳健性和选择性进行了验证。还计算了检测限和定量限。此外，对研究混合物进行了稳定性研究。统计分析证明该方法可重复使用，且对 FBX 和 MNK 混合物的测定具有选择性。
