<br>新型 TOPK 抑制剂 HI-TOPK-032 的综合理化表征、体外膜渗透和体外人皮肤细胞培养,International Journal of Molecular Sciences

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新型 TOPK 抑制剂 HI-TOPK-032 的综合理化表征、体外膜渗透和体外人皮肤细胞培养
International Journal of Molecular Sciences ( IF 4.9 ) Pub Date : 2023-10-24 , DOI: 10.3390/ijms242115515
Basanth Babu Eedara ₁ , Bhagyashree Manivannan ₁ , Wafaa Alabsi _{2,

3} , Bo Sun ₂ , Clara Curiel-Lewandrowski _{4,

5,

6} , Tianshun Zhang ₇ , Ann M Bode ₇ , Heidi M Mansour _{1,

8,

9}

Affiliation

非黑色素瘤皮肤癌 (NMSC) 是最常见的皮肤癌，美国每年约有 540 万人被诊断出来。新开发的T淋巴细胞激活杀伤细胞源性蛋白激酶（TOPK）抑制剂HI-TOPK-032可有效抑制结肠癌细胞生长、诱导结肠癌细胞凋亡和紫外线（UV）光诱导的鳞状细胞癌细胞癌（SCC）。本研究旨在研究 HI-TOPK-032 的理化性质、渗透行为和细胞毒性潜力，然后再开发适合皮肤靶向给药的外用制剂。扫描电子显微镜 (SEM)、能量色散 X 射线 (EDX) 光谱、差示扫描量热法 (DSC)、热台显微镜 (HSM)、X 射线粉末衍射 (XRPD)、卡尔费歇尔 (KF) 等技术使用库仑滴定法、拉曼光谱法、共焦拉曼显微镜 (CRM)、衰减全反射傅里叶变换红外光谱 (ATR-FTIR) 和傅里叶变换红外显微镜来表征 HI-TOPK-032。使用人皮肤角质形成细胞系 (HaCaT) 和原代正常人表皮角质形成细胞 (NHEK) 的 2D 细胞培养物评估 HI-TOPK-032 对体外细胞活力的剂量效应。在 HaCAT 人类皮肤细胞系上测量气液界面处的跨上皮电阻 (TEER) 作为剂量和时间的函数。使用 Strat-M® 合成仿生膜和体外 Franz 细胞扩散系统测试 HI-TOPK-032 的膜渗透行为。理化评价结果证实了药物的无定形性质以及具有所有特征化学峰的样品的均质性。体外细胞活力测定结果证实，HI-TOPK-032 浓度高达 10 µM 时细胞活力为 100%。此外，还开发了一种快速、特异、精确且经过验证的反相高效液相色谱 (RP-HPLC) 方法，用于定量评估 HI-TOPK-032。这是 HI-TOPK-032 的首次系统、全面的表征以及这些发现的报告。

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Comprehensive Physicochemical Characterization, In Vitro Membrane Permeation, and In Vitro Human Skin Cell Culture of a Novel TOPK Inhibitor, HI-TOPK-032

Nonmelanoma skin cancers (NMSC) are the most common skin cancers, and about 5.4 million people are diagnosed each year in the United States. A newly developed T-lymphokine-activated killer cell-originated protein kinase (TOPK) inhibitor, HI-TOPK-032, is effective in suppressing colon cancer cell growth, inducing the apoptosis of colon cancer cells and ultraviolet (UV) light-induced squamous cell carcinoma (SCC). This study aimed to investigate the physicochemical properties, permeation behavior, and cytotoxicity potential of HI-TOPK-032 prior to the development of a suitable topical formulation for targeted skin drug delivery. Techniques such as scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, differential scanning calorimetry (DSC), hot-stage microscopy (HSM), X-ray powder diffraction (XRPD), Karl Fisher (KF) coulometric titration, Raman spectrometry, confocal Raman microscopy (CRM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and Fourier transform infrared microscopy were used to characterize HI-TOPK-032. The dose effect of HI-TOPK-032 on in vitro cell viability was evaluated using a 2D cell culture of the human skin keratinocyte cell line (HaCaT) and primary normal human epidermal keratinocytes (NHEKs). Transepithelial electrical resistance (TEER) at the air–liquid interface as a function of dose and time was measured on the HaCAT human skin cell line. The membrane permeation behavior of HI-TOPK-032 was tested using the Strat-M® synthetic biomimetic membrane with an in vitro Franz cell diffusion system. The physicochemical evaluation results confirmed the amorphous nature of the drug and the homogeneity of the sample with all characteristic chemical peaks. The in vitro cell viability assay results confirmed 100% cell viability up to 10 µM of HI-TOPK-032. Further, a rapid, specific, precise, and validated reverse phase-high performance liquid chromatography (RP-HPLC) method for the quantitative estimation of HI-TOPK-032 was developed. This is the first systematic and comprehensive characterization of HI-TOPK-032 and a report of these findings.

更新日期：2023-10-24

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