Institute of Pharmacology and Toxicology, Rostock University Medical Center, 18057 Rostock, Germany.
Rudolf Zenker Institute for Experimental Surgery, Rostock University Medical Center, 18057 Rostock, Germany.
Molecules. 2022 May 9;27(9):3031. doi: 10.3390/molecules27093031.
Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3'-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin -glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin -glycosides KD87 (3-[3'-oxo-benzo[]thiophen-2'-()-ylidene]-1-(β-d-glucopyranosyl)-oxindole) and KD85 (3-[3'-oxo-benzo[]thiophen-2'-()-ylidene]-1-(β-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice.
靛玉红被鉴定为当归龙荟丸的一种有效成分，当归龙荟丸是一种中药复方制剂，在慢性白血病患者的临床试验中显示出抗癌活性。关于靛玉红抗肿瘤作用机制的研究主要集中在靛玉红衍生物吲哚美辛-3'-单肟（I3M）上。同时，几种合成的靛玉红糖苷也显示出对癌细胞的增殖抑制和细胞毒性作用。在本研究中，我们证明了噻吩类似物靛玉红糖苷 KD87（3-[3'-氧代苯并[]噻吩-2'-（）-亚基]-1-（β-d-吡喃葡萄糖基）-吲哚啉）和 KD85（3-[3'-氧代苯并[]噻吩-2'-（）-亚基]-1-（β-d-吡喃甘露糖基）-吲哚啉）对黑色素瘤和鳞状细胞癌细胞以及肺癌和神经胶质瘤细胞的细胞毒性活性。迄今为止，对靛玉红作用的临床前研究进展表明，需要从细胞、动物和人体研究中获得药代动力学数据，这需要可靠的定量分析。因此，开发并验证了一种灵敏的液相色谱-串联质谱（LC-MS/MS）法，用于同时测量血浆和细胞培养液中的 KD87、KD85 和 I3M。实验条件经过优化，用于人血浆蛋白沉淀和从血浆和细胞培养液中进行液-液萃取。该方法按照美国食品和药物管理局（FDA）的生物分析方法验证要求进行了验证，并对其选择性、灵敏度、基质效应、回收率、交叉污染、校准曲线线性、准确性、精密度和稳定性进行了评估。通过测定预先腹腔给予小鼠后小鼠血浆中的 KD87，证明了该方法的适用性。
