高效液相色谱法测定黑色素瘤小鼠尿液中达卡巴嗪

达卡巴嗪是治疗恶性黑色素瘤的一线化疗药物。它在体内主要经肝脏代谢,同时部分药物仍以原药形式经尿液排出。这就意味着可以通过监测尿液中达卡巴嗪的含量评估其在人体内的利用率和转化率,进而对其治疗效果进行评价。针对达卡巴嗪,人们发展了多种分析方法,但多基于高效液相色谱-质谱平台。然而达卡巴嗪为强极性弱碱性化合物,采用常规反相色谱法分析时会出现出峰时间过早、峰形拖尾的现象,导致定量不准确。基于此,该文建立了一种测定尿液中达卡巴嗪含量的高效液相色谱方法以克服上述问题。小鼠尿液经丙酮沉淀法去除蛋白后,采用Shimadzu-GL ODS柱（150 mm×4.6 mm,5 μm）分离,色谱条件如下：流动相为甲醇/乙腈（1：1,v/v）-0.01 mol/L磷酸二氢钠溶液（pH=6.5）（20：80,v/v）,流速1 mL/min,检测波长280 nm,柱温35℃,洗脱时间15 min。在该色谱条件下,达卡巴嗪保留时间为5.3 min且峰形良好。其在0.25~1000 μg/mL范围内线性关系良好（r ² =0.999）。基于信噪比（S /N ）=3和S /N =10,计算出检出限和定量限分别为0.12 μg/mL和0.25 μg/mL。在低、中、高（50.0、375、500 μg/mL）3个添加水平下,加标回收率分别为98.9%、102%、99.1%,相对标准偏差（RSD）分别为3.2%、1.3%、1.2%（n =5）。日内与日间RSD分别小于3.8%和4.4%。将该法应用于不同发展阶段的黑色素瘤C57BL/6小鼠尿液中达卡巴嗪的监测,结果表明该方法操作简便,结果可靠,便于推广。     

Investigating the binding measurements of human α-acid glycoprotein with chlorambucil and dacarbazine in the presence of imidazolium based -ionic liquid by affinity capillary electrophoresis

Human alpha (α1)-acid glycoprotein (AGP) is an acute phase protein whose plasma concentration increases several-folds in the presence of various diseases. The variability in AGP plasma concentration is expected to have a huge impact on the drug binding equilibrium. Therefore, a precise measurement of AGP-drug binding is of great demand for drug development. In the current study, an ionic liquid-based aqueous two-phase system combined with affinity capillary electrophoresis (ILATPS/ACE) was utilised in order to improve the accuracy of AGP-drug binding analysis through the measurements of electrophoretic mobilities. The utilisation of ILATPS has shown to have a positive impact on the stability of AGP activity solution during the storage for an extended period of time. In addition, the effect of various alkyl chains (C2-C10) of imidazolium-based ILs with concentrations ranging between 10.00 and 1000.0 μmol L⁻¹ on the AGP binding with the anti-cancer drugs chlorambucil (CHL) and dacarbazine (DAC) was examined by the system developed (ILATPS/ACE). A 100.00 μmol L⁻¹ 1-ethyl-3-methylimidazolium chloride (EMImCl) prepared in the physiological buffer conditions containing AGP (5.00–100.00 µmol L⁻¹) has provided an accurate apparent binding constant of 1.99 ± 0.11 and 6.95 ± 0.14 L mmol⁻¹ with CHL and DAC respectively. Apart from the ACE analysis, EMImCl/phosphate buffer solution was found to be a distinguished system that could lengthen the stability of AGP activity for a period of time reaching 90 days during the solution storage at 4.00 °C. This effect is thought to be due to the easy conversion of one-phase EMImCl/phosphate buffer/AGP at the ambient lab temperature into the two-phase solution at refrigerator temperature, 4.00 °C, and vice versa. Therefore, the ILATP/ACE system could be used to enhance the accuracy for other AGP-drug bindings with a fast, easy to use, and cost-effective analysis.     

ITC研究达卡巴嗪与DNA反应动力学

利用等温滴定量热(ITC)、光谱、粘度测量等方法,研究了小牛胸腺DNA与抗癌药物达卡巴嗪的相互作用。结果表明:达卡巴嗪与DNA作用后,吸收光谱会出现增色、蓝移和粘度减小等现象。采用ITC法得到了结合常数以及结合位点数,发现达卡巴嗪与DNA以非经典嵌插式及表面作用两种方式结合。对于嵌插式,ΔH10,ΔS10,K1=5.63×10~4,结合位点数0.10;对于药物分子仅与DNA表面发生作用而并未嵌入到DNA分子的疏水部分的结合方式,ΔH_20,ΔS_20,K_2=1.00×10~3,结合位点数9.99。同时发现紫外法得到的结合常数是Ka=6.70×10~4,与ITC的嵌插式吻合。     

Extra Virgin Olive Oil Secoiridoids Modulate the Metabolic Activity of Dacarbazine Pre-Treated and Treatment-Naive Melanoma Cells

Nowadays, many individuals, whether healthy or diagnosed with disease, tend to expose themselves to various easily accessible natural products in hopes of benefiting their health and well-being. Mediterranean populations have traditionally used olive oil not only in nutrition but also in cosmetics, including skincare. In this study, the phenolic profile—composed of twelve compounds altogether, including the secoiridoids oleocanthal (OCAL) and oleacein (OCEIN)—of extra virgin olive oil (EVOO) from autochthonous cultivars from Croatia was determined using ¹H qNMR spectroscopy and HPLC-DAD analysis, and its biological activity was investigated in melanoma cell lines. The EVOO with the highest OCEIN content had the strongest anti-cancer activity in A375 melanoma cells and the least toxic effect on the non-cancerous keratocyte cell line (HaCaT). On the other hand, pure OCAL was shown to be more effective and safer than pure OCEIN. Post-treatment with any of the EVOO phenolic extracts (EVOO-PEs) enhanced the anti-cancer effect of the anti-cancerous drug dacarbazine (DTIC) applied in pre-treatment, while they did not compromise the viability of non-cancerous cells. The metastatic melanoma A375M cell line was almost unresponsive to the EVOO-PEs themselves, as well as to pure OCEIN and OCAL. Our results demonstrate that olive oils and/or their compounds may have a potentially beneficial effect on melanoma treatment. However, their usage can be detrimental or futile, especially in healthy cells, due to inadequately applied concentrations/combinations or the presence of resistant cells.