DPP-4抑制剂LGT-6在不同种属血浆中蛋白结合率的比较

廖伟科 杨华丽 王忠元 陈瑞 汤磊 崔杏 朱高峰

中圖分类号 R969.1 文献标志码 A 文章编号 1001-0408（2021）14-1728-06

DOI 10.6039/j.issn.1001-0408.2021.14.11

摘 要 目的：建立二肽基肽酶4抑制剂LGT-6在不同种属血浆中蛋白结合率的测定方法并比较差异。方法：采用平衡透析法，以磷酸盐缓冲液为透析外液，将3、30、300、3 000 nmol/L的LGT-6分别在大鼠、猴、人血浆（即透析内液）中平衡48 h。以甲苯磺丁脲为内标，采用超高效液相色谱-串联质谱法测定透析内、外液中LGT-6的浓度，计算血浆蛋白结合率。以ACQUITY UPLC HSS T3为色谱柱，以水（含0.01%甲酸）-乙腈（含0.01%甲酸）为流动相进行梯度洗脱，流速为0.6 mL/min，柱温为40 ℃，进样量为2 μL;离子源为电喷雾离子源，监测模式为多反应监测模式，采集模式为正离子模式，定量分析用离子对分别为m/z 487.0→434.3（LGT-6）、m/z 271.1→172.0（内标）。结果：在3、30、300、3 000 nmol/L浓度下，LGT-6在大鼠血浆中的蛋白结合率分别为（96.25±0.97）%、（84.16±1.24）%、（78.25±0.61）%、（66.63±0.95）%，在猴血浆中的蛋白结合率分别为（98.54±0.58）%、（87.27±1.01）%、（79.35±0.86）%、（66.69±0.54）%，在人血浆中的蛋白结合率分别为（99.40±1.03）%、（84.48±1.15）%、（77.62±0.77）%、（66.93±0.48）%。在相同浓度下，LGT-6在大鼠、猴和人血浆中的蛋白结合率没有明显差异（P>0.05）;在相同种属血浆中，不同浓度LGT-6的血浆蛋白结合率组间比较差异有统计学意义（P<0.05），且有随药物浓度的升高而降低的趋势。结论：成功建立了测定LGT-6血浆蛋白结合率的方法。在相同浓度下，LGT-6在大鼠、猴、人血浆中的蛋白结合率没有明显的种属差异，但有明显的浓度依赖趋势。

关键词 二肽基肽酶4抑制剂;LGT-6;平衡透析法;超高效液相色谱-串联质谱法;血浆蛋白结合率;不同种属血浆

Comparison of Protein Binding Rate of DPP-4 Inhibitor LGT-6 in Different Species of Plasma

LIAO Weike1，2，YANG Huali3，WANG Zhongyuan3，CHEN Rui1，2，TANG Lei1，2，CUI Xing1，2，ZHU Gaofeng1，2        （1. Guizhou Provincial Engineering Technology Research Center for Chemical Drug R&D， Guiyang 550004， China; 2. College of Pharmacy， Guizhou Medical University， Guiyang 550004， China; 3. Dept. of Pharmacy， Guizhou Provincial Peoples Hospital， Guiyang 550002， China）

ABSTRACT   OBJECTIVE： To establish the method for determining protein binding rate of dipeptidyl peptidase-4 inhibitor LGT-6 in different species of plasma， and to compare their difference. METHODS： By equilibrium dialysis， LGT-6 （3， 30， 300， 3 000 nmol/L） was equilibrated in rat， monkey and human plasma （i. e. internal dialysis solution） for 48 h， using phosphate buffer as the external dialysis solution. The concentration of LGT-6 in internal and external dialysis solution was determined by UPLC-MS/MS using tolbutamide as internal standard， and the plasma protein binding rate was calculated. The determination was performed on ACQUITY UPLC HSS T3 column with water （containing 0.01% formic acid）-acetonitrile （containing 0.01% formic acid） as mobile phase at the flow rate of 0.6 mL/min. The column temperature was 40 ℃， and the sample size was 2 μL. The ion source was electrospray ion source， and the multiple ion monitoring mode was used to carry out positive ionization scanning. The ion pairs for quantitative analysis were m/z 487.0→434.3 （LGT-6）， m/z 271.1→172.0 （internal standard）， respectively. RESULTS： At the concentrations of 3， 30， 300， and 3 000 nmol/L， the protein binding rates of LGT-6 in rat plasma were （96.25±0.97）%， （84.16±1.24）%， （78.25±0.61）%， （66.63±0.95）%; the protein protein binding rates in monkey plasma were （98.54±0.58）%， （87.27±1.01）%， （79.35±0.86）%， （66.69±0.54）%; the protein binding rates in human plasma were （99.40±1.03）%， （84.48±1.15）%， （77.62±0.77）%， （66.93±0.48）%. At the same concentration， the protein binding rates of LGT-6 in rat， monkey and human plasma had no significant difference （P>0.05）. In the same species of plasma， there were significant differences in the plasma protein binding rates of different concentration of LGT-6 among those groups （P<0.05）， and it decreased with the increase of drug concentration. CONCLUSIONS： The method for the determination of plasma protein binding rate of LGT-6 is successfully established. The data revealed that the protein binding rate of LGT-6 is concentration-dependent， there was no obvious species difference on protein binding rates of LGT-6 in rat， monkey and human plasma under the same concentration.

KEYWORDS   Dipeptidyl peptidase-4 inhibitor; LGT-6; Balanced dialysis; UPLC-MS/MS; Plasma protein binding rate; Different species of plasma

随着发病率和致死率的逐年上升，糖尿病已成为全球最大的公共卫生事件之一[1]。相比于传统的双胍类、磺酰脲类、噻唑烷酮类、美脲类、α-葡萄糖苷酶抑制剂、胰岛素（易引起体质量增加、胃肠道不适、低血糖风险升高等）和新型治疗药物胰高血糖素样肽1受体激动剂（需皮下注射给药）、钠-葡萄糖协同转运蛋白2抑制剂（存在潜在的降低血压及引起生殖感染的风险）等，二肽基肽酶4（DPP-4）抑制剂以其独特的降糖机制以及较高的安全性引起了人们越来越多的关注[2-8]。经典……