抗肿瘤候选药物Gedatolisib在体外血浆和人工胃/肠液中的稳定性研究及其降解产物分析

Study on the Stability of Antitumor Candidate Gedatolisib in Plasma in vitro and Simulated Gastric/intestinal Fluid and Its Catabolites Analysis

目的:考察抗肿瘤候选药物Gedatolisib在体外血浆和人工胃/肠液中的稳定性,并分析其在体外血浆中可能的降解产物。方法:采用高效液相色谱法,以吲哚美辛为内标,分别检测Gedatolisib在SD大鼠(雄性)血浆中孵育0、0.5、1.0、1.5、2.0、3.0 h和在空白人工胃液(pH 1.3,不含酶)、空白人工肠液(pH 6.8,不含酶)、人工胃液(pH 1.3,含胃蛋白酶)、人工肠液(pH 6.8,含胰蛋白酶)中孵育0、0.5、1.0、2.0、3.0、4.0、6.0 h的含量变化,计算其剩余百分比;利用超高效液相色谱-四极杆-飞行时间质谱(UPLC-QTOF/MS)技术分析空白血浆和血浆孵育样品的总离子流图,比较差异峰并通过MS图推测降解产物。结果:Gedatolisib在大鼠血浆中孵育2.0 h时的剩余百分比约为63%,继续孵育无明显变化;在空白人工肠液中孵育不同时间的剩余百分比均在(99.18±2.15)%^(103.20±3.41)%范围内;在人工肠液中孵育2.0 h的剩余百分比降至(88.76±1.53)%,在空白人工胃液中孵育2.0 h的剩余百分比降至(85.63±1.55)%,继续孵育均无明显变化;在人工胃液中孵育的剩余百分比从0 h的(94.94±3.52)%降至6.0 h的(16.19±1.17)%。UPLC-Q-TOF/MS的总离子流图显示,血浆孵育样品与空白血浆的差异峰出现在正离子模式扫描下的6.42 min处,该峰扫描通道可见m/z 616.3351、632.3277、630.3170、602.2786[M+H]+的分子离子峰,推测Gedatolisib在大鼠血浆中可能生成1-(4-(3-(4,6-双吗啉-1,3,5-三嗪-2-基)苯基)脲基)苯甲酰基)-N,N-二甲基哌啶-4-氧化胺、1-(4-(4-(二甲基氨基)哌啶-1-羰基)苯基)-3-(4-(4-吗啉-6-(3-氧代吗啉)-1,3,5-三嗪-2-基)苯基)脲和1-(4-(3-(4-(4,6-双吗啉-1,3,5-三嗪-2-基)苯基)脲基)苯甲酰基)-N-甲基哌啶等3个降解产物。结论:Gedatolisib在大鼠血浆中不稳定,可能发生末端氮原子氧化、吗啉环氧化和末端氮脱甲基化等反应;在人工肠液和空白人工胃/肠液中稳定性良好,在胃蛋白酶存在下发生明显降解。

OBJECTIVE:To investigate the stabilities of antitumor candidate Gedatolisib in plasma in vitro and simulated gastric/intestinal fluids,and to analyze the possible catabolites in plasma. METHODS: HPLC method was adopted. Using indometacin as internal standard,the contents of Gedatolisib incubated in plasma of SD rats(male)for 0,0.5,1.0,1.5,2.0,3.0 h and blank simulated gastric fluid(p H 1.3,no enzyme),blank simulated intestinal fluid(pH 6.8,no enzyme),simulated gastric fluid(pH 1.3,containing pepsin)and simulated intestinal fluid(pH 6.8,containing trypsin)for 0,0.5,1.0,2.0,3.0,4.0,6.0 h were determined. The remaining percentage of Gedatolisib was calculated. UPLC-Q-TOF/MS was used to analyze the TIC of blank plasma and incubated samples. The differential peaks were compared, and catabolites were inferred by MS chromatograms. RESULTS: The remaining percentage in plasma of rats for 2.0 h was about 63%,and there was no significant change after continued incubation. The remaining percentage of Gedatolisib incubated in blank simulated intestinal fluid for different time ranged(99.18 ± 2.15)%-(103.20 ± 3.41)%. The remaining percentage in simulated intestinal fluids for 2.0 h ranged(88.76 ± 1.53)%. The remaining percentage in blank simulated gastric fluids for 2.0 h was ranged(85.63±1.55)%,and there was no significant change after continued incubation. The remaining percentage in simulated gastric fluid was from(94.94±3.52)%(0 h)to(16.19±1.17)%(6.0 h). TIC of UPLC-Q-TOF/MS showed that the differential peaks of incubated samples and blank plasma was 6.42 min under positive mode scanning,molecular ion peak of m/z 616.335 1,simulated 632.327 7,630.317 0,602.278 6 [M+H]+could be found in scanning channel. It was speculated that Gedatolisib could generate 1-(4-(3-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl)urea)benzoyl)-N,N-dimethylpiperidin-4-amine oxide,1-(4-(4-(dimethylamino)piperidine-1-carbonyl)phenyl)-3-(4-(4-morpholino-6-(3-oxomorpholino)-1,3,5-triazin-2-yl)phenyl)urea and 1-(4-(3-(4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl)urea)benzoyl)-Nmethylpiperidine. CONCLUSIONS:Gedatolisib is not stable in rat plasma,and it may undergo terminal N oxidation,morpholine ring oxidation and terminal N demethylation. Gedatolisib is stable in artificial intestinal fluid and blank artificial gastric/intestinal fluid,and degrades obviously in the presence of pepsin.