Faculty of Pharmacy, Charles University, Heyrovského 1203, CZ-50005 Hradec Králové, Czech Republic.
Vet Parasitol. 2012 Apr 30;185(2-4):168-74. doi: 10.1016/j.vetpar.2011.09.026. Epub 2011 Sep 22.
Biotransformation enzymes can, to a certain extent, protect parasitic worms against the toxic effects of anthelmintics and can contribute to drug-resistance development. The objective of our work was (1) to find and identify phase I and II metabolites of the anthelmintic praziquantel (PZQ) formed by the lancet fluke (Dicrocoelium dendriticum) and the rat tapeworm (Hymenolepis diminuta) and (2) to compare PZQ metabolites in helminths with PZQ biotransformation in rat as host species. Ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used for this purpose. During in vitro incubations, mitochondria-like and microsomes-like fractions (prepared from homogenates of adult worms or from rat liver homogenate) were incubated with 10 and 100 μM PZQ. Liquid/liquid extraction was used for samples during in vitro experiments. In the ex vivo study, living D. dendriticum and H. diminuta adults were incubated in RPMI-1640 medium in the presence of 50 nM or 100 nM PZQ for 24h. After incubation, the worms were removed from the medium and homogenized. Homogenates of worms, medium from the incubation of worms or rat hepatocytes and rat urine (collected during 24h after oral PZQ administration) were separately extracted using solid-phase extraction. The results showed that both D. dendriticum and H. diminuta enzymatic systems are not able to metabolize PZQ. On the other hand, thirty one different phase I and four phase II PZQ metabolites were detected in rat samples using UHPLC/MS/MS analyses. These results show that our experimental helminths, as the members of tapeworm and fluke groups of parasites, are not able to deactivate PZQ, and that the biotransformation enzymes of the studied helminths do not contribute to PZQ-resistance.
生物转化酶在一定程度上可以保护寄生虫免受驱虫药的毒性影响，并有助于耐药性的发展。我们的工作目标是：(1) 找到并鉴定由裂头绦虫（Dicrocoelium dendriticum）和大鼠带绦虫（Hymenolepis diminuta）形成的驱虫药吡喹酮（PZQ）的 I 相和 II 相代谢物；(2) 比较寄生虫与宿主物种大鼠的 PZQ 生物转化之间的 PZQ 代谢物。为此目的，使用了超高效液相色谱/串联质谱（UHPLC/MS/MS）。在体外孵育过程中，使用线粒体样和微粒体样部分（从成虫匀浆或大鼠肝匀浆中制备）孵育 10 和 100μM 的 PZQ。在体外实验中，使用液/液萃取法提取样品。在离体研究中，将活的裂头绦虫和大鼠带绦虫成虫在含有 50 nM 或 100 nM PZQ 的 RPMI-1640 培养基中孵育 24 小时。孵育后，将蠕虫从培养基中取出并匀浆。使用固相萃取法分别从蠕虫匀浆、孵育蠕虫的培养基或大鼠肝细胞和大鼠尿液（在口服 PZQ 后 24 小时收集）中提取样品。结果表明，裂头绦虫和大鼠带绦虫的酶系统都不能代谢 PZQ。另一方面，使用 UHPLC/MS/MS 分析在大鼠样品中检测到三十一种不同的 I 相和四种 II 相 PZQ 代谢物。这些结果表明，我们研究的寄生虫，作为绦虫和吸虫类寄生虫的成员，不能使 PZQ 失活，并且研究的寄生虫的生物转化酶也不会导致 PZQ 耐药性。
