Karampelas Theodoros, Skavatsou Eleni, Argyros Orestis, Fokas Demosthenes, Tamvakopoulos Constantin
Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation, Academy of Athens , 4 Soranou Ephessiou Street, 11527 Athens, Greece.
Laboratory of Medicinal Chemistry, Department of Materials Science and Engineering, University of Ioannina , 45110 Ioannina, Greece.
Mol Pharm. 2017 Mar 6;14(3):674-685. doi: 10.1021/acs.molpharmaceut.6b00961. Epub 2017 Feb 1.
Gemcitabine is a clinically established anticancer agent potent in various solid tumors but limited by its rapid metabolic inactivation and off-target toxicity. We have previously generated a metabolically superior to gemcitabine molecule (GSG) by conjugating gemcitabine to a gonadotropin releasing hormone receptor (GnRH-R) ligand peptide and showed that GSG was efficacious in a castration resistant prostate cancer (CRPC) animal model. The current article provides an in-depth metabolic and mechanistic study of GSG, coupled with toxicity assays that strengthen the potential role of GSG in the clinic. LC-MS/MS based approaches were employed to delineate the metabolism of GSG, its mechanistic cellular uptake, and release of gemcitabine and to quantitate the intracellular levels of gemcitabine and its metabolites (active dFdCTP and inactive dFdU) resulting from GSG. The GnRH-R agonistic potential of GSG was investigated by quantifying the testosterone levels in animals dosed daily with GSG, while an in vitro colony forming assay together with in vivo whole blood measurements were performed to elucidate the hematotoxicity profile of GSG. Stability showed that the major metabolite of GSG is a more stable nonapeptide that could prolong gemcitabine's bioavailability. GSG acted as a prodrug and offered a metabolic advantage compared to gemcitabine by generating higher and steadier levels of dFdCTP/dFdU ratio, while intracellular release of gemcitabine from GSG in DU145 CRPC cells depended on nucleoside transporters. Daily administrations in mice showed that GSG is a potent GnRH-R agonist that can also cause testosterone ablation without any observed hematotoxicity. In summary, GSG could offer a powerful and unique pharmacological approach to prostate cancer treatment: a single nontoxic molecule that can be used to reach the tumor site selectively with superior to gemcitabine metabolism, biodistribution, and safety while also agonistically ablating testosterone levels.
吉西他滨是一种临床上已确立的抗癌药物，对多种实体瘤有效，但受其快速代谢失活和脱靶毒性的限制。我们之前通过将吉西他滨与促性腺激素释放激素受体（GnRH-R）配体肽偶联，生成了一种代谢上优于吉西他滨的分子（GSG），并表明GSG在去势抵抗性前列腺癌（CRPC）动物模型中有效。本文对GSG进行了深入的代谢和机制研究，并结合毒性试验，强化了GSG在临床上的潜在作用。采用基于液相色谱-串联质谱（LC-MS/MS）的方法来描绘GSG的代谢、其细胞摄取机制以及吉西他滨的释放，并定量由GSG产生的吉西他滨及其代谢物（活性二氟脱氧胞苷三磷酸（dFdCTP）和非活性二氟脱氧尿苷（dFdU））的细胞内水平。通过定量每日给予GSG的动物体内的睾酮水平，研究了GSG的GnRH-R激动潜力，同时进行了体外集落形成试验和体内全血测量，以阐明GSG的血液毒性特征。稳定性研究表明，GSG的主要代谢物是一种更稳定的九肽，可延长吉西他滨的生物利用度。GSG作为一种前药，与吉西他滨相比具有代谢优势，通过产生更高且更稳定的dFdCTP/dFdU比值，而在DU145 CRPC细胞中，GSG释放吉西他滨依赖于核苷转运体。在小鼠中的每日给药表明，GSG是一种强效的GnRH-R激动剂，还可导致睾酮水平降低，且未观察到任何血液毒性。总之，GSG可为前列腺癌治疗提供一种强大且独特的药理学方法：一种单一的无毒分子，可用于选择性地到达肿瘤部位，其代谢、生物分布和安全性优于吉西他滨，同时还能激动性地降低睾酮水平。
