Enhanced Anticancer Activity Of Gemcitabine Coupling With Conjugated Linoleic Acid Against Human Breast Cance In Vitro And In Vivo

Gemcitabine(GEM)is a novel type of pyrimidine deoxy nucleoside analogues,which can effectively inhibit both DNA synthesis of tumor cells and ribonucleotide reductase,approved by FDA as a first-line therapy for non-small cell lung cancer,bladder cancer,pancreatic cancer and breast cancer.To overcome its limitation of rapid metabolism in vivo that results in short circulation time and poor antitumor efficacy,prodrug strategy of GEM has been brought forward.Long-chain polyunsaturated fatty acids have good affinity with cells and nucleic acids,in which drug molecules would quickly arrive at when they are introduced to the polyunsaturated fatty acids chain backbone.It was to exploit new amide antitumor agents,enhance antitumor activities and reduce GEM side effects.Conjugated linoleic acid(CLA)which owns anti-proliferative effects on breast cancer were covalently bonded to N4-amino group of GEM.The purpose of this study was to synthesize conjugated linoleic acid-gemcitabine(CLA-GEM)conjugate to evaluate its anti-tumor activity both in vitro and in vivo.The CLA was covalently linked with the primary GEM at the N4-amino group by the mixed anhydride method.The CLA-GEM conjugate was characterized by 1H-NMR,13C-NMR,FTIR and MALDI-TOF-MS analysis.The stability of CLA-GEM conjugate was tested in PBS and rat plasma.After 24h incubation,the results showed that pH value had a significant effect on the stability of CLA-GEM.The CLA-GEM conjugate was more stable in alkaline and neutral conditions than in acid conditions.After 24h incubation of GEM in rat plasma,GEM could not be detected any more.However,after 48h incubation of CLA-GEM in rat plasma,41.2%of GEM still could be detected which hydrolyzed from the CLA-GEM conjugate.The oil/water partition coefficient was determined by an octanol-water extraction system(logDoct=3.67).These results evidenced that compared to the parent drug GEM,CLA-GEM enjoys favorable lipophilicity and much better plasma stability.The cytotoxicities of CLA-GEM were assayed by SRB method in brain tumor cancer C6,human lung cancer NCI-H446,A549,breast cancer MCF-7 and MDA-MB-231.After 72h incubation,CLA-GEM showed significantly inhibition on various cancer cell lines,especially on NCI-H446,MCF-7 and MDA-MB-231.The MCF-7 cell line was employed as in vitro model for other experiments.The nucleoside transporter inhibition experiment showed CLA-GEM altered the transport pattern of GEM across cell membrane,evidenced by the little effect of nucleoside transporter inhibitors(NBMPR and dipyridamole)on the IC50 values of CLA-GEM,instead of the great effect on that of unmodified GEM.The cell cycle analysis of MCF-7 suggested S phase of cell cycle was significantly prolonged by CLA-GEM.Meanwhile,in vivo pharmacokinetic study of intravenous injection of CLA-GEM showed that the CLA-GEM conjugate had a longer plasma half-life and a higher bioavailability compared to that of unmodified GEM.Moreover,due to the low bioavailability and gastrointestinal toxicity of GEM,it has to be administered intravenously instead of orally.Therefore,we also conducted oral pharmacokinetics of CLA-GEM and GEM on rats.The results showed that the bioavailability of GEM had been remarkably enhanced by CLA-GEM conjugate,the relative bioavailability is 614%.Significant stronger antitumor activity was observed in the nude mice xenografted MCF-7 breast tumor after treated with CLA-GEM than that of unmodified GEM(p<0.01),while no significant body weight loss was found for CLA-GEM treated group.In conclusion,the novel CLA-GEM conjugate prepared in this study could improve the lipophility of GEM,enhance the anti-tumor effects of GEM on breast cancer.CLA-GEM conjugate could also change the transmembrane transport mechanism for GEM,which could possibly avoid the drug resistance caused by the deficiency of nucleoside transporters.After intravenous administration,CLA-GEM had much longer half-life than that of GEM.Oral absorption of GEM was greatly increased after oral administration of CLA-GEM.Taken together,CLA-GEM conjugate would be a promising prodrug of GEM for future clinical use.