| Objective Gemcitabine is a deoxycytidine(d Cyd) analog with activities in a variety of solid tumors. Despite of its wide use, the efficacy of gemcitabine is limited by a very short plasma circulation time due to deamination. In addition, its oral activity is limited by low oral bioavailability, due to the significant first-pass effect. To overcome those deficiencies, we designed a series of prodrugs and investigated the possibility of oral delivery by focusing on metabolic stability and oral bioavailability.Method The cytotoxicity of 5 prodrugs was tested in 11 different human cancer cell lines by using the MTT assay, to identify the most sensitive cell lines and potent compounds. Anti-proliferation activity was also simulated in the gemcitabine-resistant cell line. A sensitive LC-MS/MS method was developed for the quantitation of both gemcitabine and products, with a focus on metabolic stability and oral bioavailability. Pharmacokinetic study of the prodrug and gemcitabine was carried out in nude mice to evaluate oral formulations. The antitumor activity of the prodrug was investigated in H460 non-small cell lung cancer xenografts.Results In vitro cytotoxicity test showed that all of the gemcitabine prodrugs inhibited the growth of various cancer cells. Among those cancer cells, non-small cell lung cancer cells(H460, A549), pancreatic cancer cells(Panc-1), prostate cancer(DU145, PC-3), and colon cancer cell(HCT-116) were the most sensitive cell lines. Compound-3 was found to be the most potent prodrug and was thus chosen for further studies. IC50 values of compound-3 in the six sensitive cancer cells were all below 1μM, comparable to the IC50 value of gemcitabine. Analysis revealed that IC50 value of gemcitabine could increase 44.5 times in the simulated gemcitabine-resistant cancer cell line, while compound-3 only increased 1.3-fold, suggesting that compound-3 would be active against resistant cell lines. The intracellular concentration of compound-3 at 4 ℃ and 37 ℃ conditions showed significant differences, with intracellular concentration 2-3 times higher at 37 ℃ than that at 4 ℃, indicating that active transport might be involved in the compound-3 uptake. The intracellular concentration of compound-3 in sensitive cell lines were obviously higher than that in less sensitive cell lines after incubated with compound-3, and there was a good correlation between intracellular concentration and anti-proliferation activity(R2 = 0.89). Compounds 3 and gemcitabine showed reasonable plasma exposure in 1% CMC-Na containing 0.5% Tween 80 suspension and this formulation was further applied to in vivo antitumor testing. It was found that at an oral dose of 40 mg/kg twice per week, compound-3 significantly inhibited the tumor growth(65.1% as compared to the vehicle, p<0.01) after two weeks. This oral antitumor activity was comparable to gemcitabine dosed IP at 80 mg/kg twice per week.Conclusion Structural-based design lead to several prodrugs that demonstrated antiproliferation activity against numerous cancer cell lines, with compound-3 the most poten prodrug and A549, H460, HCT-116, DU-145, PC-3 the most sensitive cancer cell lines. A good correlation was noted between intracellular concentration of compound-3and its anti-proliferation activity. Based on simulation, compound-3 showed better anti-proliferation activity in gemcitabine-resistant cancer cell as compared to gemcitabine. In H460 xenograft model, compound-3 showed oral activity against tumor growth, indicating the possibility of gemcitabine-based oral treatment for various tumors. |
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