Synthesis And Biological Evaluation Of Oral Prodrugs Of Gemcitabine

Objective:In this study,small-molecule prodrug strategy has been proposed to overcome the low oral bioavailability and gastrointestinal toxicity of Gem.The fat solubility of prodrugs are enhanced through modifying the 4-position of Gem.The modification is expected to improve its bioavailability,provide a protection against deamination,reduce the toxicity of intestines caused by Gem,and make it possible for oral administration in clinics.Methods:（1）Synthesis and identification of prodrugs:the 4-amino group of Gem was introduced into three alkyl chains with different lengths via carbamate bonding to get prodrugs,the structures were identified by¹H NMR and¹³C NMR.（2）Study on physical and chemical properties:The in vitro stability of Gem and prodrugs was evaluated in different p H buffers（1.0,4.5,6.8,7.4）,simulated gastrointestinal fluids,liver homogenate,and fresh prepared rat plasma,respectively.The equilibrium solubility and the oil-water partition coefficient（Log P）of Gem and prodrugs were also investigated.（3）The cytology of Gem and prodrugs in vitro:cell toxicity of the Gem and prodrugs in Caco-2 cells were demonstrated by MTT assay.It systematically evaluated whether the uptake mechanism of Gem and prodrugs in Caco-2 cells were depended on concentration,time and temperature by cell uptake assay.And the transport mechanism was also investigated.（4）In vivo pharmacokinetic study:after oral or intravenous administration,the eyeballs of Kunming mice were taken at different time points to collect blood,and in vivo pharmacokinetic study was performed to investigate the changes of blood drug concentration over time in body after oral administration.（5）In vivo distribution experiments:after oral or intravenous administration,the mice eyeballs were taken at different time points,and the neck was taken to death immediately after the blood was collected.The heart,liver,spleen,lung,kidney,brain,pancreas and tumor were dissected to study the distribution in the body to investigate the drug distribution in different organs of mice.（6）In vivo pharmacodynamic experiments:to investigate the therapeutic effect of Gem and prodrugs,we established H22 tumor-bearing mice model.The therapeutic effect of Gem and prodrugs on tumor-bearing mice was evaluated by detecting the changes of body weight and tumor inhibition.The gastrointestinal toxicity of Gem and prodrugs were evaluated by analyzing the results of HE staining in the gastrointestinal tract of mice.Results:（1）Prodrugs were identified by¹H NMR and¹³C NMR,and the spectra showed that all prodrugs were synthesized successfully.（2）Stability experiment shows,the three prodrugs were found to have slight degradation in the buffer solution of p H 1.2,and under the conditions of p H 4.5,6.8,7.4,simulated gastric juice and intestinal fluid,they were stable.It is speculated that the prodrug will be degraded in a very small amount during gastric emptying time and can maintain good stability in the intestinal tract.Gem was rapidly degraded in the plasma and liver tissue homogenate,but prodrugs were degraded slower and effectively converted into Gem.The results of physicochemical properties showed that the liposolubility of prodrugs was significantly improved,and the Log P value was in the order of Gem<Gem-C3<Gem-C5<Gem-C7.（3）According to MTT assay,Gem and prodrugs had no obvious toxic on Caco-2cells in 4 h.The results on cellular uptake revealed that the uptake of prodrugs by Caco-2 cells was significantly higher than Gem（p<0.05）,and the uptake increased with the enhancement of lipid solubility:Gem-C7>Gem-C5>Gem-C3>Gem.Competitive inhibition assays showed that the uptake of Gem was mediated by nucleotide transporters,and other three prodrugs did not inhibited by nucleotide transporter,whereas prodrugs revealed a temperature-dependent pattern.It is concluded that the prodrug may enter Caco-2 cells via energy-dependent pathways,but the specific mechanism still needs to be explored.（4）The pharmacokinetic results showed that compared with the oral Gem,the oral prodrug showed good pharmacokinetic properties.AUC_0-∞of the three prodrugs were significantly increased,C_maxwere significantly increased（p<0.01）.The oral bioavailability of Gem-C3,Gem-C5 and Gem-C7 was 3.34,4.01 and 3.72 times of that of oral gemcitabine,respectively.The prodrug Gem-C5 were better than those of the other two prodrugs.（5）In vivo distribution experiments indicated that C_maxand AUC_0-∞of Gem-C5were significantly increased in mice after oral administration,which were 2.47 and2.12 times of those in the prodrug group,respectively,and the drug concentration in the tumor was significantly increased（p<0.05）.Targeting evaluation parameters such as the relative uptake rate（Re）,peak concentration ratio（Ce）and drug targeting index（DTI）of prodrug Gem-C5 significantly improved the distribution of drugs in tumors and had a good accumulation ability in tumor sites.（6）Pharmacodynamic experiments showed compared with Gem,the prodrug Gem-C5 can significantly reduce tumor volume at a dose of 20 mg/kg,showing excellent antitumor activity.In addition,HE staining showed that compared to Gem,the oral Gem group had serious gastrointestinal tract injury,while the prodrug had almost no side effect on the gastrointestinal tract,avoiding the damage of the prodrug on the gastrointestinal tract.Conclusion:In this study,a series of oral gemcitabine prodrugs were designed,which significantly improved oral bioavailability.In the H22 Xenograft Model,the prodrugs increased the accumulation of the drug in the tumor site,showed excellent anti-tumor efficacy,and reduced the toxic effect on the gastrointestinal tract.