Design And Evaluation Of Intestinal OCTN2 Targeting Oral Prodrugs Of Gemcitabine

Gemcitabine is a nucleoside analog that has been used as the first-line chemotherapy for pancreatic and non-small-cell lung cancers.However,the administration modality of gemcitabine is currently limited to the intravenous route,which leads to serious toxicity andvarious safety issues.Clinical studies showed that the long-time and low-dose administration of gemcitabine could significantly improve the anti-tumor effect,while prolonged intravenous infusion was very inconvenient and could bring pain to the patient.Consequently,there is an urgent need to develop high-efficacy,low-toxicity oral gemcitabine products.Unfortunately,poor membrane,low metabolic stability and gastrointestinal toxicity limited the clinical oral application of gemcitabine.For low permeability drugs,there are two kinds of strategies to improve oral absorption,including lipophilicifized prodrugs and carrier prodrugs,but there are still some problems,such as poor water solubility and limited transport capacity.The new novel organic cation transporter 2(OCTN2,SLC22A5)is an important nutrient transporter to transport carnitine in small intestine and,therefore,it may be a promising molecular target for designing oral prodrugs.We here describe the design of intestinal OCTN2-targeting prodrugs of gemcitabine by covalent coupling of L-carnitine to its N4-amino group via different lipophilic linkages.Oral absorption was improved by optimizing membrane permeability and stability simultaneously,and the toxicity of gemcitabine was reduced by blocking the pyrimidine amino group of gemcitabine.Four OCTN2-targeting prodrugs(GSC,GHC,GOC and GDC)and a lipophilized prodrug(GS)were synthesized through acylation reaction and catalytic hydrogenation.And we optimized the synthetic route for the purpose of increasing the yield of targeted products.We investigated the in vitro stability and bioactivation of prodrugs in different pH phosphate buffers,tissue homogenate and plasma.All prodrugs were more stable in pH 6.8-7.4 buffers and intestinal homogenate,indicating that the prodrugs could be delivered across intestinal epithelial cells in their intact form.Moreover,the t1/2 values of prodrugs in plasma were roughly 4-fold shorter compared with that in hepatic homogenate,indicating that the primary bioactivation site was the plasma and then the liver.The cellular uptake mechanism of prodrugs was studied in Caco-2 cells and OCTN2-HEK293 cells.The uptake of GSC and GHC by Caco-2 cells was reduced by 2-to 10-fold where Na+in medium was replaced with N-methyl-D-glucamin or at 4℃.Moreover,the cellular uptake of the targeting prodrugs was significantly reduced(by 2.2-3.7 fold)in the presence of 100 μM L-carnitine,a typical substrate of OCTN2,whereas no similar effect on GS or gemcitabine was observed.To confirm the contribution of OCTN2 on the transport of the targeted prodrugs,OCTN2 gene transfected HEK293(HEK293-OCTN2)cells were used.The uptake of GSC and GHC was,respectively,3-to 8-fold higher in HEK293/OCTN2 than HEK293 cells,but there was no significant difference in gemcitabine between the two cells.These results indicated that the targeted prodrugs were specifically taken up by the cells via interaction with OCTN2 in a Na+-dependent manner.Next,the Eadie-Hofstee cureve and molecular docking were used to study the molecular mechanism of prodrug and transporters.We found that that the fatty acid chain length has a determining effect on the affinity of the substrate toward OCTN2.The apparent transcellular permeability of gemcitabine and its prodrugs was examined in Caco-2 cells and In situ single-pass perfusion.All the targeted prodrugs increased permeability by 5-fold to 15-fold,compared with gemcitabine,and the permeability of GS was comparable with that of gemcitabine.The permeability of the targeted prodrugs was reduced by 1.6-to 3.6-fold in the presence of 100 μM L-carnitine,suggesting the improved permeability was mediated by OCTN2.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay was utilized to investigate the in vitro cytotoxicity of prodrugs against Bx-PC3 and Caco-2 cells.Compare to gemcitabine,the targeted prodrugs showed equivalent cytotoxicity,indicating that the amide-type prodrugs can rapidly release active gemcitabine molecules to produce cytotoxicity.In addition,the in vitro cytotoxicity results of GHC showed that much lower cytotoxicity in Caco-2 cells than BxPC-3 cells(IC50 40.9 VS 0.32 μM),proving that there was no toxicity of GHC in intestinal epithelial cells.The plasma concentration-time profiles of gemcitabine,a non-targeting prodrug(GS)and OCTN2-targeting prodrugs(GSC,GHC,GOC and GDC)were compared after oral administration to rats.The bioavailability of the targeted prodrugs was about 1.2-fold to 4.9-fold greater than that of gemcitabine(3.2%).The absolute bioavailability of GSC decreased from 12%to 6%in the presence of L-carnitine,demonstrating that the improved oral bioavailability was due to OCTN2-mediated delivery.To explore the bioactivation mechanism,systemic and portal vein plasma concentrations of GSC and gemcitabine were determined.The results suggested that bioactivation mainly occurred in the blood and liver.More importantly,GHC exhibited a significantly prolonged plasma half-lives and the highest oral bioavailability among the targeted prodrugs,and the longer chains(8-10 carbons)did not help improve pharmacokinetic behavior of gemcitabine through OCTN2.In conclusion,the relatively good chemical and enzymatic stability in the GI tract appears to help the oral bioavailability and to maintain the minimal intestinal toxicity of OCTN2-targeting oral prodrugs.In vivo safety studies were carried out by tissue distribution and Hematoxylin and eosin(H&E)histological analysis.No noticeable changes were observed in H&Estained tissue sections of heart,liver,spleen,lung,and kidney after continuous administration of GHC for 14 days(102 mg/kg/day).Morever,no prodrug accumulation in tissue was observed after oral GHC at dose of 102 mg/kg.These results indicated that OCTN2-targeting oral prodrugs showed no significant off-target toxicities to major organs and tissues.