Mono-carboxylate Transporter 1-targeting Cytarabine Prodrug For Enhanced Oral Delivery

Objective:Cytarabine is a first-line drug for the treatment of acute leukemia and malignant lymphoma by intravenous administration.At present,the drug is mainly administered intravenously,and its half-life is short and side effects are serious due to extensive enzyme metabolism.Studies have shown that cytarabine has low bioavailability,so it needs high dose to achieve the desired therapeutic effect,and high dose brings high toxicity,which makes the side effects of patients great.Low dose of cytarabine is helpful to improve its clinical efficacy,but long-term intravenous administration leads to inconvenient clinical use.The development of oral cytarabine can solve the above problems.However,poor membrane permeability and stability of drug in gastrointestinal tract limited its oral admistration.Intestinal mono-carboxylate transporter 1（MCT1）is high expressed with the effect of the transportation of short chain fatty acids,which supply a potential target to design oral prodrug of cytarabine to enhance the oral bioavailability.In this article,succinic acid was used as transporter recognition group to conjugate the 4-N of cytarabine for the increasing the oral bioavailability.Method:（1）Synthesis of cytarabine prodrug:The prodrug was synthesized through covalently linking cytarabine and succinic anhydride with the help of triethylamine.And the reaction was monitored by thin layer chromatography.Silica gel column chromatography（dichloromethane methanol system）and preparative chromatography（methanol acetic acid water）were used for separation,and high performance liquid chromatography（HPLC）was used to detect the purity of prodrug.The structure was confirmed by nuclear magnetic resonance（¹H-NMR）.（2）Stability of Cytarabine prodrug:Phosphate buffer with different p H s（p H1.2,6.8 and 7.4）were prepared according to the Chinese Pharmacopoeia（2020）,and then was co-incubated with cytarabine prodrug respectively.The prodrug stability in simulated gastrointestinal solution,tissue homogenates and plasma was also investigated.The contents of prodrug and cytarabine at different time points were determined by UPLC-MS/MS.（3）Cellular uptake of cytarabine prodrug:Inorder to elucidate the uptake mechanism of cytarabine prodrug,the effects of H⁺,temperature and quercetin（MCT1 Competitive inhibitors）on cellular uptake of cytarabine prodrug were investigated in human colon adenocarcinoma cell line（Caco-2）.（4）The membrane permeability of cytarabine prodrug:Caco-2 cell monolayer was used as the transport model to study the permeability of cytarabine prodrug in the lumen side（AP）→basal side（BL）and basal side（BL）→lumen side（AP）.The effect of quercetin on the transport of cytarabine prodrug was selected to clarify the mechanism of high permeability in the AP→BL side.（5）Oral pharmacokinetics of cytarabine prodrug in rats:SD[Sprague Dawley]rats were randomly divided into 4 groups with 6 rats in each group.The equivalent dose of cytarabine was selected at 50 mg/kg.After administration,blood samples were collected at the preset time points of0.083,0.15,0.25,0.5,1,2,4,8 and 12 h,respectively.And then,the samples were quickly placed in an EP tube pretreated with heparin and cytosine deaminase inhibitor（10 mg/m L）.Plasma was centrifuged at 13000 rpm and stored in a refrigerator at-80℃.The content of inosine was determined by UPLC-MS/MS,and the drug concentration-time curve was made by Origin2016.Result:（1）Synthesis of cytarabine prodrug:Cytarabine reacted rapidly with succinate in high yield with the help of triethylamine.The prodrugs were identified by HPLC,nuclear magnetic resonance（¹H-NMR）and ultra performance liquid chromatography tandem quadrupole mass spectrometry（UPLC-MS/MS）.The results indicated that the purity of prodrugs was over95%,which met the requirements of further experiments.（2）Stability of cytarabine prodrugs:Cytarabine prodrugs were stable in p H 1.2 phosphate buffers and p H 6.8 phosphate buffer with the half lives more than 10 h.The results indicated that the prodrug was stable in gastrointestinal tract,which was very helpful to target MCT1.In addition,the prodrug was rapidly degraded into cytarabine in p H 7.4 phosphate buffer,tissue homogenate and plasma with the half-life was less than 3.8 h.The results indicated that the prodrug could be rapidly activated in the circulatory system.（3）Cell uptake of cytarabine prodrug:Compared with the cellular uptake at p H 7.4,the uptake of cytarabine prodrug increased by 1.4 times at p H 6.0,while the cellular uptake of cytarabine did not change significantly.These results indicated the cellular uptake of prodrug was in H⁺dependent.Compared the cellular uptake with 37℃,the uptake was lower.Moreover,the cellular uptake of cytarabine prodrug decreased by about 70%when quercetin was added,but the cellular uptake of cytarabine did not change.And the cellular uptake of quercetin was reduced by 50%in the presence of prodrug.The above results showed that prodrug was actively absorbed into cells via MCT1.（4）The membrane permeability of cytarabine prodrug:The transport of cytarabine prodrug in AP→BL was significantly higher than that in BL→AP,but there was no significant difference of cytarabine cellular uptake between the two directions（P>0.05）.Moreover,the transport of cytarabine in AP→BL membrane was reduced by about 88%in the presence of quercetin,while the transport of cytarabine was not significantly affected（P>0.05）,indicating that the high membrane permeability of prodrug was mediated by MCT1.（5）Oral pharmacokinetics of cytarabine prodrug in rats:The absolute oral bioavailability of cytarabine prodrug in SD rats was 32.1%,which was about 3 times higher than that of the parent drug.Moreover,quercetin,an inhibitor of MCT1 transporter,could significantly reduce the area of the AUC_0-tof cytarabine prodrug by 30%,indicating that the high oral bioavailability of prodrug was mediated by intestinal MCT1.Conclusion:Succinyl cytarabine prodrug was synthesized with high yield and purity of more than 95%;Prodrugs are stable in gastrointestinal tract and can be activated rapidly in circulatory system;Cytosine arabinoside prodrug H+dependent,temperature dependent,active uptake into cells via MCT1;The high membrane permeability and oral bioavailability of prodrugs are mediated by MCT1.