邻苯二甲酸二乙酯提高华蟾素油剂体内肝细胞癌抑制活性及免疫细胞亚群应答水平

The effect of diethyl phthalate in enhancing inhibitory activity of Huachansu in oil on hepatocellular carcinoma and immune cell subset responses

目的验证邻苯二甲酸二乙酯可发挥免疫佐剂作用,适量的添加可提高华蟾素油剂抑制肝细胞癌活性以及免疫细胞亚群的应答水平。方法以高效液相色谱法比较华蟾素肠溶片与干蟾皮药材之间的化学成分差异,对华蟾素肠溶片较干蟾皮药材HPLC图谱中多出的一个色谱峰采取硅胶柱层析、Sephadex-LH20凝胶柱层析法进行分离纯化,并以液相串联质谱(LC-MS/MS)和核磁共振(1H-NMR、13 C-NMR)鉴定其化学结构。药代动力学实验以华蟾素片的油剂和水剂在给药剂量为600 mg/kg(临床折算剂量)的情况下,检测SD大鼠血浆中邻苯二甲酸二乙酯代谢产物邻苯二甲酸单乙酯的药时浓度和药代动力学参数;采用标准曲线法和外标法对华蟾素肠溶片整片、包衣层、素片中邻苯二甲酸二乙酯及蟾蜍噻咛的含量进行分析。药效学研究以鼠源肝癌Hepa1-6皮下移植瘤为模型,分别以玉米油、华蟾素肠溶整片油剂(533 mg/kg,临床折算剂量)、华蟾素素片油剂(320 mg/kg)、华蟾素肠溶片包衣层油剂(213 mg/kg)灌胃2次/d,共计21 d,测量4个实验组小鼠的肿瘤体积;并收集不同给药组小鼠的脾脏和肿瘤组织,应用流式细胞技术分析上述四组小鼠脾脏和肿瘤组织中CD8^+T细胞、调节性T细胞(Treg)、骨髓来源的抑制性细胞(MDSCs)、PD1^+CD8^+T细胞分布情况。为了考察邻苯二甲酸二乙酯本身是否具有抑瘤作用以及油剂对抑瘤效果的影响,重新剥取不含有华蟾素成分的包衣层粉末及纯度99%邻苯二甲酸二乙酯,配制成包衣层水剂组和油剂组(213 mg/kg),邻苯二甲酸二乙酯油剂组(8 mg/kg),以鼠源肝癌Hepa1-6皮下移植瘤为模型,灌胃2次/d,共计21 d,测量3个实验组小鼠的肿瘤体积。结果经鉴定,华蟾素肠溶片较干蟾皮药材HPLC图谱中多出的色谱峰为邻苯二甲酸二乙酯,推测是包衣过程中作为增塑剂辅料被引入。邻苯二甲酸二乙酯在体内转化成邻苯二甲酸单乙酯,大鼠以华蟾素油剂口服给药可提高邻苯二甲酸单乙酯在血浆中的浓度,表明油剂给药可提高邻苯二甲酸二乙酯的生物利用度。经高效液相色谱定量分析,华蟾素肠溶片整片、包衣层、素片中邻苯二甲酸二乙酯的含量分别为1.64%,2.51%,0.56%;蟾蜍噻咛的含量分别为0.033%,0.015%,0.048%。小鼠接种Hepa1-6细胞成瘤后,给药21 d后测量不同给药组小鼠的肿瘤体积发现华蟾素肠溶片包衣层油剂的抑瘤效果显著高于其他实验组,并且无毒副作用。包衣层油剂的抑瘤率可达49.2%,整片油剂的抑瘤率40.2%,素片油剂为36.2%;上述三者以油剂给药均可提高小鼠脾脏中CD8^+T细胞比重。在肿瘤组织中包衣层油剂显著提高了CD8^+T细胞的比重(t-test P<0.05)。该结果表明肿瘤免疫T细胞被活化。同时华蟾素肠溶片包衣层油剂显著降低了小鼠脾脏及肿瘤组织中的骨髓来源的抑制性细胞(Myeloid-derived suppressor cells,MDSCs)和调节性T细胞(Regulatory cells,简称Tregs)数量(t-test P<0.05),可使肿瘤细胞免疫应答抑制得以解除,从而有利于抑制肿瘤生长。当邻苯二甲酸二乙酯单独以油剂使用时,肿瘤抑制率达到了32.5%,不含华蟾素成分的包衣层(邻苯二甲酸二乙酯含量为1.27%,蟾蜍噻咛含量为0.001%)油剂抑瘤率为34.9%,而水剂给药时抑瘤率仅为25.8%;表明油剂较水剂更有利于邻苯二甲酸二乙酯发挥抑瘤作用。同时本研究显示当治疗靶点饱和后,抑瘤效果不再随邻苯二甲酸二乙酯给药剂量的增加而提高。结论邻苯二甲酸二乙酯作为增塑剂辅料添加至华蟾素肠溶片中。邻苯二甲酸二乙酯在体内可增强华蟾素激活肿瘤浸润CD8^+T细胞,降低免疫抑制细胞MDSCs和Tregs作用,提高免疫细胞亚群应答水平;并且邻苯二甲酸二乙酯本身在体内具有抑瘤作用,玉米油提高其在体内的吸收,利于发挥药效作用。所以将邻苯二甲酸二乙酯适量加入制剂处方中可提高华蟾素油剂的抑瘤作用。

Objective To verify the possibility of diethyl phthalate(DEP)as an immunologic adjuvant,which may enhance the inhibitory effects of the active ingredients in Huachansu oil solution on inhibiting liver tumor activity and the response of immune cell subsets.Methods The chemical composition difference between Huachansu enteric-coated tablets and dried toad skin medicinal materials was compared by HPLC.Silica gel column chromatography and Sephadex-LH20 gel column chromatography were used for separation and purification of an extra chromatographic peak in Huachansu enteric-coated tablets compared with dry toad skin medicinal materials,and the chemical structure of this peak was identified by liquid tandem mass spectrometry(LC-MS/MS)and nuclear magnetic resonance(1H-NMR,13 C-NMR).Pharmacokinetic studies were conducted in SD rats with Huachansu tablets in corn oil or in water at 600 mg/kg(clinically converted dose)and the concentration and pharmacokinetic indexes of DEP metabolite monoethyl phthalate in the plasma was detected;the concentration of DEP and bufothionine in Huachansu enteric-coated tablets,tablet coatings,and plain tablets were determined by using standard curve method and external standard method.In pharmacodynamics studies on Hepa1-6 subcutaneous tumor mice model,the animals were intragastrically administered with Huachansu enteric-coated tablets in corn oil(533 mg/kg,clinically converted dose),Huachansu Plain tablet oil(320 mg/kg),and Huachansu enteric-coated tablet coating in corn oil(213 mg/kg)twice daily for a total of 21 days,the tumor volumes of the mice in the four experimental groups were measured;the spleens and tumor tissues of the mice in different administration groups were collected,and the CD8^+T cells,regulatory T cells(Tregs),bone marrow-derived suppressor cells(MDSCs),and PD1+CD8^+T cells in these samples were analyzed by flow cytometry.To investigate whether DEP itself is capable to suppress tumor growth and the role of corn oil on tumor suppressing effect,animals of Hepa1-6 subcutaneous tumor mice model were intragastrically administrated with the following formulations twice a day for a total of 21 days:the re-extracted coating layers of Huachansu enteric-coated tablets,formulated together with DEP(99%purity)in either water or corn oil(213 mg/kg),and DEP in corn oil(8 mg/kg).And the tumor volumes of the treated mice were measured.Results The extra chromatographic peak in Huachansu enteric-coated tablets compared with dry toad skin medicinal materials was identified as DEP and may have been introduced as a plasticizer formulation excipient during the coating process.DEP can be converted into monoethyl phthalate in vivo.When Huachansu enteric-coated tablets in corn oil was given to animals,the plasma distribution of monoethyl phthalate was increased,which suggests that oil can increase the oral bioavailability of DEP.By HPLC analysis,DEP content in Huachansu,enteric-coated tablets,tablet coating layers,and plain tablets was determined to be 1.64%,2.51%,and 0.56%;and the bufothionine content in the three different parts of the drug was 0.033%,0.015%,and 0.048%,respectively.After 21 days of administration of different formulations in Hepa1-6 subcutaneous tumor model,it was found that the antitumor effect of the coating layer in corn oil was significantly higher than that of the other experimental groups,moreover,no toxic and other side effects were observed.The tumor inhibition rate of the coating layer in corn oil reached 49.2%,while it was 40.2%for the whole tablet in oil,and 36.2%for the plain tablet in oil,respectively;additionally,the CD8^+T proportions in the spleen of mice that were treated with the above three formulations were increased ubiquitously without significant difference.However,the coating layers in oil significantly increased the CD8^+T cells proportion in tumor tissues(P<0.05),indicating that tumor immune T cells were activated and tumor immunity is activated.Furthermore,Huachansu enteric-coated tablet coating significantly reduced the amount of bone marrow-derived suppressor cells(MDSCs)and regulatory T cells(Tregs)in mice spleen and tumor tissues(t-test,P<0.05),which relieved the suppression of tumor cell immune response and is conducive to tumor growth inhibition.The tumor inhibition rate of DEP in corn oil reached up to 32.5%,while the coating layer without the internal content of Huachansu(DEP 1.27%,bufothionine 0.001%)in oil had an anti-tumor rate of 34.9%,which was only 25.8%for Huachansu tablet in water.Apparently,corn oil was advantageous to the anti-tumor effect of DEP.Meanwhile,when the molecular target was saturated,the dose increase of DEP would not result in enhancement of the molecule′s anti-tumor effect.Conclusion DEP was used as a plasticizer excipient in Huachansu enteric-coated tablets.DEP can enhance the tumor infiltration of CD8^+T cells activated by Huachansu,reduce the effect of immunosuppressive cells MDSCs and Tregs,and improve the subset response levels of immune cells.In addition,DEP itself has anti-tumor effect in vivo,and corn oil can improve its absorption,which is beneficial to exerting its pharmacological effect.Therefore,adding an appropriate amount of DEP can improve the anti-tumor effect of Huachansu oil preparation.