【作者】 盛长城； 刘飞燕； 徐璐扬； 邱晓燕； 张明； 焦正；

【Author】 Chang-Cheng Sheng;Fei-Yan Liu;Lu-Yang Xu;Xiao-Yan Qiu;Ming Zhang;Zheng Jiao;Department of Pharmacy, Huashan Hospital, Fudan University;Department of Pharmacy, Guizhou Provincial People’s Hospital;Department of Nephropathy, Huashan Hospital, Fudan University;

【机构】 复旦大学附属华山医院药剂科； 贵州省人民医院药剂科； 复旦大学附属华山医院肾内科；

【摘要】 目的:免疫抑制剂麦考酚酸（mycophenolic acid, MPA）是一个可逆的非竞争性次黄嘌呤单核苷酸脱氢酶（inosine-5′-monophosphate dehydrogenase, IMPDH）抑制剂。本研究旨在建立、优化和验证测定IMPDH活性的高效液相色谱（high performance liquid chromatography, HPLC）方法,以探讨IMPDH在MPA个体化治疗中的作用。方法:从2 m L血样中分离外周血单个核细胞（peripheral bloodmononuclear cells, PBMC）,在37℃下进行120 min体外酶促反应,底物次黄嘌呤核苷酸（inosine monophosphate, IMP）和辅酶烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide, NAD）浓度均为0.5 mmol·L^-1。采用CNW Athena C18-WP（150×4.6mm, 3μm）色谱柱,流动相为甲醇-(6.25mmol·L^-1磷酸二氢钾+0.5mmol·L^-1四丁基硫酸氢铵)溶液（15:85,v/v）,流速1mL?min^-1,柱温35℃,紫外检测波长254nm,进样量10μL。通过该法测定产物黄嘌呤核苷酸（xanthosinemonophosphate, XMP）浓度,同时测定腺嘌呤核苷酸（adenosine monophosphate, AMP）浓度以标化细胞计数,并扣除空白样品中AMP以消除外源性干扰,从而间接测定IMPDH活性。进而通过该方法考察健康志愿者和服用MPA肾移植患者的IMPDH活性。结果:AMP和XMP的保留时间分别为9.7 min和12.0 min,线性范围均为2～100μmol·L^-1 （R2> 0.99）。AMP和XMP在低(4μmol·L^-1)、中(40μmol·L^-1)、高浓度(80μmol·L^-1)的相对回收率分别为100.29%、100.12%、99.51%和104.83%、99.25%、100.72%,批内和批间精密度相对标准差（relative standard deviation, RSD）均小于10%。平行6次测定健康志愿者IMPDH活性,精密度RSD在2%～10.7%之间,11名健康志愿者的酶活性为46.60±14.28μmol·s^-1·mol^-1AMP。4名服用MPA肾移植患的IMPDH活性与MPA浓度呈负相关（图1）,在术后第13天测定IMPDH活性的抑制率在24%～42%之间。结论:所建立的分析方法准确、灵敏、快速,可用于血中IMPDH活性测定,进行MPA的体内药效学研究。亚洲人和高加索人的MPA药效学差异可为不同种族间的用药差异提供一些依据。更多还原

【Abstract】 Objective:The immunosuppressive agent mycophenolic acid（MPA） is a reversible,non-competitive inhibitor of inosine-5′-monophosphate dehydrogenase（IMPDH）.This study aimed to establish, optimize and validate a high performance liquid chromatography（HPLC）method for quantification of IMPDH activity, and to investigate the role of IMPDH activity in MPA individualized administration as a potential pharmacodynamics biomarker.Methods:Peripheral blood mononuclear cells（PBMC） were isolated from 2 mL blood samples and an in vitro enzymatic reaction was subsequently performed at 37 ℃ for 120 min. The concentrations of substrate inosine monophosphate（IMP） and coenzyme nicotinamide adenine dinucleotide（NAD） were both 0.5 mmol·L^-1. For chromatographic separations, a CNW Athena C18-WP column（150×4.6 mm, 3 μm） was employed, and the isocratic eluent consisted of a15:85（v/v） mixture of methanol and buffer containing 6.25 mmol·L^-1 potassium dihydrogen phosphate and 0.5 mmol·L^-1 tetra-butylammonium hydrogen sulfate. A volume of 10 μL of the sample was injected into the column, and maintained at 35 °C. The flow rate and detection wavelength were set to 1 mL·min^-1 and 210 nm, respectively. By this method, the concentrations of xanthosine monophosphate（XMP） and adenosine monophosphate（AMP） were determined simultaneously, and AMP from blank samples was subtracted for eliminating exogenous AMP interference. Then the enzymatic product XMP was normalized to the amount of PBMC by measured AMP to express IMPDH enzyme activity. The method was further used to investigate IMPDH activity of kidney transplant recipients treated with MPA and healthy volunteers.Results :The retention times of AMP and XMP are 9.7 min and 12.0 min, respectively. High linearity（R²> 0.99） was observed between peak areas and concentrations of both AMP and XMP in the range of 2～100 μmol·L^-1. The relative recovery of AMP and XMP under low(4 μmol·L^-1),medium(40 μmol·L^-1) and high concentration(80 μmol·L^-1) was 100.29%, 100.12%, 99.51% and 104.83%, 99.25% and 100.72%, respectively. The relative standard deviation（RSD） of inter-and intra-day precision for AMP and XMPwas below 10%. The RSD of IMPDH activity in healthy volunteers ranged between 2% and 10.7% after measured 6 times in parallel, and the activity of 11 healthy volunteers was 46.60 ± 14.28 μmol·s^-1·mol^-1 AMP. A negative relationship between MPA concentration and IMPDH activity was observed in four kidney transplant recipients treated with MPA 13 days post-operation, while the inhibitory rate of IMPDH activity ranged between24% and 42%（Fig.1）.Conclusion:The established analytical method was proved to be accurate, sensitive and quick,which could be used to determine IMPDH activity in blood samples and therefore contribute to the MPApharmacodynamics studies. The differences in the pharmacodynamics of MPA between Asians and Caucasians may provide some evidence for dosing differences among ethnicities.更多还原