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医药导报, 2016, 35(11): 1177-1180
doi: 10.3870/j.issn.1004-0781.2016.11.005
有限采样法估算麦考酚钠药-时曲线下面积及其药动学
Limited Sampling Strategy for the Estimation of Mycophenolate Sodium Area Under Concentration-Time Curve and Pharmacokinetic Study
李纳1,, 朱振峰1,, 丰贵文2, 张俊1, 贾萌萌1, 左莉华1, 张晓坚1

摘要: 目的建立有限采样法估算霉酚酸(MPA)药-时曲线下面积的模型。方法16例肾移植患者口服麦考酚钠肠溶片(EC-MPS)720 mg,q12 h,分别于术后第4天、第7天服药前及服药后0.5,1,1.5,2,3,4,6,8,10,12 h采集静脉血样3 mL,采用超高效液相色谱-紫外(UPLC-UV)分析方法测定MPA血浆浓度。以 DAS 2.0版药动学软件进行药动学分析,MPA-AUC0-12 h按梯形法计算。用SPSS 17.0版软件进行多元线性逐步回归分析方法,建立有限采样法多元回归模型,并计算相关参数。结果术后第4天和术后第7天MPA的主要药动学参数t1/2分别为(6.93±6.51),(8.18±5.06)h(P>0.05);Cmax分别为(10.10±6.44),(17.26±11.67) mg·L-1(P<0.05);AUC0-12 h分别为(32.47±15.53),(41.88±18.30) mg·h·L-1(P<0.05)。观察到部分患者药-时曲线呈双峰,在服药后4~12 h出现第二峰,峰值较第一峰明显降低。发现任何单一时间点MPA血药浓度不能准确预测AUC0-12 h。采用多元线性逐步回归分析方法分析多个时间点MPA血药浓度与AUC0-12 h的相关性后,综合考虑选择0,4,1.5,8 h 4个时间点的MPA血药浓度来估算AUC0-12 h,计算公式为AUC0-12 h =6.207+ C0×6.881+ C4×2.388+C1.5× 0.541+C8×4.252(r2=0.888),其预测值与实测值有很好的相关性(r2=0.918),相对预测误差在可接受范围内。结论该研究建立的4点采样法多元回归模型可较好的预测MPA-AUC0-12 h,有利于进一步在临床上推广和应用,从而提高麦考酚钠合理用药水平,达到预测疗效和不良反应的目的。
关键词: 麦考酚钠 ; 霉酚酸 ; 药动学 ; 有限采样法 ; 药-时曲线下面积

Abstract:
ObjectiveTo establish a model for estimating area under the concentration -time curve(AUC) of mycophenolic acid (MPA) by a limited sampling strategy. MethodsSixteen renal allograft recipients were treated with enteric coated mycophenolate sodium (EC-MPS) twice a day respectively.The blood samples were collected on postoperative days 4 and 7 before and 0.5,1,1.5,2,3,4,6,8,10,12 h after EC-MPS oral administration.Ultra performance liquid chromatography-UV (UPLC-UV) method was employed to determine the plasma concentration of MPA.Pharmacokinetic parameters of MPA were calculated by DAS 2.0 software.Areas under curve over the period of 0 to 12 h (AUC0-12 h ) were calculated by using linear trapezoidal rule.The limited sampling model and relevant parameters were established by multivariate linear stepwise regression analysis with SPSS 17.0. ResultsThe main pharmacokinetic parameters on postoperative days 4 and 7 were as follows: t1/2 were (6.93±6.51) and (8.18±5.06) h (P>0.05); Cmax were (10.10±6.44) and (17.26±11.67) mg·L-1 (P<0.05); AUC0-12 h were (32.47±15.53) and (41.88±18.30) mg·h·L-1(P<0.05).Some patients' concentration-time curves were bimodal.The second peak appeared at 4~12 h after treatment and the peak obviously decreased compared with the first one.No single plasma concentration can accurately predict AUC0-12 h.After analyzing the correlation between multiple plasma concentrations of MPA and AUC0-12 h by the multivariate linear stepwise regression analysis method,we selected the 0,4,1.5,8 h plasma concentrations of MPA to estimate AUC0-12 h.The computational formula was AUC0-12 h =6.207+C0×6.881+C4×2.388+C1.5×0.541+C8×4.252 (r2=0.888).The predicted value has a good correlation with the measured value (r2=0.918) and the relative prediction error was in the acceptable range. ConclusionThe multivariate regression model of four points sampling method is a better predictor of MPA- AUC0-12 h.This simple method will be conducive to the further application and popularization in clinic,improve the rational use of mycophenolate sodium,and realize the purpose of curative effect and adverse reaction prediction.
Key words: Mycophenolate sodium ; Mycophenolic acid ; Pharmacokinetics ; Limited sampling strategy ; Area under concentration-time curve

麦考酚钠肠溶片(mycophenolate sodium enteric-coated tablets,EC-MPS)是霉酚酸(mycophenolic acid,MPA)的钠盐制剂,口服后在肠道直接释放出MPA,可降低胃肠道不良反应发生率[1]。MPA药动学特征有明显个体差异,服用相同剂量的药物,不同患者间MPA血药浓度和血药浓度-时间曲线下面积(area under concentration-time curve,AUC)可相差10倍[2]。因此,有必要对MPA进行血药浓度监测。由于MPA代谢中有肠肝循环现象,其谷浓度(C0)和治疗效应的相关性差。MPA免疫抑制效果及不良反应与其AUC0-12 h密切相关。常规测定一个完整的12 h给药间隔内的AUC值往往需要9~12个采血点,试验成本高,采样次数多,患者难以接受。因此,开发有限采样法,利用2~4个取样点的血药浓度来估算MPA-AUC0-12 h有重要的临床应用价值。目前已有多项研究建立了服用霉酚酸酯(mycophenolate mofetil,MMF)后估算MPA-AUC0-12 h的模型,但是针对EC-MPS的研究笔者较少见到。笔者在本实验中建立有限采样法估算MPA的AUC模型,现报道如下。

1 资料与方法
1.1 研究资料

在本院首次接受肾移植患者16例(男11例,女5例),年龄(29.06±8.53)岁,体质量(60.59±13.92) kg。患者未同时服用含有镁和铝氢氧化物的抗酸药、考来烯胺等可能影响EC-MPS吸收及肠肝循环的药物;无严重胃肠道疾病、无严重肝功能异常。在试验前所有患者均被告知试验目的和内容,均签署知情同意书。研究方案获得郑州大学第一附属医院伦理委员会批准。

1.2 免疫抑制治疗方案

术后所有患者采用EC-MPS、他克莫司及皮质类固醇激素预防排斥反应。术后第1天开始口服EC-MPS720 mg,q12 h。他克莫司起始剂量0.06~0.08 mg·kg-1·d-1,分2次空腹服用;然后加用五酯软胶囊0.5 g,bid。术前静脉滴注甲泼尼龙120 mg和抗胸腺细胞球蛋白(antithymocyte globulin,ATG)100 mg,术中分两次静脉滴注甲泼尼龙共1 g,之后连续2 d静脉输注甲泼尼龙500 mg,第3天375 mg,第4天250 mg,第5天120 mg,之后改用口服甲泼尼龙片16 mg·d-1维持。

1.3 仪器与试药

Acquity 超高效液相色谱仪(ultra performance liquid chromatography,UPLC,美国Waters公司);紫外线(ultra-violet ray,UV)光学检测器(美国Waters公司);Vortex-genie2漩涡混合器(美国Scientific Industries公司);Sorvall Legend Micro17 微量高速离心机(美国Thermo Fisher Scientific公司);BX7200HP超声波清洗器(上海新苗医疗器械制造有限公司)。

MPA对照品(含量:99.5%,美国Sigma公司,批号:011M4007V),卡马西平对照品(含量:99.7%,批号:100142-201105,中国食品药品检定研究院),试验用水均为Millipore超纯水,盐酸、磷酸均为色谱纯,磷酸二氢钾(KH2PO4)为分析纯,乙腈、甲醇均为色谱纯(美国Tedia公司)。

1.4 血样采集方法

患者服用EC-MPS达稳态后,分别于术后第4天、第7天服药前及服药后0.5,1,1.5,2,3,4,6,8,10,12 h采集静脉血样3 mL,置于乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)抗凝管中,经3 000 r·min-1 离心(r=8.6 cm)10 min后取上层血浆,置于-20 ℃冰箱,用于检测MPA浓度。

1.5 血药浓度测定方法

采用超高效液相色谱-紫外(UPLC-UV)分析方法测定血浆MPA浓度。取血浆0.2 mL,置于1.5 mL EP管中,加入1 mol·L-1盐酸(HCl)20 μL,振荡1 min,加入含内标卡马西平(1 μg·mL-1)的乙腈溶液400 μL,涡旋振荡2 min,以133 000 r·min-1 离心10 min,吸取全部上清液置于EP管中,氮气吹干。然后用50%甲醇溶液100 μL复溶,涡旋振荡2 min,以133 000 r·min-1 离心3 min,取上清液10 μL进样。色谱柱:C18柱(2.1 mm×50 mm,1.7 μm),流动相:乙腈-20 mmol·L-1 KH2PO4水溶液=30.5:69.5,流速:0.3 mL·min-1,紫外检测波长:254 nm,柱温:30 ℃,样品池温度:4 ℃。MPA在0.20~50.00 mg·L-1范围内线性良好。该方法经体内药物分析方法学指标验证均符合生物样品测定要求。

1.6 数据处理与统计学方法

以DAS 2.0版药动学软件进行药动学分析,MPA-AUC0-12 h按梯形法计算。参数tmaxCmax均为实测值。采用配对t检验方法对术后第4天和术后第7天的药动学参数进行对比分析,以P<0.05表示差异有统计学意义。

用SPSS 17.0版软件进行多元线性逐步回归分析,建立有限采样法多元回归模型,并计算相关参数。以各个时间点血MPA浓度作为自变量,以AUC0-12 h作为因变量,考察AUC0-12 h与血MPA浓度任意一点或多点间的相关性,并采用逐步多元回归方法,遴选出复相关系数(r2)较大、相对预测误差(relative prediction error,RPE)小的回归方程,用于估算MPA-AUC0-12 h。RPE、相对预测误差绝对值(absolute relative prediction error,ARPE)、平均相对预测误差(mean relative prediction error,MRPE)计算如下:RPE(%)=(AUC预测值-AUC实测值)/AUC实测值×100%,ARPE(%)=|RPE |,MRPE(%)= 1 n ∑RPEi

2 结果
2.1 EP-MPS药动学结果

16例患者口服EP-MPS 720 mg ,q12 h多剂量给药后,血MPA浓度数据使用DAS 2.0版药动学软件进行处理,计算药动学参数,其中tmaxCmax均为实测值。术后第4天和术后第7天Cmax、AUC0-12 h、MRT0-12 h差异有统计学意义,而t1/2tmax、CL/F差异无统计学意义(表1)。

表1 术后第4天和第7天MPA主要药动学参数
Tab.1 Main pharmacokinetic parameters of MPA on the postoperative day 4 and day 7 x¯±s,n=16
时间 t1/2 t max Cmax/
(mg·L-1)
CL/F
(L·h-1)
AUC0-12 h/
(mg·h·L-1)
MRT0-12 h/
h
h
术后第4天 6.93±6.51 2.72±1.66 10.10±6.44 24.91±22.73 32.47±15.53 4.81±1.20
术后第7天 8.18±5.06 2.25±2.44 17.26±11.67 15.01±8.84 41.88±18.30 4.09±1.09
t 0.804 0.771 3.024 2.037 2.482 2.528
P 0.434 0.453 0.009 0.06 0.025 0.023

表1 术后第4天和第7天MPA主要药动学参数

Tab.1 Main pharmacokinetic parameters of MPA on the postoperative day 4 and day 7 x¯±s,n=16

16例患者术后第4天和第7天MPA平均药-时曲线形态有明显差异(图1)。部分患者AUC呈双峰现象,第一峰出现在服药后1~4 h,第二峰出现在服药后4~12 h,峰值较第一峰明显降低。

图1 术后第4天和第7天MPA平均药-时曲线(x¯±s,n=16)

Fig.1 Mean concentration-time curve of MPA on the postoperative day 4 and day 7(x¯±s,n=16)

2.2 多元回归模型的分析结果

先分析单个时间点MPA血药浓度与AUC0-12 h相关性,结果发现0,8 h单个时间点MPA血药浓度与AUC0-12 h相关性较好,r2分别为0.699和0.553,MRPE分别为14.4%和18.5%,但预测的精确性都不佳,都有50%以上的预测值ARPE超过15%(表2)。任何单个时间点MPA血药浓度不能准确预测AUC0-12 h

表2 回归模型估算与实测MPA-AUC0-12 h的相关性分析
Tab.2 Correlation analysis of the estimated and factual measured MPA-AUC0-12 h by regression model
采样时
间/h
回归方程 r2 MRPE/
%
ARPE>15%
的例数(%)
0 Y=16.732+C0×12.449 0.699 14.40 18(56.25)
0.5 Y=32.247+C0.5×1.070 0.202 31.04 22(68.75)
1 Y=31.422+C1×1.042 0.155 30.90 22(68.75)
1.5 Y=28.228+C1.5×1.061 0.210 28.08 25(78.13)
2 Y=29.789+C2×1.049 0.234 28.89 23(71.88)
3 Y=21.984+C3×3.718 0.383 22.35 22(68.75)
4 Y=16.241+C4×6.462 0.471 16.44 24(75.0)
6 Y=25.968+C6×4.903 0.165 31.56 24(75.0)
8 Y=18.732+C8×10.637 0.553 18.54 20(62.5)
10 Y=24.446+C10×7.472 0.410 22.73 20(62.5)
12 Y=22.276+C12×8.225 0.474 21.05 20(62.5)
0,4 Y=9.521+C0×9.818+C4×3.559 0.811 7.76 10(31.25)
0,4,1.5 Y=7.523+C0×9.007+C4×3.432+ 0.843 5.83 8(25.0)
C1.5×0.444
0,4,1.5,8 Y=6.207+C0×6.881+C4×2.388+ 0.888 4.78 4(12.50)
C1.5×0.541+C8×4.252
0,4,1.5,8,2 Y=5.244+C0×5.240+C4×1.452+ 0.940 3.53 3(9.38)
C1.5×0.468+C8×6.100+C2×
0.582
0,4,1.5,8,2, Y=4.517+C0×3.523+C4×1.925+ 0.960 2.56 3(9.38)
0.5 C1.5×0.485+C8×5.741+C2×
0.654+C0.5×0.431

表2 回归模型估算与实测MPA-AUC0-12 h的相关性分析

Tab.2 Correlation analysis of the estimated and factual measured MPA-AUC0-12 h by regression model

采用多元线性逐步回归方法分析多个时间点MPA血药浓度与AUC0-12 h相关性,结果显示2~6个时间点MPA血药浓度所拟合的简化计算回归模型,r2明显高于单点拟合结果(表2)。随着采样时间点增加,其建立的多元回归模型与MPA-AUC0-12 h相关性越好(r2越大、MRPE越小)。但是增加采样时间点,采血次数多,成本较高,患者依从性较差,综合考虑以2~4个时间点MPA血药浓度来估算AUC0-12 h较为合适。0,4 h和0,4,1.5 hMPA血药浓度分别有31.25%和25%的ARPE>15%,预测精度欠佳。0,4,1.5,8 h的MPA血药浓度拟合效果较好,其r2为0.888,MRPE为4.78%,仅有12.50%患者的ARPE>15%,计算公式为Y=6.207+C0×6.881+C4×2.388+C1.5× 0.541+C8×4.252。用4点法估算的AUC0-12 h(eAUC0-12 h)与实际测得的AUC0-12 h(fAUC0-12 h)的相关性较好(图2),可以准确预测MPA-AUC0-12 h,同时4个采血点采样简单方便,患者易于接受。

图2 4点法估算的药-时曲线下面积(eAUC0-12 h)与实际测得的药-时曲线下面积(fAUC0-12 h)的相关性关系图(n=32)

Fig.2 Correlation of the estimation of AUC0-12 h( eAUC0-12 h) and factual measurement of AUC0-12 h(fAUC0-12 h) by four point method(n=32)

3 讨论

环孢素可以影响MPA肠肝循环,降低MPA暴露量[3],而他克莫司对MPA代谢影响较小。MPA有限采样法的建立在与他克莫司或环孢素合并用药时而有所不同。本研究主要探讨与他克莫司合用时,EC-MPS有限采样方案的建立。多剂量服用EC-MPS术后4 d和术后7 d的tmax分别为2.72和2.25 h,与MMF(tmax 0.75 h)相比,达峰时间均延迟,符合肠溶片的吸收特点。部分患者服用EC-MPS后药-时曲线上4~12 h,发现有明显的第2峰,可能与MPA的肠肝循环有关。患者术后第4天和术后第7天的MPA平均药-时曲线有明显差异,术后第7天AUC0-12 hCmax均比术后第4天明显增高,表明MPA在体内存在蓄积现象。可能与术后前4天糖皮质激素的用量大有关,因为糖皮质激素能诱导MPA代谢相关的酶,从而降低MPA的体内暴露量[4]

笔者在本研究发现,任一单个时间点MPA的血药浓度与AUC0-12 h相关度较差,不能准确预测AUC0-12 h。证实仅通过测定C0无法准确反映药物的暴露量、抗排斥反应强度及不良反应情况。通过多元线性逐步回归分析方法,笔者建立了有限采样法估算服用EC-MPS后MPA-AUC0-12 h简化计算公式,其中0,4,1.5,8 h 4个时间点MPA血药浓度拟合效果较好,预测值与实测值有很好的相关性,RPE在可接受范围内。若采用2点或3点法,虽然可减少采样次数,但RPE较大。发现随着采样点的增多,建立的公式计算精度越高,RPE也越小。但增加采样时间点后,采血次数多,采样时间较长,难以被患者接受。4点采样法已可较好预测MPA-AUC0-12 h,有利于进一步在临床推广和应用,提高合理用药水平,达到预测疗效和不良反应的目的。

笔者在本研究建立了合并使用他克莫司时估算MPA-AUC0-12 h的简化计算公式。MPA药动学个体差异较大,有限采样法估算模型的建立需要按不同给药剂型、给药方案和不同人群做进一步的研究。2006年国际会议将MPA-AUC0-12 h的目标范围界定为30~ 60 mg · h · L - 1 5 ,这主要是基于西方人群的数据。此目标范围是否也适用于中国人群,有必要结合MPA-AUC0-12 h与急性排斥反应和不良反应的关系进行更大样本的研究,以确定符合中国人群的MPA-AUC0-12 h最佳目标范围。

The authors have declared that no competing interests exist.

参考文献

[1] BUDDE K,GLANDER P,DIEKMANN F,et al.Review of the immunosuppressant enteric-coated mycophenolate sodium[J].Expert Opin Pharmacother,2004,5(6):1333-1345.
Abstract Enteric-coated mycophenolate sodium (EC-MPS; myfortic, Novartis Pharma AG) is an advanced formulation delivering mycophenolic acid (MPA). EC-MPS was designed to improve MPA-related upper gastrointestinal adverse events by delaying the release of MPA until reaching the small intestine. At a dose of 720 mg, EC-MPS exhibits equivalent MPA exposure (area under the concentration curve [AUC]) and maximal MPA concentration (C(max)) to mycophenolate mofetil (MMF; CellCept, Roche AG) 1000 mg. The time to maximal MPA concentration (T(max)) for EC-MPS is delayed relative to that for MMF, consistent with a functioning enteric coating. EC-MPS 720 mg b.i.d. has demonstrated therapeutic equivalence to MMF 1000 mg b.i.d. in renal transplant patients. Recent clinical trials have demonstrated that EC-MPS is as effective and safe as MMF in both de novo and maintenance renal transplant patients. Furthermore, studies have confirmed that maintenance patients can be safely converted from MMF to EC-MPS with no compromise of efficacy or safety. EC-MPS therefore presents physicians and patients with a valid alternative MPA therapy with a comparable efficacy and safety profile to MMF.
DOI:10.1517/14656566.5.6.1333      PMID:15163278      URL    
[本文引用:1]
[2] CIANCIO G,MILLER J,GONWA T A.Review of major cli-nical trials with mycophenolate mofetil in renal transplantation[J].Transplantation,2005,80(2 Suppl):S191-200.
Mycophenolate mofetil (MMF) was approved for the prevention of acute rejection following renal transplantation based on the results of three groundbreaking, large, clinical trials that demonstrated a significantly reduced risk of acute rejection in patients receiving MMF when compared with those receiving placebo or azathioprine. These three multicenter, prospective, double-blind trials performed at 55 transplant centers on three continents were the largest immunosuppressive drug trials ever attempted and the first prospective, randomized, double-blind trials ever performed in transplantation. These pivotal trials established a foundation for widespread acceptance of MMF in combination with cyclosporine and steroids as a maintenance regimen for renal transplant patients. The findings of these initial trials that led to the approval of MMF for renal transplantation, including long-term follow-up data, will be reviewed in this paper. The expanding scope of major trials of MMF, including trials in pediatric patients, combination regimens with novel induction therapies or other maintenance agents, and trials in special patient populations such as those at high immunological risk or with deteriorating kidney function, will also be discussed.
DOI:10.1097/01.tp.0000187035.22298.ba      PMID:16251852      URL    
[本文引用:1]
[3] 马一平. 实体器官移植患者霉酚酸的药动学特征及其影响因素[J].天津药学,2011,23(3):52-55.
目的:霉酚酸 (mycophenolic acid,MPA)已广泛用于预防实体器官移植术后的排斥反应和自身免疫性疾病的治疗。但MPA的药动学特征具有明显的个体差异。本文综述了相关文献报 导,旨在为临床开展广泛和深入的研究提供有益的参考。方法:综述霉酚酸在实体器官移植临床药动学研究及影响因素的文献报道。影响因素包括了不同器官移植、 年龄、性别、种族、血清白蛋白(ALB)、合用药物、并发症以及基因多态性的影响。结果:国内外研究结果在不同器官移植、合用药物、种族、肾功能损伤等方 面具有相似的报道,而在其他影响因素方面存在研究范围及研究结果的差异。结论:MPA药动学参数影响因素众多且不易控制,因此应继续进行多方面、大样本的 临床考察,控制药动学的影响因素,得出确定并能达成共识的临床监测指标,从而更好的取得临床疗效。
[本文引用:1]
[4] CATTANEO D,PERICO N,GASPARI F,et al.Glucocortic-oids interfere with mycophenolate mofetil bioavailability in kidney transplantation[J].Kidney Int,2002,62(3):1060-1067.
These findings indicate that steroids interfere with MPA bioavailability, and that discontinuation of the drug results in higher MPA exposure, which may compensate at least in part for the lower immunosuppressive level achieved with the remaining dual therapy with CsA and MMF.
DOI:10.1046/j.1523-1755.2002.00531.x      PMID:12164891      URL    
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[5] LE MEUR Y,BORROWS R,PESCOVITZ M D,et al.The-rapeutic drug monitoring of mycophenolates in kidney transplantation:report of the transplantation society consensus meeting[J].Transplant Rev,2011,25(2):58-64.
Le Meur Y, Borrows R, Pescovitz MD, Budde K, Grinyo J, Bloom R, Gaston R, Walker RG, Kuypers D, van Gelder T, Kiberd B.
DOI:10.1016/j.trre.2011.01.002      PMID:21454067      URL    
[本文引用:0]
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关键词(key words)
麦考酚钠
霉酚酸
药动学
有限采样法
药-时曲线下面积

Mycophenolate sodium
Mycophenolic acid
Pharmacokinetics
Limited sampling strategy
Area under concentration-...

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李纳
朱振峰
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左莉华
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