药学学报  2016, Vol. 51 Issue (11): 1666-1673   PDF    
次黄嘌呤单核苷酸脱氢酶的活性测定及临床应用
刘飞燕, 邱晓燕, 焦正     
复旦大学附属华山医院药剂科, 上海 200040
摘要: 次黄嘌呤单核苷酸脱氢酶(IMPDH)是一种鸟嘌呤从头合成途径的限速酶,在细胞增殖过程中发挥着重要作用。目前临床常用的作用于IMPDH的药物为IMPDH抑制剂,主要应用于抗免疫排斥、抗肿瘤、抗病毒或抗寄生虫等领域。IMPDH抑制剂的血药浓度与临床疗效之间存在一定关联性。但由于该关联的个体间变异通常较大,故测定IMPDH酶活性可作为一项监测其药物疗效的特异性生物标志物,与药动学监测相结合,可提高IMPDH抑制剂临床应用的安全性和有效性。本文旨在综述国内外近年来有关IMPDH酶活性的测定方法与临床应用现状,为临床IMPDH抑制剂药效学监测的开展和应用提供方法与依据。
关键词: 次黄嘌呤单核苷酸脱氢酶     次黄嘌呤单核苷酸脱氢酶抑制剂     酶活性测定     生物标志物    
The determination and clinical application of inosine 5'-monophosphate dehydrogenase activity
LIU Fei-yan, QIU Xiao-yan, JIAO Zheng     
Department of Pharmacy, Huashan Hospital, Fudan University, Shanghai 200040, China
Abstract: Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo biosynthesis of guanine and plays an important role in cell proliferation. In clinic, IMPDH inhibitors are mainly used in fields of anticancer, antiviral, anti-parasitic, and immunosuppressive chemotherapy. However, since there are usually great inter-and intra-individual variability between drug concentration and clinical effect of IMPDH inhibitors, the enzyme activity of IMPDH may be applied as a specific biomarker and combined with the pharmacokinetics (PK) monitoring to improve efficacy and safety of IMPDH inhibitors. This review aims to discuss the assay of IMPDH activity measurement and its clinical application in recent years and provide valuable insights and theoretical basis for the development of IMPDH inhibitors' pharmacodynamics monitoring.
Key words: inosine 5'-monophosphate dehydrogenase     IMPDH inhibitor     determination of IMPDH activity     biomarker    

次黄嘌呤单核苷酸脱氢酶 (inosine 5'-mono­phosphate dehydrogenase,IMPDH) 是一种烟酰胺腺嘌呤二核苷酸 (nicotinamide adenine dinucleotide,NAD+) 依赖酶,广泛存在于全身各分化细胞[1]。IMPDH是嘌呤代谢过程中的限速酶,可催化次黄嘌呤核苷酸 (inosinemonphosphate,IMP) 转化生成黄嘌呤核苷酸 (xanthosine monophosphate,XMP)[2],继而合成鸟嘌呤核苷酸,在细胞的生长、分化和凋亡中起着重要作用。同时IMPDH也是细胞免疫的一种重要酶类,可在炎症反应的应答时对黏附因子和细胞内信号进行调节[3]。此外,该酶还参与了细胞膜糖蛋白和黏附分子的合成,可影响淋巴细胞进入异体组织的募集作用与内皮渗透性[4]。IMPDH被抑制时可导致细胞内鸟嘌呤核苷三磷酸储存量的消耗,减缓炎症部位与异体组织内单核细胞和淋巴细胞的增殖与补充[3]

目前上市的IMPDH相关药物主要是IMPDH抑制剂,包括麦考酚酸 (mycophenolic acid,MPA) 制剂、咪唑立宾、利巴韦林和NAD+类似物等,应用于抗免疫排斥、抗肿瘤、抗病毒或抗寄生虫等领域[5]。该类药物的主要药理作用原理如图 1。IMPDH抑制剂的血药浓度与临床疗效之间存在一定关联性,以麦考酚酸为例,通常认为药时曲线下面积 (AUC) 为30~60 μg·h·mL-1[6]时,肾移植术后早期的免疫排斥发生率较低。但该关联的个体间变异常较大,因此测定IMPDH活性可作为一项监测其药物效应的特异性生物标志物[7]。了解患者体内IMPDH抑制水平,可有效弥补血药浓度监测的局限性,更全面地描述患者特异性的剂量-浓度-效应关系,以优化药物治疗方案[8]

Figure 1 Mechanism of IMPDH inhibitors. IMPDH: Inosine 5'-monophosphate dehydrogenase; MPA: Mycophenolic acid; IMP: Inosinemonphosphate; XMP: Xanthosine monophosphate; AMP: Adenosine 5'-monophosphate; GMP: Guanosine monophosphate; NAD+/NADH: Nicotinamide adenine dinucleotide

本文就目前常用的IMPDH酶活性测定方法,以及在临床上应用的最新进展作一综述,旨在为开展IMPDH抑制剂的药效学监测提供具体方法和依据。

1 IMPDH的基因分型

人类IMPDH有两种亚型 (Ⅰ型和Ⅱ型),分别 由基因IMPDH Ⅰ和IMPDH Ⅱ编码。两种基因分别位于7号染色体的短臂31区3带和3号染色体的 长臂21区2带[9, 10]。IMPDH Ⅰ型和Ⅱ型的氨基酸序列有85%同源性,分子量大小相似,但酶的亲和力 及催化速率常数并不一致[11]。McPhillips等[12]发现,当刺激淋巴细胞进行有丝分裂时,IMPDH Ⅰ型和Ⅱ型表达均上调,但不同细胞表达的IMPDH亚型不尽相同。IMPDH Ⅰ在肝、结肠和外周血单个核细胞 (peripheral blood mononuclear cell,PBMC) 中表达更多,而IMPDH Ⅱ在肝、肾和骨骼肌中分布更多[13]

在给予MPA治疗的肾移植患者中,有研究表 明IMPDH Ⅰ和IMPDH Ⅱ的基因多态性可影响患 者体内的酶活性,与患者的急性排斥反应的发生相关[14]。近年来已有研究发现,IMPDH Ⅰ的rs2278293、rs2278294和IMPDH Ⅱ的rs11706052等位点的突变可能增加MPA用药后的不良反应,如白细胞减少 症的发生[15],或通过对患者体内IMPDH酶活性的影响[16],从而改变MPA的治疗效果。但这些单核苷酸多态性 (single nucleotide polymorphism,SNP) 对于临床治疗效的影响,目前尚无统一结论[15, 17]

2 IMPDH活性测定方法

由于IMPDH活性难以直接测得,通常采用间接的方法,如测定IMPDH基因的表达量 (如mRNA含量[18, 19]),或在体外孵化后考察酶催化特定酶促反应的速度,如考察催化底物IMP氧化生成产物XMP或辅酶产物还原型烟酰胺腺嘌呤二核苷酸 (nicotina­mide adenine dinucleotide,NADH) 的速度[20]。测定过程一般分为3个步骤: 靶细胞的分离、细胞预处理 (如提取细胞DNA/RNA或进行体外酶促反应) 以及酶活性的测定,具体过程见图 2

Figure 2 Measurement of IMPDH activity
2.1 靶细胞的选择及分离

IMPDH虽广泛存在于体内各分化细胞,但作用于该酶的药物通常需经血红蛋白转运,且主要作用于淋巴细胞。故欲考察与药物疗效更相关的IMPDH活性,需选择合适的靶细胞[7, 21]。IMPDH 活性测定的靶细胞主要有全血或红细胞、CD4+ T细胞[22]以及PBMC[23]等。

2.1.1 全血或红细胞

因样本处理过程相对方便且操作简单,最初对IMPDH活性的测定大多选用全血。但由于全血中测定的IMPDH主要来源于红细胞,与体内实际药物效应存在差异,多不作为首选[24]

2.1.2 CD4+T细胞

CD4+ T细胞在急性排斥反应的免疫应答中发挥着重要作用。MPA可通过抑制其克隆性增长发挥药理学作用,故有研究选用该细胞作为分析样本[25]。通常CD4+ T细胞分离采用的是磁珠分选法,通过抗原与抗体的特异性结合对细胞进行有效筛选[22]。但因该类药物对体内免疫应答的影响并不仅作用于CD4+ T细胞,且其分离方法相对较繁锁,故目前并不常用。

2.1.3 PBMC

IMPDH抑制时产生药理作用的细 胞主要是淋巴细胞,但体外检测淋巴细胞首先需分离PBMC——即目前最常用于IMPDH测定的靶细胞。PBMC的分离通常采用Ficoll-hypaque密度梯度离心法,其基本操作流程为取肝素或乙二胺四乙酸 (ethylene diamine tetraacetic acid,EDTA) 抗凝全血,磷酸缓冲盐溶液 (phosphate buffer saline,PBS) 稀释后,加入淋巴细胞分离液离心,抽提淋巴细胞层、清洗离心后得到的沉淀物即为PBMC[7] (其中淋巴细胞约占90%~95%)。

在PBMC的分离过程中,血样采集后进行细胞分离的时间可对测定结果产生较大影响。时间越长,PBMC的提取率越低,其中混入的红细胞也越多,且对不同患者的最终测定结果的影响程度也不尽相同。一般以4~12 h为宜[7, 26, 27]。另外,目前的研究表明提取前保存温度对PBMC的提取率不产生影响。如Glander等[7]的研究发现: 新鲜血液在室温或4 ℃保存24 h对最终测定结果并无显著影响。分离过程的另一个重要因素则是PBS对提取细胞的清洗次数。清洗次数越多,细胞内的药物浓度越低,对IMPDH活性的影响程度也被削弱[7]

此外,Glander等[7]还分别考察了分离过程中不同影响因素,如分离时间、保存温度、内源性物质如蛋白和红细胞等对IMPDH活性测定结果的影响程 度,并改良了PBMC的分离方法。其研究结果建议,EDTA抗凝的新鲜全血须在采样后12 h内进行提取,分离后得到的PBMC最佳清洗次数为两次。

2.2 细胞预处理

根据IMPDH酶活性测定方法的不同,目前主 要有以下两种细胞预处理方法,提取细胞RNA和体外酶促反应。

2.2.1 提取细胞RNA

IMPDH的活性可用其mRNA的表达量来表示,此类方法需先提取细胞内RNA,再采用基因定量技术如实时定量PCR法[18]进行测定。目前,已有技术成熟应用广泛的商品化试剂盒,可用于实验室提取细胞RNA,如Trizol-LS(R)、RNA-STAT- 50(R) 和Ultraspec-3(R)[28]等,本文不再赘述。

2.2.2 体外酶促反应

IMPDH可在辅酶NAD+存在的条件下,将底物IMP转化生成 XMP。因此,测定细胞内IMPDH活性,可通过体外孵育的方式,模拟体内环境 (37 ℃,pH 7.4),在孵育体系中加入足量的底物和辅酶。若采用放射法,则需加入同位素标记的底物[8]。待酶促反应完全后,通过测定生成产物的含量来计算IMPDH酶活性[7] (图 1)。

IMPDH活性与孵育过程中的底物浓度、辅酶浓度以及细胞内药物的含量有关,且呈现时间依赖性。其中,底物与辅酶的浓度因不同研究中提取的血样量不同而各有差异。Daxecker等[29]分别测定了IMP和NAD+在0.034~0.546 mmol·L-1时IMPDH的活性。结果发现,若采血量为30 mL时,两种加入物质的最佳浓度均为0.273 mmol·L-1。而在Vethe等[30]的研究中,取4 mL全血进行淋巴细胞分离,结果发现: 当NAD和IMP的浓度分别为0.38 mmol·L-1和1.79 mmol·L-1时,酶促反应可达到最大速率。

同时,IMPDH活性还受体内药物浓度的影响。Daxecker等[29]的研究发现,当MPA浓度为0.5 μmol·L-1时,产物XMP的生成量将低于检测下限 (约0.72 μmol·L-1)。此外,该研究还探讨了IMPDH活性测定值与孵育时间的关系,发现在0~180 min时,120 min为最佳的孵育条件。而Vethe等[30]的研究结果也表明: 120 min可使整个酶促反应体系中的IMPDH达最大活性。

另外,酶促反应的终止多采用加入高氯酸[31, 32]或甲醇[33]的方法,充分涡旋后高速离心,并取适量碱溶液进行中和及盐析,使蛋白沉淀且pH接近中性后,进行酶活性的测定。

2.3 酶活性测定

目前用于测定IMPDH酶活性的方法主要有3 种: RT-PCR法测定IMPDH的mRNA表达量[34]、放射法检测酶促反应产物NADH[8],以及色谱法测定IMPDH催化反应产物XMP的生成量[7, 13, 21-24, 26, 27]

2.3.1 RT-PCR

RT-PCR 法是通过测定IMPDH基因的mRNA表达量来表示其最终的IMPDH活性。如Vannozzi等[34]以甘油醛-3-磷酸脱氢酶作为荧光标记物,应用实时定量PCR法检测了细胞RNA中IMPDH Ⅱ型的基因表达。该方法的最大优点在于可单独测定IMPDH某一亚型的活性,但因实验操作较为繁琐,且测定结果只能代表基因表达水平[35],无法完全代表真正的酶活性。因此,除一些机制性研究或药物基因组学研究[15]外,该法并不常用。

2.3.2 放射法

放射法是指孵育体系在外加[2,8-3H]次黄嘌呤的条件下,反应底物IMP结构中次黄嘌呤环C-2位置上的氢原子可被标记为3H,之后的酶促反应过程中,该位置的3H被羟基取代,辅酶NAD+作为电子受体,转变成为NAD3H (图 2)。因此,待酶促反应完全后,可借NAD3H的生成速度来计算IMPDH活性,如将NAD3H的生成量对反应时间进行作图,最终得到的直线斜率即代表酶活性[8],Millan等[20]的研究便是采用了此种方法。放射法的优点在于可特异性检测全血或者完整细胞中的酶反应产物,不受其他物质的干扰[36]。但由于放射性标记物的制备相对复杂,且该方法存在一定的辐射效应,故近年来应用逐渐减少。

2.3.3 色谱法

酶促反应完成后,可通过色谱法来测定产物XMP[7]的生成量,间接计算IMPDH活性 (图 1)。常用的方法是高效液相色谱法 (high performance liquid chromatography,HPLC) 及液相色谱-质谱联用技术(LC-MS/MS)。自Albrecht等[24]于2000年第一次采用HPLC方法检测IMPDH活性以来,目前HPLC法已成为应用最为广泛的方法。该法可对酶活性进行快速定量,且具较好的线性、精密度和准确度,在临床上有较好的可行性[37]

2.3.4 测定的影响因素

目前,最常用的IMPDH酶活性测定方法为色谱法。在色谱法测定过程中,为准确计算酶活性、减少实验误差,IMPDH催化反应产物的测定结果须根据提取的细胞数量进行标准化。常用的标准化方法主要有细胞计数法[13]和蛋白定量法[38],即分别通过对同一测定样本的细胞数或蛋白含量进行测定,以标化最终产物的量,计算酶活性值。

无论是自动还是人工的细胞计数法,常存在实验室间差异、观测者间的变异和重复性不佳的缺点。在提取细胞过程中,加水稀释、离心等步骤可改变渗透压致细胞破坏,使实际测定时的细胞少于计数时的细胞,增加误差。测定细胞提取物中的蛋白含量,则易受到细胞外蛋白和非PBMCs细胞的影响,如红细胞可增加最终的蛋白测定浓度[7]

另一种方法是测定IMP的细胞内源性产物AMP[7, 39]。由于AMP来源于IMPDH酶作用的同一底物,与最终测定溶液里的细胞数直接相关,故适于作为细胞数量的标准化物质。同时,AMP还可与XMP在相同条件下进行定量测定,提高了该方法的可重复性和稳定性。Glander等[7]的实验发现,用AMP进行标化后,计算得到的IMPDH酶活性相比于细胞计数和蛋白定量法,其变异更小 (< 15%)。且该方法不受内源性蛋白和红细胞等干扰物质的影响,并在多中心实验中得到了较为可信的结果。上述IMPDH酶活性的计算见公式1。

$\begin{align} & \text{IMPDHactivity(}\mu \text{mol}\cdot {{\text{s}}^{\text{-1}}}\cdot \text{mo}{{\text{l}}^{\text{-1}}}\text{AMP)=} \\ & \frac{\text{Produced XMP (}\mu \text{mol}\cdot {{\text{L}}^{\text{-1}}}\text{)}\times \text{1}{{\text{0}}^{6}}}{\text{Incubation time(s)}\times \text{measuredAMP(}\mu \text{mol}\cdot {{\text{L}}^{\text{-1}}}\text{)}} \\ \end{align}$ (1)

采用色谱法测定 IMPDH活性,应尽可能采用与样本基质相近的空白基质配制标准曲线和质控品。IMPDH活性测定最常用的靶细胞为PBMCs,而PBMC存在内源性的IMPDH和AMP干扰测定,且难以大量获取,故无法作为空白基质。现有研究中常选用的空白基质主要有水[40]、PBS缓冲液[30]和小牛血清[31]等,旨在提高实验操作性 (如水),或模拟体内环境 (如PBS缓冲液和小牛血清)。为减少IMPDH活性测定时内源性物质的干扰,常同时测定健康志愿者的IMPDH活性作为对照[7]。但尚无研究考察不同空白基质对测定结果的影响。

3 IMPDH活性测定的应用 3.1 移植与免疫

临床应用的IMPDH抑制剂中,MPA制剂是目 前使用最为广泛且研究最为系统的一类药物,主要用于移植术后抗排斥反应[41]和自身免疫性疾病的治疗[42]。MPA治疗范围窄,药动学个体间变异大且影响因素多,如与合并用药的相互作用、MPA代谢酶与转运体等的基因多态性等,现常被建议进行血药浓度监测[31]。同时,亦有研究发现[43]: 健康受试者、透析患者以及肾移植患者体内的IMPDH活性存在较大的个体间变异 (表 1),有必要进行监测。目前已有移植中心将IMPDH的活性监测纳入移植患者术后维持期的常规监测[44],作为MPA个体化给药方案设计的重要手段[45]

Table 1 IMPDH activity in different populations assayed by chromatography method: data were presented as median (range) or mean ± SD. a: Age between 2 and 11.9 years; b: Age between 12 and 18.9 years; c: Liver transplant; d: Inflammatory bowel disease; e: Hema­topoietic stem cell transplant; f: Acute lymphoblastic leukemia

多个研究表明: 移植患者IMPDH的酶活性及其抑制程度与MPA的治疗预后相关,如移植术后炎症反应、病理性急性排斥以及白细胞减少症的发生率 等[18, 46, 47]。Fukuda等[41]的研究显示: 移植术前IMPDH活性可作为临床疗效的预测因子,预测移植术后的药物毒性和移植物排斥反应。Chiarelli等[27]的研究发现: MPA治疗预后的最佳监测指标为服用其酯类前体药物 (MMF) 后1.5 h的IMPDH活性或者0~1.5 h的IMPDH活性抑制率。在Raggi等[31]的研究中,移植术后服用MPA预防急性排斥的IMPDH抑制靶水平均值约为50%。而Maiguma等[37]的研究则表明: 25%~40% 的IMPDH抑制程度是一个较为合适的范围。但因IMPDH活性的测定方法各有不同,既往研究间的数据难以直接比较 (表 1),IMPDH活性在不同人群的参考值范围仍需作进一步的考察和验证。

此外,IMPDH活性监测对免疫疾病的治疗也有积极意义。Schaier等[42]研究了MPA治疗抗中性粒 细胞胞浆抗体 (anti-neutrophil cytoplasmic antibody,ANCA) 相关血管炎的PD指标 (IMPDH活性) 与临床疗效之间的关系,发现炎症复发的患者相比于疾病状态稳定的患者而言,其基线与复发前的IMPDH酶活性水平均显著升高。由此可知预后不佳可能与IMPDH活性抑制不足有关,同时也进一步证明IMPDH活性监测可改善MPA治疗的有效性。

3.2 抗病毒与抗肿瘤

IMPDH的活性监测对于抗病毒治疗的临床应用也有一定的指导价值。Khan等[48]的研究显示: 细胞内IMPDH活性可作为基孔肯雅热病毒复制和诱导的生物标志物,表征作用于IMPDH的抗病毒药物的治疗效果,如抑制病毒复制与增殖,减少病毒引发的细胞凋亡等。

另外,在抗肿瘤治疗领域,IMPDH活性的测定还可预测疾病预后,指导临床用药方案的调整。Vethe等[49]的研究发现: IMPDH抑制药物——巯鸟嘌呤核苷酸可通过影响MOLT-4人白血病细胞中的IMPDH活性,进而调控白血病细胞的增长及疾病进展。且IMPDH活性与药物浓度关联紧密,可作为该药的药效学的生物标志物,预测疾病预后。Fellenberg等[50]研究表明: 当骨肉瘤细胞中的IMPDH Ⅱ基因过表达,其对化疗药物的耐药性将提高。故推荐IMPDH抑制剂联合化疗药物,作为治疗IMPDH Ⅱ高表达型骨肉瘤患者的优选治疗策略。以上研究均为IMPDH酶活性监测的开展提供了强有力的依据。

4 总结与展望

IMPDH酶活性测定可作为IMPDH抑制剂的药效学标志物,与血药浓度监测结合,为临床个体化给药提供更有效、安全的依据[27, 41]。如在Sommerer 等[51]的研究中发现,服用MPA肠溶制剂时,肾移植患者MPA的暴露与移植术后早期的急性排斥以及感染有关,而较低的IMPDH活性则与胃肠道不良反应的发生相关,提示结合MPA的PK与PD监测调整临床用药策略,可改善治疗效果并减少药物不良反应的发生。

目前常用的3种IMPDH活性测定方法,因其侧重点与结果的差异,实际应用各有所长。RT-PCR法因侧重于IMPDH的基因亚型,主要用于药物基因组学及作用机制研究[38]; 放射法主要检测活化的淋巴细胞,不易受到其他物质干扰,适于进行特定细胞内的药理学研究[20]; 而色谱法因其良好的准确性与精密度,以及检测过程相对前两者更方便快捷,因此更适用于临床应用[7]。但相对于血药浓度测定,IMPDH活性的测定耗时较长且操作步骤较多。随着自动化检测设备的开发,未来可有更简便、易操作的测定方法,以利于IMPDH活性的监测在临床各治疗领域的推广应用。

IMPDH活性监测对于IMPDH抑制剂的临床应用已发挥出其积极意义,可提高药物治疗效果、预测疾病预后或指导用药调整等。但目前IMPDH活性抑制的目标程度与测定时机尚无定论,未来应开展前瞻性的多中心大样本的临床研究,确定剂量-浓度-IMPDH活性和临床结局的关系,更全面深入地探讨IMPDH活性监测的临床价值,明确合适的监测方法以及与临床疗效相关的评价标准,促进该类药物的合理应用。

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