基于PKPD理论机制研究肠道菌群及维生素K对华法林抗凝作用的定量影响

Vitamin K is essential for blood coagulation. Warfarin, as vitamin K antagonists, exerts its anticoagulation through interfering the cycle of vitamin K by inhibiting the activity of vitamin K epoxide reductase (VKORC1). About 40% individual variability of warfarin also can’t be explained, even though patients administer warfarin according to patients’ demographic characteristics, drug combination, and genic factors in our previous study. Evidence indicates that variability in vitamin K influences the instability of oral anticoagulant therapies, and one vital source of vitamin K is biosynthesized by gut bacteria. So our study hypothesis: gut microbiota and/or vitamin K play an important role in anticoagulation of warfarin, and they are important factors for individual variability of warfarin, and the unexplained individual variability of warfarin would be explained partly in previous study. The present study will investigate individual medication of warfarin by PKPD method. The patients’ data will be collected in clinic. The collected data include demographic characteristics, genic factors, INR, gut microbiota, and the blood sample that the sampling time keep same with INR. The concentration of S-, R- warfarin and vitamin K will be detected by LC-MS/MS and HPLC, respectively. Then the data analysis will use NONMEM, and the warfarin individual model will be established based on mechanistic theory of PKPD. The effect of gut microbiota and vitamin K will be quantified for warfarin anticoagulation. The possible effect mechanism and quantitative influence of gut microbiota and vitamin K on warfarin anticoagulation will be explained from the aspect of model. Finally, the present study will provide the model tool for warfarin individual dosage in clinic.

维生素K(VK)是凝血过程必需维生素。华法林作为VK拮抗剂，通过抑制VK环氧还原酶(VKORC1)阻断VK体内循环而发挥抗凝作用。我们前期研究发现：即使考虑患者人口学特征、合并用药和遗传因素，华法林仍约有40%个体差异无法得到解释。体内VK主要来源之一是由肠道细菌合成，据此我们提出假说：肠道菌群和/或体内VK波动对华法林抗凝有重要影响，是华法林个体差异来源的重要因素，可在一定程度上解释当前研究无法解释的变异。本项目拟通过收集瓣膜置换术后患者人口学数据、遗传因素、INR、肠道菌群分布和与INR监测同步血样，分别采用LC-MS/MS和HPLC检测S-和R-华法林和VK血浆浓度，再用PKPD建模法分析数据，建立基于PKPD理论机制的华法林个体化给药模型，定量化肠道菌群和VK对华法林抗凝作用的影响，并从模型角度阐明肠道菌群和VK在华法林抗凝过程中的可能机制，最终为临床个体化应用华法林提供模型工具。

华法林是临床广泛应用的预防和治疗血栓栓塞性疾病的抗凝药物，在体内通过阻断维生素K的体内循环转化而产生抗凝作用。维生素K(VK)是凝血过程必需维生素。体内维生素K主要源于膳食绿叶蔬菜(维生素K1)和发酵食品、动物肝脏食品或肠道细菌生物合成(维生素K2)。据此推断，肠道菌群和/或体内VK波动对华法林抗凝有重要影响，是华法林个体差异来源的重要因素，可在一定程度上解释当前研究无法解释的变异。本项目通过收集心外科术后服用华法林患者的数据，包括了患者的基本人口学特征、华法林用药史、合并用药史、入院诊断、既往史和手术情况。同时收集了患者INR测定时的血样，用于测定S-和R-华法林、维生素K1 和MK4的血清浓度测定；以及患者入院时、抗菌药用药7天以上、抗菌药停药7天以上的粪便样本用于肠道菌群检测；患者临床常规检测CYP2C9和VKORC1基因后的DNA样本，用于华法林其他相关基因SNP的检测。本研究建立了LC-MS/MS测定血清S-和R-华法林、维生素K1 和MK4浓度方法学，基于之前建立的PKPD初步分析了维生素K和肠道菌群对华法林个体差异的影响。结果表明：之前建立PKPD模型对INR和华法林浓度预测偏倚的结果为: INR的理想预测偏差为76.4%，S -华法林为62.7%，R-华法林为70.4%。随着维生素K浓度增加，C50S-warfarin、凝血酶原复合物（PCA）半衰期增加，华法林药效(PD)减小；之前建立PKPD模型的对INR预测的高预测、理想预测和低预测之间维生素K浓度存在显著差异(P<0.05), 普氏菌属 (Prevotella)在INR预测偏差的三组之间也存在显著差异，可能与华法林抗凝有关。本研究初步证实体内维生素K含量和肠道菌群普氏菌属丰度是华法林产生个体差异来源的重要因素。