基于药效学和药动学研究三种丹参酚酸类成分对华法林抗凝作用的影响及机制

Effects of three phenolic acid components in Radix Salviae Miltiorrhizae on warfarin anticoagulation:from perspectives of pharmacodynamics and pharmacokinetics

目的研究丹酚酸B、丹酚酸A和迷迭香酸对华法林抗凝作用的影响及机制,为临床合理用药提供基础研究依据和参考。方法药效学实验:将大鼠随机分为空白对照组、丹酚酸B组、丹酚酸A组、迷迭香酸组、华法林对照组、各丹酚酸成分分别与华法林联合给药组,含丹酚酸成分组分别连续腹腔注射各丹酚酸成分14 d,在第8天灌胃给予华法林后,在0、4、8、12、24、36、48、72、96、120、144 h采血,用血凝分析仪测定相关凝血时间参数。药动学实验:将大鼠随机分为华法林对照组、各丹酚酸成分分别与华法林联合给药组,联合给药组连续14 d分别腹腔注射各丹酚酸成分,第8天灌胃给予华法林后在0.5、1、1.5、2、4、8、12、24、36、48、72、96、120、144 h采血;血浆蛋白结合率实验是将大鼠分为华法林对照组、不同剂量(低、中、高)的各丹酚酸成分分别与华法林联合给药组,联合给药组连续8 d分别腹腔注射各丹酚酸成分,第8天灌胃给予华法林后1.5 h采血。采用UPLC-MS/MS测定华法林对映体血药浓度,用DAS 2.0软件测定药动学参数。结果药效学研究表明,单独应用丹酚酸B和丹酚酸A并不影响凝血时间,但联合华法林后可延长凝血酶原时间(PT)和国际标准化比值(INR)(P<0.05),活化部分凝血活酶时间(APTT)无显著变化(P>0.05);迷迭香酸单独或联合华法林,均可延长PT、APTT和INR值(P<0.05)。药动学研究表明,丹酚酸B联合华法林后,R-华法林的药动学参数C_(max)、AUC_(0-t)、AUC_(0-∞)分别升高34.17%、53.44%和60.14%(P<0.01),t_(1/2)延长28.37%(P<0.01),CL/F降低35.48%(P<0.01),S-华法林的C_(max)、AUC_(0-t)、AUC_(0-∞)分别升高11.11%、22.13%和24.05%(P<0.05),t_(1/2)延长14.26%(P<0.05),CL/F降低19.44%(P<0.05);丹酚酸A联合华法林后,R-华法林的C_(max)、AUC_(0-t)、AUC_(0-∞)分别升高19.59%、49.98%和53.31%(P<0.01),t_(1/2)延长17.29%(P<0.05),CL/F降低34.19%(P<0.01),而S-华法林的药动学参数无显著变化;迷迭香酸联合华法林后,R-华法林的C_(max)、AUC_(0-t)、AUC_(0-∞)分别升高32.38%、67.52%和81.04%(P<0.01),t_(1/2)延长37.99%(P<0.01),CL/F降低42.58%(P<0.01),S-华法林的C_(max)、AUC_(0-t)、AUC_(0-∞)分别升高29.68%、58.08%和67.26%(P<0.01),t_(1/2)延长23.71%(P<0.01),CL/F降低39.81%(P<0.01)。此外,三种酚酸类成分均可降低华法林血浆蛋白结合率(P<0.05),使其游离浓度增加。结论丹酚酸B和迷迭香酸可同时抑制S-华法林和R-华法林代谢,丹酚酸A可抑制R-华法林代谢。三种丹酚酸成分可通过抑制华法林代谢和竞争血浆蛋白结合,增强华法林抗凝作用。华法林与含有这些成分的中药联合应用时,可能增加出血风险。

Objective To investigate the effects and mechanism of salvianolic acid B,salvianolic acid A,and rosmarinic acid on anticoagulation of warfarin in rats from perspectives of pharmacodynamics and pharmacokinetics,in order to provide the basis and reference for rational drug use.Methods Pharmacodynamic study:Wistar rats were randomly divided into different groups:blank control group,salvianolic acid B group,salvianolic acid A group,rosmarinic acid group,warfarin control group,and each phinolic acid component with warfarin group,respectively.Rats were intraperitoneal administrated corresponding phinolic acid component for 14 consecutive days,with or without warfarin treatment at day 8.Blood samples were collected at 0,4,8,12,24,36,48,72,96,120,144 h after warfarin treatment.Blood coagulation time parameters were determined by coagulation analyzer.Pharmocokinetic study:Rats were treated with phinolic acid component for 14 days with or without warfarin.Blood was collected at 0.5,1,1.5,2,4,8,12,24,36,48,72,96,120,144 h after warfarin administration.For plasma protein binding test,rats were treated with low,medium,high concentration phinolic acid component with warfarin,and blood was collected 1.5 h after warfarin administration.UPLC-MS/MS was used to determine the plasma concentrations of R-warfarin and S-warfarin.Pharmacokinetic parameters were calculated by DAS 2.0 software.Results Pharmacodynamic study showed that compared with the blank control group,single administration of salvianolic acid B or salvianolic acid A didn′t affect the coagulation parameters,but co-administrated with warfarin prolonged values of prothrombin time(PT)and international normalized ratio(INR)(P<0.05),while the value of activated partial thromboplastin time(APTT)was unaffected(P>0.05).Rosmarinic acid alone or in combination with warfarin could increase the PT,APTT,and INR(P<0.05).Pharmacokinetic study showed that salvianolic acid B+warfarin increased the pharmacokinetic parameters C_(max),AUC_(0-t),AUC_(0-∞)of R-warfarin by 34.17%,53.44%,60.14%(P<0.01),respectively,t_(1/2)extended by 28.37%(P<0.01),CL/F decreased by 35.48%(P<0.01);C_(max),AUC_(0-t),AUC_(0-∞)of S-warfarin increased by 11.11%,22.13%,24.05%(P<0.05),respectively,t_(1/2)extended by 14.26%(P<0.05),CL/F decreased by 19.44%(P<0.05).Salvianolic acid A+warfarin increased C_(max),AUC_(0-t),AUC_(0-∞)of R-warfarin by 19.59%,49.98%,53.31%(P<0.01),respectively,t_(1/2)extended by 17.29%(P<0.05),CL/F decreased by 34.19%(P<0.01),while the pharmacokinetic parameters of S-warfarin were not statistically significant.Rosmorinic acid+warfarin increased C_(max),AUC_(0-t),AUC_(0-∞)of R-warfarin by 32.38%,67.52%,81.04%(P<0.01),respectively,t_(1/2)extended by 37.99%(P<0.01),CL/F decreased by 42.58%(P<0.01);C_(max),AUC_(0-t),AUC_(0-∞)of S-warfarin increased by 29.68%,58.08%,67.26%(P<0.01),respectively,t_(1/2)extended by 23.71%(P<0.01),CL/F decreased by 39.81%(P<0.01).Moreover,the three phenolic acids competed with warfarin for binding plasma proteins,and increased its free concentration(P<0.05).Conclusion Both salvianolic acid B and rosmarinic acid can inhibit the metabolism of R-and S-warfarin,and salvianolic acid A can inhibit the metabolism of R-warfarin.The three phenolic acid components can enhance the anticoagulant effects of warfarin in vivo by inhibiting its enantiomers metabolism and competing for binding plasma proteins.When warfarin is used simultaneously with the Chinese medicine containing these ingredients,more attention should be paid to the increased bleeding risk.