多沙唑嗪对映体在动物心血管系统的手性药理学研究
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摘要
多沙唑嗪（doxazosin， DOX）是一种喹唑啉类的长效选择性α1受体阻滞剂，是治疗良性前列腺增生（BPH）所致下尿路症状的一线用药[1]，同时可扩张容量及阻力血管，具有良好的降压作用。但是，在抗高血压和降脂治疗预防心肌梗死的大规模临床试验（Antihypertensive and lipid-lowering treatment to prevent heart attacktrial, ALLHAT）中，多沙唑嗪组出现了较多的心血管事件而提前退出了临床试验[2]。为了降低多沙唑嗪的不良反应，人们着手对其手性对映体进行研究。阿夫唑嗪（alfuzosin，ALF）与DOX同属喹唑啉类高选择性α1受体阻断药，DOX及ALF的分子结构中均存在一个手性碳原子，因此，DOX和ALF分别存在(-)DOX、(+)DOX、(-)ALF和(+)ALF四个对映体，临床应用二者的消旋体[(±)DOX和(±)ALF]。目前关于(±)DOX和(±)ALF及其对映体对离体心脏的作用尚不甚清楚。一般认为，心功能受损时导致激素与促炎细胞因子分泌水平可反应心脏收缩功能。脑利钠肽（BNP）、核因子-κB（NF-κB）及白细胞介素-6（IL-6）均是心力衰竭发生的生物学标志物。本研究采用小鼠离体心房组织，观察(±)DOX、(±)ALF及其对映体对心肌收缩力和心率的影响；并观察了长期给予多沙唑嗪及其对映体对大鼠血浆氨基末端原脑利钠肽（NT-proBNP）、NF-κB及IL-6水平的不同影响。
多种信号途径可能参与药物诱发的心肌收缩力改变，为进一步探讨DOX及其光学异构体对心肌收缩力不同影响的机制，本研究使用大鼠离体心房肌标本，研究了酚苄明、阿托品及普萘洛尔、吲哚美辛、维拉帕米、亚甲蓝、H-89等不同阻断剂对多沙唑嗪对映体引起大鼠张力反应的影响。
血浆蛋白结合在药物治疗中起着重要的作用。药物与血浆蛋白结合的变化显著地影响它的药代动力学和药效学。因为对映体之间的蛋白结合率往往造成它们药代动力学的差异。对映体蛋白结合的立体选择性的研究对于理解其药理学、临床疗效及安全性是必不可少的。因此，有必要对生理条件下多沙唑嗪对映体的立体选择性进行研究。本研究采用手性固定相柱联合平衡透析法同时测定大鼠、狗和人血浆中加入消旋多沙唑嗪后其每一对映体的蛋白结合率。结果表明，多沙唑嗪对映体对大鼠，狗和人类血浆蛋白结合具有立体选择性，且种属间也存在差异。
第一部分多沙唑嗪对映体对心率、心肌收缩力及心衰生物学标志物的影响
本研究采用小鼠离体心房组织，观察(±)DOX及其两种对映体、(±)ALF及其两种对映体对小鼠离体心房心肌收缩力和心率的影响；并观察多沙唑嗪及其对映体长期给药对大鼠血浆氨基末端原脑利钠肽（NT-proBNP）、IL-6及NF-κB水平的不同影响，以期为喹唑啉类α1受体阻断药的临床安全用药和新药设计提供实验数据。
1(±)DOX和(±)ALF及二者的对映体诱发小鼠离体右心房停搏反应
(+)DOX组的16例标本中，加入30μmol·L~(-1)药物后，5例发生停搏反应；停搏率为31.3%。(±)DOX组和(-)DOX组分别在10μmol·L~(-1)和30μmol·L~(-1)浓度时，各有一例发生停搏反应。(-)ALF、(+)ALF、(±)ALF组和溶剂对照组，未发生停搏反应。
2(±)DOX和(±)ALF及二者的对映体对小鼠离体右心房心率的影响
各组离体右心房心率的药前值相同（P>0.05），溶剂对小鼠离体右心房心率无显著影响（P>0.05）。(+)DOX和(±)DOX各浓度均显著减慢小鼠右心房心率（P<0.01），并具有浓度依赖关系；30μmol·L~(-1)的(+)DOX和(±)DOX组小鼠心率减慢率分别为（83.47±12.23）%和（74.97±15.66）%，3μmol·L~(-1)(+)DOX减慢心率的作用显著强于同浓度(±)DOX（P<0.01）。(-)DOX（3μmol·L~(-1)）对离体右心房心率无显著影响（P>0.05），且(-)DOX在10和30μmol·L~(-1)浓度时减慢心率的作用显著弱于同浓度(+)DOX（P<0.01）。(-)ALF和(+)ALF（10和30μmol·L~(-1)）显著减慢小鼠心率，30μmol·L~(-1)时小鼠心率分别减慢（13.05±7.27）%和（14.27±9.75）%；(±)ALF仅在30μmol·L~(-1)浓度显著减慢小鼠心率，三者抑制心率的作用强度相同。
3(±)DOX和(±)ALF及二者的对映体对小鼠离体左心房收缩力的影响
各组左心房心肌收缩力的药前值相同（P>0.05），溶剂对小鼠离体左心房心肌收缩力无显著影响（P>0.05）。(±)DOX和(-)DOX（3、10和30μmol·L~(-1)）显著增强小鼠离体左心房心肌收缩力（P<0.05），并具有浓度依赖关系；(-)DOX（10和30μmol·L~(-1)）的正性肌力作用显著强于同浓度(±)DOX的作用（P<0.05）。相反，(+)DOX具有轻度但显著的负性肌力作用（P<0.05）。(±)ALF及其对映体（3、10和30μmol·L~(-1)）对小鼠离体左心房心肌收缩力无显著影响（P>0.05）。
4(-)DOX、(+)DOX及(±)DOX长期给药对大鼠血浆NT-proBNP浓度的影响
溶剂对照组、(-)DOX组、(+)DOX组及(±)DOX组血浆NT-proBNP浓度分别为475.55±150.21ng·L~(-1)、635.00±343.52ng·L~(-1)、375.55±127.09ng·L~(-1)及424.12±134.19ng·L~(-1)。(+)DOX组及(±)DOX组血浆NT-proBNP浓度低于对照组，但组间比较差异无统计学意义（P>0.05）。(-)DOX组血浆NT-proBNP值高于对照组，但组间比较差异亦无统计学意义（P>0.05）。(-)DOX组血浆NT-proBNP浓度高于(+)DOX组（P<0.05）。
5(-)DOX、(+)DOX及(±)DOX长期给药对大鼠血浆NF-κB浓度的影响
溶剂对照组、(-)DOX组、(+)DOX组及(±)DOX组血浆NF-κB浓度分别为888.46±343.36ng·L~(-1)、705.77±193.34ng·L~(-1)、1140.00±365.26ng·L~(-1)及910.38±276.39ng·L~(-1)。(+)DOX组及(±)DOX组血浆NF-κB浓度高于溶剂对照组，但组间比较差异无统计学意义（P>0.05）。(-)DOX组血浆NF-κB浓度高于溶剂对照组，但组间比较差异无统计学意义（P>0.05）。(-)DOX组血浆NF-κB值低于(+)DOX组（P<0.05）。
6(-)DOX、(+)DOX及(±)DOX长期给药对大鼠血浆IL-6浓度的影响
溶剂对照组、(-)DOX组、(+)DOX组及(±)DOX组血浆IL-6浓度分别为48.81±23.39ng·L~(-1)、75.13±21.58ng·L~(-1)、59.44±23.17ng·L~(-1)及70.19±37.44ng·L~(-1)。单因素方差分析结果显示：与溶剂对照组比较，各药物组大鼠血浆IL-6水平无统计学意义（P>0.05）。
研究结果表明，DOX对小鼠离体心房的心率和心肌收缩力具有明显的影响，高浓度尚可诱发心脏停博反应；DOX的手性结构对其上述活性具有明显的影响。相反，ALF仅轻度抑制小鼠心率，ALF的手性结构对其心脏效应无明显影响。长期灌胃给予DOX及其对映体未显著影响大鼠心衰生物学标志物NT-proBNP及IL-6的血浆浓度；但是，(+)DOX组大鼠的血浆NF-κB水平显著高于(-)DOX组，提示(+)DOX对机体免疫及炎症反应有一定的影响。
第二部分多沙唑嗪对映体诱发大鼠心房肌收缩力变化的作用机制
本研究采用大鼠离体心房组织研究了酚苄明、阿托品及普萘洛尔、吲哚美辛、维拉帕米、亚甲蓝、H-89等不同阻断剂对多沙唑嗪对映体引起大鼠张力反应的影响。1(-)DOX、(+)DOX对离体大鼠左心房心肌张力的影响
溶剂对大鼠心肌张力无显著影响；(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠心肌收缩力的百分比分别为（5.57±20.95）、（6.96±39.77）%、（67.46±30.09）%。(+)DOX显著抑制大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(+)DOX抑制大鼠心肌收缩力的百分比分别为（~(-1)5.61±14.15）%、（-36.99±23.58）%、（-57.48±56.01）%。
2阿托品及普萘洛尔对DOX对映体改变大鼠心房收缩力的影响
以阿托品及普萘洛尔预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力），3、10、30μmol L~(-1)的(-)DOX增强大鼠心房收缩力的百分比分别为（13.02±16.66）%、（53.65±41.02）%、（70.87±52.66）%。(+)DOX显著抑制大鼠左心房心肌收缩力，(+)DOX抑制大鼠心房收缩力的百分比分别为（-8.02±21.43）%、（-21.60±21.50）%、（-63.75±24.14）%。
3酚苄明对DOX对映体改变大鼠心房收缩力的影响
以酚苄明预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠心房收缩力的百分比分别为（13.71±14.39）%、（35.48±11.90）%、（71.62±37.20）%。(+)DOX显著抑制大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(+)DOX抑制大鼠心房收缩力的百分比分别为（~(-1)4.08±23.48）%、（-33.08±17.85）%、（-60.73±17.21）%。以乙醇预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)(-)DOX对增强大鼠心房收缩力的百分比分别为（19.05±7.14）%、（49.27±28.71）%、（71.21±35.57）%。(+)DOX显著抑制大鼠左心房心肌收缩力，(+)DOX抑制大鼠心房收缩力的百分比分别为（~(-1)0.40±14.76）%、（-23.73±26.18）%、（-65.63±6.52）%。
4吲哚美辛对DOX对映体改变大鼠心房收缩力的影响
以吲哚美辛预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠张力的百分比分别为（19.87±13.06）%、（55.73±18.00）%、（60.25±35.25）%。(+)DOX显著抑制大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(+)DOX抑制大鼠心房收缩力的百分比分别为（-7.23±13.39）%、（-31.33±18.74）%、（-70.42±8.90）%。以乙醇预处理标本后，(-)DOX显著增强大鼠左心房心肌收缩力，(-)DOX对大鼠张力的增强率为（21.01±9.75）%、（43.88±28.26）%、（59.45±24.87）%。(+)DOX显著抑制大鼠左心房心肌收缩力，(+)DOX抑制大鼠心房收缩力的百分比分别为（~(-1)5.21±13.14）%、（-29.28±23.55）%、（-66.55±6.13）%。
5维拉帕米对DOX对映体改变大鼠心房收缩力的影响
以维拉帕米预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠张力的百分比分别为（24.81±20.49）%、（75.98±23.00）%、（131.15±17.56）%。(+)DOX显著抑制大鼠左心房心肌收缩力，(+)DOX抑制大鼠心房收缩力的百分比分别为（-9.60±17.19）%、（-33.47±9.43）%、（-72.23±14.65）%。
6亚甲蓝对DOX对映体改变大鼠心房收缩力的影响
以亚甲蓝预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠张力的百分比分别为（36.26±9.82）%、（65.99±26.27）%、（16.02±35.33）%。3、10、30μmol L~(-1)的(+)DOX抑制大鼠心房收缩力的百分比分别为（-5.57±17.32）%、（~(-1)0.54±25.09）%、（-58.27±15.47）%。
7H-89对DOX对映体改变大鼠心房收缩力的影响
以H-89预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠心肌张力的百分比分别为（28.96±9.49）%、（81.93±20.75）%、（101.53±51.95）%。(+)DOX显著抑制大鼠左心房心肌收缩力，(+)DOX抑制大鼠心房收缩力的百分比分别为（~(-1)7.71±16.37）%、（-30.76±18.38）%、（-67.67±10.74）%。
研究结果表明，(-)DOX增强大鼠心房肌收缩力的作用，可能与心肌α受体、M胆碱受体、β受体以及环氧合酶无关；L型Ca~(2+)通道、细胞内cGMP以及PKA可能在某种程度上参与了(-)DOX在大鼠左心房的正性肌力作用。但是，心肌α受体、M胆碱受体、β受体、Ca~(2+)通道、环氧合酶、cGMP及PKA，可能未参与(+)DOX对大鼠左心房的负性肌力作用。
第三部分多沙唑嗪对映体在大鼠、犬和人血浆中的蛋白结合率研究
血浆蛋白结合在药物治疗中起着重要的作用。药物与血浆蛋白结合的变化显著影响其药代动力学和药效学。对映体蛋白结合率的不同往往是它们药代动力学差异的原因之一。对映体蛋白结合的立体选择性研究是明确其药理学效应、临床疗效及安全性必不可少的内容。因此，有必要对多沙唑嗪对映体蛋白结合的立体选择性进行研究。本研究采用平衡透析法利用卵粘蛋白手性色谱柱同时测定大鼠、犬和人血浆中多沙唑嗪对映体的浓度，并计算其蛋白结合率。
1高效液相色谱法的方法学验证
采用高效液相色谱法手性分离血浆及PBS中的(±)DOX，专属性良好，内标（哌唑嗪）、(-)DOX和(+)DOX达到基线分离，最低检测限为0.2ng·mL~(-1)（S/N≥3）。多沙唑嗪对映体在血浆、PBS中的浓度范围分别是50～1600ng·mL~(-1)、12.5～200ng·mL~(-1)，在上述浓度范围内，多沙唑嗪对映体的浓度与峰面积呈良好的线性关系（r>0.9990）。多沙唑嗪对映体在血浆和PBS的日内、日间精密度分别小于3.2%、6.3%，回收率为90.5%～102.4%，实验过程中稳定性良好。
2平衡透析条件的优化
在PBS中加入800ng·mL~(-1)的消旋多沙唑嗪，与混合人血浆进行平衡透析，分别在0、4、8、15及20h取样分析。试验表明，37°C时，(-)DOX和(+)DOX迅速通过半透膜，在15h内达到稳态水平。犬与大鼠的血浆平衡时间与人血浆相近。
3不同对映体间蛋白结合的差异
消旋多沙唑嗪的浓度在200～800ng·mL~(-1)时，(-)DOX在三个不同种属血浆中的蛋白结合率为89.4%～94.3%，(+)DOX为90.9%～95.4%。结果显示，无论(-)DOX还是(+)DOX的血浆蛋白结合率均较高，与之前(±)DOX的研究结果一致。在三种血浆中，加入不同浓度的消旋多沙唑嗪，(-)DOX的结合浓度（Cb）均大于(+)DOX的Cb（P<0.05），并且这种差异随多沙唑嗪浓度的增加而变得更加明显。这种立体选择性在犬血浆中比在人或鼠血浆中更为明显。犬血浆在含200、400、800ng·mL~(-1)(±)DOX的PBS中，(-)DOX与(+)DOX的Cb均值分别为200.0vs243.0、398.6vs477.3、845.1vs1006.8。因此，在人、犬、鼠的血浆中多沙唑嗪对映体的蛋白结合力均体现了立体选择性，(+)DOX的蛋白结合力均高于(-)DOX。
两对映体蛋白结合能力的不同可能会导致其药物代谢动力学的差异，药物的清除与其在血浆中的游离浓度有直接的关系。(+)DOX的蛋白结合力高于(-)DOX，因此应用消旋多沙唑嗪后人血浆中(-)DOX的非结合态浓度高于(+)DOX，可能是文献报道的人服用(±)DOX后血浆中(-)DOX浓度低于(+)DOX浓度的原因之一。事实上，我们将(±)DOX分别给予犬和鼠后，(-)DOX的浓度也低于(+)DOX的浓度。因此，(-)DOX和(+)DOX间血浆蛋白结合能力的不同可能是人、犬、鼠三个种属中二者药代动力学不同的原因之一。
4多沙唑嗪蛋白结合率的种属差异
加入200ng·mL~(-1)(±)DOX时，三个种属蛋白结合率差别无统计学意义。而在加入(±)DOX的浓度为400和800ng·mL~(-1)时，犬血浆蛋白结合率小于人的血浆蛋白结合率（P<0.05和P<0.01），此结果与以往报道的人血浆蛋白结合率高于犬一致。由于人、犬、鼠三种混和血浆的血浆蛋白浓度不同（分别为61.81、51.35和57.75mg·mL~(-1)），所以血浆蛋白结合率的值应该用血浆蛋白的浓度进行校正。校正后，多沙唑嗪对映体的蛋白结合率存在种属差异，其顺序为犬>人>鼠（P<0.01）。
以上研究结果表明，在人、犬、鼠的血浆中(+)DOX的蛋白结合力均高于(-)DOX；多沙唑嗪对映体的血浆蛋白结合力在三种血浆中均存在立体选择性；多沙唑嗪对映体的蛋白结合率具有种属差异，其顺序为犬>人>鼠。
结论
1DOX对小鼠离体心房的心率和心肌收缩力具有明显的影响，高浓度尚可诱发心脏停博反应；DOX的手性结构对其上述活性具有明显的影响。相反，ALF仅轻度抑制小鼠心率，ALF的手性结构对其心脏效应无明显影响。长期灌胃给予DOX及其对映体未显著影响大鼠心衰生物学标志物NT-proBNP及IL-6的血浆浓度；但是，(+)DOX组大鼠的血浆NF-κB水平显著高于(-)DOX组，提示(+)DOX对机体免疫及炎症反应有一定的影响。
2(-)DOX增强大鼠心房肌收缩力的作用，可能与心肌α受体、M胆碱受体、β受体以及环氧合酶无关；L型Ca~(2+)通道、细胞内cGMP以及PKA可能在某种程度上参与了(-)DOX在大鼠左心房的正性肌力作用。但是，心肌α受体、M胆碱受体、β受体、Ca~(2+)通道、环氧合酶、cGMP及PKA，可能未参与(+)DOX对大鼠左心房的负性肌力作用。
3在人、犬、鼠的血浆中多沙唑嗪对映体的蛋白结合力均体现了立体选择性，在所有三个种属中(+)DOX的蛋白结合力均高于(-)DOX。多沙唑嗪对映体的蛋白结合率存在种属差异。
Doxazosin, a quinazoline derivative, was produced firstly by Pfizer andsold in Denmark at1988. It is a new highly selective α1-adrenoceptorantagonist, with the effect of lowering blood pressure by dilating blood vesselthrough selectively and competitively block postsynaptic α1-adrenoceptor,while have no impact on postsynaptic or presynaptic α2-adrenoceptor. It couldalso relax both resistant and capacity vessels, reactively inhibit heart rate andimprove microcirculation, so decrease preload and afterload of the heart. It’susing in the treatment of lower urinary tract symptom (LUTS) due to benignprostatic hyperplasia (BPH）has been approved by FDA, furthermore, it coulddecrease the mass of prostate by inducing apoptosis. Additionally, doxazosincould decrease risk of coronary heart disease by benefiting lipid metabolismthrough decrease blood levels of total triglyceride (TG) and cholesterol (TC),simultaneously increase high density lipoprotein (HDL) and decrease lowdensity lipoprotein (LDL). However, Antihypertensive and Lipid-LoweringTreatment to Prevent Attack Trial (ALLHAT), a large clinical study carryingout recently, showed that risk of cardiovascular disease in patients randomizedto doxazosin group increased by25%; while risk of heart failure was doubled,if compared to chlorthalidone group. Due to this result, the doxazosin treatmentarm of the study was terminated prematurely. Consequently, in the SeventhReport of the Joint National Committee on Prevention, Detection, Evaluation,and Treatment of High Blood Pressure (JNC7) guidelines, alpha-blockers werenot recommended for the routine treatment of hypertension. However,alpha-blockers were still widely used in treatment of hypertension as add-onmedicine, especially for hypertension patients with BPH.
Alfuzosin, same with doxazosin, is a quinazoline compound with highlyselective α1-adrenoceptor block property. A chiral carbon atom exists in molecular structure of either doxazosin or alfuzosin, while both the twomedicines are used in clinical in their racemic enantiomers [(±)doxazosin and(±)alfuzosin]. Presently the exact effect of doxazosin and its enantiomers onisolated heart is not clear, so we studied the relationship between chiralstructure of doxazosin and alfuzosin and their effects on heart rate andcontractile force in the isolated mouse atrium. The result showed that doxazosinsignificantly affected the heart rate and contraction of the isolated mouseatrium, and induced cardiac arrest at high concentration. Chiral structure ofdoxazosin has an obvious effect on its bioactivity. laevo isomer of doxazosinhas positive inotropic action, while dextroisomer has negative effect. Alfuzosin,however, decreased the HR slightly without chiral recognition in the isolatedmouse atrium. It was reported that blood levels of hormone andpro-inflammatory cytokines related to heart function and cardiac contractility.Brain natriuretic peptide (BNP), nuclear factor-κB (NF-κB) and interleukin-6(IL-6) are all biomarkers of heart failure. BNP is a kind of hormone mainlysecreted by ventricular wall when over stretched, also has diuretic andnatriuretic effect. NF-κB belongs to the category of "rapid-acting" primarytranscription factors, play an important role in expression of inflammatorymediator and immune reaction genes. IL-6is an important proinflammatorycytokine, it`s serum concentration is independently related to myocardialcontractility. In this study we explored the impact of doxazosin enantiomers onplasma levels of NT-proBNP, IL-6and NF-κB.
Alternative pathways could mediate the inotropic or chronotropic effectsof doxazosin and its enantiomers at post-receptor levels. In order to reveal themechanism of different effect of doxazosin enantiomers on heart, we alsoinvestigated the influence of different pathway inhibitors, such as atropine,propranolol, indomethacin, phenoxybenzamine, verapamil, methylene blue andH-89, on the intropic effects of different doxazosin enantiomers in the isolatedrat left atrium strips.
Binding to plasma proteins plays a major role in drug therapy as the bounddrug is difficult to pass through blood vessel wall and cell membrane, whereas the unbound drug can cross the capillary wall to reach the cellular target as wellas metabolic tissue. A change in drug binding to plasma protein significantlyaffects its pharmacokinetic and pharmacodynamic properties. Because thedifference in the protein binding property between enantiomers often causes thedifference in their pharmacokinetic characters, enantioselective protein bindingstudy is essential to the understanding of their pharmacology, safety andclinical efficacy. Hence, it is necessary to stereoselectively detect and quantifyeach enantiomer of (±)doxazosin in biological media. In the present study, theequilibrium dialysis technique has been utilized to determine the plasma proteinbinding of the enantiomers of (±)doxazosin to the rat, dog and human plasma.Chiral HPLC-FL methods have been validated and employed to measure thedrug concentration on each side of equilibrium.
Part I Effects of doxazosin enantiomers on heart rate, contractile force andplasma heart failure biomarkers
In this part, the isolated mouse atrium strip were used to study therelationship between chiral structure of doxazosin, alfuzosin and their effectson heart, namely heart rate and contractile force. In order to understand theinfluence of different optical enantiomers of doxazosin on cardiac function ofrats, we studied the effect of long-term doxazosin or its enantiomersadministration on normal rat plasma NT-proBNP, NF-κB and IL-6levels.
1. Cardiac arrest effect of (±)doxazosin and (±)alfuzosin enantiomers onisolated mouse right atrium
Among16samples in (+)doxazosin group,5cases of cardiac arrest wereinduced at concentration of30μmol·L~(-1), so cardiac arrest rate was31.3%.(±)Doxazosin induced one cardiac arrest at30μmol·L~(-1), while (-)doxazosininduced one cardiac arrest at concentration of10μmol·L~(-1)and30μmol·L~(-1)respectively. No cardiac arrest was induced in (-)alfuzosin,(+)alfuzosin,(±)alfuzosin group and control group.
2. Impact of (±)doxazosin,(±)alfuzosin and their enantiomers on heart rate ofisolated mouse right atrium
Heart rates of isolated right atrium before administration of the drug were nearly same among all groups (P>0.05)，solvent has no effect on HR of isolatedright atrium (P>0.05).(+)doxazosin and (±)doxazosin at used concentrationssignificantly decreased the heart rate in a concentration-dependent manner(P<0.01)； HRs were decreased by (83.47±12.23)%and (74.97±15.66)%respectively induced by (+)doxazosin and (±) doxazosin at concentration of30μmol·L~(-1). The inhibition effect on HR of the isolated right atrium induced by(+)doxazosin was much stronger than (±)doxazosin at the same concentration(P<0.01).(-)Doxazosin has no effect on HR of isolated right atrium at3μmol·L~(-1)(P>0.05), while the inhibition effect on HR was significantly inferiorto (+)doxazosin at the same concentration (P<0.01).(-)Alfuzosin and(+)alfuzosin (10and30μmol·L~(-1)) decreased HR significantly, which were(13.05±7.27)%and (14.27±9.75)%respectively at concentration of30μmol·L~(-1),while (±)alfuzosin decreased HR only at30μmol·L~(-1)，and the three drugsinhibited HR at about the same degree.
3Different effect of (±)doxazosin,(±)alfuzosin and their enantiomers oncontractile force of isolated mouse left atrium.
Contractile force of left atrium in different groups were nearly same(P>0.05).The solvent has no effect on contractile force of isolated mouse leftatrium (P>0.05).(±)Doxazosin and (-)doxazosin at concentrations of3,10and30μmol·L~(-1)significantly strengthen contractile force of isolated mouse leftatrium (P<0.05) by a concentration-dependent manner. The positive inotropiceffect of (-)doxazosin (10and30μmol·L~(-1)) was stronger than (±)doxazosin ofthe same concentration (P<0.05). In contrast,(+)doxazosin has mild butsignificant negative inotropic effect (P<0.05), while (±)alfuzosin and itsenantiomers have no effect on contractile force of isolated mouse left atrium(P>0.05).
4Effect of long-term administration of doxazosin and its enantiomers onplasma NT-proBNP of rats
Plasma NT-proBNP concentrations in control group,(-)doxazosin group,(+)doxazosin group and(±)doxazosin group were475.55±150.21ng·L~(-1)、635.00±343.52ng·L~(-1)、375.55±127.09ng·L~(-1)and424.12±134.19ng·L~(-1) respectively. NT-proBNP concentration was lower in either (±)doxazosin groupor (+)doxazosin group than in control group, while concentration in(-)doxazosin group was higher than in control group, but the difference has nostatistical significance by single factor analysis of variance between groups(P>0.05). Plasma NT-proBNP concentration in (-)doxazosin group wassignificantly higher than in (+)doxazosin group (P<0.05).
5Effect of long-term administration of doxazosin and its enantiomers onplasma NF-κB of rats
Plasma NF-κB concentration in control group,(-)doxazosin group,(+)doxazosin group and (±)doxazosin group was888.46±343.36ng·L~(-1),705.77±193.34ng·L~(-1),1140.00±365.26ng·L~(-1)and910.38±276.39ng·L~(-1)respectively. NF-κB concentration in either (±)doxazosin group or(+)doxazosin group was higher than in control group, while concentration in(-)doxazosin group was lower than in control group, but there was no statisticalsignificance by single factor analysis of variance (P>0.05). Plasma NT-proBNPconcentration in (-)doxazosin group was significantly lower than in(+)doxazosin group (P<0.05).
6Effects of long-term administration of doxazosin and its enantiomers onplasms IL-6of rats
Plasma IL-6concentration of control group,(-)doxazosin group,(+)doxazosin group and (±)doxazosin group were47.1±23.9ng·L~(-1),71.5±23.7ng·L~(-1),59.4±23.1ng·L~(-1)and70.2±37.4ng·L~(-1)respectively. Difference amongthe four groups has no statistic significance (P>0.05).
These results demonstrate that the inhibit effect of (±)doxazosin and itsenantiomers on heart rate was stronger than that of (±)alfuzosin and itsenantiomers， and there exist chiral difference.(-)Doxazosin has positiveinotropic effect while (+)doxazosin has mild but significant negative inotropiceffect,(±)alfuzosin and its enantiomers with same concentration have no effecton contractile force of isolated left atrium. Chiral structure of doxazosin has anobvious effect on its bioactivity. Alfuzosin, however, decreases the HR slightlywithout chiral recognition in the isolated mouse atrium. Long-term administration of (-)doxazosin,(+)doxazosin and (±)doxazosin did not increaseor decrease plasma level of heart failure biomarker, namely NT-proBNP,NF-κB and inflammatory cytokines IL-6. Plasma NF-κB concentration in(+)doxazosin was higher than (-)doxazosin group, which showed that(+)doxazosin maybe affect immune and inflammation system.
Part II Mechanism of contractile force change induced by doxazosinenantiomers in isolated rat atrium
In this part, isolated rat left atrium strips were used to study the effect ofdifferent doxazosin enantiomers on myocardial contractile force. In order toprobe the exact mechanism of doxazosine enatiomers inotropic effects, isolatedrat left atrium were pretreated by different blocker such as atropine, propranolol,phenoxybenzamine, indomethacin, verapamil, methylene blue or H-89.1Inotropic effects of (-)doxazosin and (+)doxazosin on isolated rats left atrium
Solvent has no effect on contractile force of isolated rats right atrium.(P>0.05)；(-)doxazosin has significant positive inotropic effect on isolated ratsright arium (P<0.05)，percentages increase of contractile force induced by(-)doxazosin of3,10and30μmol·L~(-1)were (5.57±20.95)%,(6.96±39.77)%and (67.46±30.09)%respectively (P<0.05). On the contrary,(+)doxazosin hasnegative inotropic effect, the decrease in percentages of contractile forceinduced by (+)doxazosin of3,10,30μmol·L~(-1)were (~(-1)5.61±14.15)%,(-36.99±23.58)%and (-57.48±56.01)%respectively.2Impact of atropine and propranolol on cardiac inotropic effects of(-)doxazosin and (+)doxazosin
After the isolated rat atrium were pretreated with atropine and propranolol,the solvent has no effect on contractile force. The percentages increase ofcontractile force induced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (13.02±16.66)%,(53.65±41.02)%and (70.87±52.66)%respectively. The decrease in percentage of contractile force induced by(+)doxazosin at concentrations of3,10and30μmol·L~(-1)were (-8.02±21.43)%,(-21.60±21.50)%and (-63.75±24.14)%respectively.
3Impact of phenoxybenzamine on cardiac inotropic effects of (-)doxazosin and (+)doxazosin
After the isolated rat atrium were pretreated with phenoxybenzamine, thesolvent has no effect on contractile force. The percentages increase ofcontractile force induced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (13.71±14.39)%,(35.48±11.90)%and (71.62±37.20)%respectively. The decrease in percentage of contractile force induced by(+)doxazosin were (~(-1)4.08±23.48)%,(-33.08±17.85)%and (-60.73±17.21)%respectively. After pretreated with ethanol, the solvent has no effect oncontractile force. The percentages increase of contractile force induced by(-)doxazosin at concentration of3,10and30μmol·L~(-1)were (19.05±7.14)%,(49.27±28.71)%and (71.21±35.57)%respectively. The decrease in percentageof contractile force induced by (+)doxazosin were (~(-1)0.40±14.76)%,(-23.73±26.18)%and (-65.63±6.52)%respectively.
4Impact of indomethacin on cardiac inotropic effects of (-)doxazosin and(+)doxazosin
After the isolated rat atrium was pretreated with indomethacin, the solventhas no effect on contractile force. The percentages increase of contractile forceinduced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (19.87±13.06)%,(55.73±18.00)%and (60.25±35.25)%respectively. The decrease inpercentages of contractile force induced by (+)doxazosin were (-7.23±13.39)%,(-23.73±26.18)%and (-65.63±6.52)%respectively. After pretreated withethanol, the percentage increase of contractile force induced by (-)doxazosin atconcentrations of3,10and30μmol·L~(-1)were (19.05±7.14)%,(49.27±28.71)%and (71.21±35.57)%respectively. The decrease in percentage of contractileforce induced by (+)doxazosin were (~(-1)0.40±14.76)%,(-23.73±26.18)%and(-65.63±6.52)%respectively.
5Impact of verapamil on cardiac inotropic effects of (-)doxazosin and(+)doxazosin
After the isolated rat atrium was pretreated with verapamil, the solvent hasno effect on contractile force. The percentage increase of contractile forceinduced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (24.81±20.49)%,(75.98±23.00)%and (131.15±17.56)%respectively. Thedecrease in percentage of contractile force induced by (+)doxazosin atconcentration of3,10and30μmol·L~(-1)were (-9.60±17.19)%,(-33.47±9.43)%and (-72.23±14.65)%respectively.
6Impact of methylene blue on cardiac inotropic effects of (-)doxazosin and(+)doxazosin
After the isolated rat atrium was pretreated with methylene blue, thesolvent has no effect on contractile force. The percentages increase ofcontractile force induced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (36.26±9.82)%,(65.99±26.27)%and (16.02±35.33)%respectively. The decrease in percentage of contractile force induced by(+)doxazosin at concentrations of3,10μmol·L~(-1)were (-5.57±17.32)%,(~(-1)0.54±25.09)%,(-58.27±15.47)%respectively.
7Impact of H-89on cardiac inotropic effects of (-)doxazosin and (+)doxazosin
After the isolated rat atrium was pretreated with H-89, the solvent has noeffect on contractile force. The percentages increase of contractile forceinduced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (28.96±9.49)%,(81.93±20.75)%and (101.53±51.95)%respectively. The decreasein percentage of contractile force induced by (+)doxazosin was(~(-1)7.71±16.37)%,(-30.76±18.38)%and (-67.67±10.74)%respectively.
These results demonstrated that the positive inotropic effect of(-)doxazosin on isolated rat left atrium was independent of its α-receptorblocking effect, also did not related to M-receptor, β-receptor, PG enzyme.L-type Ca~(2+)channel, while intracellular cGMP and PKA maybe participatedpartly in the positive inotropic effect of (-)doxazosin. But, cardial myocyteα-receptor, M-receptor, β-receptor, L-type Ca~(2+)channel, PG enzyme, cGMP orPKA maybe not related to the negative inotropic effect of (+)doxazosin.
Part III Stereoselective binding of doxazosin enantiomers to plasmaproteins from rat, dog and human
Binding to plasma proteins plays a major role in drug therapy. A change indrug binding to plasma protein significantly affects its pharmacokinetic and pharmacodynamic properties. Because the difference in the protein bindingproperty between enantiomers often causes the difference in theirpharmacokinetic characters, enantioselective protein binding study is essentialto the understanding of their pharmacology, safety and clinical efficacy. Hence,it is necessary to stereoselectively detect and quantify each enantiomer of(±)doxazosin in biological media. Here, we report a first application of chiralHPLC methods using a chiral stationary phase column in conjunction withequilibrium dialysis for the simultaneous determination of the protein bindingof each enantiomer of doxazosin in rat, dog and human plasma after addition ofthe racemate in vitro.
1Method validation
The chiral HPLC methods for the separation of doxazosin enantiomers werevery selective.Prazosin,(-)doxazosin and (+)doxazosin were clearly wellresolved from the matrix components under the chromatographic conditionsemployed, and the two enantiomers of doxazosin were baseline resolved fromeach other.
The calibration curves (weight1/x2) of the two enantiomers of doxazosinwere linear with correlation coefficients greater than0.9990over theconcentration range of50to1600ng·mL~(-1)for plasma and12.5to200ng·mL~(-1)for PBS. The precision and accuracy of the method were also investigated. Theprecision (relative standard deviation, RSD) based on five repetitive injectionsat drug concentrations of0.2,0.5and1μg·mL~(-1)was less than3.2%(intra-day)and6.3%(inter-day) for the plasma extracts of all three species. The accuracywas found to be in the range of90.5%-102.4%for all plasma and PBS buffermatrices at three drug concentration levels. The detection limit, at asignal-to-noise ratio of3, in plasma of all three species was about0.2ng·mL~(-1)for doxazosin. The enantiomers of doxazosin were stable during the entirecourse of the study including sample preparation, centrifugation and HPLCassay.
2Optimization of equilibrium dialysis conditions
The time taken to reach equilibrium for doxazosin between plasma and isotonic PBS was investigated. Isotonic PBS spiked with (±)doxazosin (800ng·mL~(-1)) was dialyzed against pooled human plasma at0,4,8,15, and20h.(-)Doxazosin and (+)doxazosin crossed the dialysis membrane rapidly andreached a steady-state level in human plasma within15h at37°C. Theequilibrium time for dog or rat plasma was the same to that for human plasma.Therefore, equilibrium dialysis duration was15h in the subsequent equilibriumdialysis experiments at incubation temperature of37°C (body temperature).
3Difference in protein binding between the enantiomers
In the concentration range between200and800ng·mL~(-1)of (±)doxazosin,the percentage of plasma protein binding of enantiomers in the three specieswere89.4%-94.3%for (-)doxazosin and90.9%-95.4%for (+)doxazosin. Theresults showed that either (-)doxazosin or (+)doxazosin was highly bound toplasma proteins, which is consistent with the previous investigationsdetermining the binding of racemic doxazosin. Furthermore, Cb values of(-)doxazosin were significantly smaller than those of (+)doxazosin (P<0.05) ineach plasma from the three species at the used concentrations of (±)doxazosin.Difference in Cb between the two enantiomers became larger with theincreased concentration of (±)doxazosin regardless of the species used.Moreover, the stereoselectivity of doxazosin in dog plasma was moresignificant than that in rat plasma and human plasma. The mean Cb values(ng·mL~(-1)) of (-)doxazosin vs (+)doxazosin for dog plasma in the PBS contained(±)doxazosin200,400, and800ng·mL~(-1)were200.0vs243.0,398.6vs477.3,and845.1vs1006.8, respectively. Overall, enantioselective binding ofdoxazosin enantiomers to plasma proteins from rat, dog and human wasobserved, and (+)doxazosin exhibited a higher protein-binding capacity than(-)doxazosin in all three species.
A difference in plasma protein-binding capacity between the twoenantiomers may lead to their pharmacokinetic behavior being different fromeach other. As drug clearance from the blood is directly proportional to the itsfree fraction in plasma, a higher unbound (-)doxazosin concentration in humanplasma observed in the present study might partly explain the phenomenon reported by Liu et al. They found that the human plasma concentration of(-)doxazosin is lower than that of (+)doxazosin after administration of theracemate. Indeed, in our laboratory, the different plasma concentration of thetwo enantiomers in dog and rat was observed after administration of(-)doxazosin and (+)doxazosin. Therefore, the discrepancy for plasmaprotein-binding capacity between (-)doxazosin and (+)doxazosin is a commonreason for their different pharmacokinetic parameters in rat, dog and human.
4Difference in protein binding among three species
In the case of200ng·mL~(-1)of racemic doxazosin added to the PBS, nosignificant difference in percentage of plasma protein binding among threespecies was found. However, the percentage of plasma protein binding in dogplasma was significantly smaller than that in human plasma at400and800ng·mL~(-1)of racemic doxazosin (P<0.05and P<0.01), which is consistent withthe previous report that plasma protein binding of (±)doxazosin in human washigher than that in dog. Since total protein concentrations of the pooled plasmafrom rat, dog and human were61.81,51.35and57.75mg·mL~(-1)respectively;the value of percentage of plasma protein binding should be corrected with thevalue of protein assay. The corrected percentage of plasma protein binding ofthe enantiomers at a given concentrations was variable in different species:dog> human> rat (P<0.01).
These results demonstrate that either (-)doxazosin or (+)doxazosin washighly bound to plasma proteins. Moreover, protein binding of doxazosinenantiomers in human, dog and rat plasma revealed that there was a significantdifference in bound fractions, i.e., a higher protein-binding capacity of(+)doxazosin than (-)doxazosin, which might be one of the reasons for anobviously difference in plasma concentration between doxazosin enantiomersin human orally administered (±)doxazosin. Additionally, a pronounced speciesdifference in the plasma protein binding was found in the present study, with ahigher protein-binding capacity in human than dog. Since the total proteinconcentrations of the plasma were significantly different among rat, dog andhuman, the corrected percentage of plasma protein binding was calculated in the study, which indicates an order of dog> human> rat (P<0.01). Therefore,the findings of a stereoselective plasma protein binding between (-)doxazosinand (+)doxazosin and a pronounced species difference in the plasma proteinbinding of rat, dog and human should be useful for explaining thepharmacokinetic characters of chiral doxazosin.
Conclusion
Doxazosin has obvious effects on the heart rate and cardiac contractility ofisolated mouse atrium. Doxazosin could induce cardiac arrest reaction at highconcentration, chiral structure of doxazosin has obvious influence on its activity.In contrast, alfuzosin only slightly inhibited the mouse heart rate; chiralstructure of alfuzosin has no obvious effect on its cardiac effects. Long-termadministration of (-)doxazosin,(+)doxazosin and (±)doxazosin did not increaseor decrease plasma level of heart failure biomarker, namely NT-proBNP, NF-κB and IL-6. Plasma NF-κB concentration in (+)doxazosin was higher than(-)doxazosin group, which showed that (+)doxazosin maybe affect immune andinflammation system.
The positive inotropic effect of (-)doxazosin on isolated rat left atrium wasindependent of its α-receptor blocking effect, also did not related to M-receptor,β-receptor, PG enzyme. L-type Ca~(2+)channel, while intracellular cGMP andPKA maybe participated partly in the positive inotropic effect of (-)doxazosin.But, cardial myocyte α-receptor, M-receptor, β-receptor, L-type Ca~(2+)channel,PG enzyme, cGMP or PKA maybe not related to the negative inotropic effectof (+)doxazosin.
Either (-)doxazosin or (+)doxazosin was highly bound to plasma proteins.Moreover, protein binding of doxazosin enantiomers in human, dog and ratplasma revealed that there was a significant difference in bound fractions, i.e., ahigher protein-binding capacity of (+)doxazosin than (-)doxazosin, whichmight be one of the reasons for an obviously difference in plasma concentrationbetween doxazosin enantiomers in human orally administered (±)doxazosin.Additionally, a pronounced species difference in the plasma protein bindingwas found in the present study, with a higher protein-binding capacity in human than dog. Since the total protein concentrations of the plasma were significantlydifferent among rat, dog and human, the corrected percentage of plasma proteinbinding was calculated in the study, which indicates an order of dog> human>rat (P<0.01). Therefore, the findings of a stereoselective plasma protein bindingbetween (-)doxazosin and (+)doxazosin and a pronounced species difference inthe plasma protein binding of rat, dog and human should be useful forexplaining the pharmacokinetic characters of chiral doxazosin.
多种信号途径可能参与药物诱发的心肌收缩力改变，为进一步探讨DOX及其光学异构体对心肌收缩力不同影响的机制，本研究使用大鼠离体心房肌标本，研究了酚苄明、阿托品及普萘洛尔、吲哚美辛、维拉帕米、亚甲蓝、H-89等不同阻断剂对多沙唑嗪对映体引起大鼠张力反应的影响。
血浆蛋白结合在药物治疗中起着重要的作用。药物与血浆蛋白结合的变化显著地影响它的药代动力学和药效学。因为对映体之间的蛋白结合率往往造成它们药代动力学的差异。对映体蛋白结合的立体选择性的研究对于理解其药理学、临床疗效及安全性是必不可少的。因此，有必要对生理条件下多沙唑嗪对映体的立体选择性进行研究。本研究采用手性固定相柱联合平衡透析法同时测定大鼠、狗和人血浆中加入消旋多沙唑嗪后其每一对映体的蛋白结合率。结果表明，多沙唑嗪对映体对大鼠，狗和人类血浆蛋白结合具有立体选择性，且种属间也存在差异。
第一部分多沙唑嗪对映体对心率、心肌收缩力及心衰生物学标志物的影响
本研究采用小鼠离体心房组织，观察(±)DOX及其两种对映体、(±)ALF及其两种对映体对小鼠离体心房心肌收缩力和心率的影响；并观察多沙唑嗪及其对映体长期给药对大鼠血浆氨基末端原脑利钠肽（NT-proBNP）、IL-6及NF-κB水平的不同影响，以期为喹唑啉类α1受体阻断药的临床安全用药和新药设计提供实验数据。
1(±)DOX和(±)ALF及二者的对映体诱发小鼠离体右心房停搏反应
(+)DOX组的16例标本中，加入30μmol·L~(-1)药物后，5例发生停搏反应；停搏率为31.3%。(±)DOX组和(-)DOX组分别在10μmol·L~(-1)和30μmol·L~(-1)浓度时，各有一例发生停搏反应。(-)ALF、(+)ALF、(±)ALF组和溶剂对照组，未发生停搏反应。
2(±)DOX和(±)ALF及二者的对映体对小鼠离体右心房心率的影响
各组离体右心房心率的药前值相同（P>0.05），溶剂对小鼠离体右心房心率无显著影响（P>0.05）。(+)DOX和(±)DOX各浓度均显著减慢小鼠右心房心率（P<0.01），并具有浓度依赖关系；30μmol·L~(-1)的(+)DOX和(±)DOX组小鼠心率减慢率分别为（83.47±12.23）%和（74.97±15.66）%，3μmol·L~(-1)(+)DOX减慢心率的作用显著强于同浓度(±)DOX（P<0.01）。(-)DOX（3μmol·L~(-1)）对离体右心房心率无显著影响（P>0.05），且(-)DOX在10和30μmol·L~(-1)浓度时减慢心率的作用显著弱于同浓度(+)DOX（P<0.01）。(-)ALF和(+)ALF（10和30μmol·L~(-1)）显著减慢小鼠心率，30μmol·L~(-1)时小鼠心率分别减慢（13.05±7.27）%和（14.27±9.75）%；(±)ALF仅在30μmol·L~(-1)浓度显著减慢小鼠心率，三者抑制心率的作用强度相同。
3(±)DOX和(±)ALF及二者的对映体对小鼠离体左心房收缩力的影响
各组左心房心肌收缩力的药前值相同（P>0.05），溶剂对小鼠离体左心房心肌收缩力无显著影响（P>0.05）。(±)DOX和(-)DOX（3、10和30μmol·L~(-1)）显著增强小鼠离体左心房心肌收缩力（P<0.05），并具有浓度依赖关系；(-)DOX（10和30μmol·L~(-1)）的正性肌力作用显著强于同浓度(±)DOX的作用（P<0.05）。相反，(+)DOX具有轻度但显著的负性肌力作用（P<0.05）。(±)ALF及其对映体（3、10和30μmol·L~(-1)）对小鼠离体左心房心肌收缩力无显著影响（P>0.05）。
4(-)DOX、(+)DOX及(±)DOX长期给药对大鼠血浆NT-proBNP浓度的影响
溶剂对照组、(-)DOX组、(+)DOX组及(±)DOX组血浆NT-proBNP浓度分别为475.55±150.21ng·L~(-1)、635.00±343.52ng·L~(-1)、375.55±127.09ng·L~(-1)及424.12±134.19ng·L~(-1)。(+)DOX组及(±)DOX组血浆NT-proBNP浓度低于对照组，但组间比较差异无统计学意义（P>0.05）。(-)DOX组血浆NT-proBNP值高于对照组，但组间比较差异亦无统计学意义（P>0.05）。(-)DOX组血浆NT-proBNP浓度高于(+)DOX组（P<0.05）。
5(-)DOX、(+)DOX及(±)DOX长期给药对大鼠血浆NF-κB浓度的影响
溶剂对照组、(-)DOX组、(+)DOX组及(±)DOX组血浆NF-κB浓度分别为888.46±343.36ng·L~(-1)、705.77±193.34ng·L~(-1)、1140.00±365.26ng·L~(-1)及910.38±276.39ng·L~(-1)。(+)DOX组及(±)DOX组血浆NF-κB浓度高于溶剂对照组，但组间比较差异无统计学意义（P>0.05）。(-)DOX组血浆NF-κB浓度高于溶剂对照组，但组间比较差异无统计学意义（P>0.05）。(-)DOX组血浆NF-κB值低于(+)DOX组（P<0.05）。
6(-)DOX、(+)DOX及(±)DOX长期给药对大鼠血浆IL-6浓度的影响
溶剂对照组、(-)DOX组、(+)DOX组及(±)DOX组血浆IL-6浓度分别为48.81±23.39ng·L~(-1)、75.13±21.58ng·L~(-1)、59.44±23.17ng·L~(-1)及70.19±37.44ng·L~(-1)。单因素方差分析结果显示：与溶剂对照组比较，各药物组大鼠血浆IL-6水平无统计学意义（P>0.05）。
研究结果表明，DOX对小鼠离体心房的心率和心肌收缩力具有明显的影响，高浓度尚可诱发心脏停博反应；DOX的手性结构对其上述活性具有明显的影响。相反，ALF仅轻度抑制小鼠心率，ALF的手性结构对其心脏效应无明显影响。长期灌胃给予DOX及其对映体未显著影响大鼠心衰生物学标志物NT-proBNP及IL-6的血浆浓度；但是，(+)DOX组大鼠的血浆NF-κB水平显著高于(-)DOX组，提示(+)DOX对机体免疫及炎症反应有一定的影响。
第二部分多沙唑嗪对映体诱发大鼠心房肌收缩力变化的作用机制
本研究采用大鼠离体心房组织研究了酚苄明、阿托品及普萘洛尔、吲哚美辛、维拉帕米、亚甲蓝、H-89等不同阻断剂对多沙唑嗪对映体引起大鼠张力反应的影响。1(-)DOX、(+)DOX对离体大鼠左心房心肌张力的影响
溶剂对大鼠心肌张力无显著影响；(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠心肌收缩力的百分比分别为（5.57±20.95）、（6.96±39.77）%、（67.46±30.09）%。(+)DOX显著抑制大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(+)DOX抑制大鼠心肌收缩力的百分比分别为（~(-1)5.61±14.15）%、（-36.99±23.58）%、（-57.48±56.01）%。
2阿托品及普萘洛尔对DOX对映体改变大鼠心房收缩力的影响
以阿托品及普萘洛尔预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力），3、10、30μmol L~(-1)的(-)DOX增强大鼠心房收缩力的百分比分别为（13.02±16.66）%、（53.65±41.02）%、（70.87±52.66）%。(+)DOX显著抑制大鼠左心房心肌收缩力，(+)DOX抑制大鼠心房收缩力的百分比分别为（-8.02±21.43）%、（-21.60±21.50）%、（-63.75±24.14）%。
3酚苄明对DOX对映体改变大鼠心房收缩力的影响
以酚苄明预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠心房收缩力的百分比分别为（13.71±14.39）%、（35.48±11.90）%、（71.62±37.20）%。(+)DOX显著抑制大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(+)DOX抑制大鼠心房收缩力的百分比分别为（~(-1)4.08±23.48）%、（-33.08±17.85）%、（-60.73±17.21）%。以乙醇预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)(-)DOX对增强大鼠心房收缩力的百分比分别为（19.05±7.14）%、（49.27±28.71）%、（71.21±35.57）%。(+)DOX显著抑制大鼠左心房心肌收缩力，(+)DOX抑制大鼠心房收缩力的百分比分别为（~(-1)0.40±14.76）%、（-23.73±26.18）%、（-65.63±6.52）%。
4吲哚美辛对DOX对映体改变大鼠心房收缩力的影响
以吲哚美辛预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠张力的百分比分别为（19.87±13.06）%、（55.73±18.00）%、（60.25±35.25）%。(+)DOX显著抑制大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(+)DOX抑制大鼠心房收缩力的百分比分别为（-7.23±13.39）%、（-31.33±18.74）%、（-70.42±8.90）%。以乙醇预处理标本后，(-)DOX显著增强大鼠左心房心肌收缩力，(-)DOX对大鼠张力的增强率为（21.01±9.75）%、（43.88±28.26）%、（59.45±24.87）%。(+)DOX显著抑制大鼠左心房心肌收缩力，(+)DOX抑制大鼠心房收缩力的百分比分别为（~(-1)5.21±13.14）%、（-29.28±23.55）%、（-66.55±6.13）%。
5维拉帕米对DOX对映体改变大鼠心房收缩力的影响
以维拉帕米预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠张力的百分比分别为（24.81±20.49）%、（75.98±23.00）%、（131.15±17.56）%。(+)DOX显著抑制大鼠左心房心肌收缩力，(+)DOX抑制大鼠心房收缩力的百分比分别为（-9.60±17.19）%、（-33.47±9.43）%、（-72.23±14.65）%。
6亚甲蓝对DOX对映体改变大鼠心房收缩力的影响
以亚甲蓝预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠张力的百分比分别为（36.26±9.82）%、（65.99±26.27）%、（16.02±35.33）%。3、10、30μmol L~(-1)的(+)DOX抑制大鼠心房收缩力的百分比分别为（-5.57±17.32）%、（~(-1)0.54±25.09）%、（-58.27±15.47）%。
7H-89对DOX对映体改变大鼠心房收缩力的影响
以H-89预处理标本后，溶剂对大鼠心肌张力无显著影响。(-)DOX显著增强大鼠左心房心肌收缩力，3、10、30μmol L~(-1)的(-)DOX增强大鼠心肌张力的百分比分别为（28.96±9.49）%、（81.93±20.75）%、（101.53±51.95）%。(+)DOX显著抑制大鼠左心房心肌收缩力，(+)DOX抑制大鼠心房收缩力的百分比分别为（~(-1)7.71±16.37）%、（-30.76±18.38）%、（-67.67±10.74）%。
研究结果表明，(-)DOX增强大鼠心房肌收缩力的作用，可能与心肌α受体、M胆碱受体、β受体以及环氧合酶无关；L型Ca~(2+)通道、细胞内cGMP以及PKA可能在某种程度上参与了(-)DOX在大鼠左心房的正性肌力作用。但是，心肌α受体、M胆碱受体、β受体、Ca~(2+)通道、环氧合酶、cGMP及PKA，可能未参与(+)DOX对大鼠左心房的负性肌力作用。
第三部分多沙唑嗪对映体在大鼠、犬和人血浆中的蛋白结合率研究
血浆蛋白结合在药物治疗中起着重要的作用。药物与血浆蛋白结合的变化显著影响其药代动力学和药效学。对映体蛋白结合率的不同往往是它们药代动力学差异的原因之一。对映体蛋白结合的立体选择性研究是明确其药理学效应、临床疗效及安全性必不可少的内容。因此，有必要对多沙唑嗪对映体蛋白结合的立体选择性进行研究。本研究采用平衡透析法利用卵粘蛋白手性色谱柱同时测定大鼠、犬和人血浆中多沙唑嗪对映体的浓度，并计算其蛋白结合率。
1高效液相色谱法的方法学验证
采用高效液相色谱法手性分离血浆及PBS中的(±)DOX，专属性良好，内标（哌唑嗪）、(-)DOX和(+)DOX达到基线分离，最低检测限为0.2ng·mL~(-1)（S/N≥3）。多沙唑嗪对映体在血浆、PBS中的浓度范围分别是50～1600ng·mL~(-1)、12.5～200ng·mL~(-1)，在上述浓度范围内，多沙唑嗪对映体的浓度与峰面积呈良好的线性关系（r>0.9990）。多沙唑嗪对映体在血浆和PBS的日内、日间精密度分别小于3.2%、6.3%，回收率为90.5%～102.4%，实验过程中稳定性良好。
2平衡透析条件的优化
在PBS中加入800ng·mL~(-1)的消旋多沙唑嗪，与混合人血浆进行平衡透析，分别在0、4、8、15及20h取样分析。试验表明，37°C时，(-)DOX和(+)DOX迅速通过半透膜，在15h内达到稳态水平。犬与大鼠的血浆平衡时间与人血浆相近。
3不同对映体间蛋白结合的差异
消旋多沙唑嗪的浓度在200～800ng·mL~(-1)时，(-)DOX在三个不同种属血浆中的蛋白结合率为89.4%～94.3%，(+)DOX为90.9%～95.4%。结果显示，无论(-)DOX还是(+)DOX的血浆蛋白结合率均较高，与之前(±)DOX的研究结果一致。在三种血浆中，加入不同浓度的消旋多沙唑嗪，(-)DOX的结合浓度（Cb）均大于(+)DOX的Cb（P<0.05），并且这种差异随多沙唑嗪浓度的增加而变得更加明显。这种立体选择性在犬血浆中比在人或鼠血浆中更为明显。犬血浆在含200、400、800ng·mL~(-1)(±)DOX的PBS中，(-)DOX与(+)DOX的Cb均值分别为200.0vs243.0、398.6vs477.3、845.1vs1006.8。因此，在人、犬、鼠的血浆中多沙唑嗪对映体的蛋白结合力均体现了立体选择性，(+)DOX的蛋白结合力均高于(-)DOX。
两对映体蛋白结合能力的不同可能会导致其药物代谢动力学的差异，药物的清除与其在血浆中的游离浓度有直接的关系。(+)DOX的蛋白结合力高于(-)DOX，因此应用消旋多沙唑嗪后人血浆中(-)DOX的非结合态浓度高于(+)DOX，可能是文献报道的人服用(±)DOX后血浆中(-)DOX浓度低于(+)DOX浓度的原因之一。事实上，我们将(±)DOX分别给予犬和鼠后，(-)DOX的浓度也低于(+)DOX的浓度。因此，(-)DOX和(+)DOX间血浆蛋白结合能力的不同可能是人、犬、鼠三个种属中二者药代动力学不同的原因之一。
4多沙唑嗪蛋白结合率的种属差异
加入200ng·mL~(-1)(±)DOX时，三个种属蛋白结合率差别无统计学意义。而在加入(±)DOX的浓度为400和800ng·mL~(-1)时，犬血浆蛋白结合率小于人的血浆蛋白结合率（P<0.05和P<0.01），此结果与以往报道的人血浆蛋白结合率高于犬一致。由于人、犬、鼠三种混和血浆的血浆蛋白浓度不同（分别为61.81、51.35和57.75mg·mL~(-1)），所以血浆蛋白结合率的值应该用血浆蛋白的浓度进行校正。校正后，多沙唑嗪对映体的蛋白结合率存在种属差异，其顺序为犬>人>鼠（P<0.01）。
以上研究结果表明，在人、犬、鼠的血浆中(+)DOX的蛋白结合力均高于(-)DOX；多沙唑嗪对映体的血浆蛋白结合力在三种血浆中均存在立体选择性；多沙唑嗪对映体的蛋白结合率具有种属差异，其顺序为犬>人>鼠。
结论
1DOX对小鼠离体心房的心率和心肌收缩力具有明显的影响，高浓度尚可诱发心脏停博反应；DOX的手性结构对其上述活性具有明显的影响。相反，ALF仅轻度抑制小鼠心率，ALF的手性结构对其心脏效应无明显影响。长期灌胃给予DOX及其对映体未显著影响大鼠心衰生物学标志物NT-proBNP及IL-6的血浆浓度；但是，(+)DOX组大鼠的血浆NF-κB水平显著高于(-)DOX组，提示(+)DOX对机体免疫及炎症反应有一定的影响。
2(-)DOX增强大鼠心房肌收缩力的作用，可能与心肌α受体、M胆碱受体、β受体以及环氧合酶无关；L型Ca~(2+)通道、细胞内cGMP以及PKA可能在某种程度上参与了(-)DOX在大鼠左心房的正性肌力作用。但是，心肌α受体、M胆碱受体、β受体、Ca~(2+)通道、环氧合酶、cGMP及PKA，可能未参与(+)DOX对大鼠左心房的负性肌力作用。
3在人、犬、鼠的血浆中多沙唑嗪对映体的蛋白结合力均体现了立体选择性，在所有三个种属中(+)DOX的蛋白结合力均高于(-)DOX。多沙唑嗪对映体的蛋白结合率存在种属差异。
Doxazosin, a quinazoline derivative, was produced firstly by Pfizer andsold in Denmark at1988. It is a new highly selective α1-adrenoceptorantagonist, with the effect of lowering blood pressure by dilating blood vesselthrough selectively and competitively block postsynaptic α1-adrenoceptor,while have no impact on postsynaptic or presynaptic α2-adrenoceptor. It couldalso relax both resistant and capacity vessels, reactively inhibit heart rate andimprove microcirculation, so decrease preload and afterload of the heart. It’susing in the treatment of lower urinary tract symptom (LUTS) due to benignprostatic hyperplasia (BPH）has been approved by FDA, furthermore, it coulddecrease the mass of prostate by inducing apoptosis. Additionally, doxazosincould decrease risk of coronary heart disease by benefiting lipid metabolismthrough decrease blood levels of total triglyceride (TG) and cholesterol (TC),simultaneously increase high density lipoprotein (HDL) and decrease lowdensity lipoprotein (LDL). However, Antihypertensive and Lipid-LoweringTreatment to Prevent Attack Trial (ALLHAT), a large clinical study carryingout recently, showed that risk of cardiovascular disease in patients randomizedto doxazosin group increased by25%; while risk of heart failure was doubled,if compared to chlorthalidone group. Due to this result, the doxazosin treatmentarm of the study was terminated prematurely. Consequently, in the SeventhReport of the Joint National Committee on Prevention, Detection, Evaluation,and Treatment of High Blood Pressure (JNC7) guidelines, alpha-blockers werenot recommended for the routine treatment of hypertension. However,alpha-blockers were still widely used in treatment of hypertension as add-onmedicine, especially for hypertension patients with BPH.
Alfuzosin, same with doxazosin, is a quinazoline compound with highlyselective α1-adrenoceptor block property. A chiral carbon atom exists in molecular structure of either doxazosin or alfuzosin, while both the twomedicines are used in clinical in their racemic enantiomers [(±)doxazosin and(±)alfuzosin]. Presently the exact effect of doxazosin and its enantiomers onisolated heart is not clear, so we studied the relationship between chiralstructure of doxazosin and alfuzosin and their effects on heart rate andcontractile force in the isolated mouse atrium. The result showed that doxazosinsignificantly affected the heart rate and contraction of the isolated mouseatrium, and induced cardiac arrest at high concentration. Chiral structure ofdoxazosin has an obvious effect on its bioactivity. laevo isomer of doxazosinhas positive inotropic action, while dextroisomer has negative effect. Alfuzosin,however, decreased the HR slightly without chiral recognition in the isolatedmouse atrium. It was reported that blood levels of hormone andpro-inflammatory cytokines related to heart function and cardiac contractility.Brain natriuretic peptide (BNP), nuclear factor-κB (NF-κB) and interleukin-6(IL-6) are all biomarkers of heart failure. BNP is a kind of hormone mainlysecreted by ventricular wall when over stretched, also has diuretic andnatriuretic effect. NF-κB belongs to the category of "rapid-acting" primarytranscription factors, play an important role in expression of inflammatorymediator and immune reaction genes. IL-6is an important proinflammatorycytokine, it`s serum concentration is independently related to myocardialcontractility. In this study we explored the impact of doxazosin enantiomers onplasma levels of NT-proBNP, IL-6and NF-κB.
Alternative pathways could mediate the inotropic or chronotropic effectsof doxazosin and its enantiomers at post-receptor levels. In order to reveal themechanism of different effect of doxazosin enantiomers on heart, we alsoinvestigated the influence of different pathway inhibitors, such as atropine,propranolol, indomethacin, phenoxybenzamine, verapamil, methylene blue andH-89, on the intropic effects of different doxazosin enantiomers in the isolatedrat left atrium strips.
Binding to plasma proteins plays a major role in drug therapy as the bounddrug is difficult to pass through blood vessel wall and cell membrane, whereas the unbound drug can cross the capillary wall to reach the cellular target as wellas metabolic tissue. A change in drug binding to plasma protein significantlyaffects its pharmacokinetic and pharmacodynamic properties. Because thedifference in the protein binding property between enantiomers often causes thedifference in their pharmacokinetic characters, enantioselective protein bindingstudy is essential to the understanding of their pharmacology, safety andclinical efficacy. Hence, it is necessary to stereoselectively detect and quantifyeach enantiomer of (±)doxazosin in biological media. In the present study, theequilibrium dialysis technique has been utilized to determine the plasma proteinbinding of the enantiomers of (±)doxazosin to the rat, dog and human plasma.Chiral HPLC-FL methods have been validated and employed to measure thedrug concentration on each side of equilibrium.
Part I Effects of doxazosin enantiomers on heart rate, contractile force andplasma heart failure biomarkers
In this part, the isolated mouse atrium strip were used to study therelationship between chiral structure of doxazosin, alfuzosin and their effectson heart, namely heart rate and contractile force. In order to understand theinfluence of different optical enantiomers of doxazosin on cardiac function ofrats, we studied the effect of long-term doxazosin or its enantiomersadministration on normal rat plasma NT-proBNP, NF-κB and IL-6levels.
1. Cardiac arrest effect of (±)doxazosin and (±)alfuzosin enantiomers onisolated mouse right atrium
Among16samples in (+)doxazosin group,5cases of cardiac arrest wereinduced at concentration of30μmol·L~(-1), so cardiac arrest rate was31.3%.(±)Doxazosin induced one cardiac arrest at30μmol·L~(-1), while (-)doxazosininduced one cardiac arrest at concentration of10μmol·L~(-1)and30μmol·L~(-1)respectively. No cardiac arrest was induced in (-)alfuzosin,(+)alfuzosin,(±)alfuzosin group and control group.
2. Impact of (±)doxazosin,(±)alfuzosin and their enantiomers on heart rate ofisolated mouse right atrium
Heart rates of isolated right atrium before administration of the drug were nearly same among all groups (P>0.05)，solvent has no effect on HR of isolatedright atrium (P>0.05).(+)doxazosin and (±)doxazosin at used concentrationssignificantly decreased the heart rate in a concentration-dependent manner(P<0.01)； HRs were decreased by (83.47±12.23)%and (74.97±15.66)%respectively induced by (+)doxazosin and (±) doxazosin at concentration of30μmol·L~(-1). The inhibition effect on HR of the isolated right atrium induced by(+)doxazosin was much stronger than (±)doxazosin at the same concentration(P<0.01).(-)Doxazosin has no effect on HR of isolated right atrium at3μmol·L~(-1)(P>0.05), while the inhibition effect on HR was significantly inferiorto (+)doxazosin at the same concentration (P<0.01).(-)Alfuzosin and(+)alfuzosin (10and30μmol·L~(-1)) decreased HR significantly, which were(13.05±7.27)%and (14.27±9.75)%respectively at concentration of30μmol·L~(-1),while (±)alfuzosin decreased HR only at30μmol·L~(-1)，and the three drugsinhibited HR at about the same degree.
3Different effect of (±)doxazosin,(±)alfuzosin and their enantiomers oncontractile force of isolated mouse left atrium.
Contractile force of left atrium in different groups were nearly same(P>0.05).The solvent has no effect on contractile force of isolated mouse leftatrium (P>0.05).(±)Doxazosin and (-)doxazosin at concentrations of3,10and30μmol·L~(-1)significantly strengthen contractile force of isolated mouse leftatrium (P<0.05) by a concentration-dependent manner. The positive inotropiceffect of (-)doxazosin (10and30μmol·L~(-1)) was stronger than (±)doxazosin ofthe same concentration (P<0.05). In contrast,(+)doxazosin has mild butsignificant negative inotropic effect (P<0.05), while (±)alfuzosin and itsenantiomers have no effect on contractile force of isolated mouse left atrium(P>0.05).
4Effect of long-term administration of doxazosin and its enantiomers onplasma NT-proBNP of rats
Plasma NT-proBNP concentrations in control group,(-)doxazosin group,(+)doxazosin group and(±)doxazosin group were475.55±150.21ng·L~(-1)、635.00±343.52ng·L~(-1)、375.55±127.09ng·L~(-1)and424.12±134.19ng·L~(-1) respectively. NT-proBNP concentration was lower in either (±)doxazosin groupor (+)doxazosin group than in control group, while concentration in(-)doxazosin group was higher than in control group, but the difference has nostatistical significance by single factor analysis of variance between groups(P>0.05). Plasma NT-proBNP concentration in (-)doxazosin group wassignificantly higher than in (+)doxazosin group (P<0.05).
5Effect of long-term administration of doxazosin and its enantiomers onplasma NF-κB of rats
Plasma NF-κB concentration in control group,(-)doxazosin group,(+)doxazosin group and (±)doxazosin group was888.46±343.36ng·L~(-1),705.77±193.34ng·L~(-1),1140.00±365.26ng·L~(-1)and910.38±276.39ng·L~(-1)respectively. NF-κB concentration in either (±)doxazosin group or(+)doxazosin group was higher than in control group, while concentration in(-)doxazosin group was lower than in control group, but there was no statisticalsignificance by single factor analysis of variance (P>0.05). Plasma NT-proBNPconcentration in (-)doxazosin group was significantly lower than in(+)doxazosin group (P<0.05).
6Effects of long-term administration of doxazosin and its enantiomers onplasms IL-6of rats
Plasma IL-6concentration of control group,(-)doxazosin group,(+)doxazosin group and (±)doxazosin group were47.1±23.9ng·L~(-1),71.5±23.7ng·L~(-1),59.4±23.1ng·L~(-1)and70.2±37.4ng·L~(-1)respectively. Difference amongthe four groups has no statistic significance (P>0.05).
These results demonstrate that the inhibit effect of (±)doxazosin and itsenantiomers on heart rate was stronger than that of (±)alfuzosin and itsenantiomers， and there exist chiral difference.(-)Doxazosin has positiveinotropic effect while (+)doxazosin has mild but significant negative inotropiceffect,(±)alfuzosin and its enantiomers with same concentration have no effecton contractile force of isolated left atrium. Chiral structure of doxazosin has anobvious effect on its bioactivity. Alfuzosin, however, decreases the HR slightlywithout chiral recognition in the isolated mouse atrium. Long-term administration of (-)doxazosin,(+)doxazosin and (±)doxazosin did not increaseor decrease plasma level of heart failure biomarker, namely NT-proBNP,NF-κB and inflammatory cytokines IL-6. Plasma NF-κB concentration in(+)doxazosin was higher than (-)doxazosin group, which showed that(+)doxazosin maybe affect immune and inflammation system.
Part II Mechanism of contractile force change induced by doxazosinenantiomers in isolated rat atrium
In this part, isolated rat left atrium strips were used to study the effect ofdifferent doxazosin enantiomers on myocardial contractile force. In order toprobe the exact mechanism of doxazosine enatiomers inotropic effects, isolatedrat left atrium were pretreated by different blocker such as atropine, propranolol,phenoxybenzamine, indomethacin, verapamil, methylene blue or H-89.1Inotropic effects of (-)doxazosin and (+)doxazosin on isolated rats left atrium
Solvent has no effect on contractile force of isolated rats right atrium.(P>0.05)；(-)doxazosin has significant positive inotropic effect on isolated ratsright arium (P<0.05)，percentages increase of contractile force induced by(-)doxazosin of3,10and30μmol·L~(-1)were (5.57±20.95)%,(6.96±39.77)%and (67.46±30.09)%respectively (P<0.05). On the contrary,(+)doxazosin hasnegative inotropic effect, the decrease in percentages of contractile forceinduced by (+)doxazosin of3,10,30μmol·L~(-1)were (~(-1)5.61±14.15)%,(-36.99±23.58)%and (-57.48±56.01)%respectively.2Impact of atropine and propranolol on cardiac inotropic effects of(-)doxazosin and (+)doxazosin
After the isolated rat atrium were pretreated with atropine and propranolol,the solvent has no effect on contractile force. The percentages increase ofcontractile force induced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (13.02±16.66)%,(53.65±41.02)%and (70.87±52.66)%respectively. The decrease in percentage of contractile force induced by(+)doxazosin at concentrations of3,10and30μmol·L~(-1)were (-8.02±21.43)%,(-21.60±21.50)%and (-63.75±24.14)%respectively.
3Impact of phenoxybenzamine on cardiac inotropic effects of (-)doxazosin and (+)doxazosin
After the isolated rat atrium were pretreated with phenoxybenzamine, thesolvent has no effect on contractile force. The percentages increase ofcontractile force induced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (13.71±14.39)%,(35.48±11.90)%and (71.62±37.20)%respectively. The decrease in percentage of contractile force induced by(+)doxazosin were (~(-1)4.08±23.48)%,(-33.08±17.85)%and (-60.73±17.21)%respectively. After pretreated with ethanol, the solvent has no effect oncontractile force. The percentages increase of contractile force induced by(-)doxazosin at concentration of3,10and30μmol·L~(-1)were (19.05±7.14)%,(49.27±28.71)%and (71.21±35.57)%respectively. The decrease in percentageof contractile force induced by (+)doxazosin were (~(-1)0.40±14.76)%,(-23.73±26.18)%and (-65.63±6.52)%respectively.
4Impact of indomethacin on cardiac inotropic effects of (-)doxazosin and(+)doxazosin
After the isolated rat atrium was pretreated with indomethacin, the solventhas no effect on contractile force. The percentages increase of contractile forceinduced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (19.87±13.06)%,(55.73±18.00)%and (60.25±35.25)%respectively. The decrease inpercentages of contractile force induced by (+)doxazosin were (-7.23±13.39)%,(-23.73±26.18)%and (-65.63±6.52)%respectively. After pretreated withethanol, the percentage increase of contractile force induced by (-)doxazosin atconcentrations of3,10and30μmol·L~(-1)were (19.05±7.14)%,(49.27±28.71)%and (71.21±35.57)%respectively. The decrease in percentage of contractileforce induced by (+)doxazosin were (~(-1)0.40±14.76)%,(-23.73±26.18)%and(-65.63±6.52)%respectively.
5Impact of verapamil on cardiac inotropic effects of (-)doxazosin and(+)doxazosin
After the isolated rat atrium was pretreated with verapamil, the solvent hasno effect on contractile force. The percentage increase of contractile forceinduced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (24.81±20.49)%,(75.98±23.00)%and (131.15±17.56)%respectively. Thedecrease in percentage of contractile force induced by (+)doxazosin atconcentration of3,10and30μmol·L~(-1)were (-9.60±17.19)%,(-33.47±9.43)%and (-72.23±14.65)%respectively.
6Impact of methylene blue on cardiac inotropic effects of (-)doxazosin and(+)doxazosin
After the isolated rat atrium was pretreated with methylene blue, thesolvent has no effect on contractile force. The percentages increase ofcontractile force induced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (36.26±9.82)%,(65.99±26.27)%and (16.02±35.33)%respectively. The decrease in percentage of contractile force induced by(+)doxazosin at concentrations of3,10μmol·L~(-1)were (-5.57±17.32)%,(~(-1)0.54±25.09)%,(-58.27±15.47)%respectively.
7Impact of H-89on cardiac inotropic effects of (-)doxazosin and (+)doxazosin
After the isolated rat atrium was pretreated with H-89, the solvent has noeffect on contractile force. The percentages increase of contractile forceinduced by (-)doxazosin at concentrations of3,10and30μmol·L~(-1)were (28.96±9.49)%,(81.93±20.75)%and (101.53±51.95)%respectively. The decreasein percentage of contractile force induced by (+)doxazosin was(~(-1)7.71±16.37)%,(-30.76±18.38)%and (-67.67±10.74)%respectively.
These results demonstrated that the positive inotropic effect of(-)doxazosin on isolated rat left atrium was independent of its α-receptorblocking effect, also did not related to M-receptor, β-receptor, PG enzyme.L-type Ca~(2+)channel, while intracellular cGMP and PKA maybe participatedpartly in the positive inotropic effect of (-)doxazosin. But, cardial myocyteα-receptor, M-receptor, β-receptor, L-type Ca~(2+)channel, PG enzyme, cGMP orPKA maybe not related to the negative inotropic effect of (+)doxazosin.
Part III Stereoselective binding of doxazosin enantiomers to plasmaproteins from rat, dog and human
Binding to plasma proteins plays a major role in drug therapy. A change indrug binding to plasma protein significantly affects its pharmacokinetic and pharmacodynamic properties. Because the difference in the protein bindingproperty between enantiomers often causes the difference in theirpharmacokinetic characters, enantioselective protein binding study is essentialto the understanding of their pharmacology, safety and clinical efficacy. Hence,it is necessary to stereoselectively detect and quantify each enantiomer of(±)doxazosin in biological media. Here, we report a first application of chiralHPLC methods using a chiral stationary phase column in conjunction withequilibrium dialysis for the simultaneous determination of the protein bindingof each enantiomer of doxazosin in rat, dog and human plasma after addition ofthe racemate in vitro.
1Method validation
The chiral HPLC methods for the separation of doxazosin enantiomers werevery selective.Prazosin,(-)doxazosin and (+)doxazosin were clearly wellresolved from the matrix components under the chromatographic conditionsemployed, and the two enantiomers of doxazosin were baseline resolved fromeach other.
The calibration curves (weight1/x2) of the two enantiomers of doxazosinwere linear with correlation coefficients greater than0.9990over theconcentration range of50to1600ng·mL~(-1)for plasma and12.5to200ng·mL~(-1)for PBS. The precision and accuracy of the method were also investigated. Theprecision (relative standard deviation, RSD) based on five repetitive injectionsat drug concentrations of0.2,0.5and1μg·mL~(-1)was less than3.2%(intra-day)and6.3%(inter-day) for the plasma extracts of all three species. The accuracywas found to be in the range of90.5%-102.4%for all plasma and PBS buffermatrices at three drug concentration levels. The detection limit, at asignal-to-noise ratio of3, in plasma of all three species was about0.2ng·mL~(-1)for doxazosin. The enantiomers of doxazosin were stable during the entirecourse of the study including sample preparation, centrifugation and HPLCassay.
2Optimization of equilibrium dialysis conditions
The time taken to reach equilibrium for doxazosin between plasma and isotonic PBS was investigated. Isotonic PBS spiked with (±)doxazosin (800ng·mL~(-1)) was dialyzed against pooled human plasma at0,4,8,15, and20h.(-)Doxazosin and (+)doxazosin crossed the dialysis membrane rapidly andreached a steady-state level in human plasma within15h at37°C. Theequilibrium time for dog or rat plasma was the same to that for human plasma.Therefore, equilibrium dialysis duration was15h in the subsequent equilibriumdialysis experiments at incubation temperature of37°C (body temperature).
3Difference in protein binding between the enantiomers
In the concentration range between200and800ng·mL~(-1)of (±)doxazosin,the percentage of plasma protein binding of enantiomers in the three specieswere89.4%-94.3%for (-)doxazosin and90.9%-95.4%for (+)doxazosin. Theresults showed that either (-)doxazosin or (+)doxazosin was highly bound toplasma proteins, which is consistent with the previous investigationsdetermining the binding of racemic doxazosin. Furthermore, Cb values of(-)doxazosin were significantly smaller than those of (+)doxazosin (P<0.05) ineach plasma from the three species at the used concentrations of (±)doxazosin.Difference in Cb between the two enantiomers became larger with theincreased concentration of (±)doxazosin regardless of the species used.Moreover, the stereoselectivity of doxazosin in dog plasma was moresignificant than that in rat plasma and human plasma. The mean Cb values(ng·mL~(-1)) of (-)doxazosin vs (+)doxazosin for dog plasma in the PBS contained(±)doxazosin200,400, and800ng·mL~(-1)were200.0vs243.0,398.6vs477.3,and845.1vs1006.8, respectively. Overall, enantioselective binding ofdoxazosin enantiomers to plasma proteins from rat, dog and human wasobserved, and (+)doxazosin exhibited a higher protein-binding capacity than(-)doxazosin in all three species.
A difference in plasma protein-binding capacity between the twoenantiomers may lead to their pharmacokinetic behavior being different fromeach other. As drug clearance from the blood is directly proportional to the itsfree fraction in plasma, a higher unbound (-)doxazosin concentration in humanplasma observed in the present study might partly explain the phenomenon reported by Liu et al. They found that the human plasma concentration of(-)doxazosin is lower than that of (+)doxazosin after administration of theracemate. Indeed, in our laboratory, the different plasma concentration of thetwo enantiomers in dog and rat was observed after administration of(-)doxazosin and (+)doxazosin. Therefore, the discrepancy for plasmaprotein-binding capacity between (-)doxazosin and (+)doxazosin is a commonreason for their different pharmacokinetic parameters in rat, dog and human.
4Difference in protein binding among three species
In the case of200ng·mL~(-1)of racemic doxazosin added to the PBS, nosignificant difference in percentage of plasma protein binding among threespecies was found. However, the percentage of plasma protein binding in dogplasma was significantly smaller than that in human plasma at400and800ng·mL~(-1)of racemic doxazosin (P<0.05and P<0.01), which is consistent withthe previous report that plasma protein binding of (±)doxazosin in human washigher than that in dog. Since total protein concentrations of the pooled plasmafrom rat, dog and human were61.81,51.35and57.75mg·mL~(-1)respectively;the value of percentage of plasma protein binding should be corrected with thevalue of protein assay. The corrected percentage of plasma protein binding ofthe enantiomers at a given concentrations was variable in different species:dog> human> rat (P<0.01).
These results demonstrate that either (-)doxazosin or (+)doxazosin washighly bound to plasma proteins. Moreover, protein binding of doxazosinenantiomers in human, dog and rat plasma revealed that there was a significantdifference in bound fractions, i.e., a higher protein-binding capacity of(+)doxazosin than (-)doxazosin, which might be one of the reasons for anobviously difference in plasma concentration between doxazosin enantiomersin human orally administered (±)doxazosin. Additionally, a pronounced speciesdifference in the plasma protein binding was found in the present study, with ahigher protein-binding capacity in human than dog. Since the total proteinconcentrations of the plasma were significantly different among rat, dog andhuman, the corrected percentage of plasma protein binding was calculated in the study, which indicates an order of dog> human> rat (P<0.01). Therefore,the findings of a stereoselective plasma protein binding between (-)doxazosinand (+)doxazosin and a pronounced species difference in the plasma proteinbinding of rat, dog and human should be useful for explaining thepharmacokinetic characters of chiral doxazosin.
Conclusion
Doxazosin has obvious effects on the heart rate and cardiac contractility ofisolated mouse atrium. Doxazosin could induce cardiac arrest reaction at highconcentration, chiral structure of doxazosin has obvious influence on its activity.In contrast, alfuzosin only slightly inhibited the mouse heart rate; chiralstructure of alfuzosin has no obvious effect on its cardiac effects. Long-termadministration of (-)doxazosin,(+)doxazosin and (±)doxazosin did not increaseor decrease plasma level of heart failure biomarker, namely NT-proBNP, NF-κB and IL-6. Plasma NF-κB concentration in (+)doxazosin was higher than(-)doxazosin group, which showed that (+)doxazosin maybe affect immune andinflammation system.
The positive inotropic effect of (-)doxazosin on isolated rat left atrium wasindependent of its α-receptor blocking effect, also did not related to M-receptor,β-receptor, PG enzyme. L-type Ca~(2+)channel, while intracellular cGMP andPKA maybe participated partly in the positive inotropic effect of (-)doxazosin.But, cardial myocyte α-receptor, M-receptor, β-receptor, L-type Ca~(2+)channel,PG enzyme, cGMP or PKA maybe not related to the negative inotropic effectof (+)doxazosin.
Either (-)doxazosin or (+)doxazosin was highly bound to plasma proteins.Moreover, protein binding of doxazosin enantiomers in human, dog and ratplasma revealed that there was a significant difference in bound fractions, i.e., ahigher protein-binding capacity of (+)doxazosin than (-)doxazosin, whichmight be one of the reasons for an obviously difference in plasma concentrationbetween doxazosin enantiomers in human orally administered (±)doxazosin.Additionally, a pronounced species difference in the plasma protein bindingwas found in the present study, with a higher protein-binding capacity in human than dog. Since the total protein concentrations of the plasma were significantlydifferent among rat, dog and human, the corrected percentage of plasma proteinbinding was calculated in the study, which indicates an order of dog> human>rat (P<0.01). Therefore, the findings of a stereoselective plasma protein bindingbetween (-)doxazosin and (+)doxazosin and a pronounced species difference inthe plasma protein binding of rat, dog and human should be useful forexplaining the pharmacokinetic characters of chiral doxazosin.
引文
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7卢海刚,赵丁,任雷鸣.多沙唑嗪对映体对兔离体膀胱逼尿肌的作用及机制.中国药理学通报,2008,24(4):513-517
8Lefèvre-Borg F, O'Connor SE, Schoemaker H, et al. Alfuzosin, a selectivea1-adrenoceptor antagonist in the lower urinary tract [J]. Br J Pharmacol.1993,109(4),1282-1289
9卢海刚,刘丽芳,任雷鸣等.多沙唑嗪对映体对兔四种血管α受体的作用.药学学报,2007,42(2):145-151
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12田和林,任雷鸣,何东伟,等.多沙唑嗪对映体对大鼠血压和排尿功能的影响.中国药理学通报,2007,23(2):240-246
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15王荣英,任雷鸣.多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖的选择性抑制作用,中国药理学通报,2009,25(1):81-84
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19Costello-Boerrigter LC, Boerrigter G, Redfield MM, et al. Amino-terminalpro-B-type natriuretic peptide and B-type natriuretic peptide in the generalcommunity: determinants and detection of left ventricular dysfunction. JAm Coll Cardiol,2006,47(2):345-353
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29Chapman N, Chang CL, Dahlof B, Sever PS, Wedel H, Poulter NR, ASCOTInvestigators. Effect of doxazosin gastro-intestinal therapeutic system asthird-line antihypertensive therapy on blood pressure and lipids in theAnglo-Scandinavian Cardiac Outcomes Trial. Circulation,2008,118(1):42-48
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2The ALLHAT officers and coordinators for the ALLHAT collaborativeresearch group. Major cardiovascular events in hypertensive patientsrandomized to doxazosin vs chlorthalidone. JAMA,2000,283(15):1967-1975
3Packer M. The neurohormonal hypothesis: a theory to explain themechanism of disease progression in heart failure. J Am Coll Cardiol.1992,20(1):248–254
4Seta Y, Shan K, Bozkurt B, Oral H, Mann DL. Basic mechanisms in heartfailure: the cytokine hypothesis. J Card Fail.1996,2(3):243–249
5Wong SC, Fukuchi M, Melnyk P, et al. Induction of cyclooxygenase-2andactivation of nuclear factor-kappa B in myocardium of patients withcongestive heart failure. Circulation,1998,98(2):100-103
6Hatano A, Tang R, Walden PD, Lepor H. The alpha-1adrenoceptorantagonist properties of the enantiomers of doxazosin in human prostate.Eur J Pharmacol,1996,313(1-2):135-143
7卢海刚,赵丁,任雷鸣.多沙唑嗪对映体对兔离体膀胱逼尿肌的作用及机制.中国药理学通报,2008,24(4):513-517
8Lefèvre-Borg F, O'Connor SE, Schoemaker H, et al. Alfuzosin, a selectivea1-adrenoceptor antagonist in the lower urinary tract [J]. Br J Pharmacol.1993,109(4),1282-1289
9卢海刚,刘丽芳,任雷鸣等.多沙唑嗪对映体对兔四种血管α受体的作用.药学学报,2007,42(2):145-151
10Niu CQ, Zhao D, Jia XM, Ren LM. α1-Adrenoceptor antagonist profile ofdoxazosin and its enantiomers in isolated rabbit blood vessels. Chin JPharmacol Toxical,2003,17(5):354-359
11Ma SP, Ren LM, Zhao D, et al. Chiral selective effects of doxazosinenantiomers on blood pressure and urinary bladder pressure in anesthetizedrats. Acta Pharmac Sin,2006,27(11):1423-1430
12田和林,任雷鸣,何东伟,等.多沙唑嗪对映体对大鼠血压和排尿功能的影响.中国药理学通报,2007,23(2):240-246
13Martin DJ, Lluel P, Guillot E, et al. Comparative alpha-1adrenoceptorsubtype selectivity and functional uroselectivity of alpha-1adrenoceptorantagonists. J Pharmacol Exp Ther,1997,282(1):228-235
14Lee AS, Chen WP, Su MJ. Comparison of the cardiac electrophysiologicaleffects between doxazosin and bunazosin. J Biomed Sci,2008,15(4):519-528
15王荣英,任雷鸣.多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖的选择性抑制作用,中国药理学通报,2009,25(1):81-84
16Koller KJ, Lowe DG, Bennett GL, et al. Selective activation of the Bnatriuretic peptide receptor by C-type natriuretic peptide (CNP). Science,1991,252(5002):120-123
17Goetze JP. Biochemistry of pro-B-type natriuretic peptide-derived peptides:the endocrine heart revisited. Clin Chem.2004,50(9):1503-1510
18Braunwald E. Biomarkers in heart failure. N Engl J Med,2008,358(20):2148-2159
19Costello-Boerrigter LC, Boerrigter G, Redfield MM, et al. Amino-terminalpro-B-type natriuretic peptide and B-type natriuretic peptide in the generalcommunity: determinants and detection of left ventricular dysfunction. JAm Coll Cardiol,2006,47(2):345-353
20Smith JG, Newton-Cheh C, Almgren P, et al. Assessment of conventionalcardiovascular risk factors and multiple biomarkers for the prediction ofincident heart failure and atrial fibrillation. J Am Coll Cardiol,2010,56(21):1712-9171
21Baeuerle PA, Baltimore D. NF-kappa B: ten years after. Cell,1996,87(1):13-20
22Baldwin AS Jr. The NF-kappa B and I kappa B proteins: new discoveriesand insights. Annu Rev Immunol,1996,14:649-683
23Kalra D, Baumgarten G, Dibbs Z, et al. Nitric oxide provokes tumornecrosis factor-alpha expression in adult feline myocardium through acGMP-dependent pathway. Circulation,2000,102(11):1302-1307
24Mann DL, Young JB. Basic mechanisms in congestive heart failure.Recognizing the role of proinflammatory cytokines. Chest,1994,105(3):897-904
25Finkel MS, Oddis CV, Jacob TD, et al. Negative inotropic effects ofcytokines on the heart mediated by nitric oxide. Science,1992,257(5068):387-389
26Finkel MS, Hoffman RA, Shen L, et al. Interleukin-6(IL-6) as a mediatorof stunned myocardium. Am J Cardiol,1993,71(13):1231-1232
27Orus J, Roig E, Perez-Villa F, et al. Prognostic value of serum cytokines inpatients with congestive heart failure. J Heart Lung Transplant,2000,19(5):419-425
28Deswal A, Petersen NJ, Feldman AM, et al. Cytokines and cytokinereceptors in advanced heart failure: an analysis of the cytokine databasefrom the Vesnarinone trial (VEST). Circulation,2001,103(16):2055-2059
29Chapman N, Chang CL, Dahlof B, Sever PS, Wedel H, Poulter NR, ASCOTInvestigators. Effect of doxazosin gastro-intestinal therapeutic system asthird-line antihypertensive therapy on blood pressure and lipids in theAnglo-Scandinavian Cardiac Outcomes Trial. Circulation,2008,118(1):42-48
1The ALLHAT officers and coordinators for the ALLHAT collaborativeresearch group. Major cardiovascular events in hypertensive patientsrandomized to doxazosin vs chlorthalidone. JAMA,2000,283(15):1967-1975
2Movsesian MA. Beta-adrenergic receptor agonists and cyclic nucleotidephosphodiesterase inhibitors: shifting the focus from inotropy to cyclicadenosine monophosphate. J Am Coll Cardiol,1999,34(2):318-324.
3Zhao D, Duan LH, Wang FY, et al. Chiral recognition of doxazosinenantiomers in3targets for therapy as well as adverse drug reactions inanimal experiments. Can J Physiol Pharmacol,2012,90(12):1623-1633
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