【作者】 甘乐凌；

【导师】 王敏伟；

【作者基本信息】 沈阳药科大学 ， 药理学， 2003， 博士

【摘要】 SPFF（2-（4-氨基-3-氯-5-三氟甲基苯基）-2-（叔丁氨基）-乙醇盐酸盐）是沈阳药科大学药物合成室新合成的一种新型选择性β₂肾上腺素受体激动剂。前期工作表明该化合物具有明显的扩张气道作用。本文对该化合物的药效学、作用机理、受体学特征以及初步安全性进行了考察。 药效学研究表明，在离体豚鼠气管条模型中，SPFF可以剂量依赖性地降低静息气管条的张力，该作用的pD₂值7.66±0.68，而选择性β₂肾上腺素受体拮抗剂ICI-118551可竞争性阻断SPFF的这种作用，这表明SPFF为一个选择性的β₂肾上腺素受体（β₂AR）激动剂。SPFF还可抑制由组胺（His）或乙酰胆碱（Ach）引起的气管条张力升高作用，其最大抑制率分别为146％和82.6％，其IC₅₀值则分别为550±380 nmol·L^-1和3.8±2.5μmol·L^-1，进一步分析发现，SPFF对组胺的抑制作用是一种非竞争的拮抗作用；此外，SPFF(3×10^-3×10^-9mol·L^-1)对由组胺引起的离体豚鼠肺灌流量减少具有显著的抑制作用。上述离体实验结果表明SPFF对整个气道都具有显著的扩张作用。在整体动物中，SPFF（i.g）0.0625、0.125、0.25、0.5、1mg／kg对由His和Ach混合喷雾引起的豚鼠喘息具有剂量依赖性的保护作用，其作用的ED₅₀值为0.32±0.05 mg／kg，该作用强度是沙丁胺醇(SAB，ED₅₀值为2.37±0.22 mg／kg)的近8倍。在肺溢流实验（Konzett and R（?）ssler实验）中，当静脉给药时，SPFF和SAB（1、3、9μg／kg）均在给药后30s内显著降低由于Ach静注引起的溢流量升高，但SPFF的作用强度略高；十二指肠给药（i.d）时，SPFF（1、8、80μg／kg）和SAB（80μg／kg）均在给药后120 min内发挥类似的气道扩张作用，但SPFF的作用持续时间长于SAB。对肺机械功能影响实验结果表明，SPFF（i.d）20、40、80μg／kg可以显著改善由His喷雾引起的家兔肺顺应性和肺阻力的改变，但对肺顺应性的改善作用较为明显，表明本药物对小气道的扩张作用比较强。SPFF(3×10^-8-3×10^-6mol·L^-1)还可抑制卵白蛋白在致敏大鼠肺组织沈阳药科大学博士学位论文摘要中诱发的组胺释放，且该作用可为普蔡洛尔（PRO）所阻断，这表明SPFF可通过激动pZAR稳定肺大细胞膜而抑制过敏物质的释放。sPFF 1 .9 0.04、0.1、0.25 mg/kg还可以显著增加家鸽气管纤毛运动的转运速率，且同时不增加气道粘液的排泌，表明该药物对哮喘时出现的气道栓塞具有一定的清除作用。 受体学分析表明，sPFF对离体豚鼠左心房仅有很小的正性变时作用（pD:，5.41土0.38），该作用强度仅为异丙肾上腺素（150）的1/2200，通过使用离体器官功能性实验所得出sPFF对pZAR的选择系数（109一’[pDZ（traehea升pDZ（atria）]）为178，远高于150（1）。在以pZAR为主的肺组织细胞膜制备物，以及以伪AR为主的心脏组织细胞膜制备物中，以pAR特异性的氖标双氢心得舒（’H一DHA）为标记配基对SF）FF进行的竞争结合实验研究结果表明，SPFF可与标记物竞争结合膜制备物上的同一个结合位点，SPFF与肺和心脏细胞膜制备物竞争结合的工c50分别为27.3nm。1.L一1和126.1 nmol.L一l，因此该化合物在此体系中的选择系数为4.6，是:〔50（0.36）的12.8倍。SPFF 1.9（0.25、0.5、lmg/kg）还可剂量依赖性的强烈升高大鼠肺组织内的cAMP水平，其作用大大强于相同剂量的SAB。在原代培养的豚鼠气管平滑肌细胞中，sPFF 2.5x10一9、5、10一8、10一“mol几一l显示出对细胞内钙水平有显著的降低作用，此作用同样可为PRO所阻断。据此，可认为SPFF的气道扩张作用主要是通过pZAR一cAMP一ca2+这一信号转导通路而发生的。当sPFF以1、10一6mol一，浓度与离体豚鼠气管条共同孵育lh时，不能诱发明显的急性耐受作用，而SAB却可诱发明显的耐受作用。但当SPFF 1 .9 0.2 mg/kg，给药18天时，该药物在哮喘动物中的cAMP水平升高作用明显降低，而放射配基结合实验也表明，这些动物肺组织内 pZAR数量还出现了下降，同时受体结合能力也有所降低。 对SPFF的初步安全性评价结果表明，SPFF 1.9和i，v时的LD50（95%可信限）分别为378.5（331.8一425.2）mg/kg和38.7（34.1一43.4）mg/kg。与该药物的临J未剂量相比，毒性比较低。而使用5、50、500 pg/kg剂量在家猫中（1.9）所进行的一般药理实验中，该药物除能引起短暂的肌肉震颤外没有其他明显的副作用产生，对小鼠自主活动（2、20、200 09/kg，1.9）以及延长戊巴比妥诱导睡眠时间（50、100、200 09/kg，1.9）的实验则表明，该药物对神经系统可能有轻微的抑制作用。对该药物的致突变研究结果表明，SPFF在所考察的剂量范围内没有致突变作用。 综上所述，SPFF是一个高选择性的伪AR激动剂，该药物在激动氏AR后，通沈阳药科大学博士学位论文摘要过AC一cAMP一PKA途径而显著扩张气道平滑肌，稳定肥大细胞，增强气道纤毛运动能力，从而发挥抗哮喘作用。由于结构的关系，该药物的作用时间也较长。本药物还因受体选择性较高而较少引发相关的副作用，因而安全性较高。但由于SPI下仍然具有导致受体脱敏的可能性，因此本化合物不宜长期单独使用。本研究为SPI邓的进一步开更多还原

【Abstract】 SPFF, [2- (4-Amino-3-chloro-5-trifluomethyl-phenyl)-2-tert.-butylamino-ethanol chloride], was a newly synthesized β 2 adrenergic agonist by us. In this paper, we examined the bronchodilating effects, the receptorological characteristics and the action mechanism of this compound. And a part of safety evaluation work was also carried out.We studied in vitro the bronchodilating effects of SPFF and isoprenaline (ISO) on isolated guinea pig trachea strips with or without the precontraction of bronchocontractors (His and Ach). SPFF and isoprenaline dose-dependently decreased the resting tension of isolated guinea pig trachea strips with pD2 values of 7.66±0.68 and 8.79±0.19, respectively. SPFF also dose-dependently relaxed the isolated guinea pig trachea strip precontraction with His or Ach, and the maximum inhibition rate of the effects was 146 % and 82.6 %, respectively. Moreover, we confirmed that the bronchodilator effect of SPFF was due to the activation of β 2 adrenoceptor because this effect was easily antagonized by ICI-118551 (pA2 8.9010.01), a specific β 2 adrenoceptor antagonist. In addition, SPFF (3×10-3 -3×10-9mol.L-1) significantly inhibited the bronchoconstriction effect of histamine in isolated lung perfusion model in guinea pigs.The protective effects of SPFF and SAB on bronchospasm induced by bronchoconstrictor aerosol in guinea pigs in vivo were investigated, and the Konzett and Rossler experiment in rabbits in vivo was also performed. The results showed that SPFF significantly prolonged the latency time of histamine and acetylcholine induced asphyxiation collapse in guinea pigs: the ED50 value of SPFF i.g. was 0.32±0.05 mg/kgin this experiment. Meanwhile, the EDso values of SAB was 2.37±0.22, which meant that the bronchorelaxation effect of salbutamol was about 8 times less potent than that of SPFF in vivo. The result of the Konzett and Rossler experiment performed in anesthetized rabbits showed that intraduodenal administration of SPFF exerted an action of longer duration than salbutamol. SPFF (i.d) 20 , 40 , 80 ug/kg also significantly improved the alteration of lung compliance and resistance induced by His aerosol in anesthetized rabbits. From the results above we suggested that SPFF was a potent, long-acting bronchodilator with relatively higher β2 adrenoceptor selectivity. Moreover, SPFF(3×10-8 -3×10-6 mol.L-1) inhibited the release of histamine in ovalbumin-allergic rats lung fragments, which indicated a mast cell protection effect of this compound. In the investigation of the effect of SPFF on the mucus movement, it was shown that SPFF i.g 0.04,0.1, 0.25 mg/kg improved the cilium transportation function on the trachea of conscious pigeon while had no effect on the air way mucus excretion in mice, these results implied that, by prompting the clearance of airway, SPFF had an additional beneficial effect on the alleviation of asthma symptom.The positive chronotropic effects of SPFF in isolated guinea pig left atria (pD:2 , 5. 41±0. 38) was 2200 times less potent than that of ISO, and the B2AR selectivity index (log-1[pD2(trachea)-pD2(atria)]) obtained from function experiments was 178 and 1, respectively, for SPFF and ISO. Meanwhile, the selectivity index obtained from radio-ligand competitive assay using 3H-DHA for SPFF and ISO was 4.6 and 0.36, respectively. This result indicated that SPFF had a higher affinity to P2AR than ISO. As SPFF i.g (0.25, 0.5,1mg/kg) dramatically elevated the cAMP level in the lung tissue in rat, and significantly decreased the Ca2+ level in primary cultures of guinea pig trachea smooth muscle cells, we confirmed that the signal transduction pathway of SPFF should also be protein G - cAMP - Ca2+, which was the same as other p2 agonists. The desensitization characteristic of SPFF was studied in vitro and in vivo. In isolated guinea pig trachea strips, when incubated with SAB or SPFF at the concentration of 1×10-6 mol.L-1 for 1 h, severe tachyphylaxis was induced by SAB, meanwhile, SPFF did not induced tachy更多还原