5,7,3'-三甲基橙皮素的抗氣喘作用與初、後期氣喘 動物模式之探討
Anti-asthmatic action of
hesperetin-5,7,3'-O-trimethylether and investigation of asthmatic animal model with early- and late-phases of airway hyperresponsiveness
中文摘要
PART1 橙皮素 (hesperetin) 是一種選擇性 PDE4 的抑制劑，我們曾報告對氣喘治療具有潛 力，我們為提高其 PDE4H/PDE4L 比，所以合成 5,7,3'-三甲基橙皮素
(hesperetin-5,7,3'-O-trimethylether, HTME) ，並研究其抗氣喘的效果。
將雌 BALB/c 小鼠腹腔內注射卵蛋白 (ovalbumin, OVA) ，使其敏感化，再以氣化的卵蛋白 (OVA) 二次激釁 (secondary challenge)，利用整體體積描述器來分析因 methacholine (MCh) 引起的氣道過度反應 (airway hyperresponsiveness; AHR)，結果顯示 HTME 能抑 制卵蛋白 (OVA) 引起的氣道過度反應 (AHR)，HTME (10~100 μmol/kg, p.o.)能劑量依存 性且有意義地 (P < 0.05) 減少 MCh (25~50 mg/ml) 引起的 enhanced pause (Penh) 值增加。HTME (10~100 μmol/kg, p.o.) 有意義地 (P < 0.05) 減少肺泡灌洗液中總發炎 細胞數、嗜中性血球、嗜伊紅白血球、淋巴球及巨噬細胞，但最低劑量不能有意義地減少嗜中性 白血球及巨噬細胞是例外外。HTME (10~100 μmol/kg, p.o.) 也會有意義地 (P < 0.05) 降 低肺泡灌洗液中 IL-2, IL-4, IL-5, IFN- 和 TNF- 的釋放，也會有意義地 (P < 0.05) 減 少血清和肺泡灌洗液中的 total- 和 OVA-specific IgE，但最低劑量不能減少血清中 total IgE 的量是例外。
HTME 會濃度依存性地抑制 PDE1、PDE3 及 PDE4，其 IC50 值分別為 18.86、14.38、9.39 μM。HTME 取代結合在敏感化天竺鼠全腦細胞顆粒 HARBS 之 [3H]-rolipram 的 EC50 值為 171.5 μM，因此 HTME 的 PDE4H/PDE4L 比值約為 18.2。由 Lineweaver-Burk 分析發現 HTME 對 PDE1、PDE 3 及 PDE4 呈競爭性的抑制，其 Ki 值分別為 40.3, 21.9, 及 7.6 μM，
對 PDE3 和 PDE4 的 Ki 值雖然彼此間無意義差，但兩者均有意義地小於對 PDE1 的 Ki 值，顯 示對 PDE3 及 PDE4 的親和力最好，其次是 PDE1。 HTME (10~30 M) 會有意義地鬆弛敏 感化離體天竺鼠氣管的基本張力，也有意義地抑制累加 OVA (10~100 g/ml) 引起的敏感 化離體天竺鼠氣管之收縮，甚至於 HTME 3 μM 能有意義地抑制 OVA 100 μg/ml 引起的收縮。
結論，HTME 對 PDE4 的親和力最高，具有選擇性且競爭性地抑制 PDE4，也同時抑制 PDE3，
因此有加強的效果，又因其 PDE4H/PDE4L 比是 18.2，所以推測此藥物極具治療氣喘的潛力。
PART 2 本篇藉由 methylprednisolone、pyrilamine、cromolyn 三種臨床上使用的藥物，
來探討具有初、後期氣道過度反應的氣喘動物模式之可用性。
將雌 BALB/c 小鼠腹腔內注射卵蛋白 (ovalbumin; OVA) ，使其敏感化，再以卵蛋白 (OVA) 氣化噴霧激釁，利用整體體積描述器來分析因 methacholine (MCh, 50 mg/ml) 引起的氣道 過度反應 (airway hyperresponsiveness ; AHR)，結果顯示，methylprednisolone (10 mg/kg, s.c.)、pyrilamine (25 mg/kg, s.c.)或 cromolyn (2%, inhalation) 在氣道過度 反應初期會有意義地抑制 MCh 所引起的 enhanced pause (Penh) 值的增加。而在氣道過度
反應後期，則只有 methylprenisolone、pyrilamine 有意義地抑制 MCh 所引起的 Penh 值增 加。Methylprednisolone、pyrilamine、cromolyn 和對照組比較沒有意義地減少肺泡灌洗液 中的發炎細胞和細胞介素。Methyprednisolone、pyrilamine、cromolyn 有意義地 (P < 0.05) 減少肺泡灌洗液和血清中的 total IgE，methylprednisolone 和 cromolyn，但非 pyrilamine，
能有意義地 (P < 0.05)抑制肺泡灌洗液中的 OVA-specific IgE，然而三者都不能有意義地減 少血清中的 OVA-specific IgE。
本實驗結果，由於 (1) methylprednisolone 無法抑制血清中的 OVA-specific IgE，與臨床 效果不同; (2) 此三種藥物無法有意義地抑制發炎細胞的增加; (3) 對照組都無法使 IL-2、IL-4 及 TNF-α 增加，因此我們對此動物模式持保留態度，不加以推薦。
英文摘要
PART 1 Hesperetin, a selective PDE4 inhibitor, has been reported to have a potential in the treatment of asthma in our laboratory. To improve its PDE4H/PDE4L ratio, we synthesized hesperetin-5,7,3’-trimethyl ether (HTME), and investigated its
anti-asthmatic effects.
In the sensitized and OVA-secondarily challenged BALB/c mice, an asthmatic animal model, the airway hyperresponsiveness (AHR) was measured in unrestrained animals by barometric plethysmography using a whole-body plethysmograph after exposure of methacholine (MCh, 6.25~50 mg/ml) and enhanced pause (Penh) values were determined. In the present results, HTME (10~100 mol/kg, p.o.) dose-dependently and significantly suppressed the enhancement of MCh (25~50 mg/ml)-induced Penh values. It also significantly reduced the increase of total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils with an exception that at the least dose HTME did not significantly suppressed
marcrophages and neutrophils, and significantly reduced the release of IL-2, IL-4, IL-5, TNF- , and IFN- in brochoalveolar lavage fluid (BALF). It also significantly reduced total and OVA-specific IgE in serum and BALF, with an exception that at the least dose HTME did not significantly reduced total IgE in BALF.
HTME concentration-dependently inhibited PDE1, PDE3, and PDE4 with an IC50 value of 18.86, 14.38, and 9.39 M, respectively. HTME displaced [3H]-rolipram from high affinity rolipram binding sites (HARBS) of particulates of whole brains isolated from sensitized guinea pigs, with an EC50 value of 171.5 M. Therefore, the PDE4H/PDE4L ratio of HTME was 18.2. From Lineweaver-Burk analysis, HTME competitively inhibited PDE1, PDE3, and PDE4 activities, with a Ki value of 40.3, 21.9, and 7.5 M, respectively. Although the later two values did not significantly differ from each other, however, the former value was significantly greater than the later two values. It suggests that HTME has a high affinity to PDE3 and PDE4, then to PDE1. HTME (10~30 M) significantly relaxed the baseline tension and
suppressed OVA (10~100 g/ml)-induced contractions in isolated sensitized guinea pig trachealis. HTME even at 3 M significantly suppressed OVA (100
g/ml)
-induced ones.
In conclusion, HTME had high affinity to PDE4, and at the same time it also
competitively inhibited PDE3, which may potentiate anti-inflammatory effects of the former. The above results suggests that HTME, with PDE4H/PDE4L ratio of 18.2, may have potential in the treatment of asthma.
PART 2 To investigate the possibility of asthmatic animal model with early- and late-phases of airway hyperresponsiveness (AHR), we used three kinds of clinically useful drugs, methylprednisolone, pyrilamine and cromolyn in this study.
Methacholine (MCh, 50 mg/ml)-induced AHR in sensitized and challenged mice with ovalbumin (OVA) by barometric plethysmography, using a whole-body
plethymograph. In the present results, methylprednisolone (10 mg/kg, s.c. ), pyrilamine (25 mg/kg, s.c. ), and cromolyn (2%, inhalation ) significantly inhibited enhanced pause (Penh) in the early-phase of AHR. In the late-phase of AHR, only methylprednisolone and pyrilamine significantly inhibited increase of MCh-induced Penh values. When compared to control group, these three drugs did not attenuate inflammatory cells and cytokines. These three drugs significantly reduced total IgE in bronchoalveoar lavage fluid (BALF) and serum. Methylprdnisolone and cromolyn, but not pyrilamine, significantly reduced OVA-specific IgE in BALF, but not in serum.
Owing to the present results that (1) methylprednisolone, with a contrast to clinical effects, did not reduce OVA-specific IgE in serum; (2) these three drugs did not reduce inflammatory cells; and (3) IL-2, IL-4 and TNF-α did not increase in BALF of control group, we do not recommend this animal model with a conservative manner.
