新穎抗血栓藥物NP-184, NP-185和NP-313之藥理作用和機制的探討

Abstract

摘要 血管栓塞是全球罹病率與死亡率的一個主因。血小板與凝血因子媒介止血生理功能。但在病理的情形下，如動脈粥狀硬化斑塊破裂所誘發的血栓生成，會引發缺血性心臟及腦血管疾病。然而現存之抗血栓藥物常受限於他們對於止血生理功能的影響。 因此，開發抗血小板及抗凝血作用之新穎藥物仍有具迫切需求性。我們實驗室致力於天然物、植物萃取和化學合成物當中，研發少有影響止血功能之抗血栓藥物。本文即探討較安全及較有效能的NP-184, NP-185和 NP-313三個抗血栓藥物。先前報導持有benzimidazole骨幹之化學物能藉由抑制thromboxane (TX)A2合成酶活性或拮抗TXA2/prostaglandin H2 (TP)受器而抑制血小板的活化，或者干擾凝血因子活性而抑制凝血，我們篩選此系列之衍生物，發現其中以NP-184 [2-(5- methyl-2-furyl)benzimidazol和NP-185 [1-benzyl-2-(5-methyl-2-furyl)benzimidazole ]是分別俱強效抗血小板/抗凝血活性的兩個藥物。NP-184和 NP-185皆以濃度相關的方式抑制膠原蛋白(collagen) 、花生四烯酸 (arachidonic acid)和U46619 ( TXA2的類似物)所誘發之人類血小板活化和它們所誘發之TXA2 之生成。同時發現NP-184 和 NP-185皆不會影響環氧酶(cyclooxygenase -1)及細胞質磷脂水解酶A2 (cytosolic phospholipase A2) 活性， 但可選擇性的抑制TXA2 合成酶，其IC50分別為4.3 ± 0.2及 1.6 ± 0.3 μM。且發現在U46619誘發血小板凝集的濃度反應曲線圖上，NP-184和 NP-185對於TP受體呈現競爭性的拮抗作用，而NP-185強力競爭拮抗[3H] SQ-29548在TP專一性的結合(IC50 =118 nΜ)。另一方面，NP-184也以濃度相關的方式延長activated partial thromboplastin time (aPTT)，然而不會影響prothrombin time (PT)和thrombin time(TT)，因此NP-184乃選擇性的干擾凝血機制之內在路徑。而NP-185則對於凝血機制沒有任何影響。無論經由靜脈注射或口服投予NP-184或 NP-185 皆能顯著的抑制藉由激發媒介物fluorescein sodium所產生之血栓生成，但不會影響小鼠的出血時間。同時探討了NP-185在具有TXA2 合成酶基因缺陷之鼠體上效應，發現投予NP-185僅能輕微的延長血栓生成。不同於NP-185，因為NP-184之 ex vivo 抗血小板及抗凝血活性與其體內抗血栓作用有著明確之相關性，所以NP-184之抗血栓作用不只源自於抗血小板活性，且與抗凝血活性有關。綜合上述，NP-184和 NP-185皆透過抑制TXA2 合成酶活性和拮抗TP 受器來展現其抗血小板作用，在活體上且能有效的抗血栓生成而僅有極輕微的出血作用。另外，持有1,4-naphthoquinone骨幹之化學物具有廣效的藥理作用，其中了包括抗癌及抗血小板活性。我們發現了新的naphthoquinone衍生物2-acetylamino-3-chloro-1,4-naphthoquinone (NP-313) 持有較強效的拮抗血小板活化效應。NP-313以濃度相關的方式分別抑制膠原蛋白、花生四烯酸、thrombin、thapsigargin和A23187所誘發之血小板凝集，和抑制花生四烯酸所誘發的TXA2 之生成。酵素活性之分析顯示NP-313能夠抑制環氧酶、TXA2合成酶和蛋白質激酶Cα (protein kinase Cα)之活性，然而不會影響細胞質磷脂水解酶A2 和磷脂水解酶C ( phospholipase C) 之活性。更進一步發現NP-313以濃度相關的方式抑制thrombin和A23187所誘發的胞內Ca2+上升，此效應是透過其阻斷Ca2+ 從胞外influx，並非是抑制Ca2+從胞內儲存體的釋出。NP-313也抑制thapsigargin經由store-operated calcium channel (SOCC) 引發的Ca2+ influx，但它無法影響 diacylglycerol類似物1-oleoyl-2-acetyl-sn-glycerol 所引發的經由store-independent calcium channel之Ca2+ influx 。 綜合所述，NP-313能夠透過抑制TXA2合成和阻斷經由SOCC之Ca2+ influx的雙重特性進而展現其抗血小板作用。再者，靜脈注射投予NP-313在鼠體內可防止血栓生成，卻少有引發止血功能障礙。這些優勢也建議NP-313可能具有潛力進入未來的抗血栓藥物開發行列。

ABSTRACT Thrombotic disease is a major cause of morbidity and mortality worldwide. Platelet and coagulation factor-dependent thrombus formation is critical to limit post-traumatic blood loss at sites of vascular injury and it may also lead to vessel occlusion causing ischaemic cardio- and cerebro-vascular diseases under pathological conditions such as rupture of an atherosclerotic plaque. The use of existing antithrombotic agents is, however, limited by their inherent effect on primary haemostasis. Consequently, there is an ongoing search for more powerful and safer antithrombotic drugs, particularly novel antiplatelet agents and anticoagulants. In our search for new antithrombotic agents, a series of natural products, plant extracts, and chemical synthetic compounds were screened for their ability to inhibit platelet aggregation. Among them, NP-184, NP-185 and NP-313 with safer and more effective antithrombotic effect were investigated. The compounds with the chemical structure of a benzimidazole backbone have been shown to exhibit antiplatelet activities through thromboxane A2 synthase (TXAS) inhibitory, TXA2/prostaglandin H2 (TP) receptor antagonistic action or anticoagulant activities. In present study, we showed two of these compounds, NP-184 [2-(5-methyl-2-furyl)benzimidazole] and NP-185 [1-benzyl-2-(5-methyl-2-furyl)benzimidazole ], sharing common and distinct properties, display potent antiplatelet and antithrombotic activities. Both NP-184 and NP-185 concentration-dependently inhibited the human platelet aggregation induced by collagen, arachidonic acid (AA), and U46619, a thromboxane (TX)A2 mimic. In addition, TXA2 formation caused by collagen and AA was suppressed in a concentration-dependent manner by NP-184 and NP-185. NP-184 and NP-185 selectively inhibited TXAS activity, with an IC50 value of 4.3 ± 0.2 and 1.6 ± 0.3μM, respectively, but both had no effect on both cyclooxygenase-1 (COX-1) and cytosolic phospholipase A2 (cPLA2) activities. Moreover, NP-184 and NP-185 produced a right shift of the concentration–response curve of U46619, indicating a competitive antagonism on TP receptor. However, NP-185 showed a competitive inhibitory effect on the specific binding of [3H] SQ-29548 to human TP receptors expressed on HEK293 cells (IC50 =118 nΜ). On the other hand, NP-184 caused a concentration-dependent prolongation of the activated partial thromboplastin time with little effect on the prothrombin time and thrombin time, indicating that it selectively impairs the intrinsic coagulation pathway, whereas NP-185 had no influence on the coagulation pathway. We further evaluated the antithrombotic efficacy/ side effect profile of NP-184 and NP-185 in a mouse model. Both of NP-184 and NP-185, administrated by either intravenously or orally, significantly inhibited thrombus formation of the irradiated mesenteric vessels in fluorescein sodium–pretreated mice without significantly affecting the bleeding time induced by tail transaction. Moreover, aspirin was devoid of anti-thrombotic activity in TXAS -deficient mice while the occlusive time was only slightly prolonged in mice treated with NP-185 than in mice treated with vehicle. Different from NP-185, both ex vivo antiplatelet and anticoagulant activities of NP-184 was found to be positively correlated with in vivo antithrombotic action. Taken together, these results indicate that both NP-184 and NP-185 have antiplatelet activities through TP receptor blockade and TXAS inhibition while the anticoagulant activity of NP-184 may also operate. Compounds with the chemical structure of a 1,4-naphthoquinone backbone have been shown to have a wide variety of pharmacological effects including anticancer and antiplatelet activities. Among them, NP-313 [2-acetylamino-3-chloro-1,4-naphthoquinone], a newly synthesized naphthoquinone derivative, concentration-dependently inhibited human platelet aggregation induced by collagen, AA, thapsigargin, thrombin and A23187. In addition, NP-313 concentration-dependently inhibited TXA2 generation induced by AA. Enzymatic assays showed that NP-313 exhibited an inhibitory effect on COX-1, TXAS and protein kinase Cα, while it had little effect on cPLA2 or phospholipase C activity. Furthermore, NP-313 concentration-dependently inhibited thrombin- and A23187-induced [Ca2+]i increase through its inhibitory effects on Ca2+ influx, rather than blocking Ca2+ release from intracellular stores. NP-313 also inhibited thapsigargin-mediated Ca2+ influx through store-operated calcium channel (SOCC) but had no effect on Ca2+ influx through store-independent calcium channel evoked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol. These results indicate that NP-313 exerts its antiplatelet activity through dual inhibition of TXA2 synthesis and Ca2+ influx through SOCC. Also, intravenously administered NP-313 showed in vivo protection against thrombous formation with little effect on haemostasis, suggesting that it may be a potential candidate of new antithrombotic agents.