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

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.