CDP-choline: Mechanisms in Cerebral Ischemia

描述（由申请人提供）：意义和目标：CDP-胆碱（胞磷胆碱）中风在欧洲和日本的临床试验显示出显着的改善，而美国的研究提供了模糊的结果。给药途径（美国口服vs非美国静脉注射）和24小时时间窗可能阻碍了其在美国试验中的有效性。现在已经认识到，在美国试验中口服给药是不合适的，新的III期试验将很快进行。我们的研究表明，CDP-胆碱治疗延迟3小时没有提供任何神经保护。CDP-胆碱的作用模式尚未明确，了解其机制将有助于更有效地治疗缺血性脑损伤。CDP-胆碱治疗脑卒中的疗效仍有可能实现。 原理：磷脂降解是短暂性脑缺血后神经元死亡的重要促进因素。CDP-胆碱神经保护作用被认为是由于受损脑中磷脂酰胆碱（PtdCho）合成增加所致，尽管证据有限。我们在沙鼠短暂前脑缺血中表明，CDP-胆碱：（a）提供显著的神经保护（B）显著恢复PtdCho、心磷脂和鞘磷脂，（c）减弱花生四烯酸的释放和代谢，以及（d）增加谷胱甘肽水平。我们的初步结果表明，CDP-胆碱影响膜和线粒体磷脂酶A2（PLA 2）的激活，这是raM-Ca 2+依赖性（分泌型PLA 2的特征; sPLA 2），支持假设和目标1。在体外，CDP-胆碱及其组分（胞苷和胆碱）不抑制PLA 2活性，因此CDP-胆碱不是“直接PLA 2抑制剂”。相反，体内CDP-胆碱可能影响PLA 2活化。 假设：CDP-胆碱通过阻止PLA 2的活化来减弱磷脂水解。为了在短暂性脑缺血中验证这一假设，我们提出了以下具体目标：目标1：确定CDP-胆碱是否抑制PLA 2激活和蛋白表达。由于这一目标是我们的假设的核心，它将在沙土鼠短暂前脑缺血和自发性高血压大鼠短暂局灶性脑缺血进行测试。目标二：确定CDP-胆碱是否改变通过PLA 2介导的三磷酸胞苷磷酸胆碱胞苷酰转移酶（PCCT）和鞘磷脂酶活性。PCCT产生内源性CDP-胆碱，是PtdCho合成的限速酶。外源性CDP-胆碱被水解，吸收为胞苷和胆碱，并且必须由PCCT重新合成。PtdCho被PLA 2水解产生溶血PtdCho和花生四烯酸。Lyso-PtdCho抑制PCCT活性，导致PtdCho合成受损。花生四烯酸激活中性鞘磷脂酶，导致膜崩解。外源性CDP-胆碱刺激PCCT活性。目的2将在沙土鼠短暂性前脑缺血中进行测试。

DESCRIPTION (provided by applicant): Significance and goal: CDP-choline (citicoline) stroke clinical trials in Europe and Japan showed significant improvement, while US studies provided ambiguous results. The route of administration (oral in USA vs iv in non-USA) and 24-hr time window may have hindered its effectiveness in the USA trials. It has now been realized that oral administration in USA trials was inappropriate and new Phase III trials will soon be undertaken. Our studies show that CDP-choline treatment delayed by 3-hr did not offer any neuroprotection. CDP-choline mode of action has not been clearly identified, and understanding its mechanism(s) should lead to more effective treatment of ischemic brain injury. The efficacy of CDP-choline in stroke therapy might still be achieved. Rationale: Phospholipid degradation is a significant promoter of neuronal death after transient cerebral ischemia. CDP-choline neuroprotection is thought to be due to increased phosphatidyl-choline (PtdCho) synthesis in the injured brain, although the evidence is limited. We showed in gerbil transient forebrain ischemia that CDP-choline: (a) provided significant neuroprotection (b) significantly restored PtdCho, cardioliPin and sphingomyelin, (c) attenuated arachidonic acid release and metabolism, and (d) increased glutathione levels. Our preliminary results show that CDP-choline affects activation of membrane and mitochondrial phospholipase A2 (PLA2) that is raM-Ca 2+ dependent (characteristic of secretory PLA2; sPLA2), supporting the hypothesis and Aim 1. In vitro, CDP-choline and its components (cytidine and choline) did not inhibit PLA2 activity, and thus as such CDP-choline is not a "direct PLA2 inhibitor". Instead CDP-choline in vivo likely affects PLA2 activation. Hypothesis: CDP-choline attenuates phospholipid hydrolysis by preventing activation of PLA2. To test this hypothesis in transient cerebral ischemia, we propose the following specific aims: Aim 1: Determine whether CDP-choline inhibits PLA2 activation and protein expression. Since this aim is central to our hypothesis, it will be tested both in gerbil transient forebrain ischemia and transient focal cerebral ischemia of spontaneously hypertensive rat. Aim 2: Determine whether CDP-choline alters cytidine triphosphate phosphocholine cytidylyltransferase (PCCT) and sphingomyelinase activities mediated through PLA2. PCCT makes endogenous CDP-choline and is the rate-limiting enzyme in PtdCho synthesis. Exogenous CDP-choline is hydrolyzed, absorbed as cytidine and choline, and has to be re-synthesized by PCCT. PtdCho hydrolysis by PLA2 results in lyso-PtdCho and arachidonic acid. Lyso-PtdCho inhibits PCCT activity resulting in impaired PtdCho synthesis. Arachidonic acid activates neutral sphingomyelinase, resulting in membrane disintegration. PCCT activity was stimulated by exogenous CDP-choline. Aim 2 will be tested in gerbil transient forebrain ischemia.