NITROXIDES AS PROTECTORS AGAINST OXIDATIVE STRESS

已被用作EPR自旋标记的氮氧化物(如temol)已被证明具有超氧化物歧化酶(SOD)活性，并在保护细胞免受包括过氧化氢、超氧化物、有机过氧化氢、氧化还原循环化疗药物和电离辐射在内的各种氧化应激的影响方面非常有效。我们已经证明了Tempoll对体外细胞和小鼠的电离辐射都有保护作用。因此，氮氧化物代表了一种新的辐射防护剂，可能在保护人类免受辐射方面具有广泛的用途。重要的是，我们已经证明tempoll不能保护啮齿动物的肿瘤组织；我们认为其机制涉及正常组织和肿瘤组织的不同代谢还原特性。体内荷瘤动物模型的电子顺磁共振成像研究表明，与正常组织相比，肿瘤中氮氧化物的减少更快。我们已经完成了一项体外研究，以确定最有效的保护目的氮氧化物。在构效关系研究中对110多个氮氧化物进行了评价。我们已经确定了6个氮氧化物，它们提供了比坦普尔(第一个被证明具有辐射防护特性的氮氧化物)明显更好的辐射防护，并确定了3个类似物，使有氧细胞对辐射增敏。这些药物将在体内进行评估并与坦波尔进行比较。为了进一步研究这些新发现的保护性化合物，人们正在大量合成六个保护性氮氧化物中的几个。我们最近已经证明，暴露在氧化剂中的血红素蛋白形成剧毒的铁基部分，氮氧化物为这些有毒物种解毒，并赋予血红素物种更高的过氧化氢酶类活性。以类似的方式推理，我们正在研究氮氧化物作为一氧化氮合酶调节剂的影响，因为在氮氧化物的存在下，依赖于血红素氧化还原化学的酶内的中间产物可能会改变。我们还在体内模型中研究白蛋白等大分子上附加的氮氧化物的活性。由于这些药物很容易穿透细胞膜，它们可能会用于其他医学研究领域，如缺血/再灌注损伤研究、预防白内障、炎症过程和衰老。已被用作电子顺磁共振(EPR)自旋标记的氮氧化物(如temol)已被证明具有超氧化物歧化酶(SOD)活性，并在保护细胞免受包括过氧化氢、超氧化物、有机过氧化氢、氧化还原循环化疗药物和电离辐射在内的各种氧化应激的影响方面非常有效。我们已经证明，坦波尔在体外对细胞和小鼠都有保护作用，使其免受电离辐射。不同的氮氧化物类似物在给动物使用时不会影响血压，已被确认为辐射保护剂，从而消除了给药的血液动力学问题。最近的研究表明，tempoll不能保护啮齿动物的肿瘤组织；我们认为其机制涉及正常组织和肿瘤组织的不同代谢还原特性。在一个荷瘤动物模型中的体内EPR成像研究表明，与正常组织相比，肿瘤中氮氧化物的减少更快。我们目前正在使用我们新构建的用于小动物的功能性EPR成像仪器来识别和定义导致这种差异效应的细胞和生理因素。最近的研究表明，葡萄糖6-磷酸脱氢酶(G6PD)缺陷的细胞将氮氧化物还原为羟胺的速度比对照细胞慢得多，这表明这一重要的生化途径在氮氧化物还原过程中发挥了作用。我们目前正在研究肿瘤组织和正常组织中G6PD的状态。使用氮氧化物自旋探针，功能性EPR成像系统还将使我们能够绘制出组织中的氧气水平，并研究组织的各种氧化还原参数。目前正在进行研究，评估局部应用于大鼠直肠的坦普尔的辐射防护性能。由于直肠是前列腺癌和/或宫颈癌患者在放射治疗过程中受损的主要正常组织，如果我们的临床前研究证明是阳性的，我们将考虑在临床上使用TEMPOL来保护直肠。我们目前的研究是针对直肠组织的氮氧化物给药方法，以优化氮氧化物的浓度。初步研究表明，长期给药(在食物或饮用水中)对p53基因敲除小鼠延长寿命。P53基因敲除小鼠由于快速诱发肿瘤而在出生几个月后死亡。坦波尔给药后，这些动物的寿命延长了35-70%。这种效应的机制尚不清楚，目前是一个主要的焦点。最后，由于这些药物很容易穿透细胞膜，是有效的抗氧化剂，它们可能会用于其他医学研究领域，如缺血/再灌注损伤研究、预防白内障、炎症过程和衰老。最近的研究表明，在诱导大鼠脑缺血后给予坦波尔可显著缩小脑缺血/再灌流所致的脑梗塞体积。

Nitroxides (such as tempol) which have been used as EPR spin labels have been shown to exhibit superoxide dismutase (SOD) activity and are quite effective agents in protecting cells against a wide variety of oxidative stresses including hydrogen peroxide, superoxide, organic hydroperoxides, redox-cycling chemotherapy drugs, and ionizing radiation. We have demonstrated that Tempol protects both cells in vitro and mice against ionizing radiation. Thus, the nitroxides represent a new class of radiation protectors that may have widespread use in protecting humans against radiation. Importantly, we have shown that tempol does not protect rodent tumor tissue; the mechanism of which we believe involves differential metabolic reduction properties of normal versus tumor tissue. In vivo electron paramagnetic resonance imaging studies in a tumor-bearing animal model has shown more rapid reduction of nitroxides in tumor compared to normal tissue. We have completed an in vitro study to identify the most efficient nitroxide for protection purposes. Over 110 nitroxides were evaluated in a structure activity relationship study. We have identified 6 nitroxides that afford significantly more radioprotection than tempol (the first nitroxide shown to have radioprotective properties) and have also identified 3 analogs that radiosensitize aerobic cells. These agents will be evaluated and compared with tempol in vivo. Large quantities of several of the six protective nitroxides are being synthesized for further study of these newly discovered protectors. We have recently shown that heme proteins exposed to oxidants form highly toxic ferryl moieties and that nitroxides detoxify these toxic species and confer enhanced catalase-like activity to heme species. Reasoning in an analogous fashion we are investigating the affects of nitroxides as modulators of nitric oxide synthase because intermediates within the enzyme which depend on heme redox chemistry may be altered in the presence of nitroxides. We are also investigating in in vivo models, the activity of nitroxides appended to macromolecules such as albumin. Since these agents readily penetrate cell membranes, they may be of use in other areas of medical research such as ischemia/reperfusion injury studies, prevention of cataracts, inflammatory processes and aging. Nitroxides (such as tempol) which have been used as electron paramagnetic resonance (EPR) spin labels have been shown to exhibit superoxide dismutase (SOD) activity and are quite effective agents in protecting cells against a wide variety of oxidative stresses including hydrogen peroxide, superoxide, organic hydroperoxides, redox-cycling chemotherapy drugs, and ionizing radiation. We have demonstrated that tempol protects both cells in vitro and mice against ionizing radiation. Different nitroxides analogues that do not influence blood pressure when administered to animals have been positively identified as radioprotectors thus eliminating the hemodynamic concerns of tempol administration. Recent studies have shown that tempol does not protect rodent tumor tissue; the mechanism of which we believe involves differential metabolic reduction properties of normal versus tumor tissue. In vivo EPR imaging studies in one tumor-bearing animal model has shown more rapid reduction of nitroxides in tumor compared to normal tissue. We are presently seeking to identify and define cellular and physiological factors responsible for this differential effect using our newly constructed functional EPR imaging instrumentation for small animals. Recent studies have shown that cells deficient in glucose 6 phosphate dehydrogenase (G6PD) reduce the nitroxide to the hydroxylamine much slower than control cells suggesting a role for this important biochemical pathway in nitroxide reduction. We are presently studying G6PD status in tumor versus normal tissue. Using nitroxide spin probes, the functional EPR imaging system will also enable us to map out oxygen levels in tissue as well as study various redox parameters of tissue.Studies are presently underway evaluating the radioprotective properties of tempol applied topically to the rectum of rats. Since the rectum is a major normal tissue damaged during radiotherapy for patients with prostate and/or cervix cancer, we will consider using tempol clinically to protect the rectum should our pre-clinical studies prove positive. Our present studies are directed on nitroxide delivery methods to rectal tissue to optimize nitroxide concentration.Preliminary studies have indicated that long term administration of tempol (in the food or drinking water) to p53 knockout mice extends their life span. p53 knockout mice die several months after birth due to rapid tumor induction. Tempol administration extended the life span of these animals ~35-70%. The mechanism of this effect is unknown and is presently a major focus. Lastly, since these agents readily penetrate cell membranes and are potent antioxidants, they may be of use in other areas of medical research such as ischemia/reperfusion injury studies, prevention of cataracts, inflammatory processes, and aging. It has recently been shown that tempol administration after induced ischemia of rat brain markedly reduced the infarct volume associated with ischemia/reperfusion.