A MULTI-CENTER, PHASE III, RANDOMIZED, DOUBLE BLIND, PLACEBO-CONTROLLED, CLINIC

该子项目是众多利用 由美国国立卫生研究院/国家研究资源中心（NIH/NCRR）资助的中心拨款所提供资源的研究子项目之一。该子项目及其 首席研究员（PI）可能已从美国国立卫生研究院的其他资金来源获得主要资助， 因此也可能出现在其他CRISP条目中。 所列机构为 该中心所属机构，未必是研究负责人的所属机构。 本研究将验证的假设是：米诺环素可延缓肌萎缩侧索硬化症（ALS）患者整体功能的进行性恶化。主要结局指标为ALS功能评定量表 （ALSFRS-R）所测得的功能变化，即米诺环素组与安慰剂组之间的比较。本研究的统计功效为80%，旨在检测患者ALSFRS-R评分随时间推移的平均斜率是否降低18%或更多。 次要结局指标包括手动肌力测试（MMT）、用力肺活量（FVC，预测值的百分比）、生活质量（QOL）以及生存期的变化。 这是一项研究者发起的、多中心、III期、随机（1:1）、双盲、安慰剂对照试验。 研究总时长为48个月：其中24个月用于患者招募，4个月进行连续的月度评估以确定每位患者的基线进展斜率，随后是9个月的干预期（米诺环素或安慰剂），最后还有11个月用于生存随访、数据分析和论文撰写。 受试者在参与研究的13个月期间每月接受一次评估。随机分组将在第4个月的随访时进行。在干预阶段的前3周（第5个月），受试者将根据耐受情况接受剂量递增治疗，每日最高8片（400毫克），并每周进行电话随访。 本研究将招募400名早期ALS患者（用力呼气容积[FVC]≥预测值的75%，且症状持续时间少于3年）。受试者必须符合埃尔埃斯科里亚尔（El Escorial）标准，经实验室检查支持为“可能”、“可能”或“确诊”的ALS。 随机分组将按研究中心进行分层，并在每个研究中心内进一步按利鲁唑使用情况及发病部位（无球部受累的四肢受累 vs. 有或无四肢受累的球部受累）进行分层，以确保在整个试验期间，各分层中药物与安慰剂的分配均匀。 背景与初步研究 肌萎缩侧索硬化症会导致随意运动系统的退化，患者平均存活期为3年（1）。该病程呈进行性发展，功能随时间推移逐渐衰退（2,3）。 目前尚无根治方法，也无已知疗法能显著改善患者功能。该病的患病率为每10万人口4-6例，发病率为每10万人口0.4至1.8例 (4-6)。大多数ALS病例为散发性。然而，其中10%-15%呈常染色体显性遗传，称为家族性ALS（FALS）（1,7）。其中，25%与编码铜-锌超氧化物歧化酶 （SOD1）的基因缺陷（8）。在FALS中发现SOD1突变后，研究人员利用该基因构建了转基因小鼠模型。SOD1 FALS啮齿类动物模型已成为ALS病理研究和治疗药物筛选的重要方法（9）。 人类散发性与家族性ALS在临床和病理特征上相似（10）。 在散发性ALS中，发病年龄中位数为55岁（7）。男性发病率略高，年龄和性别是流行病学研究中记录的唯一反复出现的风险因素（4,11）。除关岛马里亚纳群岛发病率较高外，该病无种族或地域易感性 (4)。发病年龄较大、球部起病以及临床残疾或呼吸功能受损程度更严重的患者，生存期较短（12-14）。 确诊必须同时具备上运动神经元和下运动神经元疾病的临床体征（15,16）。肌无力及肌肉萎缩的症状通常从一侧肢体的远端非对称性开始，随后扩散至相邻的肌节，但症状也可能始于球部或肢体肌肉。 眼外肌和尿道括约肌不受累。对于肢体起病型患者，呼吸功能通常在疾病晚期才受影响，但偶尔也可能作为早期表现出现。 本研究选择米诺环素作为研究药物，是因为其作用可能通过下文概述的几个环节抑制运动神经退化。它很可能通过阻止促凋亡和促炎酶的激活来抑制细胞死亡通路。 它能抑制线粒体细胞色素c的释放（17），并抑制p38丝裂原活化蛋白（MAP）激酶（18），从而减少凋亡酶、小胶质细胞、细胞因子和氧化物的周期性激活与产生（18,19）。 它能延缓多种神经退行性疾病模型（20,21）的恶化进程，包括肌萎缩侧索硬化症（ALS）（17,22,23）。在实验性缺血模型中，它具有神经保护作用（24），并且在人类炎症性关节炎（25）中已证明其疗效，这与其抗生素特性无关。 发病机制 ALS 患者大脑、脑干和脊髓中运动神经元的丧失导致了症状的进行性恶化。尽管导致运动神经元退化的机制尚未完全阐明，但有证据表明，自由基毒性、谷氨酸兴奋性毒性、 线粒体功能障碍以及中间丝聚集，可能导致调控细胞死亡通路的基因和酶被激活。线粒体细胞色素c的释放以及应激酶（如p38 MAP激酶）的上调，可能促进促凋亡和促炎调节因子的激活（17，26-35）。 小胶质细胞中的环氧化酶-2（COX-2）和诱导型一氧化氮合酶（iNOS）被激活（36-38）， 而在人类及转基因小鼠ALS模型的神经元中，调节凋亡的半胱天冬酶被激活（31、39、40）。 自由基毒性 氧化毒性被认为源于过量的自由基产生或降解功能受损。 活性氧物种部分是通过与铁、铜等还原态金属相互作用而产生的，对大多数类型的分子和细胞器均具有毒性。在ALS患者的运动神经元中发现了细胞器氧化损伤（41-43），且周围胶质细胞中的iNOS表达上调 (38)。在某些FALS模型中，将超氧阴离子转化为过氧化氢的SOD1活性过高（毒性功能获得），并产生高于正常水平的自由基，包括超氧阴离子、过氧化氢、羟基自由基和过氧亚硝酸盐 (44)。 突变型SOD1还可能使神经元易受谷氨酸介导的兴奋性毒性影响（45）。 兴奋性毒性 兴奋性毒性可能是散发性ALS中细胞死亡的原因之一。作为中枢神经系统主要兴奋性神经递质的谷氨酸，其水平在部分ALS患者的脑脊液和脑组织中升高（46,47）。浓度升高会导致细胞内钙离子过量、中间丝磷酸化以及细胞过早死亡（48）。高于正常水平的谷氨酸浓度可能是由负责谷氨酸再摄取的胶质细胞谷氨酸转运蛋白表达下降所致（27）。 接受谷氨酸释放抑制剂利鲁唑（riluzole）治疗的ALS患者，生存期可延长约3个月（49）。 线粒体异常 在ALS患者和转基因ALS小鼠的组织中，也已发现线粒体呼吸链I复合体的异常（17,26,50）。 线粒体功能障碍会导致能量储备耗尽，从而削弱细胞清除自由基和维持细胞膜电位的 ability。细胞膜电位降低可能激活谷氨酸受体，从而增强兴奋性毒性。细胞色素功能障碍会促进促凋亡基因的激活，所有这些因素都可能导致细胞死亡 (1,48)。 无论最初的诱因是什么，这一级联事件都与细胞死亡通路的激活有关（29-30）。在实验性神经退行性变中，MAP激酶会因各种细胞应激（包括兴奋性毒性损伤）而通过磷酸化被激活（51）。 一氧化氮可在细胞培养中诱导p38 MAP激酶磷酸化（18），且p38在实验性脑缺血后会被激活（52）。 在实验性ALS脊髓中，经氧化应激诱导后，星形胶质细胞中的应激酶被激活；在其他神经退行性疾病的大脑中亦是如此（33,52,54）。MAP激酶的激活可能促进调控凋亡的酶的上调，并促进炎症介质的产生（55-57）。 已证实，一类称为半胱天冬酶（caspases）的蛋白酶家族在ALS的细胞死亡通路（很可能是凋亡）中起着核心作用（29-31,40）。半胱天冬酶参与促凋亡级联反应，最终导致特定蛋白质和DNA的裂解 （58）。启动型半胱天冬酶（包括半胱天冬酶-1）在MAP激酶的作用下从休眠的前体形式被激活，进而作用于下游半胱天冬酶（如半胱天冬酶-3）的前体，这些下游酶即为效应酶。当效应型半胱天冬酶的亚基-激活的DNA酶的亚基被裂解并降解DNA时，细胞死亡便发生了。凋亡通常是一个高度受调控的过程，在神经系统发育过程中自然发生，但也被认为与缺血及许多神经退行性疾病有关 (59)。事件发生的顺序可能因具体过程而异，但在所有情况下都会发生酶的激活，从而导致核小体内的DNA碎裂、染色质凝集、细胞收缩，并分解为被膜包裹的囊泡 （凋亡小体）(60)。最终，死亡的凋亡细胞残骸会被吞噬。 其他酶也参与凋亡过程，包括bcl-2。细胞死亡受体可通过激活凋亡小体来放大自杀信号。在体外和体内ALS模型中，半胱天冬酶均被激活（61,62）。在人类ALS中，半胱天冬酶同样被激活（39,40）。 在转基因小鼠中，突变型SOD1的表达会诱导半胱天冬酶依赖性神经元细胞死亡（62,63）。 近期研究表明，除了凋亡外，炎症机制可能在ALS的神经元破坏中起作用，且ALS中的细胞死亡通路很可能同时涉及这两个过程（28,32,64）。 应激激活的MAP激酶通过激活caspase酶来促进凋亡，同时也促进促炎介质的活化（54）。 ALS 患者脑和脊髓的尸检标本显示，运动神经元发生了变化，不仅包括线粒体肿胀、细胞膜完整性丧失、细胞内成分的氧化损伤以及细胞骨架蛋白的积累，还包括星形胶质细胞和炎性小胶质细胞数量的增加（32、35、64）。 在动物和人类ALS患者的脊髓小胶质细胞中，前列腺素E2（PGE2）、COX-2和iNOS等炎症介质的水平均升高（36、37、38）。 促炎性细胞因子会增加半胱天冬酶的转录，进而进一步增强炎症调节因子的转录（19）。因此，导致酶（可能包括p38 MAP激酶）上调的细胞应激因子，可能会通过相互关联的半胱天冬酶介导的凋亡和炎症机制促进细胞死亡 (28,32,65)。 抗凋亡剂、caspase酶抑制剂和抗炎剂可延缓ALS模型的病情进展。 当携带突变型SOD1的转基因小鼠与表达显性失活型caspase-1的小鼠杂交时，其寿命会略有延长（30），而在ALS模型中，过表达线粒体抗凋亡蛋白bcl-2可防止神经元丢失并延长寿命（31）。 外源性半胱天冬酶抑制剂也能延长携带 CuZn SOD1 突变的 ALS 小鼠模型的寿命。一种小肽半胱天冬酶抑制剂（zVAD-fmk）可将 SOD1 转基因小鼠的生存期延长 4 周，即约 20%（40）。 相比之下，利鲁唑在同一SOD1转基因品系中可将生存期延长约11%，在ALS患者中可延长约3个月。 zVAD-fmk 可阻断所有已知的半胱天冬酶，并在激活级联反应的多个环节发挥作用。其口服生物利用度较低，脑部渗透性有限，必须通过脑室输注给药。乙酰水杨酸 （一种抗炎剂）已被证实可延缓转基因SOD-1小鼠出现肌无力症状（66），而Cox-2抑制剂可在体外和体内模型中防止脊髓运动神经元丧失（67）。 米诺环素 米诺环素可抑制凋亡和炎症，在一般神经退行性疾病模型（特别是ALS模型）中能延缓疾病进展（17,20-23）。米诺环素已获FDA批准用于治疗感染，口服后对中枢神经系统的渗透性高，并能抑制p38 MAP激酶 (18)。它能在体外和体内降低半胱天冬酶-1、半胱天冬酶-3和iNOS的活性 (20,68)。其抗炎特性包括：在细胞培养中能防止谷氨酸诱导的小胶质细胞活化，并降低白细胞介素的产生（57）。在卒中/缺血性损伤的动物模型中，它具有神经保护作用（24）， 并在以caspase调控的细胞死亡为特征的神经退行性疾病动物模型中延缓疾病进展。 在亨廷顿病模型中，它能延缓疾病进展并抑制iNOS和caspase酶的活化（20），并在帕金森病的MPTP模型中预防黑质纹状体多巴胺能神经退行性变（21）。 多个实验室的研究表明，米诺环素可延缓ALS SOD1模型中疾病的进展（Serge Przedborski，个人通信）（17,22,23）， 这可能涉及p38 MAP激酶的下调。在一项针对20只SOD1啮齿动物的盲法研究中，每日腹腔注射5 mg/kg可使寿命较安慰剂组小鼠延长约11%（Serge Przedborski，个人通信）。 在另一家独立实验室的研究中，米诺环素延长了SOD1型ALS模型小鼠的寿命（17）。从5周龄开始每日注射10 mg/kg的小鼠，其运动功能障碍的出现时间推迟，且与生理盐水处理的对照组小鼠相比，生存期显著延长了11天（9%）。 从病理学角度来看，米诺环素通过对线粒体的上游作用，降低了半胱天冬酶-1、半胱天冬酶-3、诱导型一氧化氮合酶以及 p38 丝裂原活化蛋白激酶的活性。 它直接抑制了由线粒体通透性转换介导的细胞色素c释放，这是包括半胱天冬酶酶介导的凋亡在内的细胞死亡通路激活的关键早期步骤。作者利用ALS小鼠和脑缺血模型、神经元细胞以及分离的线粒体，在体内检测到了这些效应。 在针对 SOD1 肌萎缩侧索硬化症小鼠模型的另一项研究中，米诺环素提高了存活率并减少了小胶质细胞的活化 (22)。 在这项研究中，携带 G93A 人类 SOD1 突变的转基因小鼠从 70 天龄起，每个工作日均通过腹腔注射给予生理盐水或米诺环素。使用了两种不同的米诺环素剂量：25 毫克/千克和 50 毫克/千克。 米诺环素以剂量依赖性方式延缓了旋转杆测试表现的下降，其中高剂量米诺环素组与生理盐水处理组之间的差异具有统计学意义。米诺环素还以剂量依赖性方式延缓了肌肉无力的发病时间并减缓了其恶化速度，在50 mg/kg剂量下再次达到统计学意义。 两种浓度的米诺环素均显著延缓了死亡率。接受较高剂量治疗的小鼠寿命延长了16%。病理学上，在120天龄时，与对照组动物相比，接受米诺环素治疗的小鼠运动神经元丢失、空泡化和小胶质细胞活化均有所减少。 在局灶性脑缺血的啮齿类动物模型中，若在缺血发作后4小时开始给药，米诺环素可将皮质梗死体积减少63% (24)。 在此研究中，通过将尼龙线插入内颈动脉并延伸至中大脑动脉来诱发缺血。动物于第一天腹腔注射米诺环素45 mg/kg（分两次给药），随后两天每日腹腔注射22.5 mg/kg。病理学研究表明，米诺环素抑制了小胶质细胞的活化以及白细胞介素-1β转化酶的诱导，并降低了COX-2的表达和前列腺素E2的生成。 在一项针对啮齿类动物的实验性脊髓损伤研究中，全身给药米诺环素改善了功能恢复 (69)。作者报告称，在中度脊髓挫伤后1小时进行90 mg/kg的腹腔注射，与对照组动物相比，可显著改善运动功能。接受米诺环素治疗的动物，其脊髓中的神经退行性变、凋亡和半胱天冬酶活化均有所减轻。 米诺环素还能在1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）诱导的帕金森病小鼠模型中预防黑质纹状体多巴胺能神经退行性变（21）。 在这项对照研究中，小鼠在 MPTP 给药前、期间和之后，通过口服灌胃方式接受了 60-120 毫克/千克/天的米诺环素剂量。 米诺环素抑制了 p38 MAP 激酶的磷酸化，阻断了 MPTP 诱导的神经退行性变和多巴胺耗竭，并显著降低了 iNOS 和半胱天冬酶-1 的表达。 在一项使用 R6/2 亨廷顿病 (HD) 小鼠模型的盲法研究中，腹腔注射 5 毫克/千克/天的米诺环素后，小鼠的生存期得以延长 (20)。 与生理盐水处理的小鼠相比，从 6 周龄开始每天给予米诺环素治疗，显著延缓了转杆测试表现的特征性下降，并将生存期延长了 14%。 在此模型中，caspase-1 表达降低，而 caspase-3 表达上调，iNOS 活性也随之增加。四环素无法穿过血脑屏障，因此对行为表现或生存期均无影响。 米诺环素在人体中的应用 四环素类抗生素是50年前首批问世的抗生素之一，至今仍被广泛使用。米诺环素是一种第二代长效四环素 (70)。其适应症包括多种细菌感染，其中包括脑部和脑膜感染。作为治疗痤疮的全身性抗生素，米诺环素被广泛处方(70,71)，并在炎症性关节炎的治疗中显示出疗效(25)。 米诺环素的脂溶性比其他四环素高出十倍，对中枢神经系统的渗透性和生物利用度极佳。其耐受性良好，可用于门诊治疗。 其血清半衰期约为17小时。在长期治疗期间，需定期进行包括造血系统、肾脏和肝脏功能在内的器官系统实验室检查。对任何四环素类药物有过敏反应者禁用， 以及孕妇和儿童（因可能导致牙齿着色并干扰骨骼生长）禁用。 米诺环素口服剂量为每日两次、每次100毫克（大致相当于SOD1和HD模型中的5毫克/千克/天剂量），已被证实对人类类风湿性关节炎具有抗炎疗效，并用于治疗脑部和脑膜感染。 在一项为期2年的双盲研究中， 每日两次、每次100毫克的米诺环素口服剂量，在早期血清阳性类风湿关节炎患者中的疗效优于羟氯喹（25）。与对照组相比，患者达到50%改善以及逐步停用泼尼松的可能性显著更高。 在针对晚期类风湿性关节炎患者的盲法研究中，与对照组相比，每日两次、每次100毫克的剂量也显示出具有统计学意义的疗效（72）。 米诺环素的毒性与其他四环素类药物报道的相似，包括牙釉质着色、皮肤及其他组织色素沉着、光敏性、胃肠道不耐受、腹泻以及前庭系统副作用，包括头晕、共济失调和眩晕（71,73）。据报道，该药极少诱发免疫反应，导致肝炎、关节炎和药物性红斑狼疮、全身性过敏反应、类血清病反应、血管炎、假性脑瘤、过敏性肺炎、间质性肾炎及黑甲状腺综合征（74）。 肾功能衰竭患者的半衰期会延长。食物和二价阳离子对口服吸收的影响极小。米诺环素通过肝胆和胃肠道排出体外。米诺环素可能会降低口服避孕药的疗效。 由于会增强华法林引起的抗凝作用，并导致锂、地高辛和茶碱血药浓度升高，因此必须进行密切监测。常规剂量为每12小时100 mg，长期使用耐受性良好（75）。 意义 尽管近年来在部分理解导致运动神经元退化的分子机制方面取得了进展， 肌萎缩侧索硬化症（ALS）仍是一种无法治愈的疾病。在SOD1转基因小鼠中，半胱天冬酶抑制剂和抗炎药物（包括米诺环素）对生存率和运动功能的治疗益处，以及米诺环素在ALS、亨廷顿病和帕金森病动物模型中的疗效，进一步证明了应激酶介导的细胞死亡通路可能参与了神经退行性变。 这是首项针对人类ALS、研究兼具抗凋亡和抗炎作用药物的研究。任何经证实能延缓人类ALS病程的化合物，无论从临床角度还是从理解运动神经元疾病潜在生物学机制的角度来看，都将具有直接的重要性。 此外，由于成本高昂，约50%的ALS患者未服用利鲁唑——这是目前美国食品药品监督管理局（FDA）批准的唯一治疗该疾病的药物。米诺环素可作为利鲁唑的一种安全且更经济的替代治疗方案 （利鲁唑每日两次、每次50毫克的月费用为900美元；米诺环素每日两次、每次100毫克的月费用为150美元）。此外，由于两者的作用机制不同，这两种药物对该疾病可能产生协同作用，未来可进行联合用药试验进行验证。 初步研究 针对3名ALS患者开展了一项开放标签试点研究，以评估该人群对米诺环素的初步耐受性。米诺环素（每日两次，每次100毫克）与利鲁唑联合使用时耐受性良好（76）。 未出现由米诺环素引起的副作用或实验室检查异常，且经ALSFRS-R量表测得的运动功能在三个月内保持稳定。一名受试者报告在入组前服用利鲁唑期间曾出现轻度间歇性腹泻，但在研究期间未见变化。 针对该人群的两项安慰剂对照初步研究已于2003年1月完成。第一项研究由新墨西哥大学 （首席研究员保罗·戈登博士）开展，是一项关于肌萎缩侧索硬化症（ALS）患者每日两次、每次100毫克米诺环素联合利鲁唑治疗耐受性的随机、安慰剂对照研究。该项为期6个月的研究共入组19名受试者，其中11名男性，8名女性。 每月对受试者进行 ALSFRS-R、MMT、最大自愿等长收缩力 (MVIC)、用力肺活量 (FVC) 评估，并监测不良事件及实验室检查结果（肝功能、肾功能和血细胞计数）。 两名患者因路途遥远和身体残疾而停止了每月评估，但其不良事件和ALSFRS-R评分仍通过电话进行监测。 研究期间，有3名患者（2名米诺环素组；1名安慰剂组）因渐进性ALS导致的呼吸骤停而死亡。其他常见不良事件包括静脉炎（1例）、腹泻（1例）、继发感染（1例）、口干（1例）、跌倒（1例）、肺炎（1例）和流感样症状（1例）。 安慰剂组与活性药物组之间不良事件的发生率无统计学上的显著差异。 3名患者（安慰剂组2例，活性药物组1例）出现轻度肝功能轻度升高。所有患者均在服用利鲁唑。两组在肌力变化率或功能结局指标方面均无统计学上的显著差异。 在旧金山福布斯·诺里斯肌萎缩侧索硬化症研究中心开展的第二项米诺环素试点研究 （首席研究员罗伯特·米勒博士）开展的米诺环素剂量递增研究也已完成。这项随机、安慰剂对照、为期8个月的交叉研究共入组23名患者。目标剂量为400毫克米诺环素。每月进行ALSFRS-R评分、FVC测定及实验室检查。 米诺环素的平均耐受剂量为387毫克/天，即7.7片/天 （目标剂量为8片/天）。常见的不良事件包括跌倒、便秘、失眠、食欲减退和反流。仅消化不良在服用米诺环素期间发生率更高（米诺环素组与安慰剂组比例为5:1）。其他不良事件的发生率在两组之间无统计学显著差异。 服用米诺环素期间，血尿素氮（BUN）和天冬氨酸转氨酶/丙氨酸转氨酶（AST/ALT）水平升高至具有统计学意义的程度，但这些升高不被认为具有临床意义。服用米诺环素期间，ALSFRS-R评分下降速度更快（p=0.047），尽管该研究并未针对疗效设计足够的样本量。 在任何疾病中，每日剂量超过400 mg均无法耐受（Steven Projan，Wyeth Ayerst，个人通信）。 参考文献： 1. 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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The hypothesis that the current study will test is that minocycline slows the progressive deterioration of global function in patients with amyotrophic lateral sclerosis (ALS). The primary outcome measure is change in function as detected by the ALS Functional Rating Scale (ALSFRS-R) in patients taking minocycline compared to those taking placebo. The study is 80 % powered to detect an 18% or greater reduction in the average slope of patients' ALSFRS-R scores over time. The secondary outcome measures are changes in manual muscle testing (MMT), forced vital capacity (FVC, percent predicted), quality of life (QOL) and survival. This is an investigator-initiated, multi-center, phase III, randomized (1:1), double blind, placebo-controlled trial. The total study length is 48 months: Twenty-four months for patient recruitment, 4 months of serial monthly evaluations to determine baseline slopes of progression for each patient followed by 9 months of intervention (minocycline or placebo), and 11 additional months of survival follow-up, data analysis and preparation of publications. Subjects receive monthly evaluations during their 13 months of participation. Randomization will occur at the month 4 visit. During the first 3 weeks of the intervention phase (month 5) subjects receive an escalating dose of up to 8 pills (400 mg) per day as tolerated and have weekly phone contact. Four hundred patients with early ALS (FVC greater or equal to 75% predicted and symptom duration of less than 3 years) will be enrolled. Subjects must meet El Escorial criteria for laboratory supported probable, probable or definite ALS. Randomization will be stratified by center, and by riluzole use and site of onset (limb with no bulbar vs. bulbar with or without limb) within each center to assure an even distribution of drug and placebo within each stratum throughout the trial. Background and Preliminary Studies Amyotrophic lateral sclerosis leads to degeneration of the voluntary motor system and death on average in 3 years (1). The course is progressive with a decline in function with time (2,3). There is no cure or known treatment that significantly improves function. The prevalence is 4-6 cases per 100,000 with an incidence of 0.4 to 1.8 per 100,000 (4-6). The majority of ALS cases are sporadic. However, 10-15% are inherited as an autosomal dominant trait known as familial ALS (FALS) (1,7). Of these, 25% are associated with a defect in the gene encoding the enzyme copper-zinc superoxide dismutase (SOD1) (8). After discovery of mutant SOD1 in FALS, a transgenic mouse model was developed using the same gene. The SOD1 FALS rodent model has become an important method of pathological study and therapeutic drug screening in ALS (9). The sporadic and familial forms of human ALS are clinically and pathologically similar (10). In sporadic ALS, the median age of onset is 55 years (7). There is a slight male predominance, with age and gender being the only recurrent risk factors documented in epidemiological studies (4,11). There is no racial or geographic predisposition except for an increased incidence on the Marianas Islands of Guam (4). Patients with older age at onset, bulbar onset, and greater severity of clinical disability or respiratory function have shorter survival (12-14). Clinical signs of both upper and lower motor neuron disease are required for a definitive diagnosis (15,16). Symptoms of weakness and muscle atrophy usually begin asymmetrically and distally in one limb, and then spread to involve contiguous myotomes, but symptoms can begin either in bulbar or limb muscles. Extraocular muscles and the urinary sphincters are spared. Respiration is usually affected late in limb onset patients, but occasionally can be an early manifestation. Minocycline was selected for the proposed study because its effects may inhibit motor nerve degeneration at several points outlined below. It likely inhibits cell death pathways by preventing both pro-apoptotic and pro-inflammatory enzyme activation. It inhibits release of mitochondrial cytochrome c (17) and inhibits p38 mitogen activated protein (MAP) kinase (18), thus reducing cyclical activation and production of caspase enzymes, microglia, cytokines and oxidative species (18,19). It slows deterioration in several models of neurodegeneration (20,21), including ALS (17,22,23). It has neuroprotective benefit in experimental ischemia (24), and it has proven efficacy in human inflammatory arthritis (25), separate from its antibiotic properties. Pathogenesis Loss of motor neurons in the brain, brainstem and spinal cord of ALS patients causes the progressive symptoms. Although the mechanisms leading to motor neuron degeneration are incompletely understood, there is evidence that free radical toxicity, glutamate excitotoxicity, mitochondrial dysfunction and intermediate filament aggregation may lead to activation of genes and enzymes controlling cell death pathways. Release of mithondrial cytochrome c and up-regulation of stress enzymes, such as p38 MAP kinase, may promote activation of pro-apoptotic and pro-inflammatory modulators (17, 26-35). Cycolooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are activated in microglia (36-38), and caspase enzymes, which regulate apoptosis, are activated in neurons of human and transgenic mouse-model ALS (31,39,40). Free Radical Toxicity Oxidative toxicity is thought to arise from excess free radical production or impaired degradation. Reactive oxidized species are generated, in part, via interaction with reduced metals such as iron and copper, and are toxic to most types of molecules and cellular organelles. Oxidative damage to organelles is found in motor neurons of ALS patients (41-43), and iNOS is up-regulated in surrounding glial cells (38). In some FALS models, SOD1, which converts superoxide anion to hydrogen peroxide, is overactive (toxic gain of function) and produces greater than normal levels of free radicals, including superoxide, hydrogen peroxide, hydroxyl radicals, and peroxynitirite (44). Mutant SOD1 may also render neurons vulnerable to glutamate-mediated excitotoxicity (45). Excitotoxicity Excitotoxicity may contribute to cell death in sporadic ALS. Levels of glutamate, the primary excitatory neurotransmitter in the central nervous system, are elevated in spinal fluid and brain of some ALS patients (46,47). Increased concentrations lead to surplus intracellular calcium, phosphorylation of intermediate filaments and premature cell death (48). Higher than normal levels may result from decreased expression of glial glutamate transporters, responsible for glutamate reuptake (27). Survival is prolonged by approximately 3 months in ALS patients treated with riluzole, a glutamate release inhibitor (49). Mitochondrial Abnormalities Abnormalities in complex I of the mitochondrial respiratory chain have also been identified in tissues of ALS subjects and transgenic ALS mice (17,26,50). Mitochondrial dysfunction leads to energy store depletion rendering the cell less capable of detoxifying free radicals and of maintaining the cell membrane potential. Reduced cell membrane potential may activate glutamate receptors, enhancing excitotoxicity. Cytochrome dysfunction promotes activation of pro-apoptotic genes, all of which may contribute to cell death (1,48). Irrespective of the initial trigger, this cascade of events is associated with activation of cell death pathways (29-30). MAP kinases are activated by phosphorylation in response to a variety of cellular stresses, including excitotoxic injury, in experimental neurodegeneration (51). Nitric oxide induces p38 MAP kinase phosphorylation in cell culture (18), and p38 is activated after experimental cerebral ischemia (52). Stress enzymes are activated in the astrocytes of experimental ALS spinal cord after induction by oxidant stress and in brain of other neurodegenerative disorders (33,52,54). MAP kinase activation may promote up-regulation of enzymes controlling apoptosis and also promote production of inflammatory mediators (55-57). A family of proteases, called caspases, has been shown to play a central role in cell death pathways, likely apoptosis, in ALS (29-31,40). Caspase enzymes participate in the pro-apoptotic cascade that culminates in cleavage of specific proteins and DNA (58). Initiator caspases including caspase-1-enzymes activated from their dormant precursor forms in response to MAP kinases-act on the precursors of downstream caspases such as caspase-3, which are the effector enzymes. Cell death occurs when subunits of effector caspase-activated DNAase are cleaved and degrade DNA. Apoptosis is normally a highly regulated process that occurs naturally during the development of the nervous system, but it has also been implicated in ischemia and many neurodegenerative diseases (59). The sequence of events may differ depending on the process, but in all cases there is enzyme activation, resulting in intranucleosomal DNA fragmentation, chromatin condensation, cell shrinkage, and disassembly into membrane-enclosed vesicles (apoptosomes) (60). Finally, the remnants of the dead apoptotic cell are phagocytized. Other enzymes are also involved in apoptosis including bcl-2. Cell death receptors may amplify suicide signals by activating the apoptosome. Caspase enzymes are activated in in vitro and in vivo ALS models (61,62). Caspase enzymes are activated in human ALS (39,40). In transgenic mice mutant SOD1 expression induces caspase dependent neuronal cell death (62,63). Recent studies demonstrate that, in addition to apoptosis, inflammatory mechanisms may play a role in neuronal destruction in ALS, and that cell death pathways in ALS likely involve both processes (28,32,64). Stress-activated MAP kinases promote apoptosis via caspase enzyme activation, and also promote activation of pro-inflammatory mediators (54). Autopsy specimens of brain and spinal cord of ALS patients demonstrate motor neuron changes that include not only swelling of mitochondria, loss of integrity of cellular membranes, oxidative damage to intracellular constituents, and accumulation of cytoskeletal proteins, but in addition, increased numbers of astrocytes and inflammatory microglia (32,35,64). The inflammatory mediators prostaglandin E2 (PGE2), COX-2 and iNOS are elevated in the spinal cord microglia of animal and human ALS (36,37,38). Pro-inflammatory cytokines increase transcription of caspase enzymes, which in turn further augment transcription of inflammatory modulators (19). Hence, cell stressors leading to up-regulation of enzymes, possibly including p38 MAP kinase, may promote cell death pathways via interrelated caspase enzyme-mediated apoptotic and inflammatory mechanisms (28,32,65). Anti-apoptotic agents, caspase enzyme inhibitors and anti-inflammatory agents slow progression in ALS models. When transgenic mice carrying mutant SOD1 are crossed with mice expressing a dominant inactive form of caspase-1, they have a slight increase in lifespan (30), and over-expression of bcl-2, a mitochondrial inhibitor of apoptosis, protects against neuronal loss and prolongs life in the ALS model (31). Exogenous caspase inhibitors also prolong life in ALS mouse models with CuZn SOD1 mutations. A small peptide caspase inhibitor (zVAD-fmk) prolongs the survival of SOD1 transgenic mice by 4 weeks or about 20% (40). By comparison, riluzole extends survival by about 11% in the same SOD1 transgenic line and by about 3 months in ALS patients. zVAD-fmk blocks all known caspases and acts at several points in the activation cascade. It has low oral bioavailability and limited brain penetrance, and must be delivered by infusion into the cerebral ventricles. Acetylsalicylate (an anti-inflammatory agent) has been shown to delay the appearance of weakness in transgenic SOD-1 mice (66), and Cox-2 inhibitors protect against loss of spinal motor neurons in vitro and in vivo (67). Minocycline Minocycline, which inhibits apoptosis and inflammation, slows disease progression in models of neurodegeneration in general and of ALS in particular (17,20-23). Minocycline is FDA approved for treatment of infection, has high CNS penetration when taken orally and inhibits p38 MAP kinase (18). It reduces caspase-1, caspase-3 and iNOS activity in vitro and in vivo (20,68). Its anti-inflammatory properties include prevention of glutamate-induced activation of microglia and reduction of interleukin production in cell culture (57). It has neuroprotective effects in animal models of stroke/ischemic injury (24), and it delays disease progression in animal models of neurodegenerative disorders marked by caspase-regulated cell death. It slows disease progression and prevents activation of iNOS and caspase enzymes in the Huntington disease model (20), and prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson disease (21). Several laboratories have shown that minocycline delays disease progression in the ALS SOD1 model (Serge Przedborski, Personal Communication) (17,22,23), possibly involving down-regulation of p38 MAP Kinase. Intraperitoneal injections of 5 mg/kg/day provided an increase in lifespan of approximately 11% compared to placebo-treated mice in a blinded study of 20 SOD1 rodents (Serge Przedborski, personal communication). In an independent laboratory, minocycline prolonged life in the SOD1 ALS model (17). Mice injected with 10 mg/kg per day beginning at five weeks of age had delayed onset of impaired motor performance and had statistically significant extended survival of 11 days (9%) compared to saline-treated control mice. Pathologically, minocycline reduced the activation of caspase-1, caspase-3, inducible nitric oxide synthetase and p38 mitogen-activated protein kinase activity secondary to upstream effects on mitochondria. It directly inhibited mitochondrial permeability-transition-mediated cytochrome c release, a critical early step in the activation of cell death pathways including caspase-enzyme-mediated apoptosis. The authors detected these effects in vivo using ALS mice and cerebral ischemia models, in neuronal cells and in isolated mitochondria. In a separate study in the SOD1 ALS mouse model, minocycline improved survival and reduced microglial activation (22). In this study, transgenic mice with the G93A human SOD1 mutation were treated every weekday with an intraperitoneal injection of saline or minocycline starting at 70 days of age. Two different minocycline doses were used: 25 mg/kg and 50 mg/kg. Minocycline dose-dependently delayed decline in rotarod performance, which met statistical significance between high dose minocycline and saline-treated mice. Minocycline also delayed the onset and slowed decline in muscle weakness in a dose-dependent manner, again meeting statistical significance at the 50 mg/kg dose. Both minocycline concentrations delayed mortality to a significant degree. Mice treated with the higher dose had a prolonged life span of 16%. Pathologically, at 120 days of age, mice treated with minocycline had reduced motor neuron loss, vacuolization and microglial activation compared to control animals. In a rodent model of focal cerebral ischemia, minocycline reduced cortical infarction volume by 63% when started 4 hours after the onset of ischemia (24). In this study, ischemia was induced by inserting nylon thread into the internal carotid artery up to the middle cerebral artery. Animals received intraperitoneal injections of minocycline at 45 mg/kg twice the first day and 22.5 mg/kg for the subsequent 2 days. Pathologic studies indicated that minocycline inhibited activation of microglia and induction of interleukin-1beta converting enzyme, and reduced COX-2 expression and prostaglandin E2 production. In a study of experimental spinal cord injury in rodents, systemic administration of minocycline improved functional recovery (69). The authors reported that 90 mg/kg intraperitoneal injections one hour following moderate spinal cord contusion provided significant improvement in motor function when compared to control animals. Those animals that received minocycline had reduced neurodegeneration, apoptosis and caspase activation in the spinal cord. Minocycline also prevents nigrostriatal dopaminergic neurodegeneration in the 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease (21). In this controlled study, mice received doses of minocycline ranging from 60-120 mg/kg/day by oral gavage before, during and after MPTP administration. Minocycline inhibited phosphorylation of p38 MAP kinase, blocked MPTP-induced neurodegeneration and dopamine depletion, and was associated with marked reductions in iNOS and caspase-1 expression. In a blinded study using the R6/2 Huntington disease (HD) mouse model survival was prolonged following intraperitoneal injections of minocycline at 5 mg/kg/day (20). Daily minocycline treatment beginning at 6 weeks of age significantly delayed the characteristic decline of Rotarod performance and extended survival by 14% when compared to saline-treated mice. In this model there was reduction of caspase-1, and caspase-3 upregulation and of iNOS activity. There was no effect of tetracycline, which does not cross the blood-brain barrier, on performance or survival. Minocycline in Man The tetracyclines, among the first of the antibiotics to become available 50 years ago, remain widely used. Minocycline is a second-generation, long-acting tetracycline (70). It is indicated in the treatment of a variety of bacterial infections, including brain and meningeal infections. It is a widely prescribed systemic antibiotic for the management of acne (70,71), and has demonstrated benefit in inflammatory arthritis (25). Minocycline is ten times more lipid-soluble than other tetracyclines and has excellent CNS penetration and bioavailability. It is well tolerated and is used in outpatients. The serum half-life is approximately 17 hours. In long-term therapy, periodic laboratory evaluations of organ systems, including hematopoietic, renal and hepatic studies are performed. It is contraindicated in persons who have shown hypersensitivity to any of the tetracyclines, and during pregnancy and childhood because of dental staining and interference with bone growth. Minocycline, at oral doses of 100 mg twice daily, which roughly equates to the 5 mg/kg/day doses in the SOD1 and HD models provides proven anti-inflammatory benefit in human rheumatoid arthritis and is used to treat brain and meningeal infections. In a 2-year, double-blind protocol, oral minocyline at 100 mg twice per day, was superior to hydroxychloroquine in patients with early seropositive rheumatoid arthritis (25). Patients were significantly more likely to achieve 50% improvement and to be tapered off prednisone than controls. In blinded studies of subjects with advanced rheumatoid arthritis, 100 mg twice daily also provided statistically significant benefit when compared to control subjects (72). Toxicities of minocycline are similar to those reported with other tetracyclines and include staining of dental enamel, hyperpigmentation of skin and other tissues, photosensitivity, gastrointestinal intolerance, diarrhea and vestibular side effects, including dizziness, ataxia and vertigo (71,73). It has been reported to rarely induce immune reactions resulting in hepatitis, arthritis and drug-induced lupus, generalized hypersensitivity, serum sickness-like reactions, vasculitis, pseudotumor cerebri, hypersensitivity pneumonitis, interstitial nephritis and black thyroid syndrome (74). The half-life is prolonged in patients with renal failure. Food and divalent cations interfere minimally with oral absorption. Minocycline is eliminated through the hepatobiliary and gastrointestinal tracts. Minocycline may reduce oral contraceptive efficacy. Potentiation of warfarin-induced anticoagulation, and elevation of lithium, digoxin and theophylline levels necessitates close monitoring. The usual dosage is 100 mg every 12 hours, which has been well tolerated in chronic use (75). Significance Despite recent advances in partial understanding of molecular events leading to motor neuron degeneration, ALS remains an incurable disease. The therapeutic benefit of caspase inhibitors and anti-inflammatory agents, including minocycline, on survival and motor function in SOD1 transgenic mice and of minocycline in animal models of ALS, Huntington disease and Parkinson disease provides further evidence that stress enzyme-mediated cell death pathways may contribute to neurodegeneration. This is the first study in human ALS of a medication that acts as both an anti-apoptotic and anti-inflammatory agent. Any compound proven to slow the course of human ALS will be of immediate importance both clinically and from the perspective of understanding the underlying biology of motor neuron diseases. Additionally, approximately 50% of patients with ALS do not take riluzole, the only currently FDA approved drug for this disease, because of its high cost. Minocycline would provide a safe and less expensive treatment alternative to riluzole (monthly cost of riluzole 50 mg twice daily = $900; monthly cost of minocycline 100 mg twice daily = $150). Additionally, because of differing mechanisms of action, the drugs could have a synergistic effect upon the disease, and be tested in future combination trials. Preliminary Studies An open-label pilot study in 3 ALS patients was performed to test initial tolerability in this population. Minocycline, 100 mg twice daily, was well tolerated in combination with riluzole (76). There were no side effects due to minocycline or laboratory abnormalities, and motor function as measured by the ALSFRS-R was stable over three months. One subject reported mild intermittent diarrhea while taking riluzole prior to enrollment and noted no change during the study period. Two placebo controlled pilot studies of this population were completed in January 2003. The first, at the University of New Mexico (PI Dr. Paul Gordon), was a randomized placebo controlled study of the tolerability of minocycline 100 mg twice daily in combination with riluzole in patients with ALS. There were nineteen subjects, 11 men and 8 women, enrolled in the 6-month study. Subjects were evaluated using ALSFRS-R, MMT, maximum voluntary isometric contraction (MVIC), forced vital capacity (FVC), adverse events and laboratory studies (liver function, renal function and blood count) monthly. Two patients stopped the monthly evaluations because of travel distance and disability, but had AE and ALSFRS-R monitored by telephone. Three patients (2 minocycline; 1 placebo) died during the study of respiratory arrest due to progressive ALS. Other common adverse events included phlebitis (1), diarrhea (1), superinfection (1), dry mouth (1), fall (1), pneumonia (1), and flu-like symptoms (1). There were no statistically significant differences in occurrence of adverse events between placebo and active drug groups. Three patients (2 placebo, 1 active drug group) had mild elevation of liver function. All were taking riluzole. There were no statistically significant differences in the rate of change in strength, or functional outcome measures between groups. A second pilot study of minocycline at the Forbes Norris ALS Research Center in San Francisco (PI Dr. Robert Miller), a dose escalation study, is also completed. There were 23 patients enrolled in this randomized, placebo-controlled, 8-month crossover study. The target dose was 400 mg of minocycline. ALSFRS-R, FVC and laboratory evaluations were completed monthly. The mean tolerated dose of minocycline was 387 mg/day, or 7.7 pills/day (target 8/day). The common adverse events encountered were falls, constipation, insomnia, appetite loss and reflux. 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