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. Only dyspepsia occurred more often while taking minocycline (5:1 minocycline: placebo). There were no statistically significant differences between groups in occurrence of other adverse events. BUN and AST/ALT were elevated to a statistically significant degree while taking minocycline, though the elevations were not considered clinically significant. The ALSFRS-R declined at a greater rate while taking minocycline (p=0.047), though the study was not powered for efficacy. Doses above 400 mg/day have not been tolerated in any disease (Steven Projan, Wyeth Ayerst, personal communication). References: 1. Rowland LP, Shneider NA. Amyotrophic lateral sclerosis. N Engl J Med 2001;344:1688-1700. 2. Munsat TL, Andres PL, Finison L, Conlon T, Thibodeau L. The natural history of motor neuron loss in ALS. Neurology 1988;38:452-458. 3. The ALS CNTF Treatment Study (ACTS) Phase I-II Study Group. The amyotrophic lateral sclerosis functional rating scale. 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