Protective Effects of Estrogen via Nanoparticle Delivery to Attenuate Myelin Loss and Neuronal Death after Spinal Cord Injury.

Spinal cord injury (SCI) is associated with devastating neurological deficits affecting more than 11,000 Americans each year. Although several therapeutic agents have been proposed and tested, no FDA-approved pharmacotherapy is available for SCI treatment. We have recently demonstrated that estrogen (E2) acts as an antioxidant and anti-inflammatory agent, attenuating gliosis in SCI. We have also demonstrated that nanoparticle-mediated focal delivery of E2 to the injured spinal cord decreases lesion size, reactive gliosis, and glial scar formation. The current study tested in vitro effects of E2 on reactive oxygen species (ROS) and calpain activity in microglia, astroglia, macrophages, and fibroblasts, which are believed to participate in the inflammatory events and glial scar formation after SCI. E2 treatment decreased ROS production and calpain activity in these glial cells, macrophages, and fibroblast cells in vitro. This study also tested the efficacy of fast- and slow-release nanoparticle-E2 constructs in a rat model of SCI. Focal delivery of E2 via nanoparticles increased tissue distribution of E2 over time, attenuated cell death, and improved myelin preservation in injured spinal cord. Specifically, the fast-release nanoparticle-E2 construct reduced the Bax/Bcl-2 ratio in injured spinal cord tissues, and the slow-release nanoparticle-E2 construct prevented gliosis and penumbral demyelination distal to the lesion site. These data suggest this novel E2 delivery strategy to the lesion site may decrease inflammation and improve functional outcomes following SCI.

Nanoparticle-Based Estrogen Delivery to Spinal Cord Injury Site Reduces Local Parenchymal Destruction and Improves Functional Recovery.

Spinal cord injury (SCI) patients sustain significant functional impairments; this is causally related to restricted neuronal regeneration after injury. The ensuing reactive gliosis, inflammatory cascade, and glial scar formation impede axonal regrowth. Although systemic anti-inflammatory agents (steroids) have been previously administered to counteract this, no current therapeutic is approved for post-injury neuronal regeneration, in part because of related side effects. Likewise, therapeutic systemic estrogen levels exhibit neuroprotective properties, but dose-dependent side effects are prohibitive. The current study thus uses low-dose estrogen delivery to the spinal cord injury (SCI) site using an agarose gel patch embedded with estrogen-loaded nanoparticles. Compared to controls, spinal cords from rodents treated with nanoparticle site-directed estrogen demonstrated significantly decreased post-injury lesion size, reactive gliosis, and glial scar formation. However, axonal regeneration, vascular endothelial growth factor production, and glial-cell-derived neurotrophic factor levels were increased with estrogen administration. Concomitantly improved locomotor and bladder functional recovery were observed with estrogen administration after injury. Therefore, low-dose site-directed estrogen may provide a future approach for enhanced neuronal repair and functional recovery in SCI patients.

Targeting Enolase in Reducing Secondary Damage in Acute Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a complex debilitating condition leading to permanent life-long neurological deficits. The complexity of SCI suggests that a concerted multi-targeted therapeutic approach is warranted to optimally improve function. Damage to spinal cord is complicated by an increased detrimental response from secondary injury factors mediated by activated glial cells and infiltrating macrophages. While elevation of enolase especially neuron specific enolase (NSE) in glial and neuronal cells is believed to trigger inflammatory cascades in acute SCI, alteration of NSE and its subsequent effects in acute SCI remains unknown. This study measured NSE expression levels and key inflammatory mediators after acute SCI and investigated the role of ENOblock, a novel small molecule inhibitor of enolase, in a male Sprague-Dawley (SD) rat SCI model. Serum NSE levels as well as cytokines/chemokines and metabolic factors were evaluated in injured animals following treatment with vehicle alone or ENOblock using Discovery assay. Spinal cord samples were also analyzed for NSE and MMPs 2 and 9 as well as glial markers by Western blotting. The results indicated a significant decrease in serum inflammatory cytokines/chemokines and NSE, alterations of metabolic factors and expression of MMPs in spinal cord tissues after treatment with ENOblock (100 µg/kg, twice). These results support the hypothesis that activation of glial cells and inflammation status can be modulated by regulation of NSE expression and activity. Analysis of SCI tissue samples by immunohistochemistry confirmed that ENOblock decreased gliosis which may have occurred through reduction of elevated NSE in rats. Overall, elevation of NSE is deleterious as it promotes extracellular degradation and production of inflammatory cytokines/chemokines and metabolic factors which activates glia and damages neurons. Thus, reduction of NSE by ENOblock may have potential therapeutic implications in acute SCI.

Low-Dose Pulsatile Interleukin-6 As a Treatment Option for Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) remains one of the most common and serious complications of diabetes. Currently, pharmacological agents are limited to treating the pain associated with DPN, and do not address the underlying pathological mechanisms driving nerve damage, thus leaving a significant unmet medical need. Interestingly, research conducted using exercise as a treatment for DPN has revealed interleukin-6 (IL-6) signaling to be associated with many positive benefits such as enhanced blood flow and lipid metabolism, decreased chronic inflammation, and peripheral nerve fiber regeneration. IL-6, once known solely as a pro-inflammatory cytokine, is now understood to signal as a multifunctional cytokine, capable of eliciting both pro- and anti-inflammatory responses in a context-dependent fashion. IL-6 released from muscle in response to exercise signals as a myokine and as such has a unique kinetic profile, whereby levels are transiently elevated up to 100-fold and return to baseline levels within 4 h. Importantly, this kinetic profile is in stark contrast to long-term IL-6 elevation that is associated with pro-inflammatory states. Given exercise induces IL-6 myokine signaling, and exercise has been shown to elicit numerous beneficial effects for the treatment of DPN, a causal link has been suggested. Here, we discuss both the clinical and preclinical literature related to the application of IL-6 as a treatment strategy for DPN. In addition, we discuss how IL-6 may directly modulate Schwann and nerve cells to explore a mechanistic understanding of how this treatment elicits a neuroprotective and/or regenerative response. Collectively, studies suggest that IL-6, when administered in a low-dose pulsatile strategy to mimic the body's natural response to exercise, may prove to be an effective treatment for the protection and/or restoration of peripheral nerve function in DPN. This review highlights the studies supporting this assertion and provides rationale for continued investigation of IL-6 for the treatment of DPN.

Neuron specific enolase: a promising therapeutic target in acute spinal cord injury.

Enolase is a multifunctional protein, which is expressed abundantly in the cytosol. Upon stimulatory signals, enolase can traffic to cell surface and contribute to different pathologies including injury, autoimmunity, infection, inflammation, and cancer. Cell-surface expression of enolase is often detected on activated macrophages, microglia/macrophages, microglia, and astrocytes, promoting extracellular matrix degradation, production of pro-inflammatory cytokines/chemokines, and invasion of inflammatory cells in the sites of injury and inflammation. Inflammatory stimulation also induces translocation of enolase from the cytosolic pool to the cell surface where it can act as a plasminogen receptor and promote extracellular matrix degradation and tissue damage. Spinal cord injury (SCI) is a devastating debilitating condition characterized by progressive pathological changes including complex and evolving molecular cascades, and insights into the role of enolase in multiple inflammatory events have not yet been fully elucidated. Neuronal damage following SCI is associated with an elevation of neuron specific enolase (NSE), which is also known to play a role in the pathogenesis of hypoxic-ischemic brain injury. Thus, NSE is now considered as a biomarker in ischemic brain damage, and it has recently been suggested to be a biomarker in traumatic brain injury (TBI), stroke and anoxic encephalopathy after cardiac arrest and acute SCI as well. This review article gives an overview of the current basic research and clinical studies on the role of multifunctional enolase in neurotrauma, with a special emphasis on NSE in acute SCI.

Molecular Changes in Sub-lesional Muscle Following Acute Phase of Spinal Cord Injury.

To clarify the molecular changes of sublesional muscle in the acute phase of spinal cord injury (SCI), a moderately severe injury (40 g cm) was induced in the spinal cord (T10 vertebral level) of adult male Sprague-Dawley rats (injury) and compared with sham (laminectomy only). Rats were sacrificed at 48 h (acute) post injury, and gastrocnemius muscles were excised. Morphological examination revealed no significant changes in the muscle fiber diameter between the sham and injury rats. Western blot analyses performed on the visibly red, central portion of the gastrocnemius muscle showed significantly higher expression of muscle specific E3 ubiquitin ligases (muscle ring finger-1 and muscle atrophy f-box) and significantly lower expression of phosphorylated Akt-1/2/3 in the injury group compared to the sham group. Cyclooxygenase 2, tumor necrosis factor alpha (TNF-α), and caspase-1, also had a significantly higher expression in the injury group; although, the mRNA levels of TNF-α and IL-6 did not show any significant difference between the sham and injury groups. These results suggest activation of protein degradation, deactivation of protein synthesis, and development of inflammatory reaction occurring in the sublesional muscles in the acute phase of SCI before overt muscle atrophy is seen.

Inhibition of Calpain Activation Protects MPTP-Induced Nigral and Spinal Cord Neurodegeneration, Reduces Inflammation, and Improves Gait Dynamics in Mice.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, resulting in dopaminergic (DA) neuronal loss in the substantia nigra pars compacta (SNpc) and damage to the extranigral spinal cord neurons. Current therapies do not prevent the disease progression. Hence, developing efficacious therapeutic strategies for treatment of PD is of utmost importance. The goal of this study is to delineate the involvement of calpain-mediated inflammation and neurodegeneration in SN and spinal cord in MPTP-induced parkinsonian mice (C57BL/6 N), thereby elucidating potential therapeutic target(s). Increased calpain expression was found localized to tyrosine hydroxylase (TH(+)) neurons in SN with significantly increased TUNEL-positive neurons in SN and spinal cord neurons in MPTP mice. Inflammatory markers Cox-2, caspase-1, and NOS-2 were significantly upregulated in MPTP mouse spinal cord as compared to control. These parameters correlated with the activation of astrocytes, microglia, infiltration of CD4(+)/CD8(+) T cells, and macrophages. We found that subpopulations of CD4(+) cells (Th1 and Tregs) were differentially expanded in MPTP mice, which could be regulated by inhibition of calpain with the potent inhibitor calpeptin. Pretreatment with calpeptin (25 µg/kg, i.p.) attenuated glial activation, T cell infiltration, nigral dopaminergic degeneration in SN, and neuronal death in spinal cord. Importantly, calpeptin ameliorated MPTP-induced altered gait parameters (e.g., reduced stride length and increased stride frequency) as demonstrated by analyses of spatiotemporal gait indices using ventral plane videography. These findings suggest that calpain plays a pivotal role in MPTP-induced nigral and extranigral neurodegenerative processes and may be a valid therapeutic target in PD.

Nanoparticle Estrogen in Rat Spinal Cord Injury Elicits Rapid Anti-Inflammatory Effects in Plasma, Cerebrospinal Fluid, and Tissue.

Persons with spinal cord injury (SCI) are in need of effective therapeutics. Estrogen (E2), as a steroid hormone, is a highly pleiotropic agent; with anti-inflammatory, anti-apoptotic, and neurotrophic properties, it is ideal for use in treatment of patients with SCI. Safety concerns around the use of high doses of E2 have limited clinical application, however. To address these concerns, low doses of E2 (25 µg and 2.5 µg) were focally delivered to the injured spinal cord using nanoparticles. A per-acute model (6 h after injury) was used to assess nanoparticle release of E2 into damaged spinal cord tissue; in addition, E2 was evaluated as a rapid anti-inflammatory. To assess inflammation, 27-plex cytokine/chemokine arrays were conducted in plasma, cerebrospinal fluid (CSF), and spinal cord tissue. A particular focus was placed on IL-6, GRO-KC, and MCP-1 as these have been identified from CSF in human studies as potential biomarkers in SCI. S100ß, an additional proposed biomarker, was also assessed in spinal cord tissue only. Tissue concentrations of E2 were double those found in the plasma, indicating focal release. E2 showed rapid anti-inflammatory effects, significantly reducing interleukin (IL)-6, GRO-KC, MCP-1, and S100ß in one or all compartments. Numerous additional targets of rapid E2 modulation were identified including: leptin, MIP-1α, IL-4, IL-2, IL-10, IFNγ, tumor necrosis factor-α, etc. These data further elucidate the rapid anti-inflammatory effects E2 exerts in an acute rat SCI model, have identified additional targets of estrogen efficacy, and suggest nanoparticle delivered estrogen may provide a safe and efficacious treatment option in persons with acute SCI.

Recent advances in the pharmacologic treatment of spinal cord injury.

A need exists for the effective treatment of individuals suffering from spinal cord injury (SCI). Recent advances in the understanding of the pathophysiological mechanisms occurring in SCI have resulted in an expansion of new therapeutic targets. This review summarizes both preclinical and clinical findings investigating the mechanisms and cognate pharmacologic therapeutics targeted to modulate hypoxia, ischemia, excitotoxicity, inflammation, apoptosis, epigenetic alterations, myelin regeneration and scar remodeling. Successful modulation of these targets has been demonstrated in both preclinical and clinical studies with agents such as Oxycyte, Minocycline, Riluzole, Premarin, Cethrin, and ATI-355. The translation of these agents into clinical studies highlights the progress the field has made in the past decade. SCI proves to be a complex condition; the numerous pathophysiological mechanisms occurring at varying time points suggests that a single agent approach to the treatment of SCI may not be optimal. As the field continues to mature, the hope is that the knowledge gained from these studies will be applied to the development of an effective multi-pronged treatment strategy for SCI.

8-Tetrahydropyran-2-yl chromans: highly selective beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors.

A series of 2,3,4,4a,10,10a-hexahydropyrano[3,2-b]chromene analogs was developed that demonstrated high selectivity (>2000-fold) for BACE1 vs Cathepsin D (CatD). Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Guided by structure based design, changes to P2' and P3 moieties were explored. A conformationally restricted P2' methyl group provided inhibitors with excellent cell potency (37-137 nM) and selectivity (435 to >2000-fold) for BACE1 vs CatD. These efforts lead to compound 59, which demonstrated a 69% reduction in rat CSF Aß1-40 at 60 mg/kg (PO).

Discovery of a Novel Class of Imidazo[1,2-a]Pyridines with Potent PDGFR Activity and Oral Bioavailability.

The in silico construction of a PDGFRß kinase homology model and ensuing medicinal chemistry guided by molecular modeling, led to the identification of potent, small molecule inhibitors of PDGFR. Subsequent exploration of structure-activity relationships (SAR) led to the incorporation of a constrained secondary amine to enhance selectivity. Further refinements led to the integration of a fluorine substituted piperidine, which resulted in significant reduction of P-glycoprotein (Pgp) mediated efflux and improved bioavailability. Compound 28 displayed oral exposure in rodents and had a pronounced effect in a pharmacokinetic-pharmacodynamic (PKPD) assay.

Discovery of 7-tetrahydropyran-2-yl chromans: ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors that reduce amyloid ß-protein (Aß) in the central nervous system.

In an attempt to increase selectivity vs Cathepsin D (CatD) in our BACE1 program, a series of 1,3,4,4a,10,10a-hexahydropyrano[4,3-b]chromene analogues was developed. Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Using structure-based design, substitutions to improve binding to both the S3 and S2' sites of BACE1 were explored. An acyl guanidine moiety provided the most potent analogues. These compounds demonstrated 10-420 fold selectivity for BACE1 vs CatD, and were highly potent in a cell assay measuring Aß1-40 production (5-99 nM). They also suffered from high efflux. Despite this undesirable property, two of the acyl guanidines achieved free brain concentrations (Cfree,brain) in a guinea pig PD model sufficient to cover their cell IC50s. Moreover, a significant reduction of Aß1-40 in guinea pig, rat, and cyno CSF (58%, 53%, and 63%, respectively) was observed for compound 62.

Qualification and application of a liquid chromatography-tandem mass spectrometric method for the determination of human Aß1-40 and Aß1-42 peptides in transgenic mouse plasma using micro-elution solid phase extraction.

A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and applied for the determination of human Aß1-40 and Aß1-42 peptides in transgenic mouse plasma to support preclinical pharmacodynamics studies. The method consisted of micro-elution solid phase extraction for sample preparation and LC-MS/MS analysis in the negative ion mode using electrospray ionization for analysis. (15)N53-Aß1-40 and (15)N55-Aß1-42 peptides were used as internal standards. A quadratic regression (weighted 1/concentrations), with an equation y = ax(2) + bx + c, was used to fit calibration curves over the concentration range of 0.500-100 ng/mL for both Aß1-40 and Aß1-42 peptides. For quality control samples at 6.00, 40.0 and 80.0 ng/mL from the qualification experiment, the within-run accuracy ranged from -2.69 to 0.583 % with precision values ≤8.23 % for Aß1-40. Within-run accuracy ranged from -4.83 to 10.1 % with precision values ≤8.87 % for Aß1-42. Samples from a pharmacodynamics study using Tg2576 transgenic mice were analyzed by this qualified LC-MS/MS method and concentrations were compared to those generated by ELISA. The two methods were shown to be comparable for Aß1-40 quantification of samples from the Tg2576 amyloid precursor protein transgenic mouse model, but varied slightly for Aß1-42.

Spirocyclic ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors: from hit to lowering of cerebrospinal fluid (CSF) amyloid ß in a higher species.

A hallmark of Alzheimer's disease is the brain deposition of amyloid beta (Aß), a peptide of 36-43 amino acids that is likely a primary driver of neurodegeneration. Aß is produced by the sequential cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of BACE1 represents an attractive therapeutic target to slow or prevent Alzheimer's disease. Herein we describe BACE1 inhibitors with limited molecular flexibility and molecular weight that decrease CSF Aß in vivo, despite efflux. Starting with spirocycle 1a, we explore structure-activity relationships of core changes, P3 moieties, and Asp binding functional groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood-brain barrier (BBB) penetration. Using wild type guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aß lowering. Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aß in rodents and in monkey.

ARRY-520, a novel KSP inhibitor with potent activity in hematological and taxane-resistant tumor models.

AIM: Profiling the efficacy and pharmacodynamic activity of the kinesin spindle protein (KSP) inhibitor ARRY-520 will aid the identification of responsive tumor types and pharmacodynamic profiles that correlate with activity. MATERIALS AND METHODS: In vivo activity was evaluated in a diverse panel of 16 different tumor xenograft models. Pharmacodynamic activity was evaluated in selected models. RESULTS: ARRY-520 had low nanomolar antiproliferative activity in tumor cell lines. Monopolar spindles were formed at active potencies. Partial or complete responses were observed in 13/16 xenograft models. Hematological tumors were particularly sensitive, with a 100% complete response rate in some models. Maintenance of mitotic block for a sufficient length of time for cells to lose survival signals and progress to apoptosis was a key component of the mechanism of activity. ARRY-520 was also active in several taxane resistant models. CONCLUSION: The data provide a rationale for clinical evaluation of the activity of ARRY-520 in hematological carcinomas and taxane-resistant tumors.

Oxygen treatment triggers cognitive impairment in Alzheimer's transgenic mice.

An association between major surgery in the elderly and precipitation of Alzheimer's disease has been reported. As 100% oxygen (hyperoxia) is commonly administered after surgery, we exposed cognitively unimpaired Alzheimer's transgenic mice to hyperoxia typical of human exposure in a hospital setting. Three-hour hyperoxia treatments to young adult Alzheimer's transgenic mice: (i) triggered cognitive impairment that was not otherwise present at that age, (ii) increased aberrant brain synaptophysin staining, and (iii) increased brain levels of isofurans (products of lipid peroxidation sensitive to hyperoxia). Thus, hyperoxia-induced synaptic dysfunction and brain oxidative stress are likely the triggering mechanisms of cognitive dysfunction in Alzheimer's mice. These results may suggest that exposure of elderly patients to excessive amounts of oxygen perioperatively hastens the development of Alzheimer's disease.

GRK5 deficiency leads to early Alzheimer-like pathology and working memory impairment.

G-protein coupled receptor kinase-5 (GRK5) deficiency has been linked to early Alzheimer's disease in humans and mouse models of the disease. To determine potential roles of GRK5 in the disease pathogenesis, the GRK5 knockout mouse was evaluated at pathological and behavioral levels. We found that these mice displayed an age-dependent increase in hippocampal axonal defects characterized by clusters of axonal swellings that accumulated abnormal amounts of molecular motor proteins, microtubule-associated proteins, intracellular beta-amyloid, and subcellular organelles. In severe cases, extracellular beta-amyloid fibrillar deposits were occasionally observed, along with degenerating axonal components, and were tightly surrounded by reactive astrocytes. Moreover, significant loss of synaptic proteins and early signs of cholinoceptive neurodegeneration were evident in the hippocampus as well. Consistent with the moderate level of pathologic change, aged GRK5 knockout mice displayed selective working memory impairment, with other cognitive domains unaffected. Taken together, these findings not only strongly support an important role of GRK5 deficiency in early Alzheimer's pathogenesis, but also promote the GRK5 knockout mouse as an additional model for early Alzheimer-related studies.