Drug Repurposing: A Systematic Approach to Evaluate Candidate Oral Neuroprotective Interventions for Secondary Progressive Multiple Sclerosis

Objective

To develop and implement an evidence based framework to select, from drugs already licenced, candidate oral neuroprotective drugs to be tested in secondary progressive multiple sclerosis.

Design

Systematic review of clinical studies of oral putative neuroprotective therapies in MS and four other neurodegenerative diseases with shared pathological features, followed by systematic review and meta-analyses of the in vivo experimental data for those interventions. We presented summary data to an international multi-disciplinary committee, which assessed each drug in turn using pre-specified criteria including consideration of mechanism of action.

Results

We identified a short list of fifty-two candidate interventions. After review of all clinical and pre-clinical evidence we identified ibudilast, riluzole, amiloride, pirfenidone, fluoxetine, oxcarbazepine, and the polyunsaturated fatty-acid class (Linoleic Acid, Lipoic acid; Omega-3 fatty acid, Max EPA oil) as lead candidates for clinical evaluation.

Conclusions

We demonstrate a standardised and systematic approach to candidate identification for drug rescue and repurposing trials that can be applied widely to neurodegenerative disorders.     

Volumetric Analysis of the Hypothalamus in Huntington Disease Using 3T MRI: The IMAGE-HD Study

Huntington disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin gene. Non-motor symptoms and signs such as psychiatric disturbances, sleep problems and metabolic dysfunction are part of the disease manifestation. These aspects may relate to changes in the hypothalamus, an area of the brain involved in the regulation of emotion, sleep and metabolism. Neuropathological and imaging studies using both voxel-based morphometry (VBM) of magnetic resonance imaging (MRI) as well as positron emission tomography (PET) have demonstrated pathological changes in the hypothalamic region during early stages in symptomatic HD. In this investigation, we aimed to establish a robust method for measurements of the hypothalamic volume in MRI in order to determine whether the hypothalamic dysfunction in HD is associated with the volume of this region. Using T1-weighted imaging, we describe a reproducible delineation procedure to estimate the hypothalamic volume which was based on the same landmarks used in histologically processed postmortem hypothalamic tissue. Participants included 36 prodromal HD (pre-HD), 33 symptomatic HD (symp-HD) and 33 control participants who underwent MRI scanning at baseline and 18 months follow-up as part of the IMAGE-HD study. We found no evidence of cross-sectional or longitudinal changes between groups in hypothalamic volume. Our results suggest that hypothalamic pathology in HD is not associated with volume changes.     

Pancreatic polypeptide inhibits somatostatin secretion

Pancreatic polypeptide (PP) is a major agonist for neuropeptide Y4 receptors (NPY4R). While NPY4R has been identified in various tissues, the cells on which it is expressed and its function in those cells has not been clearly delineated. Here we report that NPY4R is present in all somatostatin-containing cells of tissues that we tested, including pancreatic islets, duodenum, hippocampus, and hypothalamus. Its agonism by PP decreases somatostatin secretion from human islets. Mouse embryonic hippocampal (mHippo E18) cells expressed NPY4Rs and their activation by PP consistently decreased somatostatin secretion. Furthermore, central injection of PP in mice induced c-Fos immunoreactivity in somatostatin-containing cells in the hippocampus compared with PBS-injected mice. In sum, our results identify PP as a pivotal modulator of somatostatin secretion.     

The neutral lipid composition present in the digestive vacuole of Plasmodium falciparum concentrates heme and mediates β-hematin formation with an unusually low activation energy

In eukaryotic cells, neutral lipids serve as major energy storage molecules; however, in Plasmodium falciparum, a parasite responsible for causing malaria in humans, neutral lipids may have other functions during the intraerythrocytic stage of the parasite life cycle. Specifically, experimental data suggest that neutral lipid structures behave as a catalyst for the crystallization of hemozoin, a detoxification byproduct of several blood-feeding organisms, including malaria parasites. Synthetic neutral lipid droplets (SNLDs) were produced by depositing a lipid blend solution comprised of mono- and diglycerides onto an aqueous surface. These lipid droplets are able to mediate the production of brown pigments that are morphologically and chemically identical to hemozoin. The partitioning of heme into these SNLDs was examined by employing Nile Red, a lipid specific dye. Soluble ferriprotoporphyrin IX was observed to spontaneously localize to the lipid droplets, partitioning in a pH-dependent manner with an estimated log P of 2.6. Interestingly, the pH profile of heme partitioning closely resembles that of β-hematin formation. Differential scanning calorimetry and kinetic studies demonstrated that the SNLDs provide a unique environment that promotes hemozoin formation. SNLD-mediated formation of the malaria pigment displayed an activation energy barrier lower than those of individual lipid components. In particular, lipid droplets composed of diglycerides displayed activation barriers lower than those composed of monoglycerides. This difference was attributed to the greater fluidity of these lipids. In conjunction with the known pattern of lipid body proliferation, it is suggested that neutral lipid structures within the digestive vacuole not only are the location of in vivo hemozoin formation but are also essential for the survival of the parasite by functioning as a kinetically competent and site specific mediator for heme detoxification.     

Healthy Human T-Cell Responses to Aspergillus fumigatus Antigens

Background

Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (T_H1–T_H17) and destructive allergic (T_H2) immunity. How Aspergillus allergens (Asp f proteins) participate in the development of allergic sensitization is unknown.

Methodology/Principal Findings

To determine whether Asp f proteins are strictly associated with T_H2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-γ, IL-4 or IL-17) to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-γ more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 T_H1-responses to Asp f3 (a putative peroxismal membrane protein), Asp f9/16 (cell wall glucanase), Asp f11 (cyclophilin type peptidyl-prolyl isomerase) and Asp f22 (enolase). Strong IFN-γ responses were reproduced in most subjects tested over 6 month intervals.

Conclusions

Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases.     

Comparison of hot water immersion at self-adjusted maximum tolerable temperature,with or without the addition of salt,for rapid weight loss in mixed martial arts athletes

Hot water immersion is used by athletes in weight category sports to produce rapid weight loss (RWL) by means of passive fluid loss, and often is performed with the addition of Epsom salts (magnesium sulphate). This study investigated the magnitude of body mass losses during hot water immersion with or without the addition of salt, with the temperature commencing at 37.8°C and being self-adjusted by participants to their maximum tolerable temperature. In a crossover design, eight male MMA athletes (29.4 ± 5.3 y; 1.83 ± 0.05 m; 85.0 ± 4.9 kg) performed a 20 min whole-body immersion followed by a 40 min wrap in a warm room, twice in sequence per visit. During one visit, only fresh water was used (FWB), and in the other visit, magnesium sulphate (1.6% wt/vol) was added to the bath (SWB). Prior to each visit, 24 h of carbohydrate, fibre and fluid restriction was undertaken. Water temperatures at the end of the first and second baths were ~39.0°C and ~39.5°C, respectively. Body mass losses induced by the hot bath protocols were 1.71 ± 0.70 kg and 1.66 ± 0.78 kg for FWB and SWB, respectively (P = 0.867 between trials, d = 0.07), and equivalent to ~2.0% body mass. Body mass lost during the entire RWL protocol was 4.5 ± 0.7%. Under the conditions employed, the magnitude of body mass lost in SWB was similar to FWB. Augmenting passive fluid loss during hot water immersion with the addition of salt may require a higher salt concentration than that presently utilised.     

Differences in the electrostatic surfaces of the type III secretion needle proteins PrgI, BsaL, and MxiH

Gram-negative bacteria use a needle-like protein assembly, the type III secretion apparatus, to inject virulence factors into target cells to initiate human disease. The needle is formed by the polymerization of approximately 120 copies of a small acidic protein that is conserved among diverse pathogens. We previously reported the structure of the BsaL needle monomer from Burkholderia pseudomallei by nuclear magnetic resonance (NMR) spectroscopy and others have determined the crystal structure of the Shigella flexneri MxiH needle. Here, we report the NMR structure of the PrgI needle protein of Salmonella typhimurium, a human pathogen associated with food poisoning. PrgI, BsaL, and MxiH form similar two helix bundles, however, the electrostatic surfaces of PrgI differ radically from those of BsaL or MxiH. In BsaL and MxiH, a large negative area is on a face formed by the helix alpha1-alpha2 interface. In PrgI, the major negatively charged surface is not on the "face" but instead is on the "side" of the two-helix bundle, and only residues from helix alpha1 contribute to this negative region. Despite being highly acidic proteins, these molecules contain large basic regions, suggesting that electrostatic contacts are important in needle assembly. Our results also suggest that needle-packing interactions may be different among these bacteria and provide the structural basis for why PrgI and MxiH, despite 63% sequence identity, are not interchangeable in S. typhimurium and S. flexneri.     

DeltaF508 mutation results in impaired gastric acid secretion

The cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as a multifunctional protein that is involved in Cl(-) secretion, as well as acting as a regulatory protein. In order for acid secretion to take place a complex interaction of transport proteins and channels must occur at the apical pole of the parietal cell. Included in this process is at least one K(+) and Cl(-) channel, allowing for both recycling of K(+) for the H,K-ATPase, and Cl(-) secretion, necessary for the generation of concentrated HCl in the gastric gland lumen. We have previously shown that an ATP-sensitive potassium channel (K(ATP)) is expressed in parietal cells. In the present study we measured secretagogue-induced acid secretion from wild-type and DeltaF508-deficient mice in isolated gastric glands and whole stomach preparations. Secretagogue-induced acid secretion in wild-type mouse gastric glands could be significantly reduced with either glibenclamide or the specific inhibitor CFTR-inh172. In DeltaF508-deficient mice, however, histamine-induced acid secretion was significantly less than in wild-type mice. Furthermore, immunofluorescent localization of sulfonylurea 1 and 2 failed to show expression of a sulfonylurea receptor in the parietal cell, thus further implicating CFTR as the ATP-binding cassette transporter associated with the K(ATP) channels. These results demonstrate a regulatory role for the CFTR protein in normal gastric acid secretion.