The mechanism of action of a novel neuroprotective low molecular weight dextran sulphate: New platform therapy for neurodegenerative diseases like Amyotrophic Lateral Sclerosis

Abstract

Background: Acute and chronic neurodegenerative diseases represent an immense socioeconomic burden that drives the need for new disease modifying drugs. Common pathogenic mechanisms in these diseases are evident, suggesting that a platform neuroprotective therapy may offer effective treatments. Here we present evidence for the mode of pharmacological action of a novel neuroprotective low molecular weight dextran sulphate drug called ILB^®. The working hypothesis was that ILB^® acts via the activation of heparin-binding growth factors (HBGF). Methods: Pre-clinical and clinical (healthy people and patients with ALS) in vitro and in vivo studies evaluated the mode of action of ILB^®. In vitro binding studies, functional assays and gene expression analyses were followed by the assessment of the drug effects in an animal model of severe traumatic brain injury (sTBI) using gene expression studies followed by functional analysis. Clinical data, to assess the hypothesized mode of action, are also presented from early phase clinical trials. Results: ILB^® lengthened APTT time, acted as a competitive inhibitor for HGF-Glypican-3 binding, effected pulse release of heparin-binding growth factors (HBGF) into the circulation and modulated growth factor signaling pathways. Gene expression analysis demonstrated substantial similarities in the functional dysregulation induced by sTBI and various human neurodegenerative conditions and supported a cascading effect of ILB^® on growth factor activation, followed by gene expression changes with profound beneficial effect on molecular and cellular functions affected by these diseases. The transcriptional signature of ILB^® relevant to cell survival, inflammation, glutamate signaling, metabolism and synaptogenesis, are consistent with the activation of neuroprotective growth factors as was the ability of ILB^® to elevate circulating levels of HGF in animal models and humans. Conclusion: ILB^® releases, redistributes and modulates the bioactivity of HBGF that target disease compromised nervous tissues to initiate a cascade of transcriptional, metabolic and immunological effects that control glutamate toxicity, normalize tissue bioenergetics, and resolve inflammation to improve tissue function. This unique mechanism of action mobilizes and modulates naturally occurring tissue repair mechanisms to restore cellular homeostasis and function. The identified pharmacological impact of ILB^® supports the potential to treat various acute and chronic neurodegenerative disease, including sTBI and ALS.

Keywords: amyotrophic lateral sclerosis; glutamate; heparin-binding growth factors; inflammation; low molecular weight-dextran sulphate; metabolism; neurodegeneration; traumatic brain injury.

FIGURE 1

ILB^® structure.

FIGURE 2

The effect of ILB^® on Glypican-3:HGF binding. (A) Concentration-effect curves of Glypican-3 binding to HGF in the absence or presence of various concentrations of ILB^® (µg/ml). (B). Regression analysis of Log (DR-1) values for different ILB^® concentrations results in a linear regression with a slope not significantly different from 1 (with the given variability of the assay—see in Supplementary Material S1).

FIGURE 3

Anticoagulant effect of ILB^® measured by APTT and anti-FXII activity. (A) At relatively low concentrations ILB^® increases the Activated Partial Thromboplastin Time (APTT), yet (B) higher ILB^® concentrations are required to impact anti-FXII activity.

FIGURE 4

ILB^® impacts the intracellular signalling of heparin-binding growth factors. (A) Concentration-dependent TGFß-induced responses in human embryonic kidney cells (HEK-Blue™) stably transfected with human TGFßR1, Smad3 and Smad4 genes, along with Smad3/4-binding elements (SBE)-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene were differentially modulated by ILB^® in a concentration-dependant manner over a range of concentrations (10–600 μg/ml). The data presented was pooled from 7 independent experiments. (B) The potentiation of the TGFß response (illustrated at 3.0 ng/ml) by ILB^® at 10 μg/ml (*p < 0.05; mean + SEM, n = 7) and the inhibition of the TGFß (100 ng/ml) response by ILB^® at 600 μg/ml (*p < 0.05; mean - SEM, n = 7).

FIGURE 5

Impact of ILB^® upon the secretion of IL-6 from human monocytes. Monocytes purified from human PBMCs were cultured in the absence of stimulation (media) or stimulated with LPS (0.01 ng/ml) in the absence (Vehicle) or presence of either ILB^®, dexamethasone or heparin for 24 h. Levels of IL-6 were quantified in the cell culture supernatant by ELISA. Data presented as mean + SEM, n = 10. * indicates IL-6 levels below the limit of detection from monocytes from at least one donor (5.0 pg/ml). +p < 0.05, +++p < 0.001 Significant difference to stimulation (Mann Whitney U Test).

FIGURE 6

Similarities and differences between the functional signature of human neurodegenerative conditions and the sTBI model. (A) Overlap of the top 20 significantly affected canonical pathways. (B) Overlap of the top 20 significantly affected functions. (C) Overlap of the top 20 significantly affected diseases. (D) Overlap of the top 20 upstream regulator growth factors and cytokines. The complete dataset from the brain transcriptome analysis is available on application to the authors.

FIGURE 7

The HGF concentration in mouse peripheral blood measured 30 min after ILB^® administration. Mean ± SEM is shown as well as statistically significant data compared to vehicle (student t-test, **p < 0.01, ***p < 0.001).

FIGURE 8

The HGF concentration was measured in mouse peripheral blood at 10, 30 and 60 min after either subcutaneous (s.c.) or intravenous (i.v.) administration of 10 mg/kg ILB^®. Mean ± SEM is shown as well as statistically significant increases compared to vehicle (student t-test, *p < 0.05, **p < 0.01, ***p < 0.001) and significant differences between the two routes of administration (student t-test, ^†† p < 0.01, ^††† p < 0.001).

FIGURE 9

Time course of HGF release into the plasma of healthy humans after ILB^® administration.

FIGURE 10

HGF release following s. c. injection of 1 mg/kg ILB^® in patients with ALS. PK of HGF release in response to ILB^® administration. Error bars represent the standard deviation.

FIGURE 11

Relationship between HGF and ILB^® concentration in plasma of patients with ALS. Linear regression within therapeutic range of ILB^®, regression function: y = 11733x-1197.2; R ² = 0.7014.

FIGURE 12

Effect of ILB^® on APTT ratio in patients with ALS. Linear regression within therapeutic range of ILB^®, regression function: y = 0.0844x+0.9999; R ² = 0.5202.

FIGURE 13

Summary diagram of ILB^® mechanism of action.