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OUR LEAD ASSET

NT-101 shown preclinically to be safe and reduce gliosis in TBI-induced mice (similar to sham mice)

The company’s principal strategic focus is developing our lead candidate (NT-101) which is a non-toxic solution of modified streptolysin O. Streptolysin O (SLO) is a 60 kDa exotoxin from Group A beta-hemolytic streptococci. While historically used as a permeabilizing agent in diagnostic tests, we have formulated a 55 kDa recombinant form of SLO (rSLO) which, when packaged after exposure to oxygen, becomes  a therapeutic agent.

 

Since the 1960s the world’s literature has described the toxicity of SLO in laboratory animals occurring only at doses which are several logs higher than those that we use.  That same literature has also described oSLO as an even less toxic version of SLO.  On-going research is expected to elucidate the impact of (a) significantly lower dosing and (b) oxidation on the toxicity of NT-101.  Until that research is completed, we use interchangeably the terms oSLO and rSLO to describe NT-101.

NT-101’s first indication is for the treatment of chronic Traumatic Brain Injury, a condition which can be described as concussions that have not completely healed.

Traumatic Brain Injury

Traumatic injury to the brain elicits physical damage to the vascular networks and neural circuit architecture alike. Gliosis is a reactive cellular process that occurs after injury. As with scarring in other organs and tissues, the glial scar is the body's mechanism to protect and begin the healing process in the nervous system. Unfortunately, the glial scar also serves as a major barrier to regenerating axons, and therefore, its emergence in evolution amongst higher vertebrates appears counterproductive (Rolls 2009).
As part of scar formation in the brain, astrocytes form a dense network of gap junctions that generate a physical barrier to axon extensions. (Cregg 2014). Coupled with many neuro-development inhibitor molecules that are secreted by the cells within the glial scar, physical and functional recovery from chronic injury is inhibited. Consequently, we believe that the recovery can be attributed to one or more of the following hypothesized mechanisms of action:
  • Reductions in neuroinflammation being achieved through down-regulation of pro-inflammatory genes and up-regulation of anti-inflammatory genes (Teng-Chao 2021, Mamber 2011)
  • Reversal of gliosis associated with post-concussive syndrome.
  • Reduced scarring in damaged areas of the cerebrovascular system potentially leading to improved brain hemodynamics.
  • Lowered fibrin levels lessening the physical interference of scar tissue with normal transmission of brain signals and restoring the extracellular matrix (Mamber 2004).
  • Inhibition of microglial infiltration of the wound area decreasing inflammation and gliosis (Beech Tree Labs / Harvard Medical School).
  • Induction of neuron remyelination; observations of remyelination in peripheral nerves (B.E.T.) may be broadened to encompass all neurons.
  • Stimulation of dendritic cells signaling macrophages to shift from their active state (which can be destructive if uncontrolled) to a resting phase which facilitates healing (Sanders, Mitchell – personal communication).

Genomic Profiling

Extensive genomic profiling has been completed on oSLO, starting with early efforts with qPCR and more recently with NanoString. Genomic profiling suggests that in 48 hours of exposure to oSLO, statistically significant changes in gene expression occur  (Alira 2022).  The biochemical pathways primarily involved include cytokine signaling, integrated stress response, autophagy and growth factor signaling.  We also discovered that PLA2G4A, a gene associated with delirium, dementia, amnesia and cognitive disorders, and CD209, which is associated with psychogenic disorders, is also downregulated.

 

oSLO suppresses the inflammatory phase of dendritic cells, which play a crucial role in modulating the detrimental conditions associated with cTBI. Genes associated with certain cytokines were down-regulated while genes associated with selected growth factors were up-regulated.


We have demonstrated that oSLO can change microglia and macrophage gene expression.  By triggering intra- and inter-cellular signaling and signaling via the extra-cellular matrix, oSLO can induce dendritic cell alterations and allow passage across the blood-brain barrier. Genomic data showcases that oSLO is acting to change the gene expression of microglia and macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) cellular phenotypes.
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The Role of Microglia 

Increased Density associated with TBI 

Preclinical Efficacy

oSLO Reduces Inflammatory Microglia

Effects of oSLO were studied in mice models for acute TBI (concussion) (Beech Tree Labs/Harvard Medical School).  The concussed mice were treated with subcutaneous (SC) administration of microdose oSLO (2.0 IUe) or a vehicle control.  Sham-concussed mice (no treatment) served as normal positive control.  The subcutaneous dosing route was used due to the difficulty of administering a dose to a mouse sublingually.  The mice were tested for memory retention in the standard water maze as time to reach the target platform.  The mice were euthanized and brains examined histologically.  Vehicle treated mice water maze performance was slower than those of oSLO-treated mice.  oSLO-treated concussed mice water maze performance was similar to the sham-concussed (normal) mice performance.
It has been shown that mild repetitive head impacts affect microglia density. Adult male rats subjective to sham or two mild head impacts were compared. Significant increase in microgliosis was demonstrated in rats with head impact versus sham control (Cai, 2021)
BrainComms
SLO Mouse Study

Preclinical Safety 

The safety of NT-101 has been evaluated preclinically in:
  • (2) single dose acute toxicity studies (rat and dog)
  • (2) 7-day repeat dose studies (rat)
  • (2) 28-day repeat dose studies (rat and sheep)
  • oSLO was shown to be safe, well-tolerated with no toxicity observed in all species in both single and multiple dose studies.
 
The No Observed Effect Level (NOEL) was demonstrated to be 140.8 ng/day (22 times higher than the projected human dose). The ongoing 180-day chronic rat study will be completed in Q2, 2024.
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