Acute ischemic stroke accounts for around 6.5 million deaths per year and is one of the leading causes of death and disability in the world, and therapeutic treatment is urgently needed. Currently, thrombotic brain stroke is treated with thrombolysis to break up blood clots, but most patients receive no treatment at all and less than 40% of stroke patients have good clinical outcome. During a stroke, the glutamate concentration in the brain may increase to a level that can induce serious damage. Glutamate is quantitatively the most important neurotransmitter in the brain, and excess stimulation of glutamate receptors may lead to excitotoxicity and substantial neuronal death, leading to brain damage and loss of neurological functions. Thus, a treatment strategy to reduce the extracellular glutamate concentration in the brain during and after a stroke may improve the outcome of the disease. Glutamate-oxaloacetate transaminase (GOT) is an enzyme that facilitates degradation of glutamate in the blood. Systemically administered GOT has been demonstrated to deplete blood glutamate levels, which in turn causes an increased efflux of excess glutamate from the brain followed by a reduction of the ischemic lesions and improved recovery.
One major shortcoming is, however, that the effect of administered GOT is short-lived. The project, therefore, developed a GOT nanoparticle (GOT NP), with increased circulatory half-life and enhanced efficacy. The work aimed to provide a new perspective on the treatment of excitotoxic brain injuries and offer new opportunities for nanomedicine-based therapy. The specific benefits for ischemic stroke were assessed in the project, but the concept is applicable for many other neurodegenerative disorders, as traumatic brain injury, Parkinson s disease and Alzheimer s disease. In addition to investigating the therapeutic and mechanistic effects of GOT-NP, the safety of GOT-NP was assessed by in vitro toxicity testing, with main emphasis on genotoxicity, which is crucial endpoint for pharmaceuticals used for treatment of diseases and required for regulatory risk assessors. The safe-by-design approach was followed, with toxicity testing in parallel with development of the GOT-NP.
Partners in this project were from Canada (coordinator), Spain, Turkey, and Norway. The Canadian partner was responsible for the synthesis and characterization of GOT-NP. The Spain partner was responsible for testing of the neuroprotective effects of GOT-NP on animals. The Turkish partner was responsible for elucidating the neuroprotective mechanism of the GOT-NP, whereas the Norwegian partner was responsible for the toxicity testing in addition to mechanistic studies. The toxicity testing and mechanistic work with GOT-NP were performed on relevant cell models of ischemic stroke, with two types of nerve cells and astrocytes.
An in vitro model of ischemic stroke was applied to test the neuroprotective effect of GOT and GOT-NP. GOT-NP showed enhanced efficacy compared with GOT, in line with the results from the animal studies. Toxicity testing showed that GOT-NP did not induce toxicity in neurons or astrocytes, and did not induce genotoxicity (DNA strand breaks, mutations nor damage on chromosomes).
This project proposes a concept for the treatment of ischemic stroke. A bio-conjugated glutamate oxaloacetate transaminase (GOT) enzyme (GOT-NP) was developed with enhanced efficacy compared with GOT. The GOT-NP had a more sustained therapeutic effect in the brain, and remained longer in the blood stream. As drug transport across the blood-brain-barrier (BBB) is a great challenge, the demostrated therapeutic strategy represents a promising paradigm as it does not require BBB transport, but near-brain accumulation by targeting. This will improve the efficacy of future treatments, and as the GOT-NP did not show any toxicity, it is expected to have limited adverse effects. This work provide a new perspective on the treatment of brain injuries, and offer new opportunities for using therapies based on polymer-modified proteins. The GOT-NP was found to be neuroprotective in an in vitro model of brain ischemia, and, in consistency, reduced the infarct size in an animal model of brain stroke. The concept should potentialle be applicable for many other diseases implicating such as traumatic brain injury, Parkinson’s disease and Alzheimer’s disease. This therapeutic approach will benefit a large proportion of population in addition to reducing a huge socio-economic burden by contibuting to new knowledge and treatment strategy for brain stroke by nanomedicine.
We prepared a video to explain the aim of the project and which can be seen here: https://www.youtube.com/watch?v=E0H4J6kJPlE
The project will develop and test the first targeted and long-acting nanomedicine with neuroprotective properties for
ischemic stroke, with potential application in other neurological diseases. The Team will demonstrate that the targeted delivery of a long-acting glutamate oxaloacetate transaminase (GOT) nanoparticle to the brain in order to
enhance the neuroprotective character of GOT (i.e., prevention of neuronal apoptosis and cell death) in a model ischemic stroke. Systemically-administered GOT has been demonstrated to deplete blood glutamate levels, which in turn causes an efflux of excess glutamate from the brain. One major shortcoming of this approach is that the systemic effect of GOT on brain glutamate concentration is short-lived (~1 h), mainly because of its rapid elimination from the body. The project will: i) increase the circulatory half-life of GOT and ii) target GOT to- or near to- the ischemic region of the brain where GOT can exert its therapeutic catalytic activity. These objectives will be met by preparing a Blood-Brain-Barrier (BBB)-targeted nano-formulation of GOT (GOT-NP). What is particularly original in this strategy is that accumulation of GOT-NP at the blood-side of the BBB will promote the efflux of glutamate from the brain by increasing the glutamate gradient on either side of the BBB. As such, GOT-NP does not actually have to cross the BBB to produce an enhanced neuroprotective effect. Crossing the BBB, which is substantially more challenging, would represent an added bonus of selectively depleting glutamate in the cerebrospinal fluid. In addition to the design and synthesis of GOT-NP, this project will investigate and validate iii) the mechanism of in vitro neuroprotection as well as iv) the in vivo biodistribution and neuroprotective effect of GOT-NP in an animal model of ischemic stroke, in order to conclude pre-clinical studies and place the Team in a position to embark on clinical testing.