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 bran during and after a stroke may improve the outcome of the disease. Glutamate-oxaloacetate transaminase (GOT) is an enzyme that facilitate 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, aims to develop a GOT nanoparticle (GOT NP), with increased circulatory half-life and enhanced efficacy. The work aims 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 will be 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 GOT-NP will be tested for potential toxic effects 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 will be followed, with toxicity testing in parallel with development of the GOT-NP.
Partners in this project are from Canada (coordinator), Spain, Turkey, and Norway. The Canadian partner is responsible for the synthesis and characterization of GOT-NP. The Spain partner is responsible for testing of the neuroprotective effects of GOT-NP on animals. The Turkish partner is responsible for elucidating the neuroprotective mechanism of the GOT-NP, whereas the Norwegian partner is responsible for the toxicity testing. The toxicity testing and mechanistic work with GOT-NP will be performed on relevant cell models of ischemic stroke, such as nerve cells, endothelial and astrocytic cells resembling the blood brain barrier (BBB).
NILU has participated in the consortium meetings arranged online, to discuss results and experimental planning. GOT-NP is being tested in an ischemic rat model. There have been some delays in the project due to COVID 19 and laboratories lock down. NILU has received and tested GOT and GOT-NP for cytotoxicity and DNA damage in a neuronal cell line, and the results are promising as, no cyto- and genotoxicity were detected yet. Further testing is ongoing and will be performed also in a hippocampal cell line and in glial cells. Hippocampal cells were received, but this work is delayed due to mycoplasma infection in the cells upon arrival. A battery of genotoxicity test will be applied, and work is ongoing with more advanced test methods for detection of potential mutagenic and carcinogenic effects. An in vitro model for brain stroke by oxygen and glucose deprivation is being set up at NILU (equipment has recently been installed), to be applied for mechanistical investigations and mapping of key events.
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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.
