Authors :
Presenting Author: Christina Meyer, MS – Iowa State University
Elizabeth Grego, BS – Iowa State University; Suraj S. Vasanthi, DVM, M.V.Sc. – Iowa State University; Nikhil S. Rao, DVM, M.V.Sc. – Iowa State University; Nyzil Massey, DVM, PhD – Iowa State University; Claire Holtkamp, BS – Iowa State University; Joselyn Huss, BS – Iowa State University; Mihir Kharate, BS – Iowa State University; Balaji Narasimhan, PhD – Iowa State University; Thimmasettappa Thippeswamy, DVM, M.V.Sc., PhD – Iowa State University
Rationale:
DFP-induced seizures can cause neurodegeneration and neuroinflammation, which is exacerbated by oxidative stress. Mitoapocynin (MPO), an NADPH oxidase (NOX) inhibitor, has been shown to reduce oxidative stress by decreasing the production of free radicals. We have shown that a 10 mg/kg (oral) MPO dose reduces proinflammatory cytokines and oxidative stress markers in the periphery but not in the brain. To enhance MPO bioavailability, we encapsulated MPO into polyanhydride nanoparticles (NP) for blood-brain-barrier penetration and extended drug release. We explored the pharmacokinetics and toxicity of MPO-encapsulated nanoparticles (MPO-NP) and its efficacy in mitigating neurodegeneration and neuroinflammation in a rat DFP model.
Methods:
Adult male and female Sprague Dawley rats were used in all experiments. To test NP toxicity, three groups of animals (n=10) received one dose (4 mg, i.m.) of one of two empty polyanhydride NP formulations: CPH:SA, CPTEG:CPH, or a cocktail. Visceral organs were collected after eight days and processed for H&E staining. For pharmacokinetic studies, four rats received two doses of CPH:SA MPO-NP (6 mg, i.m.), 12 h apart. Blood was collected at multiple timepoints. LC-MS was performed on hippocampi to determine MPO concentrations. To test the efficacy of MPO-NP on DFP toxicity, 32 animals were randomized into control, DFP+VEH, DFP+MPO-NP, or DFP+MPO (oral). DFP (4 mg/kg, s.c.) was administered to induce status epilepticus (SE), immediately followed by 2-PAM (25 mg/kg, i.m.) and atropine sulfate (2 mg/kg, i.m) to reduce mortality. SE was monitored and scored for an hour and midazolam (3 mg/kg, i.m.) was given to terminate SE. Animals received MPO-NP (CPH:SA; 3 mg, i.m.), MPO oral (30 mg/kg), or vehicle 1 h post-DFP challenge, then every other day for five days (three total doses) or once a day, respectively. Brain immunohistochemistry was performed to measure neuroinflammation and neurodegeneration.
Results:
All NP formulations showed short-term pulmonary fibrosis and mononuclear infiltration in the lungs and spleens which is likely to resolve in the long term. Blood MPO concentrations of MPO encapsulated in CPH:SA particles increased steadily over 24 h, an improvement compared to kinetics of the 10 mg/kg oral dose. Neither MPO-NP nor MPO oral had an effect on microgliosis and neurodegeneration induced by DFP exposure. In contrast, astrogliosis was attenuated in DFP-challenged animals administered MPO-NP or MPO oral.
Conclusions:
The NPs caused minor toxicity, which is expected to resolve in the long term. The serum MPO concentrations were improved when delivered via NPs vs oral dosage. MPO-NP significantly reduced astrogliosis but had minimal effect on neurodegeneration and no effect on microgliosis. Further MPO-NP studies are necessary to optimize efficacy and to evaluate NP toxicity over time.
Funding:
This study is funded by the NIH/NINDS CounterACT program (R21 NS120916-01).