Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (Medical Science)

First Advisor

Nasser Zawia

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disease that is responsible for up to 80% of all dementia cases. Hallmarks of AD include the presence of beta-amyloid (Aβ) deposits and twisted strands of abnormal hyperphosphorylated tau forming neurofibrillary tangles (NFTs), which ultimately lead to damage and death of nerve cells in the brain. Symptoms of the disease include impairments in cognitive and functional abilities that gradually worsen as the disease progress. Finally, in advanced AD, patients become unable to perform basic daily activities, become more susceptible to infections, and ultimately die. Only five drugs are FDA-approved for use in AD, which only provide temporary symptomatic relief without capabilities of modifying the progression of the disease. Tolfenamic acid is a non-steroidal antiinflammatory drug that has the potential for slowing the progression of AD by inducing the degradation of the transcription factor specificity protein 1 (Sp1). Sp1 regulates the expression of several AD-related genes including amyloid precursor protein (APP), tau and beta-site APP-cleaving enzyme 1 (BACE1).

To investigate the ability of tolfenamic acid to cross the blood brain barrier (BBB), multiple approaches were utilized. In silico computational models to predict logBB and logPS for CNS penetration indicated that tolfenamic acid could transfer passively through the BBB. In addition, immobilized artificial membrane (IAM) chromatography using a phosphatidylcholine column was utilized for the in vitro determination of the brain penetration capacity factor [(KIAM/MW4)x1010] of tolfenamic acid. The results showed that the [(KIAM/MW4)x1010] for tolfenamic acid was 2.73 indicating its ability to cross the BBB and penetrate into the brain. Upon intravenous (IV) administration of tolfenamic acid to guinea pigs and mice, LC-MS analysis revealed the presence of tolfenamic acid in the extracted brain tissue. This in vivo approach was also used to estimate the brain-to-plasma ratio (Kp) for tolfenamic acid, which was 0.11. Thus, using in silico, in vitro and in vivo methods, we confirmed the ability of tolfenamic acid to cross the BBB. The study offers a guide of using multiple approaches to predict the ability of different compounds to penetrate the brain.

Next, we investigated the ability of tolfenamic acid to attenuate the cognitive deficits in a transgenic mouse model of AD, namely R1.40. First, we demonstrated that hemizygous R1.40 mouse model exhibits spatial working and long-term memory deficits when tested in the Morris water maze and Y-maze. We found that short-term administration of tolfenamic acid for 34 days was able to reverse the observed cognitive deficits in hemizygous R1.40 mice. These mnemonic improvements were parallel to reductions in SP1 protein, APP expression and protein, and soluble and insoluble Aβ40-42 levels. These findings suggest that the reductions in the biochemical markers of AD were also accompanied by functional improvements in the treated mice.

We further examined the ability of tolfenamic acid to improve spatial learning and memory as well as to reduce Aβ plaque pathology in old homozygous R1.40. Homozygous R1.40 mice exhibit cognitive deficits that are accompanied by fibrillar Aβ deposition in different regions of the brain by the age of 14-15 months. Our

immunohistochemical results indicated that short-term tolfenamic acid treatment for 34 days was able to produce a drastic reduction in Aβ plaque pathology. In addition, spatial working memory, assessed by the spontaneous alternations in the Y-maze, was improved by tolfenamic acid treatment. Further, we investigated if tolfenamic acid would improve the age-related learning and memory impairments in senescent C57BL/6 mice and we found that treatment with tolfenamic acid for 34 days resulted in improvements in the spatial reference memory functions as well. The major findings from these studies suggest that tolfenamic acid crosses the BBB into the brain, is able to decrease AD-related proteins including Aβ plaques, and is able to produce mnemonic functional improvements in AD transgenic mouse model and senescent wild type mice as well. Thus, tolfenamic acid could be proposed as a disease-modifying therapy for AD via its unique mechanism.

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