Running head: NSAID TREATMENT FOR ALZHEIMER’S DISEASE 1

NSAID Treatment Targeting Microglial Inflammation

and Amyloid Beta Plaques in Alzheimer’s Disease

Mackenzie M. Andrews & Kari Severeid

University of Washington – Bioengineering

Need Statement:

We propose to design a novel drug treatment to solve microglial inflammation and

amyloid beta build-up in Alzheimer’s Disease.

NSAID TREATMENT FOR ALZHEIMER’S DISEASE 2

Abstract

Alzheimer’s disease (AD) is a highly prevalent neurodegenerative disease affecting over

5 million Americans over the age of 65. The disease presents with many cognitive

symptoms including difficulty learning, difficulty performing tasks, and memory loss.

While the root cause is unknown, AD has been attributed to the build up of amyloid beta

(Aβ) plaques and the chronic activation of proinflammatory microglia which lead to

synaptic interference and neurodegeneration. Nonsteroidal anti-inflammatory drugs

(NSAIDs) have been shown to reduce microglial associated inflammation and the

production of Aβ plaques. However, the low blood brain barrier (BBB) permeability of

NSAIDs have made them ineffective in clinical trials. To address this issue, we propose

a novel drug treatment in which we synthesize Flurbiprofen to a polycyclic cage

structure to produce a prodrug compound capable of crossing the BBB. In addition, we

would co-administer the prodrug with EPPS, a molecule shown to break down Aβ

aggregates.

Keywords: Alzheimer’s, microglia, amyloid beta, NSAID, Flurbiprofen

Abbreviations Used

Alzheimer’s Disease AD
Amyloid Beta Aβ
Blood Brain Barrier BBB
Nonsteroidal Anti-Inflammatory Drug NSAID
Central Nervous System CNS
Reactive Oxygen Species ROS
Electroencephalogram EEG
Computed Tomography Scan CT
Non-Human Primate NHP
4-(2-hydroxyethyl)-1- EPPS
piperazinepropanesulphonic acid
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 3

NSAID Treatment Targeting Microglial Inflammation

and Amyloid Beta Plaques in Alzheimer’s Disease

Epidemiology

Since AD was discovered in 1909, it has rapidly become a household name. In

the United States, AD is in the top ten causes of death, leading to about 94,000 deaths
in 2014 (Centers for Disease Control and Prevention, 2017). In 2013, over five million
Americans had been diagnosed with AD (DeFina, P.A., et al., 2013). In the world,
roughly 3.9% of people over the age of sixty have been diagnosed. Of those people
affected by AD, the region affected the most is North America, which is broken down
and shown in part by Figure 1 (Qiu, C., Kivipelto, M., & von Strauss, E., 2009). From the
beginning, when Alois Alzheimer first discovered this disease, he saw “distinctive
plaques and neurofibrillary tangles in the brain histology” (Hippius, H., & Neundörfer, G.,
2003). Since then, advances have been made in understanding the cause of this
disease, but it is not fully understood yet. Currently, it is hypothesized that in patients
with AD, there is buildup of Aβ protein that induces the activity of microglia (the brain’s
macrophages) and can lead to neurodegeneration (Mandrekar, S., & Landreth, G.E.,
2010). With the increasing prevalence, studies are being done in the hopes of fully
understanding the cause of this disease so that better treatments can be found. Current
treatments such as acetylcholinesterase inhibitors and N-methyl d-aspartate receptor
antagonist drugs work to slow down the progression and severity of symptoms, but do
not treat the root cause (Kumar, A., Singh, A., & Ekavali., 2014).

Normal Physiology

AD is a degenerative disease of the central nervous system (CNS) and

specifically affects the learning and memory capabilities of the brain. In the normal adult
brain, neurons act as the fundamental information processing and storage units. In
order to successfully transmit information through the brain, neurons rely on cell-cell
signaling via neurotransmitters at cell junctions or synapses.
This signaling process is reliant on the ability of the brain to remain free of debris
and harmful plaque buildup that can clog synapsis and lead to neuronal death. A fine
balance of activational molecules and immune cells regulate neurotransmission and the
breakdown of debris. One of these activational molecules is the Aβ protein which has
been shown to activate kinase enzymes, protect against oxidative stress, regulate
cholesterol transport, and regulate anti-microbial activity (Bogoyevitch, M.A., et al.,
2004; Zou, K., et al., 2002; Maloney, B. & Lahiri, D.K., 2011). In normal function, Aβ
forms oligomer clusters, however, misfolding of these oligomers can lead to excessive
protein aggregation forming fibrous plaques (Hamley, I.W., 2012).
To regulate the buildup of harmful debris, the brain has immune molecules called
microglia which are a type of CNS macrophage. Microglia account for 10–15% of all
cells found within the brain and are found in two forms: ramified (resting) and activated
(Lawson, L.J., Perry, V.H. & Gordon, S., 1992). When activated, microglia are
responsible for overall brain maintenance which includes scavenging for and
phagocytosing plaque buildups, damaged or unnecessary neurons, and infectious
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 4

agents (Gehrmann, J., Matsumoto, Y., & Kreutzberg, G.W., 1995). Activation of
microglia results in the secretion of proteases, cytokines, and reactive oxygen species
(ROS). The released cytokines then activate and recruit more microglia, rapidly
escalating the immune response (Aloisi, F., 2001). However, if too many microglia are
recruited or if the microglia remain in their activated state too long, the proteases,
cytokines and ROS can reach neurotoxic levels resulting in neurodegeneration (Streit,
W.J. & Kincaid-Colton, C.A., 1995).

Pathophysiology

In the case of AD, the Aβ oligomers misfold and form plaques (Figure 2). This
process has been shown to affect the membrane receptors of neurons and astrocytes.
Astrocytes help stabilize the internal environment and tissues (Li, Y., Tan, M-S., Jiang,
T., & Tan, L., 2014), thus Aβ disruption of astrocyte signaling causes a number of
negative effects, including a greater production of ROS which can encourage additional
aggregation of Aβ (Kumar, A., Singh, A., & Ekavali., 2014; Mrak, R.E., Sheng, J.G., &
Griffin, W.S.T., 1995).
This increased Aβ buildup is especially destructive because it also chronically
activates microglia (Kumar, A., Singh, A., & Ekavali., 2014). Chronically activated
microglia release proinflammatory cytokines and other toxic products that can lead to
neural degeneration. More inflammatory molecules are made when those cytokines
encounter glial cells and those additional inflammatory molecules activate more
microglia, leading to more neural degradation (Li, Y., Tan, et al., 2014).
AD is difficult to control because a number of compounding positive feedback
loops are formed (Figure 3). The formation of Aβ plaques leads to additional Aβ
production. As plaques build up, microglia are activated in an attempt to return to
homeostatic levels. Chronic microglial activation leads to recruitment of more microglia
causing a buildup of toxic inflammation molecules. These processes are compounding
because Aβ activates microglia, which leads to greater production of proinflammatory
cytokines which induce a greater production of Aβ protein and can decrease a main
enzyme used to degrade Aβ, so the cycle continues (Mandrekar, S., & Landreth, G.E.,
2010). In order to slow down the progression of AD, an interruption of these positive
feedback loops could have significant impact on the neurodegeneration in patients.

Clinical Presentation

The symptoms presented in AD are fairly well known (Figure 4), but can vary
somewhat with what is expressed and the severity in individual cases. Most commonly,
“neuropsychiatric symptoms such as apathy, depression, aggression, agitation, sleep
disruption, and psychosis” are characteristic of AD (Li, X-L., et al., 2014). These
symptoms often start with memory impairment. In the case of elderly patients, this is
often brushed off as a normal part of aging. As the brain undergoes neurodegeneration,
there is more cognitive decline in a stage called Mild Cognitive Impairment. Patients can
still function fairly independently at this stage, but often present with amnesia. After this
stage, some patients progress to dementia, with 5 to 15% of patients going from Mild
Cognitive Impairment to dementia annually (DeFina, P.A. et al., 2013).
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 5

Diagnosing AD has come a long way, but is imperfect as the diagnosis can only
be confirmed during an autopsy (Li, X-L., et al., 2014; McKhann, G., et al., 1984).
Diagnosis is somewhat based on ruling out other neurological diseases or causes of
dementia. Most criteria are based on behavior or psychiatric evaluations, but some lab
tests can be done to aid in determining a diagnosis. The key labs are lumbar punctures,
examination of differences in an EEG, or cerebral atrophy shown on a CT (McKhann, G.
et al., 1984).

Clinical Outcomes

Currently, the treatments for Alzheimer’s can only slow down neurodegeneration,
so the quality of life for patients does not get better over time. Buildup of plaques can
persist for roughly ten to fifteen years before symptoms of cognitive impairment appear
(Perrin, R.J., Fagan, A.M., & Holtzman, D.M., 2009). Once symptoms are present,
patients start treatment to slow cognitive decline and other symptoms. The most
common drugs given to Alzheimer’s patients can help the patients’ quality of life, but
haven’t been proven to extend the lifespan of patients (Rountree, S.D., et al.,
2012). Determining the average lifespan of patients mostly relies on the age at the time
of diagnosis. If the patient is 65 when diagnosed, the average lifespan is about 8.3
years, while patients at age 90 when diagnosed live an average of 3.4 years (Figure 5).
There does not appear to be a strong correlation between gender and expected lifespan
(Brookmeyer, R., et al., 2002). The treatments available can slow the progression of
symptoms present, but there is no current way to stop the neurodegeneration that
plagues these patients.

Treatment by Design

Our proposed solution to treat AD targets the microglial inflammation and Aβ

protein buildup responsible for the ‘positive feedback’ hypothesis. Studies suggest that
NSAID’s are able to decrease the microglial production of proinflammatory cytokines
(Dokmeci, D., 2004). This reduction in microglial inflammation has been shown to
decrease Aβ production in studies targeting primary neurons (Gasparini, L., et al.,
2004). Flurbiprofen has been shown to be the most effective NSAID in the reduction of
microglial inflammation via direct nucleus basalis injection in rats (Prosperi, et al., 2004).
While NSAIDs have been tested as a treatment for AD in clinical trials, the effective
doses of NSAIDs, in order to have Aβ-lowering properties, were typically shown to be
above the physiological doses that can be used to treat patients (Walker, D., & Lue, L.-
F., 2007). This is due to the hydrophilic nature of most NSAIDs which drastically
decreases the permeability of the drug to cross the BBB (Manilla et al., 2000). In order
to overcome the permeability barrier and achieve clinically effective doses in the CNS
without reaching toxic levels in systemic circulation, we propose an ester prodrug
modification to Flurbiprofen which would make Flurbiprofen lipid soluble and capable of
crossing the BBB.
In addition to the prodrug conjugate of Flurbiprofen, we propose the
coadministration of 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS), a
molecule that has been shown to breakdown Aβ aggregates into non-harmful
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 6

monomers via oral administration (Kim, H.Y., et al., 2015). The administration of EPPS
is intended to break down existing Aβ plaques in attempt to reduce the neurotoxic
effects of the plaques and prevent further aggregation and inflammation. EPPS has
been shown to be orally safe and successful at penetrating the blood brain barrier.

Prodrug Synthesis

We would be using the polycyclic cage ester prodrug modification method

outlined by Marli van den Berg (Van den Berg, M., 2010). Using the Cookson diketone
(pentacyclo[5.4.0.02 ,6.03.1o.05,9]undecane-8,11-dione), we would link 2-aminoethanol
to one of the ketone oxygens and then esterify the product to Flurbiprofen (Figure 6).
The ester prodrug version of Flurbiprofen would be co-administered orally with EPPS.

Testing and Success Criteria

The prodrug would first be tested on stem cell derived microglia in vitro. The
cultured microglia could be chronically activated via IL-4 cytokines. The co-
administration with EPPS could be tested by adding Aβ aggregates to the cell culture. If
the in vitro model shows significant reduction in microglial activation and significant Aβ
aggregate breakdown, the drugs could then be administered systemically into a mouse
model of AD such as those described by Gregory Elder (Elder, G.A., Gama Sosa, M.A.,
& De Gasperi, R., 2010). In the mouse model, we would test for four metrics: microglial
activation, Aβ plaque levels, neurodegeneration, and cognitive impairment. If the mouse
model shows significant reduction in microglial activation, reduced Aβ aggregates,
reduced neurodegeneration, and improved cognitive function over control mice, as well
as no other significant side effects or health impairments, we would perform the same
examinations in non-human primate (NHP) models such as those described by Debby
Van Dam (Van Dam, D. & Deyn, P.P.D, 2016).
Pending the success in NHP trials, we would move to clinical trials of the
prodrug/EPPS treatment. The clinical trials would be targeted to individuals in the
‘preclinical’ and ‘mild cognitive impairment’ stages of the disease. Since our treatment is
not intended to repair pre-existing neurodegeneration, we would not target patients in
the most advanced stage of the disease. In clinical trials, we would assess the patients
on the diagnostic criteria outlined by the National Institute on Aging-Alzheimer’s
Association (Albert, M.S., et al. 2011; Sperling, R.A., et al. 2011). Those criteria would
be monitored for disease progression in the trial group and compared to control groups
undergoing the most effective current therapy.
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 7

Figures

Figure 1:Prevelance of AD Across Four Populations – Across populations, the

prevalence of AD increases with age. Roughly 3.9% of people 65 and older have been
diagnosed with AD. The prevalence is highest in the US with over 30% of the population
aged 90+ having the disease. (Qiu, C., Kivipelto, M., & von Strauss, E., 2009)
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 8

Figure 2: Aβ Formation and Interaction with Neurons – Aβ forms from precursor

proteins and eventually folds into oligomers which can become fibrous structures that
can aggregate into plaques. Aβ oligomers can cluster on the dendrites and synapses of
neurons. Plaque formations can encompass parts of or the entirety of neurons.
(The AD Plan, 2017)
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 9

Figure 3: Positive Feedback Loop Hypothesis – Aβ oligomers can form Aβ plaques

which cause the activation of ramified microglia. The activated microglia produce
cytokines and ROS. The ROS catalyze the production of more Aβ plaques and the
cytokines activate more microglia. The cycle produces increasing amounts of Aβ
plaques, ROS, and cytokines which all contribute to neurodegeneration.
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 10

Figure 4: Symptoms of AD – AD presents with a number of symptoms of varying

degrees of severity equating to progressive stages of the disease. Preclinical symptoms
(green) progress towards dementia (red), the most advanced stage of the disease.
(Senior Care Resources, 2015)
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 11

Figure 5: Survival Outcomes Following Diagnosis In Three Age Demographics –

The survival time of patients with AD increases the earlier the disease is diagnosed.
20% of patients diagnosed under 75 years of age live for 12 years post diagnosis
compared to a 20% survival of only 5 years in patients diagnosed over 85 years of age.
(Brookmeyer, R., et al., 2002)
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 12

Figure 6: Synthesis of Flurbiprofen Ester Prodrug – Reaction [1] – Diels-Alder

adduct formation and photocyclization of Cookson’s diketone polycyclic cage structure
(1). Reaction [2] – Polycyclic cage linkage to 2-aminoethanol (2) yielding compound (3).
Compound (3) esterification to Flurbiprofen (4) to yield prodrug compound (5).
(Adaptation of Van den Berg, M., 2010)
NSAID TREATMENT FOR ALZHEIMER’S DISEASE 13

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Individual Contributions:

Mackenzie – Normal Physiology, Treatment by Design, Abstract, Figures/Formatting

Kari – Epidemiology, Pathophysiology, Clinical Presentation, Clinical Outcomes