Evaluation of calpain inhibitor III as a therapeutic intervention for traumatic brain injury

2/29/16
SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11210
Dr. Douglas Ling, Rachelle Dugue, and William Stewart

I.

Proposal Summary/Abstract
Traumatic brain injury is an impact of the brain that accounts for 1.7 million cases each
year. Clinical therapy for traumatic brain injury is currently limited to skull fracture repair and
supportive treatment, such as rest and maintaining blood pressure and oxygen levels. There is no
current therapeutic treatment for traumatic brain injury. This experiment seeks to find out whether
calpain inhibitor III is an effective therapeutic intervention for traumatic brain injury. This will be
determined successful if it represents a cheaper, more easily synthesized alternative to Gabadur, a
novel calpain inhibitor currently under investigation at SUNY Downstate that links a calpain
inhibitor to the antiepileptic drug, pregabalin. Both calpain inhibitor III and Gabadur are capable
of crossing the blood-brain barrier, but Gabadur is significantly more costly and difficult to
produce. Young Sprague-Dawley rats will be subjected to traumatic brain injury using the
controlled cortical impact injury model. Controlled cortical impact injured rats will be
administered calpain inhibitor III after injury. Untreated (sham-drug) control rats will also be
subjected to controlled cortical impact but will only receive a dose of the saline vehicle. This
study will use both histological and behavioral assessments such as the Morris Water Maze and
Active Place Avoidance to assess learning and memory. Motor skill tasks such as the balance
beam, and histochemical analysis using Fluoro-Jade B staining will be used to determine where
postinjury administration of calpain inhibitor III inhibits injury-induced neuronal cell loss and
prevents motor and memory deficits to controlled cortical impact injured rats. The data from this
study will be compared to the findings of a parallel study of Gabadur currently being conducted
in Dr. Lings laboratory at SUNY Downstate. If rats that are treated with calpain inhibitor II are
able to perform better than untreated rats on learning and memory tasks as well as motor skill
tasks, this will suggest that calpain inhibitor III may represent a potential neuroprotective therapy
against cortical traumatic brain injury.
II.

Background
Traumatic Brain Injury (TBI) is defined as an impact, penetration, or rapid movement of
the brain within the skull that results in altered mental state (Prins & Greco, et al. 2013). TBI is
the most common disease, with more known cases worldwide than breast cancer, AIDS, and
Parkinsons disease (Prins, Greco, et al. 2013). In the US, there are 1.7 million new cases of TBI
each year, which result in 50,000 deaths and 80,000 permanent disabilities. TBI results in many
cognitive deficits, which include deficits in attention, learning and memory, and higher-order
executive functions. Currently, treatment is limited to skull fracture repair and supportive
treatment, such as rest and maintaining blood pressure and oxygen levels. There are no treatments
to prevent neuronal death and the development of secondary injury effects.
Calpain is a calcium-activated cysteine protease, meaning that it is an enzyme that
degrades proteins (Saatman, et. al, 2011). Calpain overactivation triggers a massive release of
glutamate in the brain, leading to cell dysfunction and death (Saatman, et. al, 2011). Calpain
inhibitor III is hypothesized to protect against neuronal death and inhibit apoptosis (Kawamura,
Nakajima, et al., 2004). It is also hypothesized to be able to penetrate the brain rapidly, meaning it
can be administered to patients within a large time window after injury (Kawamura, Nakajima, et
al., 2004). By studying the ability of calpain inhibitor III, we can use the information gathered to
help the millions of people who suffer from TBI every year.

III.

Project Description
This study aims to assess calpain inhibitor III, also known as the calpain inhibitor drug
MDL-28170) as a potential therapeutic solution to traumatic brain injury (TBI) and whether it
may provide a cheaper, more easily synthesized alternative to Gabadur. Gabadur is a novel
calpain inhibitor attached to pregabalin, an anti-seizure medication. Both Gabadur and calpain
inhibitor III are capable of crossing the blood-brain barrier to enter the central nervous systems,
but Gabadur is hypothesized to have greater neuroprotective potential, as it links the calpain
inhibitor to the antiepileptic drug, pregabalin. However, Gabadur is significantly more difficult
and expensive to synthesize than calpain inhibitor III. Thus, it remains to be determined whether
the additional cost of Gabadur is justified over other, more readily available and less expansive
calpain inhibitor agents.
Calpain is an intracellular, calcium-activated cysteine protease, and is a major player in
most forms of apoptosis (Saatman et al., 2011). Massive increases of intracellular calcium in TBI
lead to the overactivation of calpain (Saatman et al., 2011). Calpain overactivation is a major
contributor to cell dysfunction and neurodegeneration following TBI (Saatman et al., 2011).
Therefore, a calpain-inhibitor has been actively investigated as a viable therapeutic strategy to
stop neurodegeneration and preserve neurological function post-TBI (Saatman et al., 2011). It is
hypothesized that calpain inhibitor III will help to restore and protect motor function and learning
and memory ability post-injury, by preventing apoptosis and general neuronal cell death. The
independent variable for the project as a whole will be the dose of the calpain inhibitor III, and
the dependent variable will be the rat's performance on each task. The rats performance on each
task will help to identify whether the calpain inhibitor III drug has successfully blocked the overactivation of calpain, and thus, helped to prevent brain cell death.
IV.

Research Strategy
This study will be conducted on young Sprague-Dawley rats, using the Controlled
Cortical Impact (CCI) model to administer the traumatic brain injury (TBI). The CCI produces
visible anatomical damage to the cortical and hippocampal areas of the brain (Yang et al., 2010)
leading to disruption in cognitive function (Brody et al., 2007). The motor assessment and
behavioral tasks will consist of a beam walk test and tactile adhesive task. The beam walk will
consist of three wooden beams of various widths; rats walk along a 15mm, 18mm, and 25mm
wide beam for 60 seconds and are scored based on their ability to travel completely across the
beam without slipping, falling, or dragging limbs. The tactile adhesive task will measure a rats
ability to remove stickers from their paws; time to remove from left versus right forepaw is
measured. Spatial ability and learning will also be measured using the Morris Water Maze, and
Active Place Avoidance. For the morris water maze, rats will be placed at different coordinates
along the circumference of a circular pool. They will be given 2 minutes to find a clear platform
placed 1 cm below the water. Rats must use spatial cues placed around the room to find the
platform. After 10 days of 2 trials per day, the rats will be given a 10-minute trial without the
platform present. The amount of time spent inside the quadrant where the platform was
previously located will be measured against the total time (30 seconds). The data will be
measured as a ratio of time spent in correct quadrant to the total time of the trial. Active place
avoidance will test the rats associative learning. The rat must continuously move away from a
45 shock zone placed in a fixed location across all trials. The circular arena will rotate, and the
rat will need to use spatial cues placed around the room to avoid the shock zone. The amount of
times entered the shock zone, number of shocks, and distance traveled will be measured.
V.

Bibliography

Brody DL et al. Electromagnetic Controlled Cortical Impact Device for Precise, Graded
Experimental Traumatic Brain Injury. J. Neurotrauma 24: 657-673 (2007).
Kawamura, M., Nakajima, Wako., Isida, Akira. et all. (2004). Calpain inhibitor MDL
28170 protects hypoxic-ischemic brain injury in neonatal rats by inhibition of both apoptosis and
necrosis. Science Direct, 1037 (2005) 59-69.
Andaluz, Norberto. Traumatic Brain Injury - Overview. Mayfield Brain & Spine. N.p.,
Feb. 2013. Web. 15 Oct. 2015. Retreived from http://www.mayfieldclinic.com/PE-TBI.htm
Prins, Mayumi., Greco, Tiffany., et al. (2013). The pathophysiology of traumatic brain
injury at a glance. Science at a Glance. 6, 1307-1315.
Saatman et. al, Calpain as a Therapeutic Target in Traumatic Brain Injury. 2011,
Neurotherapeutics.
Schmued LC and Hopkins KJ. Fluoro-Jade B: a high affinity fluorescent marker for the
localization of neuronal degeneration. Brain Research 874: 123-130 (2000)
Yang L, Afroz S, Michelson HB, Goodman JH, Valsamis HA, and Ling DSF.
Spontaneous Epileptiform Activity in Rat Neocortex after Controlled Cortical Impact Injury. J.
Neurotrauma 27: 15411548 (2010).