The document summarizes the research of Dr. Thomas Hudson Sanderson and his team on using non-invasive light therapy to modulate mitochondria and reduce reactive oxygen species production after brain ischemia. They have found specific infrared light wavelengths that can inhibit cytochrome c oxidase activity and mitochondrial respiration in cellular and animal models of brain injury. Further pre-clinical studies in a pig model of cardiac arrest showed inhibitory light therapy reduced neuronal injury and improved functional outcomes. Based on this research, Dr. Sanderson co-founded Mitovation, Inc. to develop a medical device for treating post-ischemic brain injury in humans using non-invasive mitochondrial modulation therapy.

1. Mitochondria in Post-Ischemic Brain Injury:
Mechanistic Insights and Novel Therapeutics
Thomas Hudson Sanderson, Ph.D.
Associate Professor
Departments of Emergency Medicine and Molecular and Integrative Physiology
University of Michigan Medical School, Ann Arbor MI
Grant/Research Support
NIH - NINDS R42NS105238
NIH - NINDS R01NS091242
NIH - NINDS R01NS076715
DoD - W81XWH-16-0175
Kellogg Foundation
NEI/MTRAC
American Heart Association
Disclosures
Patents
8945196- “Light Therapy Treatment”
9610460- “Light Therapy Treatment –Method”
Company Interests
Co-Founder and CSO of Mitovation, Inc.

2. Understanding Brain Ischemia: From Biochemistry to the Pig
Molecular Interrogation
• Cellular models – primary neurons or immortalized cells
• Genetic manipulation – viral constructs, plasmids, Cas9/CRISPR,
transgenic mice
Small Animal Models of Brain Ischemia
• Cardiac arrest/resuscitation – mouse
• Adult global brain ischemia – rat: 2VOH
• Stroke - focal ischemia – rat: MCAO
• Perinatal hypoxia/ischemia – rat: Vannucci model of HIE
Large Animal Translational Models
• Pig model of cardiac arrest/resuscitation
• Piglet model of perinatal hypoxia/asphyxia
Sanderson Lab: Basic and Translational Neuroscience
EMERGENCY MEDICINE

3. Non-Invasive Mitochondrial Modulation Therapy
• Therapeutic discovery→ development → translation
• Isolated enzyme studies on CcO activity
• Cellular, mechanistic, and efficacy investigations
• Pre-clinical studies: cardiac arrest/resuscitation in pigs.
Overview
EMERGENCY MEDICINE

4. Design: Utilize a combined O2
electrode/ spectrophotometer to
find IRL wavelengths that modulate
CcO and mitochondrial respiration
Mechanistic Discovery: The Foundation
EMERGENCY MEDICINE

5. Design: Utilize a combined O2
electrode/ spectrophotometer to
find IRL wavelengths that modulate
CcO and mitochondrial respiration
• Two wavelengths of IRL, 750nm
and 950nm, were identified that
inhibited O2 consumption
(attenuated CcO activity).
• Effect translates to intact
mitochondria.
• 810nm (used in previous
studies) activates CcO.
Mechanistic Discovery: The Foundation
EMERGENCY MEDICINE

6. How Does IRL Modulate Mitochondrial Respiration?
EMERGENCY MEDICINE
It is the scientific consensus that
mitochondrial CcO is the primary
cellular photo-acceptor of IRL.
CcO contains two copper centers:
CuA and CuB (blue and green
arrows)
• Involved in enzyme catalysis and
have been shown to function as
the photo-acceptors for IRL

7. Problem: Generation of ROS during early reperfusion precipitates
significant neuronal injury and cell death.
Barriers to Previous Therapeutic Approaches:
• ROS act rapidly (nano to milliseconds) and irreversibly.
• Scavenging compounds must be at the appropriate subcellular
targets at the necessary concentration at the time of reflow.
• Pretreatment needed to deliver compounds to ischemic tissue.
Solution: Non-Invasive Mitochondrial Modulation.
Hypothesis: Non-Invasive mitochondrial modulation therapy can
overcome these barriers to ROS therapy and reduce post-ischemic
brain injury.
Potential for Therapeutic Use
EMERGENCY MEDICINE

8. Balance Between Energy and ROS
EMERGENCY MEDICINE
• The mitochondrial
membrane potential (m)
is critical for energy
production (ATP) and
reactive oxygen species
(ROS) generation.
• Dynamic control of
mitochondrial membrane
potential is disrupted during
ischemia/reperfusion.

9. Balance Between Energy and ROS
EMERGENCY MEDICINE
• The mitochondrial
membrane potential (m)
is critical for energy
production (ATP) and
reactive oxygen species
(ROS) generation.
• Dynamic control of
mitochondrial membrane
potential is disrupted during
ischemia/reperfusion.
During ischemia mitochondrial
membrane potential depolarizes

10. Balance Between Energy and ROS
EMERGENCY MEDICINE
• The mitochondrial
membrane potential (m)
is critical for energy
production (ATP) and
reactive oxygen species
(ROS) generation.
• Dynamic control of
mitochondrial membrane
potential is disrupted during
ischemia/reperfusion.
During reperfusion mitochondrial
membrane potential is rapidly restored

11. Balance Between Energy and ROS
EMERGENCY MEDICINE
• The mitochondrial
membrane potential (m)
is critical for energy
production (ATP) and
reactive oxygen species
(ROS) generation.
• Dynamic control of
mitochondrial membrane
potential is disrupted during
ischemia/reperfusion.
During reperfusion
increased mitochondrial
membrane potential
levelslead to excessive
ROS production.
During reperfusion mitochondrial
membrane potential is rapidly restored

12. Balance Between Energy and ROS
EMERGENCY MEDICINE
Mild mitochondrial modulation
can provide significant
reduction in ROS production.
• The mitochondrial
membrane potential (m)
is critical for energy
production (ATP) and
reactive oxygen species
(ROS) generation.
• Dynamic control of
mitochondrial membrane
potential is disrupted during
ischemia/reperfusion.
During reperfusion
increased mitochondrial
membrane potential
levelslead to excessive
ROS production.

13. In Vitro – Mitochondrial ROS and Cell Death
EMERGENCY MEDICINE
Glutamate Exposure:
• Glutamate promotes
mitochondrial ROS
generation (Red).
• IRL treatment minimized
mitochondrial ROS.
Oxygen Glucose
Deprivation (OGD):
• OGD causes loss of live
neurons (green) and
increases dead neurons
(red).
• Inhibitory IRL increases
neuronal survival.
• Excitatory IRL is ineffective.
In Vitro Models of Neuronal Injury
Sanderson TH, Hüttemann M. et al. Sci Rep 2018.

14. Efficacy Trials in Small Animal Model
EMERGENCY MEDICINE
Sanderson TH, Hüttemann M. et al. Sci Rep 2018.
Global Brain Ischemia Protocol
• Random enrollment, blinded treatment
and analysis.
• IRL treatment with LED diodes for a 2
hour duration.
• No increase in brain temperature.
Brain Injury with Histology
• Global brain ischemia caused loss of
neurons in the hippocampus.
• Inhibitory IRL prevented neuronal injury.
• Excitatory IRL was ineffective.
• Functional improvement with treatment
detected with RAM.
• Delayed treatment effective, but efficacy
reduced.

15. Pre-Clinical Evaluation of Mitovation Technology
EMERGENCY MEDICINE
Pig Cardiac Arrest/Resuscitation – Survival Studies
• Random enrollment, blinded treatment and analysis.
• IRL treatment with bench prototype - LED diodes for a 2 hour
duration. Device provides light penetration through pig brain.
Post-Ischemic Brain Injury. Cardiac arrest/resuscitation
resulted in loss of neurons in the hippocampus 4 days after
ROSC.
• Inhibitory IRL reduced neuronal injury.
• Early evidence of functional improvement.

16. Mitovation, Inc. is a medical device company
developing a non-invasive platform technology
for the treatment of post-ischemic brain injury.
• A start-up company founded by:
Thomas H. Sanderson, Ph.D.
Maik Hüttemann, Ph.D.
Mark Morsfield, M.B.A.
The Mitovation Device:
• Disposable human interface - Fiber optic
light delivery system.
• Therapeutic light generator - Compact,
portable, and powered by a battery or
electrical outlet.
MTRAC and the DoD funded the alpha
prototype design through Tekna, Inc.
• Regulatory and efficacy testing ongoing
through Mitovation, Inc. and the
Sanderson lab.
Human Device Prototyping
EMERGENCY MEDICINE Mitovation, Inc.

17. Research Teams
SANDERSON LAB
Kathleen Maheras, PhD
Postdoctoral Fellow - Genetics, molecular
biology, mouse cardiac/arrest resuscitation
Joseph Wider, PhD
Postdoctoral Fellow - Large/small animal
models of brain and heart ischemia, cell
culture/biochemistry
Erin Gruley, BS
Research Specialist - Large animal
surgery, porcine behavioral studies,
neonatal brain injury
Sarita Raghunayakula, MS
Research Specialist - Molecular biology,
cell culture, primary neuron
isolation/culturing, genetic manipulation
Anthony Anzell, BS
PhD candidate - Primary neuron culture, in
vitro hypoxia, live cell imaging
Christos Strubakos, MS
PhD candidate - Ischemic stroke, MRI
EMERGENCY
MITOVATION, INC
Thomas Sanderson, PhD
Co-Founder and Chief Scientific Officer,
Mitovation, Inc.
Maik Hüttemann, PhD
Co-Founder and Chief Technology
Officer, Mitovation, Inc.
Professor of Molecular Medicine, Wayne
State University
Mark Morsfield, MBA
Chief Executive Officer, Mitovation, Inc.
Christian Reynolds, PhD
Director of Translational Research,
Mitovation, Inc. Assistant Professor,
Wayne State University
Joseph Wider, PhD
Research Fellow, University of Michigan
Lola Tatum
Grants Management Specialist,
Controller, IBT
Kimberly Johnston, DVM
Veterinary Surgeon, IBT