An updated short deck for Neurocarrus Inc and its new pain therapeutic.

1. Targeted, Non-Opioid
Pain Therapeutics
Treating Pain
Without Addiction

2. 2
2019: The opioid epidemic rages on…
NEW approaches to pain management
are needed.

3. 3
Annual Patient Population
Post operative acute pain (limited dosing) 50 M
Chronic pain (repeated dosing) 46.3 M
Shingles, Diabetes, Osteoarthritis
Post Operative Acute Pain Market
At market entry, 1% of patient population
0.5 M doses at projected price point $500
Annual Initial Sales $250M
Potential 100-fold growth with market takeover
Domestic Market Analysis

4. 4
BOTOX freezes motor neurons…
…what if we could freeze pain
neurons?
With N-001, we can.

5. 5
N-001 Profile
• A rationally designed protein
that targets pain (sensory)
neurons and active only inside
them
• Disrupts pain signaling by the
neuron axon through
modification of cytoskeletal
actin that alters the action
potential
• Locally administered at the site
of pain by injection or topical
application, it is not systemic
• Performs like a very long-acting
local anesthetic that could delay
or prevent opioid prescription
Figure 1 N-001 Mode of Action. A. model of N-001 domains. B Uptake
of fluorescently labeled full length N-001 or isolated domains by neuronal
cell lines (N2A) was measured by cell sorting. Pre-treatment with GT1b
promoted significant uptake relative to untreated controls. C. Mode of
action. The C2II domain binds neuron target cells via the C1 binding
domain and translocates the payload C2I into the cytoplasm. C2II binds
the cell surface via interactions with asparagine-linked glycans on the cell
membrane. The C2I/C2II complex is internalized by clathrin and Rho-
dependent mechanisms within endosomes. Acidification of the endosome
causes membrane pore formation by C2II oligomers and C2I dissociates
and is transported into the cytoplasm where it acts on G actin.

6. 6
N-001 Reversibly Remodels the
Actin Cytoskeleton
Inhibition and reversibility of actin
polymerization. Polymerized F-actin
was measured using an alexa fluor
conjugated phalloidin stain and
imaged by confocal microscopy using
chicken or mice primary sensory
neurons (a) or quantitated using a
microtiter plate reader and SH-SY5Y
neuroblastoma cells (b).

7. 7
N-001 Reversibly Inhibits Calcium
influx Blocking Action Potential
Inhibition and reversibility of calcium
influx. Calcium influx was measured
using Fluo 4 calcium indicator and a
fluorescent microtiter plate reader.
The effect of C2C treatment relative
to latrunculin A was examined using
primary chicken sensory neurons (a).
The effect of C2C treatment relative
to latrunculin A was tested in GT1b-
positive SH-SY5Y cells (b). The effect
of removal of C2C and latrunculin was
also examined with primary chicken
sensory neurons (c) along with the
effect of removal from SH-SY5Y cells
(d). Error bars represent the standard
deviation between replicates.

8. 8
N-001 Targets Sensory
Nerves In Vivo Subcutaneous injection of
fluorescent N-001 in mouse back
muscle shows colocalization with
sensory neuron markers not motor
neuron markers. Latissimus dorsi
samples post injection were treated
with antibody reporters of whole-
mounts (A, B) or cryosections (C, D).
Panel A. N-001 (green), sensory
neuron marker anti-Calcitonin gene
related peptide (CGRP, blue). White
arrows show colocalized N-001/CGRP.
Panel B. N-001 (green), motor neuron
marker anti-choline acetyltransferase
(ChAT, Red), no colocalization.
Panel C. N-001 (green), CGRP (blue),
DNA/DAPI (white), and Neurofilament
(red). Inset arrows, colocalized N-
001/CGRP.
Panel D, as for panel C but ChAT
(red), no colocalization.

9. 9
N-001 Targets Sensory Nerves
and Nerve Fibers In Vivo
N-001 also colocalized with CGRP-
positive afferent sensory nerve fibers in
1.2 µm optical slices of whole mount
tissues [Fig. a] where N-001 is green
and CGRP is blue. In addition, N-001
(green) colocalized with CGRP-positive
(blue) afferent nerve fiber in compiled
optical slices of whole mount tissue [Fig
S4]. N-001 was also observed
throughout the length of axons of
sensory neurons [Fig. S5].

10. 10
N-001 Blocks Acute Pain
Longer Than Anesthetics**
Dose response and duration of N-001 in
mouse formalin inflammatory pain test.
Using a double blinded procedure, inbred
female mice (BALB/c) were treated (sc) with
either placebo (PBS), Buprenorphine (641.5
µM), or N-001 (amounts indicated) of the
hindpaw (a). After 6 hours, mice were
treated with formalin. Total duration of pain-
like behaviors (hindpaw licking) was video-
recorded for 30 min after the 5 min acute
response phase. The minimum effective
dose (MED) was 0.6 pmol. No pain relief
using either N-001 subunit alone.
A constant N-001 dose (2.2 pmol) vs PBS
was administered and animals tested for
pain-like behavior after formalin challenge at
the times indicated (b). The onset of full
efficacy was 2 hours. One dose lasted more
than 1 week. No benefit was evident after 2
weeks. All values were highly significant as
calculated by power analysis, bars indicate
variation between replicates.
**Independently confirmed by
CRO using double blinded
procedures

11. 11
Patent No. 10,633,643 “Engineered toxin adapted to deliver
molecules into selected cells” issued by the USPTO on April
28, 2020.
Neurocarrus has an exclusive license for this patent for all fields of
use. The license is between Neurocarrus Inc., and Nutech
Ventures the technology transfer office of the University of
Nebraska Lincoln. This patent has a priority date of May 15, 2015.
Paul Blum and coworkers invented the technology at the University
of Nebraska Lincoln. The patent is held by the regents of University
of Nebraska.
PCTUS1632573 covering the same technology was filed on May
14, 2016 in seven countries (Canada, China, Europe, India,
Israel, Japan, South Korea) based on manufacturing
considerations. These separately prosecuted filings are in process.
A divisional application has also been filed with the USPTO
expanding on related claims. This divisional application will have
the same priority date as 10,633,643.
Intellectual Property

12. 12
Allen et al 2020 Nature Sci
Reports (in press)
Pavlik et al 2017 Current
Topics in Peptide and
Protein Research 2017
18:1-15
Pavlik et al 2016 Nature Sci
Reports 6:23707 | DOI:
10.1038
Publications
Repurposed bacterial toxins for human therapeutics
Benjamin J. Pavlik1
, Kevin E. Van Cott1
and Paul H. Blum2
1
Department of Chemical and Biomolecular Engineering, 207 Othmer Hall,
University of Nebraska-Lincoln, Lincoln, NE 68588-0643; 2
School of Biological Sciences,
1901 Vine Street, University of Nebraska-Lincoln, Lincoln, NE 68588-0665, USA.
ABSTRACT
Pathogenic bacterial toxins can be repurposed
as therapeutics. Binary bacterial toxins are
macromolecular complexes that are a current focus
of therapeutic development. These proteins bind
to surfaces of specific human cell populations and
transport enzymes across membranes. Basic research
has characterized bacterial toxin mechanisms and
structure so that protein domains can be “shuffled”
for a variety of applications. This approach delivers
an already characterized enzyme to new cell types,
specified by binding affinity. Separated protein
components from holotoxins are also repurposed
into drug delivery applications to form composite
multifunctional drug delivery units. Enzymatic
domains are used for cancer diagnosis and treatment,
influence of intracellular trafficking, and for providing
relief from pain, autonomic disorders, movement
disorders, spasticity, and HIV. Technical challenges
to this field are the immunogenicity, solubility and
stability of therapeutic fusion proteins. Clinical
intervention and predictive computational approaches
identify, prevent, and remove known and predicted
immunogenicity without a significant loss of
efficacy. Unrealized medical potential exists in a
wealth of bacterial diversity that may be captured
by the repurposing of bacterial toxins.
KEYWORDS: bacterial AB exotoxins, protein
engineering, human therapeutics, biologics, drug
delivery
INTRODUCTION
Therapeutic repurposed bacterial toxins (repTox)
are derived from naturally occurring proteins that
target, enter, and disrupt the biological structures and
processes of cells. Many repTox are sophisticated
membrane-associated proteins with high aqueous
solubility, capable of targeted molecular transport to
specific human cell types and intracellular locations.
Treatments for cancer and neurological disorders
have been the focus of several clinical trials [1-4],
but only two have been approved for therapeutic use.
Clostridium botulinum neurotoxin serotypes A and
B (Botox®
, Dysport®
, Xeomin®
, Myobloc®
) can be
purified directly from the microorganism and are
locally administered cosmetics and therapeutics
that cause neuromuscular paralysis. Engineered
interleukin-2-diphtheria toxin (Ontak®
) is an
intravenously injected cancer therapeutic produced
by recombinant biotechnology that combines the
biomolecular functions of interleukin-2 and diphtheria
toxin from Corynebacterium diphtheria to target
IL-2 receptors on the cancer cell surface and gain
entry into the cytoplasm to disrupt a vital protein
translation factor. The potential of these functionally
efficient bacterial components has not yet been
fully realized in the context of human health, and
may provide many new treatments and biomedical
research tools [5-13]. Development of repTox into
therapeutics has been enabled by advances in
protein engineering and functional proteomics. This
minireview considers current human therapeutic
applications of repurposed bacterial toxins using
protein engineering and biotechnology.
Structure/function of bacterial toxins
Pathogenic bacteria have evolved to produce a
swarm of proteins (Table 1), lipopolysaccharides
and effectors to increase virulence [14, 15], but
Current Topics in
Peptide & Protein
R e s e a r c h

13. 13
Research and Administrative Tasks
Summary Preparatory to IND FiIing
• Optimize Process
• ID & Develop Analytical Tests
• Formulation & Stability
Manufacturing
(Underway)
• Proof of Principle Efficacy
• Acute Safety and Toxicity
In Vivo Animal Testing
(Underway)
• MED, No AE Dose, MTD
• Definitive Dose-Dependent Efficacy
• Systemic and Local Toxicity
• Systemic and Local PK
• Predictive Immunogenicity Testing
• GLP Acute and Subchronic Toxicity
In Vivo Animal Testing
2021 Q3
• CMC Preparation
• Package Insert, Investigators’ Brochure
• Phase I Clinical Protocol
• Investigation Plan, Other Supporting Data
Regulatory Activities
2021 Q3
• Begin Phase I Clinical Trial: Postoperative Pain in
Thoracotomy
IND Submission 2021
Q4

14. 14
Paul Blum, Ph.D. CEO
University of Nebraska – Lincoln
25 years of protein development
U. Nebraska Inventor of the Year 2015
Marina Blum Operations
University of California - Berkeley
BA – Public Health
Team
Jianguo Cheng, M.D., Ph.D.
Professor of Anesthesiology
Cleveland Clinic, President Elect
American Academy of Pain Medicine
Richard L. Stieg, M.D., MHS
Former President of American Academy of Pain
Medicine
Founding Member, National Pain Foundation
Stephan B. Abramson, Ph.D.
LifeSci Partners, LLC.
Formerly Director of Clinical
Affairs, Alpha Therapeutic Corp.
Board
Partners

15. For more information
please contact:
paul@neurocarrus.com