Next-generation small molecule neuro regeneration therapy Dual actions of increasing endogenous stem cells and suppressing glial differentiation of neural stem cells to regenerate neurons by peripheral administration of low molecular weight compounds.

1. Next generation small molecule
neuro regeneration therapy
PROGENICYTE JAPAN Co., Ltd.
【Concept】
Dual actions of increasing endogenous stem cells and
suppressing glial differentiation of neural stem cells to
regenerate neurons by peripheral administration of low
molecular weight compounds.

2. 【Core Technology】
Small molecular compound to increase
endogenous stem cells, KS-217
(Patented)
Inhibit glial differentiation of neural stem
cells and increase neurogenesis
APP production inhibitor (Patented)
Neurogenesis to treat
APC-100 (Patented)
We found that mechanism of action of
APP function inducing glial
differentiation of neural stem cells
Neurotoxicity Research. 18(3,4) 328-38
Combination treatment of KS-217 and APP
signaling inhibitor increase neurogenesis.
（Patent filed in 2020, unpublished）
Combination of
KS-217
APP production
inhibitor
Further research

3. 【Issues with the current neuroregeneration therapies】
☑Neural stem cell transplantation
Neural stem cell transplantation is very effective to
treat neurodegenerative diseases. However, almost
impossible to get the transplantable cells because of
their location.
☑Transplantation of adult stem cells in peripheral
tissue, such as mesenchymal stem cells.
Those adult stem cells do not differentiate into
neural cells.
☑Transplantation of pluripotent stem cells
including embryonic stem cells and iPS cells.
Teratoma and other tumor formation prevents the
clinical use of these cells.

4. 【Evidence data1】KS-217 increase proliferation of neural and
mesenchymal stem cells
• Human neural stem cells were cultured with ¼
concentration of growth factor (FGF and EGF).
• Human mesenchymal stem cells were cultured
with ¼ concentration of FBS.
• EC50 for increasing effect of neural stem cell
proliferation was 5.2nM. This is about 70 times
lower than that for mesenchymal stem cells.
• Cell toxicity was not observed up to 30mM.
• There was no effect on primary cancer and
tumor cell lines.

5. 【Evidence data 2-1】Peripherally administered KS-217 increased neural
stem cells in vivo
ü KS-217 increases cell proliferation in the cortex (D) of aged-memory unimpaired rats compared to the
control (A).
ü The number of proliferating cells was increased by KS-217 treatment in sub ventricular zone of both
aged-memory impaired rats (E) and aged-memory unimpaired rats (F) compared to the controls (B,C). In
treated animals, newly generated stem cells appear to migrate into the white matter of the cortex (arrows
in F).
Aged rats were tested for memory
impairment and treated with KS-217
(3mg/kg/day i.p.) for 3 days.

6. 【Evidence data 2-2】 Peripherally administered KS-217 increased neural
stem cells in vivo
Aged rats were tested for memory impairment
in a water maze and treated with KS-217
(3mg/kg/day i.p.) for 3 days.
ü Stereological analysis shows 7-fold
increased in proliferation of endogenous
BSCs in the cortex of aged-impaired
animals treated with KS-217.
Antidepressant (SSRI), currently known as the only drug that increases neural stem cells, has
an increase rate of 20% (Vichier-Guerre C, et.al., Toxicol Lett. 2017 Nov 5;281:20-25)

7. 【Evidence data3】 Peripheral KS-217 treatment increased rotarod performance of
MPTP Parkinson’s disease model mice
• 8 weeks old male C57BL/6J mice
(n=12) were used. Behavior of the
animal was tested by Rotarod test
(Initial speed５rpm, Last speed 40prm,
300 seconds).
• After base line of the behavior was
taken, PD model animals were made by
MPTP (25mg/kg/day i,p,) injections for 3
days.
• Behavior to stay on the rotating rod was
assessed at 5, and 6 weeks after
treatment with KS-217 (3 mg/kg/day for
5 days).
ü KS-217 treatment significantly reduced Parkinson’s disease symptoms in the treated
animals. (**：P<0.01、ANOVA followed by Tukey's HSD test ）

8. 【Evidence data 4-1】Secreated type amyloid precursor protein (APP)
induced glial differentiation of neural stem cells
Human neural stem cells were cultured in non-serum media
and differentiated by taking proliferation factors (FGF, EGF)
from the media. During the differentiation secreted type of
amyloid precursor protein (APP, 0-100ng/ml) was added to the
media. Cells were immunostained for βIII-tubulin (green),and
GFAP(red), neuronal and glial markers, respectively. Nuclei
were counter stained with DAPI (blue)
ü APP(25ng/ml) increased both neural and glial differentiations of neural stem cells.
ü APP dose dependently increase glial differentiation of neural stem cells.
ü APP(>50ng/ml) suppress neuronal differentiation of neural stem cells.
Biochem Biophys Res Commun, 344 (1) 431-7.

9. 【Evidence data 4-2】Over expression of amyloid precursor protein (APP)
induced glial differentiation
ü Human neural stem cells were cultured in
non-serum media and differentiated by
taking proliferation factors (FGF, EGF) from
the media. Cells were immunostained for
βIII-tubulin (green),and GFAP(red),
neuronal and glial markers, respectively.
Nuclei were counter stained with DAPI
(blue)
ü The cells received empty vector
differentiated into neurons and glial cell
50/50. The cells over expressing APP
mostly differentiated into glia and
neuronally differentiated cell showed
peculiar morphology.
ü The differentiation pattern and morphology
were very similar to the neural cells
differentiated from Down’s syndrome
patient neural stem cells. (THE LANSET
359, 9303, P310- 315)
Biochem Biophys Res Commun, 344 (1) 431-7.

10. 【Evidence data 5】Neural stem cells were able to differentiated after
phenserine treatment, which reduces APP level in vivo
Control
APP23
Saline (+)-Phenserine
APP23 transgenic mice were treated with
phenserine (0, 25mg/kg/day i.p.) for 1
week, then human neural stem cells were
transplanted. Then the animals were
continued to be treated the phenserine
another 3 weeks. The hippocampus slices
(20µm) were immunostained for neuronal
marker, βIII-tubulin (green) Nuclei were
counter stained with DAPI (blue).
ü Neural stem cells were able to
differentiate into neurons in control
animal w/wo phenserine.
ü In APP transgenic animal expressing
excess APP, neurogenesis were only
observed after the phenserine
treatment.
Proc Natl Acad Sci U S A.104(30):12506-11

11. 【Evidence data 6】APC-100, combination of KS-217 and phenserine
increased neural stem cells and neurogenesis in 5xFAD transgenic mice
• 8 months old male B6SJL-Tg(APPSwFlLon,PSEN1
*M146L*L286V) 6799Vas/Mmjax (5xFAD,n=6) mice
were used.
• The animals received KS-217 (3mg/kg/day i.p. ) and
phenserine (25mg/kg/day i.p.) for 3 weeks. Control
received saline i.p. injection.
• Immunohistochemistry for CD133 as a neural stem
cell marker and doublecortin as a marker for
neurogenesis.
ü Neural stem cells and neurogenesis were significantly reduced in 5xFAD mice compare to the wild
type mice. This may be because of premature glial differentiation of neural stem cells by a high
concentration of APP.
ü Treatment with APC-100 significantly increased both neural stem cells and neurogenesis in 5xFAD
mice. Neurogenesis was more than the control level; this may be because the increased neural
stem cells migrated and differentiated by responding factors in these animals’ brain similarly to the
memory impaired animal seen in Evidence data 2-1.

12. 【Evidence data 7】APC-100 improved cognition of５xFAD in radial water maze
We have tested special memory of 8-month-old male 5XFAD mice
and wild mice (n=12) in radial water maze.
The mice were treated with APC-100, combination of KS-217
(3mg/kg/day i.p.) and phenserine (25mg/kg/day i.p.) for 3 weeks and
tested in the water maze one week after the treatment.
We conducted 10 trials per day for 2 days. The first day the goal was
visible to the animals and the second day the goal was hides under
the water. The data shows only for the 2nd day results.
We counted error, animal turn into the arm without the goal, till they
reached to the goal.
ü Wild type mice reached to the goal almost without error the the final trial.
ü 5XFAD did not show an improvement after 4 trials in the second day, and they still made error at the final trial.
ü KS-217, which increase endogenous neural stem cells, treated 5xFAD mice did not show any improvement in
their cognition.
ü APC-100, combination of KS-217 and phenserine treated 5xFAD mice behave like wild type mice.

13. 【Conclusion】
• KS-217 increased proliferation of human neural stem cells at EC50=5.2nM, which is 70 time lower than that for
mesenchymal stem cells.
• KS-217 increased proliferation of endogenous neural stem cells up to 600％ by peripheral injection.
• KS-217 did not increase proliferation of cancer and it did not have cytotoxicity up to 30 mM
• APP increased glial differentiation of human neural stem cells 50ng/ml or more.
• APP over expression in neural stem cells induced glial differentiation similar to the neural stem cells from Down’s
syndrome patient. This may be because APP gene is on the chromosome 21, which Down’s syndrome patient has
trisomy.
• Treatment of phenserine, which reduce APP expression, allowed neural stem cells to differentiated into neurons in APP
transgenic mice expressing high level of APP.
• Peripheral administration of APC-100, combination of KS-217and phenserine increased neural stem cells and
neurogenesis in the 5xFAD transgenic mice. APC-100 improved the cognition of the animal to the wild type animal level.
• Since APP level is known to increase in not only Down’s Syndrome but also aging, neurodegeneration and traumatic
brain, APC-100 and the successor drug may be good treatments for these conditions.
APC-100 and the successor drug may dramatically acerate the development of neuroregenerative
medicine as they do not depend on the cell transplantation like the current regenerative medicine.
【 Target diseases 】
Down’s Syndrome, Parkinson's Disease, Alzheimer's Disease, Chronic Stroke, Spinal Cord Injury

14. 【 Patent 】
◇Granted
A method of biasing implanted human neural stem cells away from differentiation into glial cells by(+)phenserine
to modulate the concentration of soluble βapp in tissue or csf（U S 9,095,573）
Use of Modified Pyrimidine Compounds to Promote Stem Cell Migration and Proliferation
(U S 7,687,505,U S 8,273,756、U S 9,084,789、1/14/2003, 7/21/2015）
Drug Treatment to Increase Stem Cell Population
(CA2473503、EP1471918,603 50 413.2, 1471918FR, JP4623967B, 5414626JPD）
Combination therapy to improve brain function or promote neurogenesis for treating neurodegenerative conditions
(U S 62/346,166, PCT/US17/36220)
◇Pending
Combination therapy for neurogenesis
Filed 8/27/2020
◇Other related patents
Compositions for Treating or Delaying the Onset of Hair Loss
(US 8,791,128, PCT/US09/57134, CA2,736,018, EP2326330, 602009028877.6, 2326330FR, 2324023UK, 5555922JP)
Vigor Enhancement Via Administration of Pyrimidine Derivatives
(CA2,670,341, PCT/US07/85765, EP2324023, 602007047544.9, 2324023FR, 2324023UK, HK1158204B)

15. 【Progenicyte Japan Co., Ltd.】
Progenisyte Japan is a venture company spin off from the University of Central Florida School of Medicine in the United States. It
aims to commercialize regenerative medicine with a new technology that increases endogenous stem cells with low-molecular-
weight compounds without relying on cell transplantation as in the past.
Representative Directors Kiminobu Sugaya, Ph.D.
Neung Suh
Established January the 24th 2020
URL https://www.progenicytejapan.com/en-us/
Address Kobe International Conference Center 606
6 Chome-9-1 Minatojima Nakamachi, Chuo Ward, Kobe,
Hyogo 650-0046, Japan
【Progenicyte LLC.】
Progenicyte is a holding company providing innovative revolutionizing technologies developed by Dr. Kiminobu Sugaya,
Representative Director of Progenicyte Japan and Professor of Medicine at the University of Central Florida.
CEO Kiminobu Sugaya, Ph.D.
Established May 2008
URL http://progenicyte.com/
Address 19501 Paddock St, FL 32833, USA