/

University of Southern California Dissertations and Theses

/

The opening of the blood brain barrier by homogenized perillyl alcohol

(USC Thesis Other)

The opening of the blood brain barrier by homogenized perillyl alcohol

PDF

Download

Open document

Flip pages

Contact Us

Copy asset link

Request this asset

Transcript (if available)

Content

THE
OPENING
OF
THE
BLOOD
BRAIN
BARRIER
BY
HOMOGENIZED
PERILLYL

ALCOHOL

by

Samantha
Stack

A
Thesis
Presented
to
the

FACULTY
OF
THE
USC
KECK
SCHOOL
OF
MEDICINE

UNIVERSITY
OF
SOUTHERN
CALIFORNIA

In
Partial
Fulfillment
of
the

Requirements
for
the
Degree

MASTER
OF
SCEINCE

(MOLECULAR
MICROBIOLOGY
AND
IMMUNOLOGY)

May
2020

Copyright
2020

Samantha
Stack

ii

Acknowledgements

I
would
like
to
thank
Dr.
Axel
Schönthal
for
giving
me
the
opportunity
to
work
in
his

lab.
He
gave
me
the
guidance
and
support
I
needed.
He
pushed
me
in
the
right
direction
but

also
gave
me
the
freedom
to
take
the
project
in
the
direction
I
wanted
to.

I
would
also
like
to
thank
Dr.
Steve
Swenson.
He
has
helped
me
tremendously
with

learning
new
things
in
lab
and
the
best
techniques
to
use.
He
also
allowed
me
to
work
in
his

lab
as
an
Undergrad
student
and
for
that
I
will
always
be
grateful.
He
was
the
one
to
open

my
eyes
to
the
research
field.

Next
I
would
like
to
thank
Robert
Herrera,
my
fellow
masters
student
and
friend.

Our
collaboration
gave
me
insight
into
new
experiments
and
when
I
needed
help
he
was

always
there.
Our
projects
both
dealt
with
the
blood
brain
barrier
and
his
collaborations

really
helped
me
gain
results
and
troubleshoot
issues.

I
would
also
like
to
thank
Franny
Ferri.
She
was
always
there
to
help
and
she
is
a

great
friend
who
got
me
through
the
tough
days.
It
was
nice
having
a
good
friend
to
turn
to

when
needed.

Lastly,
I
would
like
to
give
a
special
thanks
to
my
husband
XanTh
Stack.
Without
him

these
last
two
years
would
not
have
been
possible.
He
worked
2
jobs
and
countless
hours

so
I
could
pursue
my
dreams
in
this
program.
I
would
not
be
here
without
him.

iii

Table
of
Contents

Acknowledgements
..........................................................................................................................................
ii

List
of
Figures
......................................................................................................................................................
v

List
of
Abbreviations
.......................................................................................................................................
vi

Abstract
...............................................................................................................................................................
vii

Chapter
1
–
Introduction
..................................................................................................................................
1

1.1
The
blood
brain
barrier
..............................................................................................................
1

1.1.1
Introduction
..................................................................................................................
1

1.1.2
Structure
and
function
..............................................................................................
1

1.1.2.1
Brain
endothelial
cells
............................................................................
2

1.1.2.2
Tight
junctions
...........................................................................................
3

1.1.2.3
Pericytes
.......................................................................................................
3

1.1.2.4
Astrocytes
....................................................................................................
4

1.2
Brain
metastasis
............................................................................................................................
4

1.3
Mannitol
as
a
current
therapy
to
open
the
BBB
...............................................................
4

1.4
Trastuzumab
to
treat
metastatic
breast
cancer
...............................................................
5

1.5
Perillyl
alcohol/NEO100
............................................................................................................
6

1.6
Hypothesis
........................................................................................................................................
6

Chapter
2
–
Material
and
Methods
...............................................................................................................
8

2.1
Pharmacological
agents
..............................................................................................................
8

2.2
Homogenization
.............................................................................................................................
8

2.3
In
vitro
analysis
..............................................................................................................................
9

2.3.1
Cell
lines
and
maintenance
.....................................................................................
9

2.3.2
MTT
cell
viability
assays
.......................................................................................
10

2.3.3
Trans
endothelial
electrical
resistance
(TEER)
..........................................
11

2.4
In
vivo
analysis
.............................................................................................................................
12

2.4.1
Evans
blue
analysis
.................................................................................................
12

2.5
Immunohistochemistry
...........................................................................................................
13

2.6
Western
Blotting
.........................................................................................................................
15

Chapter
3
–
Results
..........................................................................................................................................
16

3.1
Homogenized
POH
at
higher
concentrations
does
not
affect
cell
viability
.......
16

3.1.1
Purpose
of
Study
......................................................................................................
16

3.1.2
Effect
of
homogenized
POH
on
cell
viability
compared
to
POH

in
DMSO
or
EtOH/Gly
on
MDCK
cells
..........................................
16

3.1.3
Effect
of
homogenized
POH
on
cell
viability
to
POH
in
DMSO

on
BBB
cells
............................................................................................
22

3.2
POH
decreases
trans
endothelial
resistance
..................................................................
25

3.2.1
Purpose
of
Study
......................................................................................................
25

iv

3.2.2
POH
in
DMSO
lowers
TEER
in
MDCK
..............................................................
26

3.2.3
Homogenized
POH
lowers
TEER
in
BBB
cells
.............................................
29

3.3
Homogenized
POH
opens
the
BBB
in
mice
.....................................................................
32

3.3.1
Purpose
of
Study
......................................................................................................
32

3.3.2
Homogenized
POH
allows
EB
into
brain
........................................................
32

3.3.3
Homogenized
POH
increases
levels
of
EB
in
the
brain
............................
35

3.4
Homogenized
POH
shows
no
change
in
claudin-‐5
expression
...............................
36

3.4.1
Purpose
of
Study
......................................................................................................
36

3.4.2
Homogenized
POH
does
not
change
claudin-‐5
expression
...................
36

Chapter
4
–
Discussion
...................................................................................................................................
39

References
...........................................................................................................................................................
43

v

List
of
Figures

Figure
1.1:
Structure
of
normal
capillaries
vs.
brain
capillaries
...................................................
2

Figure
2.1:
Visualization
for
the
process
of
homogenization
.........................................................
9

Figure
2.2:
Blood
brain
barrier
model
set-‐up
on
inserts
...............................................................
12

Figure
2.3:
Intracardiac
injection
of
mice
............................................................................................
13

Figure
3.1:
Effect
of
POH
in
DMSO
on
cell
viability
in
MDCK
cells
............................................
17

Figure
3.2:
Effect
of
POH
in
DMSO
vs.
EtOH/Gly
on
cell
viability
in
MDCK
cells
................
18

Figure
3.3:
Effect
of
old
vs.
new
homogenized
POH
on
cell
viability
in
MDCK
cells
..........
20

Figure
3.4:
Effect
of
homogenized
POH
+
DMSO
on
cell
viability
in
MDCK
cells
................
21

Figure
3.5:
Effect
of
homogenized
POH
at
high
concentrations
on
cell
viability
in

MDCK
cells
..........................................................................................................................................
22

Figure
3.6:
Effect
of
homogenized
POH
on
cell
viability
in
BECs,
ACs
and
PCs
...................
23

Figure
3.7:
Effect
of
homogenized
POH
and
mannitol
on
cell
viability
in
BECs
at

different
time
points
.......................................................................................................................
25

Figure
3.8:
Change
in
resistance
of
MDCK
cells
by
Nagore
Marin
Ramos
..............................
26

Figure
3.9:
Change
in
resistance
of
MDCK
cell
model
with
POH
in
DMSO
treatment
.......
27

Figure
3.10:
Change
in
resistance
of
MDCK
cell
model
with
homogenized
POH

treatment
.............................................................................................................................................
28

Figure
3.11:
Change
in
resistance
of
BBB
cell
model
......................................................................
30

Figure
3.12:
Change
in
resistance
of
BBB
cell
model
with
recovery
........................................
32

Figure
3.13:
Effect
of
POH
on
opening
the
BBB
in
vivo
..................................................................
34

Figure
3.14:
Levels
of
EB
in
the
brain
after
treatment
...................................................................
35

Figure
3.15:
Change
in
claudin-‐5
expression
.....................................................................................
37

Figure
3.16:
Expression
levels
of
claudin-‐5
........................................................................................
38

vi

List
of
Abbreviations

AC:
Astrocytes

BBB:
Blood
brain
barrier

BEC:
Brain
endothelial
cells

BM:
Brain
metastasis

CNS:
Central
nervous
system

DAPI:
Diamidino
phenylindole

DMSO:
Dimethyl
sulfoxide

EC:
Endothelial
cell

FBS:
Fetal
bovine
serum

HER2:
Human
epidermal
growth
factor
receptor

homPOH:
homogenized
POH

IHC:
Immunohistochemistry

IP:
Intraperitoneal

MDCK:
Madine
Darby
canine
kidney

MTT:
methylthiazoletetrazolium

NEO
100:
pure
form
of
POH

PC:
Pericytes

POH:
Perillyl
alcohol

TEER:
Trans
endothelial
electrical
resistance

TJ:
Tight
Junction

vii

Abstract

Background:
Cancer
was
the
second
highest
cause
of
death
in
the
United
States
in
2017

and
10%
to
26%
of
cancers
will
metastasize
to
the
brain
(Amsbaugh
and
Kim
2020).
The

blood
brain
barrier
(BBB)
acts
as
a
block
to
treating
these
metastatic
cancers.
An
estimated

98%
of
all
therapeutics
for
metastatic
and
primary
brain
cancers
are
not
able
to
cross
the

BBB.

Methods:
We
investigated
the
effect
of
homogenized
perillyl
alcohol
(homPOH)
on
opening

the
BBB
both
in
vitro
and
in
vivo.
POH
is
a
naturally
occurring
monoterpene
that
can
be

isolated
from
several
plants.
In
vitro
experiments
used
MDCK
cells
and
BBB
cell
lines
as

models
to
mimic
the
BBB.

The
BBB
cells
included
human
brain
endothelial
cells,
astrocytes

and
pericytes.
These
cell
lines
were
treated
with
homPOH
and
we
measured
cell
viability

and
changes
in
trans
endothelial
electrical
resistance
(TEER).
In
vivo
experiments

measured
the
effect
of
POH
in
allowing
a
large
molecule
of
Evans
blue
albumin
to
cross
the

BBB.

Results:
Homogenized
POH
displayed
low
toxicity
in
both
MDCK
cells
and
BBB
cells,

resulting
in
a
significant
drop
in
TEER
after
treatment
in
these
cell
lines.
In
vivo
results

indicated
an
increase
of
Evan
blue
albumin
uptake
into
the
brain
through
qualitative
and

quantitative
analyses.
One
key
player
contributing
to
the
leakiness
in
the
BBB
appears
to
be

Claudin-‐5,
a
tight
junction
marker.

Conclusions:
Homogenized
POH
displayed
promising
results
in
opening
the
BBB.
The

decrease
in
Claudin-‐5
expression
seems
to
play
a
major
role
in
opening
of
the
BBB.
We

propose
that
POH
should
be
investigated
further
toward
clinical
testing
in
conjunction
with

other
chemotherapeutics
to
treat
metastatic
brain
cancers.

1

Chapter
1
–
Introduction

1.1
The
Blood
Brain
Barrier

1.1.1
Introduction

The
blood
brain
barrier
(BBB)
is
important
for
normal
brain
function
and

maintaining
homeostasis.
There
is
a
strict
regulation
of
components
that
are
permitted
to

enter
the
brain.
Regulation
and
homeostatic
control
is
maintained
in
the
brain
by
a
physical

barrier
formed
in
the
Central
Nervous
System
(CNS)
[Serlin
et
al.
2015].
The BBB is not
one physiology, but a series of physiological properties that need to be induced or
inhibited, i.e., tight junctions, transporters, and metabolic enzymes in endothelial cells of
the central nervous system” [Daneman and Prat 2015]. Manipulating the BBB is an
important aspect in treatments that need to enter the brain.

1.1.2
Structure
and
function

The
BBB
is
composed
of
three
main
cell
types.
These
include
brain
endothelial
cells

(BEC),
pericytes
(PC)
and
astrocytes
(AC).
BECs
grow
tightly
together
and
form
tight

junctions
making
up
the
luminal
side
of
the
vasculature.
ACs
and
PCs
form
the
abluminal

side
and
associate
with
the
endothelial
cells.
The
presence
of
these
three
cell
types
and

tight
junctions
contribute
to
the
tightness
of
the
BBB
and
the
association
between
cells

influences
the
ability
of
the
BBB
to
highly
regulate
passage
through
the
cranial
vasculature.

2

1.1.2.1
Brain
Endothelial
Cells

The
BECs
compose
the
blood
vessel
wall
and
establish
the
main
properties
of
the

BBB,
but
maintenance
of
these
properties
are
provided
by
cells
that
interact
with
the
BECs

[Daneman and Prat 2015].
Figure
1.1
shows
the
difference
between
capillaries
in
the
brain

and
other
capillaries.

Capillaries,
in
general,
are
comprised
of
endothelial
cells
with
intercellular
spaces.

This
allows
for
exchange
of
both
large
and
small
molecules
between
the
blood
and
tissues.

Brain
capillaries
consist
of
astrocytes,
pericytes
and
tight
junctions
in
addition
to
the

endothelial
cells.
BECs
have
unique
properties
of
tightly
regulating
movement
of
ions,

molecules
and
cells
between
the
blood
and
the
brain
tissue.
Mitochondria
are
seen
in

higher
amounts
in
BECs
compared
to
general
endothelial
cells.
The
increase
in

Figure
1.1
Comparison
of
brain
capillaries
vs.
capillaries
in
general

(outside
of
the
brain)
to
show
the
Blood
Brain
Barrier
structure.
(Adapted
from:
Prasad
et
al.,
2012)

3

mitochondria
aids
in
ATP
production
needed
for
transport.
Tight
junctions
hold
BECs

together,
limiting
the
paracellular
flux
in
between
cells
[Daneman and Prat 2015].

1.1.2.2
Tight
Junctions

Tight
junctions
(TJ)
seal
the
endothelium
and
represent
the
core
structure
of
the

BBB
[Bauer
et
al.
2014].

The
molecular
interactions
of
TJs
are
indicative
of
low
ion

permeability,
providing
high
transcellular
electrical
resistance
in
the
BBB
endothelial
cells

[Bauer
et
al.
2014].

There
are
three
major
TJ-‐
associated
proteins:
claudins,
TAMPs
(TJ-‐
associated
MARVEL
proteins),
and
immunoglobulin
superfamily
membrane
proteins.

Claudins
comprise
27
different
members,
claudin-‐3
and
claudin-‐5
are
expressed
in
the
BBB

and
claudin-‐5
is
the
most
abundant
[Bauer
et
al.
2014].
TAMPs
are
a
group
of
proteins

containing
the
MARVEL
motif,
occludin
being
among
them
[Bauer
et
al.
2014].
The

immunoglobulin
superfamily
membrane
proteins
are
JAMs,
CARs
(coxsackie-‐
and

adenovirus
receptor)
and
ESAMs
(endothelial
cell
selective
adhesion
molecules).
Tight

junctions
function
in
maintaining
homeostasis
in
the
brain
and
it
is
suggested
that
claudins

are
essential
for
the
paracellular
barrier
[Daneman and Prat 2015].

1.1.2.3
Pericytes

Pericytes
(PC)
sit
on
the
ablumenal
surface
of
the
endothelial
tube
and
extend
long

cellular
processes
along
this
surface
covering
multiple
endothelial
cells. The
ratio
of
ECs
to

PCs
in
the
CNS
is
1:1
or
3:1,
compared
to
muscles
having
a
100:1
ratio
[Daneman and Prat
2015].
This
creates
higher
coverage
of
ECs
by
PCs,
which
is
believed
to
be
instrumental
for

BBB
function
and
regulation
[Daneman and Prat 2015].

4

1.1.2.4
Astrocytes

Astrocytes
are
a
glial
cell
type
and
provide
a
cellular
link
between
neuronal
circuitry

and
blood
vessels
[Daneman et al. 2015].
Mature
astrocytes
maintain
the
BBB
by
secreting

growth
factors
(GF)
such
as
VEGF,
glial
cell
line-‐derived
neurotrophic
factor
(GDNF),
basic

fibroblast
growth
factor
(bFGF),
and
ANG-‐1.
Tight
junction
formation
relies
on
secretion
of

these
growth
factors,
as
well
as
promotion
of
polarization
of
transporters
[Cabezas
et
al.

2014].

1.2
Brain
Metastasis

The
most
common
brain
tumors
in
adults
are
metastatic
brain
tumors.
Metastatic

brain
tumors
occur
10
times
more
frequently
than
primary
brain
tumors
[Ostrom
et
al.

2018].
The
most
common
cancer
types
to
metastasize
to
the
brain
are
Lung(44%),

Breast(11.9%)
and
Melanoma(10.2%)
[Text
book
of
Neuro-‐Oncology
2005].
Breast
to
brain

metastasis
is
more
common
in
triple
negative
or
HER2/neu
positive
primary
breast

cancers
[Ostrom
et
al.
2018].

There
is
an
incidence
of
up
to
50%
of
patients
with
HER2+

breast
cancer
developing
intracranial
metastases
(Venur
and
Leone
2016).

1.3
Mannitol
as
a
current
therapy
to
open
the
BBB

Mannitol
is
a
six-‐carbon
linear
simple
sugar
that
is
not
fully
metabolized
by
the
body

and
can
be
used
as
a
sugar
substitute
(Tenny
et
al.
2020).
Mannitol
solution
is

hyperosmolar
and
can
be
used
to
disrupt
the
BBB
by
causing
a
loss
of
water
in
cells.
This

loss
of
water
loosens
the
cell-‐cell
junction
interaction
[Brown
at
al.
2004].
The
method
is

considered
to
be
an
osmotic
opening
of
the
BBB
mediated
by
vasodilation
and
shrinkage
of

5

BECs,
creating
an
increase
in
the
interendothelial
space
and
loosening
TJs.
(Rapoport

2000).
Mannitol
administration
occurs
by
intracarotid
infusion
and
the
effects
only
last
for

a
short
time,
approximately
ten
minutes
(Rapoport
2000).
Current
therapies
use
mannitol

in
conjunction
with
chemotherapies
to
open
the
BBB
for
a
brief
time.
It
has
been
shown

that
mannitol
increases
permeability
of
sucrose
but
not
of
Evans
blue
albumin,
which
has
a

molecular
weight
of
~66kDa.

(Brown
et
al.
2004).
Current
mannitol
therapies
are
limited

in
that
they
do
not
allow
penetrability
of
larger
molecules
such
as
antibodies.

1.4
Trastuzumab
to
treat
for
metastatic
breast
cancer

Treatment
for
HER2+
metastatic
breast
cancer
is
done
with
administration
of

Trastuzumab,
or
Herceptin.
Herceptin
is
a
recombinant
humanized
IgG
monoclonal

antibody
against
the
extracellular
domain
of
the
HER2(EGF)
receptor
(Boekhout
et
al.

2011).
Herceptin
binds
to
the
extracellular
domain
of
HER2
preventing
activation
of
the

receptor
and
the
down
stream
cleavage
of
the
intracellular
domain,
which
in
turn
results
in

cell
mediated
cytotoxicity
(Boekhout
et
al
2011).
Herceptin
has
shown
promising
result
in

treatment
of
HER2+
breast
cancers
but
once
these
cancers
metastasize
to
the
brain,
there
is

no
current
approach
in
the
clinic
to
administer
Herceptin
to
allow
passage
through
the

BBB.
Brain
metastasis
is
a
major
cause
of
morbidity
in
patients
with
advanced
breast

cancer
(Venur
and
Leone
2016)
and
leads
to
the
need
for
a
necessary
therapy
that
allows

large
drug
molecules,
such
as
antibodies
to
cross
the
BBB.

6

1.5
Perillyl
alcohol/NEO100

Neo
100
is
a
synthetic
highly
pure
form
of
Perillyl
alcohol
(POH),
manufactured

under
good
laboratory
practice
conditions
(Chen
et
al.
2015).
POH
is
a
naturally
occurring

monocyclic
terpene
that
can
be
isolated
from
several
plants:
e.g.,
lavender,
peppermint,

cherries,
caraway,
lilac
oil,
cranberries,
sage,
celery
seeds,
and
certain
other
plants
(Chen
et

al.
2015).
It
is
a
metabolite
of
limonene
and
is
thus
derived
from
the

mevalonate/isoprenoid
pathway
(Chen
et
al.
2018).
The
impact
of
POH
is
pleiotropic
on

many
cellular
targets.
POH
was
shown
to
be
a
cytotoxic
agent
by
functioning
as
a
Ras

inhibitor,
cell-‐cycle
inhibitor,
and
upregulator
of
the
proapoptotic
protein
Bax
(Cho
2012).

It
has
also
been
shown
that
POH
induces
apoptosis
in
human
glioblastoma
multiforme
cells

in
vitro
by
exhibiting
morphological
alterations
in
treated
cells
(Fernandez
et
al.
2005).

Further
studies
now
show
that
POH
may
play
a
role
in
opening
the
BBB
thus
increasing

permeability
of
substances.
POH
exhibits
physical
properties
that
are
similar
to
oil,

meaning
that
POH
is
not
miscible
with
water.
This
leads
to
a
need
for
a
vehicle
that
will

completely
solubilize
POH
and
allow
for
a
uniform
mixture.

1.6
Hypothesis

Based
on
current
data
suggesting
Trastuzumab
increases
the
overall
survival
of

patients
with
HER2+
metastatic
breast
cancer
(Venur
and
Leone
2016),
there
is
a
push
to

find
a
way
to
administer
the
drug
into
the
brain.
The
extensive
use
of
POH
has
been

widespread
and
in
particular,
intranasal
delivery
to
glioblastoma
patients
showed

promising
results
as
a
chemotherapeutic
with
low
toxic
effects
compared
to
oral

administration
(Chen
et
al.
2015).

7

After
continued
research
and
administration
of
POH
we
concluded
that
POH
might

have
an
advantage
in
altering
the
BBB.
After
using
many
vehicles
we
hypothesize
that
using

homogenized
POH
is
effective
in
disrupting
the
BBB
allowing
for
larger
molecules
to
cross.

8

Chapter
2
–
Materials
and
Methods

2.1
Pharmacological
Agents

NEO
100
or
POH,
purchased
from
Sigma
Aldrich
(St.
Louis,
MO).
Will
be
referred
to

as
POH.
POH
was
dissolved
in
DMSO
(Santa
Cruz
Biotechnology,
Dallas,
TX)
or
a
50:50(v:v)

solution
of
ethanol/glycerol
(EtOH/Gly)
(Sigma,
St
Louis,
Missouri).
POH
was
diluted
from

6.3M
to
a
concentration
of
800
mM
or
homogenized
at
3%
(~189
mM).
POH
was
diluted

further
in
cell
culture
medium
to
get
experimental
concentrations.
DMSO
percentage
was

calculated
based
on
the
amount
of
DMSO
in
total
solution.
The
20%
OSMITROL
Injection
or

20%
Mannitol
(Baxter,
Deerfield,
IL)
were
heated
in
a
water
bath
and
then
diluted
to
a
final

working
concentration
in
cell
culture
medium.

2.2
Homogenization

POH
was
homogenized
by
pushing
liquid
at
a
speed
of
400m/s
and
at
10,000-‐
20,000psi
through
a
sharp
filter
with
10,000,000
pores.
The
homogenizer
machine
is
a

model
M110L
from
Microfluidics
(Westwood,
Massachusetts).
Figure
2.1
shows
the
process

of
homogenization.
POH
samples
were
homogenized
at
1,
2.2,
or
3%(~63,
138.6
or
189
mM

respectively).
The
homogenized
samples
were
used
immediately
for
in
vivo
experiments.

For
in
vitro
analysis,
fetal
bovine
serum
(FBS),
from
Omega
Scientific
(Tarzana,
CA),
was

added
to
samples
allowing
them
to
sit
for
an
extended
amount
of
time
at
4
deg
C.

9

2.3
In
vitro
analysis

2.3.1
Cells
and
maintenance

Madin-‐Darby
Canine
Kidney
(MDCK)
cells
(Science
Cell
Research
Labratories,

Carlsbad,
CA)
were
propagated
in
Dulbecco’s
Modification
of
Eagle’s
Medium
(DMEM)
with

4.5
g/L
glucose,
L-‐glutamine,
sodium
pyruvate
supplemented
with
10%
fetal
bovine
serum

(FBS),
100
u/mL
penicillin,
and
0.1
mg/mL
streptomycin.
FBS,
penicillin
and
streptomycin

were
obtained
from
the
USCs
Cell
Culture
Core
(Los
Angeles,
CA).

Human
brain
endothelial
cells
(HBEC)
transfected
with
SV40,
human
pericytes

transfected
with
SV40
and
human
astrocytes
transfected
with
SV40
were
obtained

Science

Cell
Research
Labratories,
Carlsbad,
CA.

The
human
cell
lines
were
used
to
reconstitute
the

Figure
2.1:
Visualization
on
how
the
homogenization
process
works.

POH
is
added
to
medium
then
run
through
the
machine
under
high

pressure,
pushing
it
through
a
sharp
filter
that
evenly
distributes
POH

molecules
in
liquid.

Un-‐
homogenized

POH
Homogenized

POH

Sharp
Filter

High

Pressure

10

BBB
in
vitro
and
were
grown
in
special
medium
consisting
of:

50%
Gibco
Advanced
DMEM/F12
purchased
from
Thermo
Fisher
(Grand
Islands,
NY)

50%
Nerobasal
A
purchased
from
Fisher
Scientific
(Grand
Island,
NY)

2.5%
Gibco
B-‐27
Supplement
-‐
serum
free
(50X)
purchased
from
Thermo
Fisher
(Grand

Island,
NY)

1%
Antibiotic-‐Antimycotic
(100X)
purchased
from
Thermo
fisher
(Grand
Island,
NY)

1%
Gibco
GlutaMAX
Supplement
purchased
from
Thermo
fisher
(Grand
Island,
NY)

5%
FBS

All
cells
were
kept
in
a
humidified
incubator
at
37°C
and
a
5%
CO2
atmosphere.

2.3.2
MTT
Cell
Viability
Assays

Methylthiazoletetrazolium
(MTT)
assays
were
performed
as
follow:
cells
were

seeded
into
96-‐well
plates
in
a
volume
of
50
μL
per
well.
Cell
densities
varied
from
1.0-‐8.0

×
10
5

cells/mL.
An
additional
50
μL
of
medium
containing
various
concentrations
of
drug

was
added
24
hours
later
to
allow
cells
to
completely
adhere.
Cells
were
incubated
for
the

various
time
intervals
with
drug
depending
on
each
experimental
design,
this
could
be
5

min,
10
min,
30
min,
60
min
or
24h.
This
was
followed
by
the
addition
of
thiazolyl
blue

tetrazoliumbromide
(methylthiazoletetrazolium,
MTT;
Sigma–Aldrich,
St.
Louis,
MO)
at

10%
for
4
hours
at
37°C
(stock
solution
of
MTT
is
5
mg/mL
in
PBS).

Cells
uptake
the
MTT

dye
and
mitochondrial
succinate
dehydrogenase
reduces
MTT
to
MTT
formazan,
a
purple

product.
The
reaction
was
stopped
at
4
hours
and
the
cell
cultures
lysed
by
the
addition
of

11

100
μL
of
solubilization
solution
(10%
sodium
dodecyl
sulfate
(SDS),
in
0.01
M

hydrochloric
acid,
HCl).
The
96-‐well
plate
was
left
in
the
cell
culture
incubator
over
night

for
complete
solubilization
of
the
MTT
crystals,
and
the
optical
density
(OD)
of
each
well

was
determined
in
a
Varioskan
Lux
Reader
(Thermo
Scientific,
Waltham,
MA)
at
570
nm.

The
background
value
(OD
of
control
well
containing
medium
without
cells
+
MTT
+

solubilization
solution)
was
subtracted
from
all
measured
values.
Cell
viability
percentages

were
calculated
by
dividing
all
samples
by
untreated.
In
individual
experiments,
each

treatment
condition
was
set
up
in
duplicates
or
triplicates.

2.3.3
Trans
endothelial
electrical
resistance

MDCK
and
human
BBB
cells
were
used
to
create
a
blood
brain
model
and
measure

resistance.
MDCK
cells
(1.0-‐1.5
×
10
5

cells/mL)
were
plated
on
the
inside
of
inserts
(8um

Thincert™
12
Well
Plates
Inserts
from
Greiner
Bio-‐One).
MDCK
cells
took
approximately
6-‐
8
days
to
reach
resistance.
Human
astrocytes
and
pericytes
were
plated
in
a
1:1
ratio
at
3.3

×
10
5

cells/mL
in
300μL
on
the
bottom
of
inserts
(8um
Thincert™
12
Well
Plates
Inserts

from
Greiner
Bio-‐One).
Cells
were
allowed
to
attach
overnight,
then
inserts
were
flipped

and
HBEC
cells
were
plated
on
other
side
of
insert
at
1.3
×
10
5

cells/mL
in
1mL.
Human

cells
took
approximately
10-‐14
days
to
reach
resistance.
Treatments
with
POH
at
different

concentrations
were
then
started
and
resistance
was
measured
with
an
epithelial
ohm

meter
(MERSSTX01
Electrode
from
Millipore).
A
blank
well
was
measured
with
medium

alone
and
no
cells.
This
blank
was
subtracted
from
all
measurements
and
final

measurements
were
multiplied
by
1.1
to
account
for
area
of
the
insert,
giving
final

ohmscm
2
.
Figure
2.2
shows
the
set
up
of
MDCK
cells
and
BBB
cells
to
measure
resistance.

12

2.4
In
vivo
analysis

2.4.1
Evans
blue

Opening
of
BBB
was
visualized
in
vivo
with
IV
injection
of
2%
Evans
blue
(EB)
into

the
tail
vein
of
mice
(Sigma
St
Louis,Missouri).

EB
was
injected
at
4mL/kg.
Immediately

following
IV
injection
was
intracardiac
injection
(40
μL)
of
POH
or
controls.
The

intracardiac
injection
can
be
visualized
in
Figure
2.3.
Mice
were
anesthetized
with
2%

isoflurane
before
the
start
of
any
injections.
Intracardiac
injection
protocol
was
performed

by
Ivetta
Vorobyova
at
the
imaging
center
at
USC.
After
injections
mice
were
left
to
awaken.

Perfusions
were
performed
1-‐2
hours
later.
Mice
were
injected
intraperitoneally
(IP)
with

150uL
of
xylazine
(Sigma,
St
Louis,
Missouri)
and
allowed
to
become
fully
unconscious
with

no
pain
perception,
tested
by
squeezing
the
feet
of
the
mice.
Mice
were
then
perfused

MDCK
cells

Electrode

Brain
endothelial

cells
Astrocytes
and

Pericytes

Figure
2.2
Visualization
of
set-‐up
for
measuring
resistance.
The
left
depicts
set
up

for
MDCK
cells
and
the
right
represents
the
model
set
up
for
the
BBB
cells.
MDCK

cells
are
plated
directly
in
the
inserts.
Astrocytes
and
Pericytes
are
plated
on
the

bottom
of
the
inserts
and
are
left
to
adhere
overnight.
The
brain
endothelial
cells

are
plated
on
the
inside
of
the
inserts.
Electrode

13

according
to
standard
protocol
(Devraj
et
al.
2018).
Brains
and
Kidneys
were
dissected
out

and
imaged.
Brain
and
kidneys
were
also
homogenized
in
1
mL
pure
methanol
using
a
glass

tissue
homogenizer.
After
tissue
was
completely
emulsified
samples
were
centrifuged
at

8,000
g
for
15
min.
Supernatant
was
collected
and
absorbance
was
measure
at
620nm.

2.5
Immunohistochemistry
(IHC)

To
determine
levels
of
claudin-‐5
expression,
HBEC
cells
and
mouse
brain
samples

were
used.
HBEC
cells
were
plated
onto
glass
discs
and
allowed
to
propagate
until

confluent.
Cells
were
treated
with
POH
and
then
fixed
in
1%
formaldehyde
for
10
minutes.

Figure
2.3
Mice
intracardiac
injection.
Top
image
shows
set
up
for
how
mouse
is

staged
and
how
needle
is
set
to
inject.
The
bottom
images
depict
the
sonogram

of
the
mouse
heart.
The
red
circle
indicates
the
needle
that
is
being
pushed
into

the
left
ventricle
of
the
heart.

14

Paraffin
sections
obtained
from
mouse
brains
treated
with
POH
were
stained
with
a

fluorescent
antibody.
Slides
were
initially
dewaxed
in
xylene
2
times,
for
5
minutes
each.

Then
the
slides
were
hydrated
through
decreasing
concentrations
of
ethanol,
beginning

with
two
100%
ethanol
washes
for
three
minutes.
Hydration
continued
through
a
single

wash
in
95%
and
80%
for
one
minute
each,
then
a
final
wash
in
distilled
water
for
5

minutes.
Afterwards,
slides
were
processed
for
antigen
recovery
and
placed
in
a
10
mM,
pH

6.0
sodium
citrate
(Sigma
Aldrich
–
St.
Louis,
Missouri,
USA)
buffer
at
approximately
95-‐
100°C
for
30
minutes.
The
slides
remained
in
the
buffer
but
the
container
was
removed

from
the
hot
plate
and
placed
on
the
bench
to
cool
for
an
additional
20
minutes.
Tissues

were
then
washed
with
isotonic,
phosphate-‐buffered
saline
(PBS)
for
5
minutes.

Afterwards,
tissues
were
washed
three
times
in
PBS
for
5
minutes
each.
Tissue
samples

were
then
blocked
for
an
hour
with
50%
SEA
block
(Thermo
Scientific
-‐
Waltham,

Massachusetts,
USA).

For
immunostaining,
fixed
HBEC
cells
or
mouse
brain
tissues
were
incubated
with

the
primary
antibody
overnight
at
4°C.
Sections
were
stained
with
anti-‐claudin-‐5
(1:200,

Invitrogen
–polyclonal
antibody
from
Thermo
Fisher
Scientific,
catalog
#
34-‐1600).
The

day
after,
samples
were
quickly
rinsed
then
washed
three
times
with
PBS
for
5
minutes

each.
Next,
the
samples
were
incubated
with
the
secondary
antibody
for
an
hour
at
room

temperature.
Secondary
antibodies
used
were
Rhodamine(cat#
sc-‐2091
from
Santa
Cruz

Biotech)
.
Coverslips
were
then
mounted
with
Immunogold+DAPI
(Fisher
Scientific
-‐

Waltham,
Massachusetts,
USA)
and
sealed
with
nail
polish.

15

2.6
Western
blotting

Total
cell
lysates
were
prepared
by
disrupting
cells
with
RIPA
buffer(Thermo

Scientific,
Rockford,
IL)
+protease
inhibitors(Thermo
Scientific,
Rockford,
IL);
protein

concentrations
were
determined
using
the
Pierce
BCA
protein
assay
reagent
(Thermo

Scientific,
Waltham
MA).
Fifty
μg
of
total
cell
lysate
was
added
to
each
lane
of
10%
SDS-‐

PAGE
gels.
Trans-‐blot
(BioRad,
Hercules,
CA)
was
used
for
the
semi-‐dry
transfer.
For
the

detection
of
claudin-‐5,
I
used
a
polyclonal
antibody
(Thermo
Fisher
Scientific,
catalog
#34-‐
1600).
Horseradish
peroxidase-‐antibody
conjugates
(i.e.,
secondary
antibodies)
were

obtained
from
Jackson
ImmunoResearch
Laboratories
Inc
(West
Grove,
PA).
All
antibodies

were
used
according
to
the
suppliers’
recommendations.
For
detection
Prometheus
Pro

Signal
Pico
was
used
(Genesee
Scientific,
El
Cajon,
CA).

16

Chapter
3
-‐
Results

3.1
Homogenized
POH
at
higher
concentrations
does
not
affect
cell
viability
in
MDCK

cells
or
the
three
BBB
cell
lines:
Human
brain
endothelial
cells,
pericytes
and

astrocytes

3.1.1
Purpose
of
Study

The
purpose
of
this
study
was
to
use
MTT
assays
to
determine
the
highest

concentrations
of
POH
that
could
be
used
in
either
MDCK
cells
or
BBB
cells
without

creating
cell
toxicity.
MTT
assays
are
an
efficient
way
to
measure
the
metabolic
activity
of

living
cells
by
indirectly
measuring
cell
viability
based
on
the
amount
of
formazan
blue
that

is
present.
Mitochondria
of
living,
healthy
cells
will
reduce
MTT
dye
to
formazan
blue,
an

insoluble
dye
that
can
be
measured.
When
opening
the
BBB
the
goal
was
to
use

concentrations
of
drug
that
showed
minimal
cell
death.
We
want
to
be
able
to
open
the
BBB

and
then
once
the
drug
is
removed
allow
recovery
of
cells
in
re-‐forming
tight
junctions
and

continuing
with
normal
metabolic
activity.

3.1.2
Effect
of
homogenized
POH
on
cell
viability
compared
to
POH
in
DMSO
or
EtOH/Gly
on

MDCK
cells

The
first
few
MTT
assays
used
Madin-‐Darby
Canine
Kidney
(MDCK)
cells.
These
cells

are
an
expedient
model
system
and
can
mimic
properties
of
the
BBB
and
are
widely
used
to

represent
a
model
for
in
vitro
analysis.
MDCK
cells
create
a
monolayer
after
7
days
of

growth
and
they
exhibit
high
TEER
values.
MDCK
cells
are
of
epithelial
origin
instead
of

endothelial
and
they
are
derived
from
dog
kidney
cells
(Wang
et
al.
2005),
indicating
the

cells
do
exhibit
some
limitations
when
trying
to
study
effects
on
the
BBB.
We
wanted
to

determine
cell
viability
in
MDCK
cells
treated
with
POH
diluted
in
DMSO.
Pure
POH
was

diluted
from
6.3M
to
800mM
in
DMSO.
This
was
then
further
diluted
to
obtain
differing

17

POH
concentrations
that
correlated
with
different
DMSO
percentages,
as
indicated
in

Figure
3.1.

DMSO,
or
dimethyl
sulfoxide,
is
a
clear
odorless
liquid
that
can
be
used
as
a

polar,
aprotic
solvent
that
is
miscible
with
water
and
can
easily
dissolve
many
polar
and

non-‐polar
small
molecules
(Capriotti
2012).
We
can
see
complete
cell
death
starting
at
2

mM
POH
and
0.65%
DMSO
(Figure
3.1).
Tests
were
done
with
DMSO
alone
in
cell
culture

medium
up
to
concentrations
of
1%
and
displayed
no
effect
on
cell
viability
and
are
not

displayed
in
the
figures.
There
was
a
drastic
drop
in
cell
viability
from
1
mM
to
2
mM.
The

percentage
of
DMSO
in
1
mM
and
2
mM
POH
were
0.3%
and
0.65%
respectively.
The

different
cell
densities
tested
show
similar
toxicities.
The
smaller
input
of
5k
cells
show
an

IC50
at
1
mM
while
the
other
two
cell
inputs
show
very
little
cell
death.
We
determined

treating
with
POH
diluted
in
DMSO
displayed
high
cell
toxicity
at
low
concentrations.
These

findings
were
unexpected
and
as
2mM
had
been
used
in
previous
studies,
this
indicated
the

experiment
needed
to
be
repeated.

Figure
3.1
Effect
of
POH
diluted
in
DMSO
on
MDCK
cell
viability.
MTT
assay
with

increasing
concentrations
of
POH
in
DMSO
treated
for
24hrs.
Cell
viability

percentage
was
calculated
using
the
untreated
sample
as
the
divisor.
Three

different
cell
inputs
were
plated
in
a
96
well
plate:
15k,
10k
and
5k.
POH

concentrations
used
were
0.3,
1,
2,
4
and
8
mM
with
corresponding
DMSO

percentages
at
0.1,
0.3,
0.65,
1.3,
and
2.5
%
respectively.

-‐50.000

0.000

50.000

100.000

150.000

0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
8.000

Cell
Viability
(%)

Concentration
of
POH
(mM)

MDCK
cells:
POH
in
DMSO

15k
cells
10k
cells
5k
cells

DMSO
%

0.1

0.3

0.65

1.3

18

The
next
idea
was
to
perform
a
comparison
in
MDCK
cells
to
determine
if
there
was

any
difference
in
using
DMSO
verse
EtOH/Gly
as
a
vehicle.
EtOH/Gly
or
ethanol/glycerol
is

a
solvent
that
can
be
used
for
lipophilic
drugs.
Glycerol
shows
low
toxicity
in
cells
but
was

not
sufficient
by
itself
as
a
solvent
for
POH.
Previous
experiments,
not
displayed,
showed

that
the
best
vehicle
combination
for
a
POH
solvent
was
50%
ethanol
and
50%
glycerol.

Experiments
indicated
that
POH
dissolved
in
both
vehicles
have
a
similar
effect
on

MDCK
cell
viability
revealing
over
50%
cell
death
at
2
mM
(Figure
3.2).
The
different
cell

densities
show
a
similar
curve
in
toxicity
as
concentrations
increase,
indicating
that
the

results
cause
a
constant
decrease
in
viability
regardless
of
cell
volume.

These
results

indicated
that
diluting
POH
in
either
DMSO
or
EtOH/Gly
was
unfavorable
on
cell
viability.

0.000

50.000

100.000

150.000

0
0.5
1
1.5
2
2.5

Cell
Viability
(%)

Concentrations
(mM)

POH
in
DMSO
[mM]

10k
cells
5k
cells
2.5k
cells

0.000

50.000

100.000

150.000

0
0.5
1
1.5
2
2.5
3
3.5
4
4.5

Cell
Viabilty
(%)

Concentrations
(mM)

POH
in
EtOH/Gly
[mM]

Figure
3.2
Comparison
of
POH
in
two
different
vehicles:
DMSO
vs
EtOH/Gly.
MTT

assay
of
MDCK
cells
treated
with
increasing
concentrations
of
POH
for
24hrs.

MDCK
cells
were
plated
at
different
inputs:
10k,
5k,
or
2.5k.
Vehicles
of
DMSO
and

50:50
Ethanol/Glycerol
were
used.
Percent
of
vehicle
was
the
same
for
DMSO
and

EtOH/Gly:
0.15%
0.325%
0.485%
0.65%
respectively
for
0.5,
1,
1.5,
2
mM.
Cell

viability
percentage
was
calculated
using
the
untreated
sample
as
the
divider.

19

After
completing
the
first
few
assays
with
MDCK
cells,
experimental
data
indicated

that
diluting
POH
in
DMSO
had
an
adverse
side
effect
due
to
cell
toxicity.
This
led
us
to

develop
a
new
method
that
would
not
require
the
use
of
a
solvent.
We
came
up
with
the

idea
to
homogenize
POH
in
an
aqueous
solvent,
known
as
homogenized
POH
(homPOH).

This
method
allowed
us
to
increase
drug
concentration
without
the
adverse
effect
of

lowering
cell
viability.

We
then
were
able
to
satisfactorily
dilute
POH
and
not
use
the
previously
mentioned

vehicles
as
solvents.
We
first
homogenized
POH
in
PBS,
then
moved
to
homogenizing
in

DMEM
medium.
Homogenizing
POH
in
PBS
was
hard
to
visualize
if
POH
was
actually
being

mixed
into
PBS.
By
homogenizing
POH
in
medium
we
could
visualize
the
particles
in

solution,
as
the
medium
would
turn
a
lighter
pink
color
to
indicate
a
colloidal
mixture
was

occurring.
HomPOH
concentrations
up
to
6
mM
indicated
very
little
cell
toxicity
in
MDCK

cells
(Figure
3.3).

A
comparison
was
done
between
POH
homogenized
21
days
prior
to
use

(old)
and
POH
that
was
immediately
homogenized
and
used.
Our
results
indicated
that
POH

used
immediately
after
homogenization
or
weeks
after
showed
no
significant
difference
in

activity.
We
can
also
see
that
using
homPOH
resulted
in
lower
cell
toxicity
at
higher

concentrations,
up
to
6
mM.

20

Knowing
that
diluting
POH
in
DMSO
resulted
in
toxicity
at
low
concentrations,
we

wanted
to
test
if
adding
DMSO
to
homogenized
POH
right
before
treatment
showed
similar

effects.
This
was
done
by
first
homogenizing
POH,
followed
by
dilution
to
the
desired
final

concentration,
and
then
mixing
with
DMSO
right
before
treatment.
We
observed
very

similar
response
curves
in
Figure
3.4
as
in
Figure
3.3.
Homogenized
POH
up
to
2
mM
at

lower
cell
densities,
showed
no
significant
change
in
cell
viability
with
a
slight
drop
to
70%

survival
at
6
mM.
At
high
cell
density
there
was
no
decrease
in
cell
viability
up
to
6
mM

(Figure
3.4).
These
results
indicated
that
adding
DMSO
to
our
treatments
were
not
having

an
effect
on
cell
viability,
but
diluting
POH
in
DMSO
did
result
in
cell
toxicity.

0

20

40

60

80

100

120

140

0
Hom
Med
1
2
4
6

Cell
Viability
(%)

Concentration
of
homPOH
(mM)

MDCK
cells:
homogenized
POH

10k
cells
(old)
10k
cells

Figure
3.3
Cell
viability
of
MDCK
cells
treated
with
increasing
concentrations
of

homogenized
POH
for
24hrs.
Cells
were
plated
at
a
density
of
10k.
Old

represents
POH
that
was
homogenized
a
few
weeks
prior.
Cell
viability

percentage
was
calculated
using
the
untreated
sample
as
the
divider.

21

After
testing
different
cell
densities
I
tested
toxicity
of
homPOH
in
confluent
MDCK

cells
at
increasing
drug
concentrations.
When
working
with
a
BBB
model,
cells
will
be
at

maximum
confluency
so
as
to
allow
for
tight
junction
formation
and
polarization
of
the

cells.
The
goal
was
to
mimic
a
BBB
model
for
drug
treatment.
After
the
cells
were
plated

they
were
left
to
grow
for
a
few
days
until
90%
confluence
was
seen.
After
treatment
with

homPOH
we
observed
that
treatment
with
up
to
6
mM
homPOH
showed
little
toxicity
in

MDCK
cells
(Figure
3.5).

The
results
indicated
that
homPOH
can
be
used
at
higher

concentrations
compared
to
POH
diluted
in
DMSO.

0

50

100

0
1
2
3
4
5
6
7

Cell
Viability

(%)

Concentration
of
homPOH
(mM)
+
1%
DMSO

MDCK
cells:
homPOH
plus
1%
DMSO

10k
cells
5k
cells

Figure
3.4
Cell
viability
of
MDCK
cells
treated
with
homogenized
POH
plus
DMSO

added
right
before
treatment.
MTT
assay
was
performed
after
cells
were
treated

with
increasing
concentrations
of
homPOH
for
24hrs.
DMSO
at
1%
was
added

separately
to
homPOH.
Cells
were
plated
at
two
separate
inputs
of
10k
and
5k.

Cell
viability
was
expressed
as
a
percentage
using
the
untreated
sample
as
the

divisor.

22

3.1.3
Effect
of
homogenized
POH
on
cell
viability
on
BBB
cells

A
comparison
was
done
to
determine
the
toxicity
of
homogenized
POH
on
the
three

main
cell
types
of
the
BBB:
HBEC,
pericytes
and
astrocytes.
The
results
showed
that
all

three
cell
lines
displayed
the
same
trends
with
increasing
concentration
of
homogenized

POH.
I
found
all
three
cells
lines
showed
an
IC50
of
approximately
6
mM
of
homogenized

POH
and
complete
cell
death
at
~13
mM
(Figure
3.6).
Student
t
tests
showed
no
statistical

differences
between
each
of
the
individual
BBB
cell
types.
These
results
indicated
that
all
of

the
BBB
cells
reacted
similarly
to
homPOH.
This
also
showed
that
the
BBB
cells
are
more

sensitive
to
homPOH
compared
to
MDCK
cells
as
these
cells
showed
no
cell
death
at
6
mM

homPOH.

Figure
3.5
Affect
of
homogenized
POH
on
cell
viability
in
MDCK
cells
at

increasing
homPOH
concentrations.
MTT
assay
of
confluent
MDCK
cells

treated
for
24
hours.
Cell
viability
percentage
was
calculated
using
the

untreated
sample
as
the
divider.
0.000

20.000

40.000

60.000

80.000

100.000

120.000

0
20
40
60
80
100

Cell
Viability
(%)

Concentration
of
homPOH
(mM)

MDCK
cells:
homPOH

23

The
next
consideration
was
to
investigate
treatments
at
shorter
time
intervals,
using

the
same
concentrations
but
up
to
one
hour
would
be
the
longest
drug
treatment
when

working
with
the
BBB
model
in
inserts.
Results
from
this
study
this
showed
the
same

trends
but
indicated
lower
toxicities
with
higher
concentrstions.
After
a
24
hour
treatment

we
saw
complete
cell
death
at
approximately
13
mM
(Figure
3.6),
and
when
the
treatment

-‐50

0

50

100

150

0
10
20
30
40
50
60

Cell
Viability
%

homPOH
Concentrations
(mM)

BBB
cells:
homPOH

Brain
endothelial
cells

Pericytes

Astrocytes

0

20

40

60

80

100

120

140

0
5
10
15
20

Cell
Viabilty
(%)

homPOH
Concentration
(mM)

Figure
3.6
Effect
of
homogenized
POH
on
cell
viability
in
cells
that
make
up
the
blood

brain
barrier.
All
cells
were
grown
to
confluence
and
treatments
were
done
for
24h.
The

top
graph
shows
homPOH
concentrations
up
to
63
mM,
while
the
bottom
graph
is
a

zoomed
in
version
of
concentration
up
to
20
mM.

24

time
was
reduced
we
saw
that
complete
cell
death
occured
around
20
mM
homPH
for
the

30
and
60
minutes
treatment
times(Figure
3.7).
When
treatment
lasted
for
5
and
15

minutes,
complete
cell
death
was
observed
at
homPOH
concentrations
over
30
mM
(Figure

3.7).
This
graph
was
important
because
I
determined
the
working
concentration
I
will
use

in
TEER
experiments
to
be
3.15
mM.
At
this
concentration
in
any
time
point
there
was
no

indication
of
any
decrease
in
cell
viability.
These
results
indicated
the
best
homPOH

concentrations
for
use
in
follow
up
experiments.

A
comparison
was
also
done
with
mannitol
treatments
for
5,
15
and
60
minutes.

Currently,
mannitol
is
used
in
clinic
to
open
the
BBB.
So
we
wanted
to
have
a
comparison
of

what
is
currently
being
used
to
our
new
drug
homPOH.
Mannitol
showed
very
different

trends
on
cell
viability.
Even
up
to
concentrations
of
20%
cell
viability
did
not
reach
an

IC50
value(Figure
3.7).

We
use
20%
mannitol
as
that
is
what
is
currently
administered
in

the
clinic.
The
molar
concentration
of
20%
mannitol
is
1M.
The
treatment
periods
of
5
and

15
minutes
do
not
show
any
significant
difference
but
a
1
hour
treatment
shows
a

statistical
difference
and
decline
in
cell
viability.
Viability
dropped
to
approximately
80%

after
this
treatment
time.
This
indicated
that
mannitol
has
very
low
toxicity
to
cells
but
was

likely
not
useful
when
we
need
to
open
the
BBB
to
allow
entry
of
large
molecules.

25

3.2
POH
decreases
trans
endothelial
resistance
(TEER)

3.2.1
Purpose
of
Study

After
establishing
that
homPOH
showed
lower
cell
toxicities
at
higher
drug

concentrations,
the
next
step
was
to
determine
if
homogenized
POH
had
an
effect
on

cellular
resistance
at
non-‐toxic
levels.
The
MDCK
cells
were
used
as
an
initial
test
for

barrier
resistance
then
we
switched
to
using
the
more
physiological
BBB
model
with

HBECs,
ACs
and
PCs.

0

50

100

150

0
5
10
15
20

Cell
Viability
(%)

Mannitol
(%)

BBB
cells:
Mannitol

5min

15min

60min

Figure
3.7
Cell
viability
of
HBECs
treated
with
homPOH
or
mannitol
for
different
time

intervals.
Cell
viability
percentage
was
calculated
by
using
the
untreated
sample
as

the
divisor.
Cells
were
treated
at
5,
15,
30
or
60
minutes.
*p-‐value
<
0.05;
**p-‐value
<

0.001.

-‐50

0

50

100

150

0
10
20
30
40
50
60

Cell
Viability
(%)

homPOH
Concentration
(mM)

BBB
cells:
homPOH

5min

15min

30min
60min

ns

ns
**

*

*

26

3.2.2
POH
in
DMSO
lowers
TEER
in
MDCK

The
first
step
was
to
look
at
POH
diluted
in
DMSO
as
a
positive
control
in
MDCK

cells.
Previous
data
showed
that
POH
in
DMSO
lowered
resistance
in
MDCK
cells
(Figure

3.8).

After
10
minutes
of
treatment
there
was
a
drastic
drop
from
1000
to
500
ohmscm
2
.

The
next
drop
was
seen
at
120
minutes,
where
we
see
almost
a
complete
loss
in
resistance.

Using
these
data
I
ran
TEER
using
1
mM
POH
in
DMSO
as
a
positive
control.
I
used
1

mM
because
my
MTTs
revealed
2mM
showed
toxicity
to
MDCK
cells
(Figure
3.2).
The

percent
of
DMSO
used
was
0.3%.
DMSO
alone
showed
no
change
to
resistance
(data
not

shown).

With
1
mM
POH
in
DMSO
treatment,
there
was
a
significant
drop
in
resistance
at

1hr
(Figure
3.9).
Resistance
dropped
from
1500
to
under
200
ohmscm
2

with
1
mM
POH.

The
p-‐values
for
1
mM
were
less
than
0.001
indicating
a
highly
significant
drop
in

Figure
3.8
Effect
of
POH
in
DMSO
on
TEER
for
MDCK
cells.
NEO
100
(POH)
was
used

at
a
concentration
of
2mM.
Time
point
measurements
were
done
at
0,
10,
20,
40,
60,

80,
120
and
180
minutes.
The
resistance
was
measured
in
Ohmcm
2

.

Figure
by
Nagore
Marin
Ramos

27

resistance
from
the
untreated
to
1
hour
after
treatment
and
the
0.5
mM
treatment
had
p-‐
values
less
than
0.05,
also
indicating
a
significant
difference.
After
3
hours
the
drug
was

removed
to
see
if
the
cells
would
recover
and
increase
resistance.
There
was
no
significant

recovery
of
resistance
for
the
0.5
mM
or
the
1
mM
drug
treatments.
This
could
have
been

due
to
allowing
treatment
for
up
to
three
hours.
In
later
tests
we
use
a
shorter
treatment

time.

0

200

400

600

800

1000

1200

1400

1600

1800

MDCK
cells:
POH
in
DMSO

1
mM
POH
in
DMSO

0.5
mM
POH
in

DMSO

**

*

ns

ns

Figure 3.9 Disruption of the BBB in MDCK cells. TEER procedure with resistance
measured before and after treatment with POH in DMSO at two concentrations of 1 mM
and 0.5 mM. Recovery was tested for after 3 hours. *p-value < 0.05; **p-value < 0.001.
Resistance
Ohmscm
2

28

The
next
goal
was
to
use
homPOH
on
MDCK
cells.
When
looking
at
homPOH
in

MDCK
cells
we
saw
no
drop
in
resistance
after
2
hours,
with
concentrations
up
to
4
mM

(Figure
3.10).
There
was
no
significant
difference
between
any
of
the
treatments.
One
issue

with
this
experiment
was
that
the
POH
was
homogenized
in
PBS
instead
of
cell
culture

medium.
We
had
just
started
working
with
the
homogenization
process
and
we
were

testing
different
formulation
approaches.
We
also
assayed
homogenates
passed
through

the
machine
multiple
times
to
determine
the
optimum
number
of
homogenization
cycles.

Results
showed
that
running
the
sample
through
10
times
sufficiently
homogenized
the

POH
and
vehicle;
theses
conditions
will
be
used
later
on.
We
later
discovered
that

homogenizing
in
cell
culture
medium
was
better
as
we
were
able
to
qualitatively
visualize

the
mixture.
Another
hypothesis
was
that
MDCK
cells
did
not
realistically
represent
the

BBB
and
are
not
an
applicable
model.
MDCK
are
epithelial
while
BECs
are
endothelial,

which
could
be
responsible
for
the
observed
results
in
Figure
3.10.

0

500

1000

1500

2000

2500

3000

Before
Treatment

1h
2h

Resistance
Ohmscm
2

After
Treatment

MDCK
cells:
homPOH

1
mM
homPOH

2
mM
homPOH

4
mM
homPOH

Figure
3.10
Disruption
of
resistance
in
MDCK
cells.
TEER
procedure
with
resistance

measured
before
and
after
treatment
with
homogenized
POH
at
1
mM,
2
mM
and
4

mM.
There
was
no
significant
drop
in
resistance.

29

3.2.3
Homogenized
POH
lowers
TEER
in
BBB
cells

The
TEER
experiment
was
repeated
using
the
three
co
cultured
BBB
cell
types.

Homogenized
POH
at
3.2
mM
and
1
mM
POH
in
DMSO
were
used
for
a
comparison.
The

first
significant
drop
in
resistance
values
were
seen
at
45
minutes
for
1
mM
POH
in
DMSO

and
60
minutes
for
3.2
mM
homPOH
(Figure
3.11).
The
p-‐values
for
each
treatment
were

below
0.05
starting
at
these
two
time
points.
The
p-‐values
were
obtained
by
doing
a

Student,
paired
t-‐test
between
the
untreated
and
the
individual
time
points.
The
interesting

result
that
was
not
expected
was
that
at
each
of
the
45
and
60
min
time
points
there
was

not
a
significant
difference
in
values
compared
to
the
control
samples.

The
p-‐values
for

these
tests
were
determined
using
a
Student,
independent
t-‐test.
These
results
end
up

being
inconclusive.
A
big
problem
that
may
have
occurred
during
this
experiment
was
the

cell
inserts
grew
longer
than
the
standard
8
days.
The
barrier
became
leaky
as
evident
from

medium
flowing
slowly
from
top
to
bottom.
This
could
have
indicated
a
problem
with
this

test.

30

The
TEER
experiment
was
repeated
but
also
including
a
recovery
step
after
drug

removal.
We
observed
a
significant
drop
in
resistance
compared
to
the
untreated
cells
at
45

min
for
both
the
1
mM
POH
in
DMSO
and
3.2
mM
homPOH
treatments
(Figure
3.12).
We

saw
that
resistance
in
cells
treated
with
homPOH
started
above
40
ohmscm
2

and
after

treatment
for
one
hour,
the
resistance
dropped
to
below
10
ohmscm
2
.
All
p-‐values
were

calculated
using
Student
t-‐test.
We
also
saw
that
the
treated
samples
were
statistically

different
from
the
untreated
samples
indicating
a
significance
to
the
drop
of
resistance.

Figure
3.11
Disruption
of
resistance
in
BBB
model.
TEER
procedure
with
resistance

measure
before
and
after
treatment
of
POH
in
DMSO
at
1
mM
and
homPOH
at
3.2

mM.
The
3.2
mM
concentration
was
obtained
from
MTT
results
Measurements
were

done
before
treatment
and
in
time
intervals
of
5,
10,
15,
30
45
and
60
min
after

treatment.
There
was
an
untreated
measurement
at
each
time
interval
which
is

depicted.
*p-‐value
<
0.05;
**p-‐value
<
0.001.

0

5

10

15

20

25

30

35

40

45

Before

treatment

5
min
10
min
15
min
30
min
45
min
60
min

After
Treatment

BBB
cells:
HPOH

UT

3.2
mM
POH
Homogenized

1mM
POH
in
DMSO

*

*

ns

ns

Resistance
Ohmscm
2

31

Two
hours
after
drug
removal
we
also
observed
a
significant
difference
in
resistance
and

recovery
of
the
3.2
mM
treatments.
The
1
mM
homPOH
in
DMSO
treatments
did
not

indicate
a
significant
recovery.
This
correlated
with
findings
from
Figure
3.9
that
MDCK

cells
do
not
show
significant
recovery
after
treatment
as
well.
The
BBB
cells
may
have
the

same
trend
indicating
another
reason
why
POH
in
DMSO
may
not
be
the
best
option.
The

last
thing
to
note
is
that
at
all
recovery
stages
the
untreated
and
treated
samples
were
not

significantly
different.
At
the
2
hour
and
19
hour
recoveries
we
can
see
the
untreated

samples
and
treatment
samples
were
all
near
the
same
levels
but
the
1
mM
POH
in
DMSO

never
reached
a
full
recovery.
By
48
hours
the
resistance
had
substantially
dropped
in

treated
and
untreated
samples.
The
thought
for
this
occurring
is
that
these
inserts
were

taken
out
multiple
times
to
measure
resistance
and
an
electrode
was
inserted
into
the
well

at
each
time
point.
The
physical
harshness
of
this
on
the
cells
could
be
physically
creating
a

break
in
the
barrier
over
time.
These
results
still
indicated
that
with
3
mM
homPOH
there

was
a
significant
drop
in
resistance
with
treatment
and
recovery
of
resistance
after

treatment.
The
1
mM
POH
in
DMSO
showed
a
significant
drop
in
resistance
but
not
a

significant
recovery.

32

3.3
Homogenized
POH
opens
the
BBB
in
mice

3.3.1
Purpose
of
Study

The
purpose
was
to
use
a
mouse
model
to
show
penetrance
of
the
blood
brain

barrier
with
homogenized
POH.
This
was
done
so
as
to
determine
if
a
larger
molecule,
like

Evans
blue
albumin,
was
able
to
cross
the
BBB
into
the
brain.

3.3.2
Homogenized
POH
allows
EB
into
brain

Mice
were
first
injected
with
Evans
blue
(EB)
followed
by
injection
of
intracardiac

POH
in
different
vehicles.
Intracardiac
injection
needed
to
be
done
so
that
POH
reached
the

Figure
3.12
Disruption
of
resistance
in
BBB
cells.
TEER
procedure
with
resistance

measure
before
and
after
treatment
of
homogenized
POH.
Recovery
of
cells
was

measured
after
removal
of
drug.
Cells
were
treated
and
measurements
were
done
at

5,
15,
30,
45,
and
60minute
intervals.
There
was
an
untreated
measurement
at
each

time
interval
which
is
depicted
.

*p-‐value
<
0.05;
**p-‐value
<
0.001.

0

10

20

30

40

50

60

After
treatment

BBB
cells:
HPOH

UT
3.2
mM
homPOH
1mM
POH
in
DMSO

*
*
*
ns
ns
ns
ns
ns
*
*

Resistance
Ohmscm
2

33

brain
without
being
metabolized
by
the
liver.
After
treatments
and
perfusions
we
saw
that

with
homPOH
treatment,
there
was
presence
of
EB
in
the
brain
compared
to
the
controls

(Figure
3.13).

Blocks
A,
B
and
C
represent
three
separate
negative
controls.
Column
A

represents
a
normal
untreated
mouse,
column
B
shows
a
negative
control
with

homogenized
medium
and
EB
and
column
C
shows
only
2%
EB.
In
each
of
the
negative

controls
we
see
no
presence
of
EB
in
the
brain.
Column
D
and
E
represent
homPOH
at
two

different
concentrations
of
1%
and
3%;
we
can
see
presence
of
EB
in
the
brain
at
both
of

these
concentrations.

Column
F
represents
1%
POH
in
DMSO
and
column
G
represents
1%

POH
in
saline.
Both
columns
F
and
G
also
confirm
that
EB
was
in
the
brain.
The
presence
of

EB
indicated
that
there
was
a
break
in
the
BBB
to
allow
EB
to
cross.
The
POH
in
saline
was

a
technique
used
as
an
alternative
to
injecting
mice
with
DMSO
as
to
avoid
the
use
of
DMSO

if
possible.
The
issue
with
POH
in
PBS
is
that
the
two
are
not
miscible.
This
creates
a
bolus

of
drug
that
was
injected
in
the
mouse,
which
could
have
an
effect
on
the
brain
and
other

organs.
The
use
of
homPOH
showed
a
positive
result
and
is
a
good
indicator
that
this
future

technique
may
be
used.

34

Figure
3.13
Effect
of
POH
opening
the
BBB
in
different
vehicles
with
presence
of
Evans
blue.

All
mice
were
perfused
before
dissection.
The
kidneys
shown
are
positive
controls
for
each

respective
mouse
to
show
presence
of
Evans
Blue.
Column
A,
B
and
C
are
negative
controls.

POH
in
saline
was
used
as
a
positive
control
as
previous
experimental
results
have
shown

POH
in
saline.

1%

homPOH
+

2%
EB
3%
homPOH

+
2%
EB
1%
POH
in

DMSO

+
2%
EB
1%
POH
in

saline

+
2%
EB
D E F G
Normal

Brain
And
Kidney

Homogenized

medium
only
+

2%
EB
2%
EB

only
A B C

35

3.3.3
Homogenized
NEO
100
increases
levels
of
EB
in
the
brain

Another
in
vivo
experiment
looked
at
the
amount
of
EB
that
was
able
to
get
into
the

brain
quantitated
by
tissue
homogenization
and
measuring
the
absorbance
of
EB
in
the

positive
control
brains
compared
to
the
negative
controls.
I
compared
absorbance
values
to

a
standard
curve
of
EB
dilutions
to
calculate
the
actual
micrograms
of
EB
in
each
tissue.
The

final
microgram
amounts
were
calculated
by
dividing
the
amount
in
the
brain
by
the

amount
in
the
kidney,
to
use
the
kidney
as
the
reference
control.
Results
indicated
that

mice
treated
with
1%
homPOH
had
more
EB
in
the
brain
compared
to
mouse
with
no

treatment
(Figure
3.14).
There
was
a
significant
difference
in
μg
amounts
of
EB
in
the
brain

compared
to
that
in
the
untreated.
There
was
also
no
significant
difference
in
amount
of
EB

between
medium
only
treatment
and
untreated.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

No
treatment
2%
EB
only
1%
homPOH
homogenized

medium
only

Ratio
of
ug
EB
in
brain/

EB
in

kidney

Amount
of
EB
in
the
Brain

Figure
3.14:
Amount
of
Evans
blue
in
the
brain
of
a
mouse
after
treatments.
There
were

three
negative
control
of
homogenized
medium
only,
2%
EB
only
and
untreated
mouse.

Treatment
was
1%
homPOH.
Values
were
calculated
by
dividing
μg
EB
levels
in
the
brain

by
μg
levels
in
the
kidney.
*p-‐value
<
0.05;
**p-‐value
<
0.001.

*
ns μg

36

3.4
Homogenized
POH
shows
a
change
in
claudin-‐5
expression

3.4.1
Purpose
of
Study

The
purpose
of
this
study
was
to
determine
if
claudin-‐5
expression,
a
tight
junction

marker,
was
affected
by
addition
of
homPOH.
The
idea
was
to
determine
a
possible

molecular
mechanism
for
the
breakdown
of
the
BBB.

3.4.2
Treatment
with
Homogenized
POH
shows
a
change
in
claudin-‐5
expression
in
HBEC
and

in
mouse
brain
tissue
samples

The
first
step
was
to
measure
claudin-‐5
expression
in
HBEC
cells
in
vitro.
Results
for

this
indicated
that
expression
of
claudin-‐5
in
homPOH
treated
cells
had
decreased
(Figure

3.15).
Compared
to
the
untreated
samples,
it
appeared
that
claudin-‐5
expression
in
both

1mM
POH
in
DMSO
and
1mM
homPOH
had
decreased.
Diamidino
phenylindole
(DAPI)
was

used
for
nuclear
staining
as
it
binds
to
DNA.
When
comparing
DAPI
nuclear
staining
to

claudin-‐5
as
a
ratio,
the
expression
of
claudin-‐5
appears
to
decrease
with
treatment.
One

problem
with
this
experiment
is
the
lower
numbers
of
nuclei
in
the
untreated
samples.
This

could
have
been
due
to
the
percent
of
paraformaldehyde
used.
I
used
1%
when
a
4%

solution
would
have
been
a
better
solution,
as
it
would
have
made
the
cells
more

permeable
to
DAPI.

37

The
next
step
was
to
confirm
claudin-‐5
expression
in
HBEC
cells
after
treatment

with
homPOH.
To
confirm
claudin-‐5
expression
I
ran
a
western
blot
using
3.15
mM

homPOH
and
treated
cells
for
different
times.
Claudin
5
expression
began
to
decrease

starting
at
15
minutes
(Figure
3.16).
By
45
and
60
minute
treatment
times
we
saw
a

substantial
decrease
in
expression.
This
coincided
with
data
from
TEER
showing
a

significant
drop
in
resistance
at
45
and
60
minutes
(Figure
3.12).
The
45
minute
and
60

minute
treatment
times
represent
that
there
is
a
significant
break
in
the
BBB
at
these
time

points.

1mM
POH
in

DMSO

Untreated

1mM

homPOH

DAPI Claudin-‐5 Merge
Figure
3.15
Claudin-‐5
expression
in
HBEC
cells
after
treatment
for
1
hour

with
1
mM
POH
in
DMSO
or
1
mM
homPOH.
DAPI
is
used
for
nuclear

staining.
All
images
were
taken
at
60X.

38

Claudin-‐5
Actin
HBEC
cells:
homPOH
at
3.15
mM

0

5

15

45

60

min
Figure
3.16
Expression
of
claudin-‐5
in
Human
BEC
cells
after

treatment
with
homPOH
using
Western
Blot
analysis.

Concentration
of
homPOH
was
the
same
for
each
treatment
at

3.15
mM.
There
were
four
treatments
for
5,
15,
45
and
60

minutes.
Cells
were
collected
at
the
end
of
each
time
point.

Actin
was
used
as
loading
control.

39

Chapter
4
–
Discussion

Current
therapies
for
brain
metastasis
(BM)
are
surgical
resection
and
radiation

therapy
with
some
therapies
that
are
directed
towards
alleviating
symptoms
but
not

targeting
the
cancer
itself
(Dagogo
et
al.
2017).
The
inability
to
successfully
penetrate
the

BBB
is
limiting
treatments
and
leads
to
lower
survival
rates
in
patients
that
end
up
with

BMs.
The
median
survival
rate
for
BMs
is
6
months
and
the
one-‐year
survival
rate
is
8.3%,

dropping
to
1.4%
for
two-‐year
survivals
(Rastogi
2018).

In
this
study
we
aimed
to
find
a
drug
that
could
open
the
BBB
and
have
minimal

cytotoxic
effects.
The
idea
for
using
POH
for
brain
tumors
was
first
introduced
during
the

treatments
of
glioblastomas.
Because
POH
is
seen
to
have
a
pleiotropic
effect,
it
has
been

tested
in
several
different
cancer
types.
During
the
testing
of
glioblastoma
multiformes

(GMB),
results
indicate
that
POH
is
able
to
enter
the
brain
(Cho
et
al.
2012).
This
discovery

has
led
to
further
testing
of
POH
within
the
brain
microenvironment.

The
first
approach
to
using
POH
in
vitro
involved
diluting
it
in
DMSO.
My
data

indicated
that
DMSO
may
be
having
an
adverse
side
effect
on
cells
(Figure
3.1
and
3.2).
At

concentrations
as
low
as
1.5
mM,
we
began
to
see
a
drop
in
cell
viability.
The
goal
was
to

stray
away
from
using
DMSO
as
a
vehicle;
however
further
research
into
why
we
were

seeing
this
adverse
side
effect
was
not
pursued.
After
determining
DMSO
was
not
a

practical
option,
we
tested
a
combination
of
different
vehicles
so
as
to
be
able
to
administer

the
drug
intravenously.
Previous
testing
found
that
POH
had
an
unfavorable
effect
in

patients
when
it
is
taken
orally
as
it
causes
gastrointestinal
side
effects
(Cho
et
al.
2012).

40

After
testing
the
solvent
capability
of
multiple
vehicles
we
came
up
with
the
idea
to

homogenize
the
drug,
which
turned
out
to
be
very
promising
and
could
effectively
be

administered
to
mice.
Previous
studies
by
our
lab
group
indicated
that
injecting
the
drug

into
the
heart,
intracardiac
injection,
gave
better
results
compared
to
an
IV
tail
injection
of

the
drug.
The
intracardiac
injection
is
used
as
a
model
for
intra-‐arterial
injections
(Chen
et

al.
2018).
By
contract,
an
intravenous
tail
injection
led
to
the
drug
being
filtered
through

the
liver
and
diluted
before
reaching
the
brain,
while
injecting
directly
into
the
left

ventricle
of
the
heart
allowed
a
higher
concentration
of
drug
to
reach
the
brain.

Once
we
had
the
homogenized
version
of
the
drug
we
were
able
to
find

concentrations
of
POH
that
exhibited
no
toxicity
to
BBB
model
cells.
Our
assays
indicated

that
we
could
go
up
to
10
mM
of
homPOH
for
1
hour
with
no
adverse
effect
to
the
BBB
cells

(Figure
3.7).
The
next
step
was
to
test
the
homogenized
drug
on
a
BBB
model
to
determine

if
there
was
a
change
to
electrical
current
resistance.
We
arbitrarily
decided
to
use
3.2
mM

drug
concentration.
At
this
concentration
there
was
no
decline
in
cell
viability,
making
it
a

good
candidate.
Our
data
indicated
that
homPOH
at
3.2
mM
was
able
to
disrupt
the
barrier

and
lower
the
electrical
resistance
(Figure
3.11).
This
drop
was
concomitant
with
a
break

in
the
barrier
and
opening
of
the
BBB.
We
also
were
able
to
show
that
the
cells
were
able
to

substantially,
fully
recover,
once
the
drug
was
removed.
These
results
indicated
that

whatever
mechanism
POH
has,
it
can
be
reversed
once
the
drug
was
removed.
The

capability
of
POH
to
be
reversed
is
essential
to
treatments
especially
when
it
comes
to

disrupting
the
BBB.
The
BBB
has
an
important
physiological
function
in
the
brain
and
it
can

not
be
permanently
altered.
These
results
are
important
as
they
indicate
that
there
is
some

41

type
of
mechanism
that
POH
has
that
is
lowering
the
tightness
of
the
cells
comprising
the

brain
barrier,
but
once
the
drug
is
removed
the
tightness
is
able
to
re-‐establish.

In
conjunction
with
in
vitro
testing,
in
vivo
testing
was
an
integral
part
of
the
puzzle.

Results
from
this
testing
indicated
that
the
homPOH
was
effective
in
disrupting
the
BBB.

We
saw
the
presence
of
Evans
blue
albumin
in
the
brain
after
the
perfusion
of
mice

indicating
that
the
BBB
was
altered
by
homPOH
allowing
EB
to
enter
(Figure
3.12).
Seeing

the
entry
of
EB
into
the
brain
is
an
important
aspect
in
determining
if
POH
is
able
to
open

the
BBB
enough
to
allow
entry
of
large
molecules.
Mannitol
treatment
does
not
allow
for

passage
of
large
molecules,
so
POH
being
able
to
facilitate
the
entry
of
EB
can
be
a
big
step

in
finding
a
new
technique
for
opening
the
BBB.

The
last
step
was
trying
to
find
a
molecular
mechanism
for
the
break
down
of
the

BBB.
Claudin-‐5
was
a
good
candidate
to
be
tested,
as
it
is
a
prominent
tight
junction
protein

in
the
brain.
Our
results
indicated
that
there
was
a
decrease
in
claudin-‐5
expression
after

treatment
with
homPOH
(Figure
3.15
and
3.16).
We
saw
through
western
blot
analysis
and

IHC
that
there
is
decreased
presence
of
claudin-‐5
after
treatment
with
homPOH.
With
the

western
blot
analysis
we
saw
that
starting
at
15
minutes
there
began
a
change
in
claudin-‐5

expression.
A
repeat
of
this
experiment
would
be
essential
in
furthering
this
research
as

well
as
additional
information
showing
RNA
expression
of
claudin-‐5.

Our
results
show
promise
in
the
ability
of
POH
opening
the
BBB
but
more
studies

need
to
be
done
to
confirm
that
homPOH
is
the
best
treatment
for
BMs.
Testing
of

antibodies
would
be
a
vital
piece
of
information,
and
in
vitro
and
in
vivo
analysis
could
be

done
to
determine
the
capability
of
antibodies
to
cross
the
BBB.
Antibody
treatment
is
a

current
prevalent
focus
of
cancer
care,
so
it
would
be
important
to
further
this
study.

It

42

would
also
be
a
good
idea
to
test
a
variety
of
different
sized
substances
for
their
ability
to

cross
the
BBB
to
validate
claims
that
POH
is
truly
opening
the
BBB.

Further
investigation
would
also
need
to
go
into
the
mechanism
for
why
POH
is
disrupting

the
BBB.
Finding
the
molecular
mechanism
of
how
POH
is
altering
endothelial
cells
and
all

other
aspects
of
the
BBB
is
important
to
understanding
long
term
consequences
of
opening

the
BBB.
Additional
knowledge
can
lead
to
future
treatments
of
patients
with
BMs
and
a

possibility
of
longer
life
expectancies.

43

References

Amsbaugh,
M.J.,
and
Kim,
C.S.
(2020).
Cancer,
Brain
Metastasis.
In
StatPearls,
(Treasure

Island
(FL):
StatPearls
Publishing),
p.

Anatomy
and
Physiology
of
the
Blood-‐Brain
Barrier.
Semin
Cell
Dev
Biol
38,
2–6.

Bauer,
H.-‐C.,
Krizbai,
I.A.,
Bauer,
H.,
and
Traweger,
A.
(2014).
“You
Shall
Not
Pass”—tight

junctions
of
the
blood
brain
barrier.
Front
Neurosci
8.

Boekhout,
A.H.,
Beijnen,
J.H.,
and
Schellens,
J.H.M.
(2011).
Trastuzumab.
Oncologist
16,

800–810.

Brown,
R.C.,
Egleton,
R.D.,
and
Davis,
T.P.
(2004).
Mannitol
opening
of
the
blood–brain

barrier:
regional
variation
in
the
permeability
of
sucrose,
but
not
86Rb+
or
albumin.

Brain
Research
1014,
221–227.

Cabezas,
R.,
Ávila,
M.,
Gonzalez,
J.,
El-‐Bachá,
R.S.,
Báez,
E.,
García-‐Segura,
L.M.,
Jurado

Coronel,
J.C.,
Capani,
F.,
Cardona-‐Gomez,
G.P.,
and
Barreto,
G.E.
(2014).
Astrocytic

modulation
of
blood
brain
barrier:
perspectives
on
Parkinson’s
disease.
Front
Cell

Neurosci
8.

Coronel,
J.C.,
Capani,
F.,
Cardona-‐Gomez,
G.P.,
and
Barreto,
G.E.
(2014).
Astrocytic

modulation
of
blood
brain
barrier:
perspectives
on
Parkinson’s
disease.
Front
Cell

Neurosci
8.

Capriotti,
K.,
and
Capriotti,
J.A.
(2012).
Dimethyl
Sulfoxide.
J
Clin
Aesthet
Dermatol
5,
24–
26.

Chen,
T.C.,
Cho,
H.-‐Y.,
Wang,
W.,
Nguyen,
J.,
Jhaveri,
N.,
Rosenstein-‐Sisson,
R.,
Hofman,
F.M.,

and
Schönthal,
A.H.
(2015).
A
novel
temozolomide
analog,
NEO212,
with
enhanced

activity
against
MGMT-‐positive
melanoma
in
vitro
and
in
vivo.
Cancer
Lett.
358,

144–151.

Chen,
T.,
Wang,
W.,
Hofman,
F.,
and
Schonthal,
A.
(2018).
CADD-‐56.
INTRA-‐ARTERIAL

NEO100
TRANSIENTLY
OPENS
THE
BLOOD
BRAIN
BARRIER.
Neuro
Oncol
20,

vi283.

Chen,
T.C.,
da
Fonseca,
C.O.,
and
Schönthal,
A.H.
(2018).
Intranasal
Perillyl
Alcohol
for

Glioma
Therapy:
Molecular
Mechanisms
and
Clinical
Development.
Int
J
Mol
Sci
19.

44

Chen,
T.C.,
Fonseca,
C.O.D.,
and
Schönthal,
A.H.
(2015).
Preclinical
development
and
clinical

use
of
perillyl
alcohol
for
chemoprevention
and
cancer
therapy.
Am
J
Cancer
Res
5,

1580–1593.

Cho,
H.-‐Y.,
Wang,
W.,
Jhaveri,
N.,
Torres,
S.,
Tseng,
J.,
Leong,
M.N.,
Lee,
D.J.,
Goldkorn,
A.,
Xu,

T.,
Petasis,
N.A.,
et
al.
(2012).
Perillyl
alcohol
for
the
treatment
of
temozolomide-‐
resistant
gliomas.
Mol.
Cancer
Ther.
11,
2462–2472.

Dagogo-‐Jack,
I.,
Gill,
C.M.,
Cahill,
D.P.,
Santagata,
S.,
and
Brastianos,
P.K.
(2017).
Treatment

of
brain
metastases
in
the
modern
genomic
era.
Pharmacology
&
Therapeutics
170,

64–72.

Daneman,
R.,
and
Prat,
A.
(2015).
The
Blood–Brain
Barrier.
Cold
Spring
Harb
Perspect
Biol

7.
Serlin,
Y.,
Shelef,
I.,
Knyazer,
B.,
and
Friedman,
A.
(2015).

Fernandes,
J.,
Fonseca,
C.,
Teixeira,
A.,
and
Gattass,
C.
(2005).
Perillyl
alcohol
induces

apoptosis
in
human
glioblastoma
multiforme
cells.
Oncology
Reports
13,
943–947.

Neearti,
Prasad
&
Mohammada,
Rokjahabegam
&
Bangarua,
Raju
&
Devde,
Raju
&
R,
Jagat.

(2012).
The
effects
of
verapamil,
curcumin,
and
capsaicin
pretreatments
on
the
BBB

uptake
clearance
of
digoxin
in
rats.
Journal
of
Pharmacy
Research.
5.
2126-‐2133.

Ostrom,
Q.T.,
Wright,
C.H.,
and
Barnholtz-‐Sloan,
J.S.
(2018).
Chapter
2
-‐
Brain
metastases:

epidemiology.
In
Handbook
of
Clinical
Neurology,
D.
Schiff,
and
M.J.
van
den
Bent,

eds.
(Elsevier),
pp.
27–42.

Rapoport,
S.I.
(2000).
Osmotic
opening
of
the
blood-‐brain
barrier:
principles,
mechanism,

and
therapeutic
applications.
Cell.
Mol.
Neurobiol.
20,
217–230.

Rastogi,
K.,
Bhaskar,
S.,
Gupta,
S.,
Jain,
S.,
Singh,
D.,
and
Kumar,
P.
(2018).
Palliation
of
Brain

Metastases:
Analysis
of
Prognostic
Factors
Affecting
Overall
Survival.
Indian
J
Palliat

Care
24,
308–312.

Serlin,
Y.,
Shelef,
I.,
Knyazer,
B.,
and
Friedman,
A.
(2015).
Anatomy
and
Physiology
of
the

Blood-‐Brain
Barrier.
Semin
Cell
Dev
Biol
38,
2–6.

Tenny,
S.,
Patel,
R.,
and
Thorell,
W.
(2020).
Mannitol.
In
StatPearls,
(Treasure
Island
(FL):

StatPearls
Publishing),
p.

Textbook
of
Neuro-‐Oncology
(2005)
(Elsevier).

Venur,
V.A.,
and
Leone,
J.P.
(2016).
Targeted
Therapies
for
Brain
Metastases
from
Breast

Cancer.
Int
J
Mol
Sci
17.

45

Wang,
Q.,
Rager,
J.D.,
Weinstein,
K.,
Kardos,
P.S.,
Dobson,
G.L.,
Li,
J.,
and
Hidalgo,
I.J.
(2005).

Evaluation
of
the
MDR-‐MDCK
cell
line
as
a
permeability
screen
for
the
blood–brain

barrier.
International
Journal
of
Pharmaceutics
288,
349–359.

Abstract (if available)

Abstract Background: Cancer was the second highest cause of death in the United States in 2017 and 10% to 26% of cancers will metastasize to the brain (Amsbaugh and Kim 2020). The blood brain barrier (BBB) acts as a block to treating these metastatic cancers. An estimated 98% of all therapeutics for metastatic and primary brain cancers are not able to cross the BBB. ❧ Methods: We investigated the effect of homogenized perillyl alcohol (homPOH) on opening the BBB both in vitro and in vivo. POH is a naturally occurring monoterpene that can be isolated from several plants. In vitro experiments used MDCK cells and BBB cell lines as models to mimic the BBB. The BBB cells included human brain endothelial cells, astrocytes and pericytes. These cell lines were treated with homPOH and we measured cell viability and changes in trans endothelial electrical resistance (TEER). In vivo experiments measured the effect of POH in allowing a large molecule of Evans blue albumin to cross the BBB. ❧ Results: Homogenized POH displayed low toxicity in both MDCK cells and BBB cells, resulting in a significant drop in TEER after treatment in these cell lines. In vivo results indicated an increase of Evan blue albumin uptake into the brain through qualitative and quantitative analyses. One key player contributing to the leakiness in the BBB appears to be Claudin-5, a tight junction marker. ❧ Conclusions: Homogenized POH displayed promising results in opening the BBB. The decrease in Claudin-5 expression seems to play a major role in opening of the BBB. We propose that POH should be investigated further toward clinical testing in conjunction with other chemotherapeutics to treat metastatic brain cancers.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Corisol's role in breast-to-brain metastasis

PDF

Cytotoxic effect of NEO212, a novel perillyl alcohol-temozolomide conjugate, on canine lymphoma

PDF

Neuronal master regulator SRRM4 in breast cancer cells facilitates CNS-acclimation and colonization leading to brain metastasis

PDF

The role of serotonergic receptor, HTR2B, on myeloid-derived suppressor cells in the brain metastatic environment

PDF

MGMT in emergence of melanoma resistance to temozolomide

PDF

Development of a temozolomide-perillyl alcohol conjugate, NEO212, for the treatment of hematologic malignancies

PDF

Effect of NEO212, a novel perillyl alcohol-temozolomide conjugate, on mycosis fungoides and Sézary syndrome

PDF

Reactivation of the Epstein Barr virus lytic cycle in nasopharyngeal carcinoma by NEO212, a novel temozolomide-perillyl alcohol conjugate

PDF

Radiosensitization of nasopharyngal carcinoma cells by NEO212, a perillyl alcohol-temozolomide conjugate drug

PDF

Analysis of human brain endothelial cells versus human tumor associated brain endothelial cell tight junction and adherens junction expression differences in glioblastoma multiforme

PDF

Role of a novel transmembrane protein, MTTS1 in mitochondrial regulation and tumor suppression

PDF

Novel approaches of mobilizing human iNKT cells for cancer immunotherapies

PDF

Understanding the role of APP and DYRK1A in human brain pericytes

PDF

Medulloblastoma, the GABA shunt, and possible insight on metastatic capabilities

PDF

Targeting glioma cancer stem cells for the treatment of glioblastoma multiforme

PDF

The role of GRP78 in the regulation of apoptosis and prostate cancer progression

PDF

Effect of reductants on sodium chromate-induced cytotoxicity and morphological transformation to C3H/10T1/2 Cl 8 mouse embryo cells