Phyllanthin and hypophyllanthin inhibit function of P-gp but not MRP2 in Caco-2 cells


Sukhaphirom, N.; Vardhanabhuti, N.; Chirdchupunseree, H.; Pramyothin, P.; Jianmongkol, S.

Journal of Pharmacy and Pharmacology 65(2): 292-299

2013


The purposes of this study were to investigate the inhibitory effects of two lignans, phyllanthin and hypophyllanthin, on the function of P-glycoprotein (P-gp) and multidrug resistance protein 2 (MRP2), using the in-vitro model of Caco-2 cells. In addition, the effect of prolonged exposure to these two compounds on the expression of active P-gp was also determined. The activity of P-gp and MRP2 was determined in the uptake assays by monitoring the intracellular accumulation of their specific substrates (calcein acetoxymethyl ester and 5-carboxy-2?,7?-dichlorofluorescein diacetate, respectively) with fluorescence spectroscopy.Key findingsHypophyllanthin and phyllanthin inhibited P-gp function with comparable potencies, but neither compound affected MRP2 activity. When the lignans were washed out before addition of substrate, the inhibitory action of both compounds against P-gp function was lost. These results suggested the reversibility of the inhibition. Moreover, prolonged exposure of the Caco-2 cells to both lignans (up to 7 days) had no effect on P-gp function. Phyllanthin and hypophyllanthin directly inhibited P-gp activity and did not interfere with MRP2 activity. It was likely that both phyllanthin and hypophyllanthin could reversibly inhibit P-gp function.

JPP
JOURNAL
OF
Pharmacy
and
Pharmacology
ROYAL
PHARMACEUTICAL
SOCIETY
JPP
Journal
of
Pharmacy
And
Pharmacology
Research
Paper
Phyllanthin
and
hypophyllanthin
inhibit
function
of
P-gp
but
not
MRP2
in
Caco-2
cells
Naphassamon
Sukhaphiroma,
Nontima
Vardhanabhutib,
Hemvala
Chirdchupunsereea,
Pornpen
Pramyothina
and
Suree
Jianmongkola
Departments
of
'Pharmacology
and
Physiology,
b
Pharmaceutics
and
Industrial
Pharmacy,
Faculty
of
Pharmaceutical
Sciences,
Chulalongkorn
University,
Bangkok,
Thailand
Abstract
Objectives
The
purposes
of
this
study
were
to
investigate
the
inhibitory
effects
of
two
lignans,
phyllanthin
and
hypophyllanthin,
on
the
function
of
P-glycoprotein
(P-gp)
and
multidrug
resistance
protein
2
(MRP2),
using
the
in-vitro
model
of
Caco-2
cells.
In
addition,
the
effect
of
prolonged
exposure
to
these
two
com-
pounds
on
the
expression
of
active
P-gp
was
also
determined.
Methods
The
activity
of
P-gp
and
MRP2
was
determined
in
the
uptake
assays
by
monitoring
the
intracellular
accumulation
of
their
specific
substrates
(calcein
acetoxymethyl
ester
and
5(6)-carboxy-2',7'-dichlorofluorescein
diacetate,
respec-
tively)
with
fluorescence
spectroscopy.
Key
findings
Hypophyllanthin
and
phyllanthin
inhibited
P-gp
function
with
comparable
potencies,
but
neither
compound
affected
MRP2
activity.
When
the
lignans
were
washed
out
before
addition
of
substrate,
the
inhibitory
action
of
both
compounds
against
P-gp
function
was
lost.
These
results
suggested
the
reversibil-
ity
of
the
inhibition.
Moreover,
prolonged
exposure
of
the
Caco-2
cells
to
both
lignans
(up
to
7
days)
had
no
effect
on
P-gp
function.
Conclusions
Phyllanthin
and
hypophyllanthin
directly
inhibited
P-gp
activity
and
did
not
interfere
with
MRP2
activity.
It
was
likely
that
both
phyllanthin
and
hypophyllanthin
could
reversibly
inhibit
P-gp function.
Keywords
Caco-2
cells;
hypophyllanthin;
MRP2;
P-glycoprotein;
phyllanthin
Correspondence
Suree
Jianmongkol,
Department
of
Pharmacology
and
Physiology,
Faculty
of
Pharmaceutical
Sciences,
Chulalongkorn
University,
Payathai
Rd.,
Pathumwan,
Bangkok
10330,
Thailand.
E-mail:
sureejmk@yahoo.com
Received
January
28,
2012
Accepted
August
12,
2012
doi:
10.1111/j.2042-7158.2012.01593.x
Introduction
P-glycoprotein
(P-gp)
and
multidrug
resistance
protein
2
(MRP2)
are
major
drug
efflux
pumps
in
the
ATP
binding
cassette
(ABC)
transporters.
These
efflux
proteins
play
a
sig-
nificant
role
in
the
translocation
of
certain
drugs
and
xeno-
biotics
through
restrictive
barriers
such
as
the
intestinal
absorptive
barrier.
[1-31
The
presence
of
P-gp
and
other
efflux
transporters
on
the
apical
membrane
of
enterocytes
can
limit
absorption
and
thus
oral
bioavailability
of
their
sub-
strates.
1451
Inhibition
of
the
activity
of
these
transporters
may
increase
plasma
levels
of
various
drugs
that
are
their
substrates,
leading
to
changes
in
drug
efficacy
and
adverse
reactions.
Phyllanthus
amarus
Schum.
et
Thonn.
(Euphorbiaceae;
synonym,
Phyllanthus
niruri)
has
been
used
as
a
tropical
medicinal
plant
for
various
diseases
including
hepatitis,
jaundice,
kidney
stones,
dyspepsia,
viral
and
bacterial
infec-
tion
and
cancer."'
Two
major
pharmacologically
active
compounds
isolated
from
P.
amarus
are
the
lignans
phyllan-
thin
and
hypophyllanthin
(Figure
1)."'
111
It
has
been
sug-
gested
that
these
two
lignans
might
be
able
to
prevent
hepatotoxicity
caused
by
carbon
tetrachloride,
ethanol
or
galactosamine.m
-141
In
addition
to
the
hepatoprotective
and
antioxidative
actions,
we
recently
reported
that
both
lignans
affected
calcium
movement
across
the
plasma
membrane
as
well
as
intracellular
calcium
release
from
the
sarcoplasmic
reticulumPl
Considering
the
potential
therapeutic
value
of
P.
amarus,
evaluation
of
these
two
bioactive
lignans
for
their
drug
interaction-related
problems
is
needed
to
ensure
their
safe
use.
Drug
interactions
may
involve
interference
with
either
the
metabolizing
enzymes
or
the
efflux
transporter
proteins.
A
number
of
studies
have
demonstrated
the
inhibitory
actions
of
both
phyllanthin
and
hypophyllanthin
on
cyto-
chromes
P450.
[16
'
171
On
the
other
hand,
reports
on
the
2012
The
Authors.
JPP
2012
292
Royal
Pharmaceutical
Society
2013
Journal
of
Pharmacy
and
Pharmacology,
65,
pp.
292-299
Naphassamon
Sukhaphirom
et
al.
P-gp
inhibition
by
lignans
of
P
amarus
H
3
C0
H
3
C0
H
H
H
3
0C
OCH
3
OCH
3
-OCH
3
OCH
3
H
'
OCH
3
OCH
3
OCH
3
OCH
3
Phyllanthin
Figure
1
Chemical
structures
of
phyllanthin
and
hypophyllanthin.
effects
of
these
two
lignans
on
the
function
of
efflux
trans-
porters
are
quite
limited.
Leite
et
al.
reported
that
phyllan-
thin
and
other
lignans
purified
from
P.
amarus
were
able
to
increase
the
accumulation
of
rhodamine
in
vincristine-
resistant
Lucena-1
cell
line,
suggesting
their
inhibitory
action
on
P-gp
activitel
However,
there
has
been
no
report
regarding
the
effect
of
hypophyllanthin,
another
major
lignan
of
P.
amarus,
on
P-gp
function.
Long-term
uses
of
medicinal
plants,
in
particular
P.
amarus,
have
been
anticipated
due
to
their
hepatopro-
tective
outcome.
Though
phyllanthin
is
known
to
interfere
with
the
function
of
P-gp,
such
an
effect
has
not
been
con-
firmed
for
hypophyllanthin.
In
addition,
prolonged
expo-
sure
to
several
natural
substances,
such
as
capsaicin
and
piperine,
can
possibly
affect
P-gp
function."'"I
No
research
has
been
focused
on
investigating
the
modulating
effects
of
these
two
lignans
on
P-gp
activity
after
pro-
longed
exposure.
The
substrates
and
modulators
of
P-gp
and
MRP2
efflux
pumps
are
diverse
with
a
broad
spectrum
of
chemical
struc-
tures.
[451
It
has
been
reported
that
certain
substrates
of
P-gp,
including
vinblastine,
[21
I
ritonavir
[22
I
and
tetramethylpyra-
zine,
[231
can
be
transported
out
of
the
cells
by
MRP2.
Any
compound
that
can
interact
with
more
than
one
type
of
transporter
might
be
able
to
escalate
drug
interaction
prob-
lems.
[241
Hence,
it
would
be
beneficial
to
investigate
whether
phyllanthin
or
hypophyllanthin
was
able
to
interfere
with
the
activity
of
MRP2.
This
study
determined
the
modulating
effects
of
phyllan-
thin
and
hypophyllanthin
on
the
activity
of
P-gp
and
MRP2
expressed
in
human
colon
cancer
Caco-2
cells.
We
also
examined
the
reversibility
of
the
inhibitory
effects
of
both
lignans
on
the
transporters.
Moreover,
the
effect
of
pro-
Hypophyllanthin
longed
exposure
of
the
cells
to
the
compounds
on
the
func-
tion
of
P-gp
was
also
evaluated.
Materials
and
Methods
Materials
Phyllanthin
and
hypophyllanthin
were
isolated
from
P.
amarus
using
the
purification
and
identification
processes
previously
described."'
12141
Briefly,
the
air-dried
aerial
parts
of
the
plant
were
pulverized
and
mixed
with
slaked
lime
and
10%
sodium
carbonate
solution.
The
mixture
was
allowed
to
dry
overnight.
The
dried
mass
was
then
extracted
with
hexane
in
a
Soxhlet
apparatus.
The
solvent
was
evapo-
rated
under
reduced
pressure
at
40°C
to
give
a
semi-solid
residue.
The
residue
was
separated
by
column
chromatogra-
phy
over
silica
gel
using
n-hexane
with
an
increasing
amount
of
ethyl
acetate
as
the
eluent
to
yield
phyllanthin
and
hypophyllanthin.
Both
compounds
were
identified
by
infrared
(IR)
and
high-performance
liquid
chromatography
(HPLC)
analyses.
IR
spectra
were
collected
on
a
Fourier
transform
infrared
spectrometer
(PerkinElmer-Spectrum
One;
PerkinElmer
Inc.,
Waltham,
MA,
USA).
The
HPLC
analysis
was
performed
on
a
HPLC
system
(Shimadzu
LC-20AD
Prominence
series;
Kyoto,
Japan)
equipped
with
a
BDS
Hypersil
column
(4.6
mm
x
250
mm,
5µm)
and
an
ultraviolet
(UV)
detector.
The
mobile
phase
was
acetonitrile—water
(55:45
v/v)
with
a
flow
rate
of
1.4
ml/min
at
30
±
1°C.
The
UV
detector
was
set
at
230
nm.
The
IR
spectra
and
the
HPLC
chromatograms
were
compared
with
those
of
the
standard
references
(ChromaDex
Inc.,
Irvine,
CA,
USA).
The
purity
of
each
lignan
was
at
least
98%.
Both
lignans
were
dissolved
in
dimethyl
sulfoxide
(DMSO)
(99.9%)
and
stored
at
—20°C
until
used.
The
final
2012
The
Authors.
JPP
2012
Royal
Pharmaceutical
Society
2013
Journal
of
Pharmacy
and
Pharmacology,
65,
pp.
292-299
293
(1)
Caco2
cells
(2)
(21
days
after
seeding)
(3)
P-gp
inhibition
by
lignans
of
P
amarus
Naphassamon
Sukhaphirom
et
al.
concentration
of
DMSO
to
which
the
cells
were
exposed
was
less
than
0.5%
(v/v)
in
all
cases.
Calcein
acetoxymethyl
ester
(Calcein-AM)
and
5(6)-
carboxy-2',7'-dichlorofluorescein
diacetate
(CDCFDA)
were
purchased
from
Fluka
(Buchs,
Switzerland).
Other
chemicals,
such
as
verapamil,
indometacin,
penicillin
G
sodium,
Triton
X-100
and
streptomycin
sulfate,
were
pur-
chased
from
Sigma
Chemical
Company
(St
Louis,
MO,
USA).
Dulbecco's
Modified
Eagle's
Medium
(DMEM)
and
L-glutamine
were
obtained
from
Gibco
Life
Technologies
(Grand
Island,
NY,
USA)
and
fetal
bovine
serum
(FBS)
was
obtained
from
Biochrome
AG
(Berlin,
Germany).
Cell
cultures
The
Caco-2
cells
were
obtained
from
American
Type
Culture
Collection
(ATCC,
Rockville,
MD,
USA).
The
cells
were
cultured
in
complete
DMEM
medium
supplemented
with
10%
FBS,
1%
nonessential
amino
acid,
1%
L-glutamine
and
1%
penicillin—streptomycin.
The
cells
were
grown
in
a
humidified
atmosphere
of
5%
CO
2
at
37°C,
and
sub-cultured
using
0.25%
trypsin
in
1
mm
EDTA
solution
every
3-4
days
(at
approximately
70%
confluency).
For
the
experiments,
the
Caco-2
cells
(passage
numbers
50-78)
were
seeded
onto
24-well
plates
at
a
density
of
1.3
x
10
4
cells/cm
2
.
The
cells
were
fed
with
fresh
medium
every
2
days
until
they
were
used
in
the
assay
at
21
days
after
seeding.
Inhibitory
action
of
the
lignans
on
the
transporters
To
determine
the
activity
of
P-gp
and
MRP-2
transporters,
the
accumulation
of
specific
fluorescent
probes
was
applied.
The
inhibitory
effects
of
the
test
compounds
were
deter-
mined
at
the
condition
in
which
the
compounds
were
pre-
incubated
with
the
cells
for
30
min
before
the
addition
of
specific
substrate
(the
standard
pre/co-treatment
condition)
(Figure
2).
In
brief,
after
washing
twice
with
Hanks'
balanced
salts
solution
(HBSS),
the
cells
were
pre-incubated
for
30
min
at
Inhibitor
Substrate
+
Inhibitor
30
min
30
min
>
>
Inhibitor
Substrate
30
min
30
min
Fluorescence
Intensity
Substrate
+
Inhibitor
30
m
i
n
>
>
Figure
2
Time
schedule
of
inhibition
studies
in
the
standard
pre/co-
treatment
condition
(1);
the
pre-treatment
condition
(2);
and
the
co-treatment
condition
(3).
37°C
with
HBSS
in
the
absence
and
presence
of
the
test
compounds.
Then,
a
specific
substrate
of
each
transporter
was
added
to
the
assay
medium
and
the
cells
were
further
incubated
for
another
30
min.
In
this
study,
calcein-AM
(0.4
gm)
and
CDCFDA
(5
gm)
were
selected
to
evaluate
the
function
of
P-gp
and
MRP2,
respectively.
In
the
cells,
nonfluorescent
calcein-AM
and
CDCFDA
were
con-
verted
into
fluorescent
calcein
and
5(6)-carboxy-2',7'-
dichlorofluorescein
(CDCF)
after
cleavage
by
intracellular
esterases.
[25
.
261
At
the
end
of
this
co-incubation
period,
cells
were
washed
with
ice-cold
phosphate-buffered
saline
(PBS)
three
times
and
lysed
with
0.1%
Triton
X-100.
The
fluores-
cence
intensity
of
calcein
or
CDCF
was
determined
on
a
microplate
reader
(Wallac
1420
Perkin-Elmer
Victor
3;
Perkin
Elmer
Inc.)
at
an
excitation
wavelength
and
an
emis-
sion
wavelength
of
485
nm
and
535
nm,
respectively.
The
cellular
proteins
were
spectrophotometrically
quantified
with
Bradford
reagent
at
595
nm.
Reversibility
of
the
inhibitory
action
To
test
the
reversibility
of
the
inhibitory
action
against
the
transporters,
the
Caco-2
monolayers
were
treated
with
the
lignans
in
two
conditions
in
addition
to
the
standard
pre/
co-treatment
described
above:
(1)
the
cell
culture
was
exposed
to
the
test
compound
for
30
min
before
replace-
ment
with
the
P-gp
substrate
(the
pre-treatment
condition);
(2)
the
test
compounds
and
the
P-gp
substrate
were
concomitantly
incubated
(the
co-treatment
condition)
(Figure
2).
Effect
of
prolonged
exposure
on
P-gp
function
The
effect
of
prolonged
exposure
to
the
lignans
on
P-gp
function
was
evaluated.
On
day
14
after
seeding,
the
Caco-2
cell
monolayers
were
treated
with
the
DMEM
complete
medium
containing
either
phyllanthin
or
hypophyllanthin.
The
P-gp
activity
was
determined
with
the
calcein-AM
accumulation
assay
at
2
and
7
days
after
the
treatment
(equivalent
to
day
16
and
day
21
after
seeding).
On
the
day
of
P-gp
function
determination,
the
culture
medium
con-
taining
the
test
lignan
was
replaced
with
fresh
medium
without
the
lignan
for
3
h
before
the
uptake
assay.
In
this
experiment,
the
estimation
of
residual
P-gp
activity
in
the
lignan-treated
Caco-2
monolayers
was
carried
out
using
verapamil
(100
gm)
as
the
P-gp
inhibitor.
The
highest
concentrations
of
both
phyllanthin
and
hypophyllanthin
used
in
each
experiment
were
non-
cytotoxic
to
the
Caco-2
cells,
as
determined
by
trypan
blue
extrusion
assay.
In
addition,
treatment
with
the
test
com-
pounds
had
no
effects
on
cell
morphology
and
cell
attach-
ment
to
the
culture
plates
when
monitored
under
an
optical
microscope.
2012
The
Authors.
JPP
2012
294
Royal
Pharmaceutical
Society
2013
Journal
of
Pharmacy
and
Pharmacology,
65,
pp.
292-299
Naphassamon
Sukhaphirom
et
al.
P-gp
inhibition
by
lignans
of
P
amarus
Statistical
analysis
All
experiments
were
performed
at
least
three
times
on
dif-
ferent
days.
Results
were
expressed
as
mean
±
SEM.
Statisti-
cal
analyses
were
performed
by
the
Kruskal—Wallis
test
followed
by
the
Dunn's
test,
where
appropriate.
The
signifi-
cant
difference
between
two
groups
was
tested
by
the
Mann—Whitney
U-test.
Differences
were
considered
statisti-
cally
significant
at
P
<
0.05.
Results
HPLC
chromatograms
of
the
isolated
phyllanthin
and
hypophyllanthin
showed
a
single
peak
with
the
retention
time
corresponding
to
that
of
the
standards
(Figure
3).
The
Caco-2
monolayers
at
21
days
post
seeding
expressed
an
appreciable
level
of
P-gp
and
MRP2
activity.
As
shown
in
Figure
4a,
both
the
P-gp
inhibitor
verapamil
(100
gm)
and
60
-
(a)
the
MRP2
inhibitor
indometacin
(500
µm)
increased
accu-
mulation
of
calcein
by
approximately
three
fold.
When
the
substrate
was
CDCFDA,
indometacin,
but
not
verapamil,
significantly
increased
the
accumulation
of
CDCF
by
five
fold
(Figure
4b).
As
the
concentrations
of
phyllanthin
and
hypophyllan-
thin
increased
from
1
to
100
pi,
the
amount
of
calcein
accumulated
inside
the
cells
increased
accordingly
(Figure
4a).
The
effects
of
these
two
compounds
at
equimo-
lar
concentration
on
intracellular
calcein
accumulation
were
comparable.
At
the
concentration
of
100
phyllan-
thin
and
hypophyllanthin
inhibited
P-gp
function
by
3.7
and
3.2
fold,
respectively.
On
the
other
hand,
neither
lignan
caused
intracellular
accumulation
of
CDCF,
a
known
sub-
strate
of
MRP2
(Figure
4b).
These
findings
indicated
that
neither
phyllanthin
nor
hypophyllanthin
affected
MRP2
activity.
60
-
(b)
50
-
40
-
m
N
is
30
E
20
CO
0
50
-
N
-
40
-
is
30
E
20
-
10
-
0
-
10
0
5
60
-
(C)
50
-
10
15
20
Time
(min)
25
30
0
5
10
15
20
Time
(min)
25
30
(d)
100
80
-
13
E
a
60-
40
-
20
-
40
-
<
30
-
E
20
-
10
-
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time
(min)
Time
(min)
Figure
3
HPLC
chromatograms
of
standard
phyllanthin
7514/m1(a),
phyllanthin
isolated
from
P.
amarus
5014/m1
(b),
standard
hypophyllanthin
5014/m1(c)
and
hypophyllanthin
isolated
from
P.
amarus
100
µg/m1(d).
2012
The
Authors.
JPP
2012
Royal
Pharmaceutical
Society
2013
Journal
of
Pharmacy
and
Pharmacology,
65,
pp.
292-299
295
30000
(a)
25000
-
20000
15000
10000
-
5000
0
*
T
300000
250000
200000
150000
100000
50000
0
(b)
Ca
lce
in
flu
orescence
(
FU)
/
mg
p
ro
te
in
T
CDCF
flu
orescence
(
FU)
/mg
p
ro
te
in
P-gp
inhibition
by
lignans
of
P
amarus
Naphassamon
Sukhaphirom
et
al.
s
e
4'
isi
s?
\
s? s? s?
se se
\
4
)
o
QI
.4
6
b
o
4z
r
,z
\
,A9
\
if
q
,ZA
,z,A90
(Ns
(Ns
(Nse_INse
CO
N
e
(rjr
\Cr
A6
60
Figure
4
Intracellular
accumulation
of
calcein
(a)
or
CDCF
(b)
in
Caco-2
cells.
The
Caco-2
cells
were
pre-incubated
with
the
test
compounds
(vera-
pamil
(Ver),
indometacin
(Indo);
phyllanthin
(Phyll),
hypophyllanthin
(Hypo))
for
30
min
followed
by
another
30-min
incubation
period
with
either
calcein-AM
(0.4
µm)
or
CDCFDA
(5
µm)
and
the
test
compound.
Calcein
or
CDCF
fluorescence
was
normalized
per
mg
of
protein.
Data
are
means
±
SEM,
n
=
5-6
separated
experiments.
*P<
0.05
vs
control.
Ca
lce
in
flu
orescence
(
FU)
/
mg
p
ro
te
in
(a)
40000
-
(b)
30000
-
20000
-
10000
-
40000
-
30000
-
20000
-
10000
-
0 0
Control
Ver
Phyll
Hypo
Control
Ver
Phyll
Hypo
Figure
5
Intracellular
accumulation
of
calcein
in
the
pre-treatment
condition
(a)
and
in
the
co-treatment
condition
(b).
The
Caco-2
cells
were
treated
with
the
test
compounds
(verapamil
(Ver),
indometacin
(Indo);
phyllanthin
(Phyll),
hypophyllanthin
(Hypo))
at
a
concentration
of
100
µm
under
different
conditions
of
calcein-AM
(0.4
µm)
availability,
as
described
in
Materials
and
Methods.
Data
are
means
±
SEM,
n
=
4
separated
experiments.
*P<
0.05
vs
control.
The
reversibility
of
the
lignan-mediated
inhibition
of
P-gp
activity
was
further
examined
by
comparing
the
inhibitory
effects
in
two
other
experimental
conditions
in
addition
to
the
standard
pre/co-treatment
condition
(Figure
2).
In
the
pre-treatment
condition
in
which
the
lignans
were
present
only
in
the
pre-incubation
period,
neither
phyllanthin
nor
hypophyllanthin
exert
an
inhibitory
action
against
P-gp
activity
(Figure
5a).
The
inhibitory
action
of
the
known
P-gp
competitive
inhibitor
verapamil
was
not
observed
under
this
experimental
condition.
Fur-
thermore,
the
co-existence
of
the
lignan
and
calcein-AM
in
the
co-treatment
condition
appeared
to
increase
the
accu-
mulation
of
intracellular
calcein
with
a
high
variation
(Figure
5b).
Taken
together,
both
lignans
could
elicit
their
maximal
inhibitory
activity
against
P-gp
in
the
pre/co-
treatment
standard
condition.
The
inhibitory
action
was
abolished
after
the
compounds
were
removed
from
the
assay
system
in
the
pre-treatment
condition.
Thus,
both
2012
The
Authors.
JPP
2012
296
Royal
Pharmaceutical
Society
2013
Journal
of
Pharmacy
and
Pharmacology,
65,
pp.
292-299
Naphassamon
Sukhaphirom
et
a/.
P-gp
inhibition
by
lignans
of
P
amarus
Increase
in
ca
lce
in
-
AM
up
ta
ke
(
Fo
ld)
o
Sample
w/o
Ver
Control
(a)
(b)
10
pm
4
50
pm
3
2
w/o
Ver
Baseline
2
days
7
days
w/o
Ver
Baseline
2
days
7
days
Exposure
duration
Figure
6
The
activity
of
P-gp
in
Caco-2
cells
after
long-term
exposure
to
phyllanthin
(a)
and
hypophyllanthin
(b).
The
Caco-2
monolayers
were
cul-
tured
in
DMEM
medium
for
14
days
before
exposure
to
either
phyllanthin
or
hypophyllanthin
for
2
or
7
days
duration.
The
activity
of
P-gp
was
assessed
by
calcein-AM
uptake
assay
on
day
14
(baseline),
day
16
(after
a
2-day
exposure
period)
and
day
21
(after
a
7-day
exposure
period)
follow-
ing
seeding.
The
bar
graphs
represent
the
fold
increase
of
intracellular
calcein
accumulation
in
the
presence
of
verapamil
(Ver,
100
µm)
compared
with
that
in
the
absence
of
verapamil
(open
bar).
Data
are
means
±
SEM,
n
=
3
separate
experiments.
compounds
were
likely
to
interfere
with
P-gp
function
in
a
reversible
fashion.
The
effect
of
duration
of
exposure
to
the
lignans
on
P-gp
function
was
determined
in
the
Caco-2
cells
at
2
and
7
days'
exposure
duration
(Figure
6a
and
6b).
At
14-day
post
seeding,
the
Caco-2
monolayers
exhibited
the
2.61-fold
increase
in
calcein
accumulation
upon
treatment
with
vera-
pamil
(100
p.m),
indicating
the
expression
of
P-gp
activity.
On
days
16
and
21
after
seeding,
calcein
accumulation
levels
were
2.86
and
2.90
fold,
respectively.
Exposure
of
the
cells
to
each
lignan
(at
the
non-cytotoxic
concentrations
of
10
and
50
gm)
either
for
2
days
or
7
days
did
not
result
in
any
changes
in
the
degree
of
calcein
accumulation
(expressed
as
the
fold
increase
in
calcein
fluorescence
in
the
presence
of
verapamil)
relative
to
those
of
the
control
groups.
These
findings
suggested
that
prolonged
exposure
to
phyllanthin
or
hypophyllanthin
up
to
7
days
had
no
appreciable
effect
on
P-gp
activity.
Discussion
Interference
with
the
action
of
efflux
transporters
such
as
P-gp
and
MRP2
can
cause
problems
regarding
drug
efficacy,
toxicity
and
interactions.
In
this
study,
we
examined
the
effects
of
phyllanthin
and
hypophyllanthin
isolated
from
P.
amarus
on
the
function
of
P-gp
and
MRP2.
Based
on
well-established
extraction
and
identification
proc-
esses,
[1
°'
12
'
14]
our
isolated
compounds,
phyllanthin
and
hypophyllanthin,
were
demonstrated
to
be
highly
pure
compounds.
We
demonstrated
the
inhibitory
action
of
phyllanthin
and
hypophyllanthin
against
P-gp
activity,
but
not
MRP2
activity,
in
Caco-2
monolayers.
The
inhibitory
potencies
of
these
two
lignans
were
quite
comparable.
The
effect
of
hypophyllanthin
on
P-gp
function
was
similar
to
that
previously
reported
for
phyllanthinm
and
other
lignans
such
as
macelignan,
[271
nirtetralin,
niranthin
and
phyltetralin.
[181
It
has
been
known
that
calcein-AM
is
a
good
substrate
of
P-gp
whereas
its
hydrolysis
product,
the
fluorescent
calcein,
is
a
substrate
of
MRP2.
[25
.
28291
Thus,
increase
in
accumula-
tion
of
intracellular
calcein
can
be
expected
in
the
presence
of
an
inhibitor
of
either
P-gp
or
MRP2.
As
evident
in
this
study,
both
verapamil
(a
known
P-gp
inhibitor)
and
indometacin
(a
known
MRP2
inhibitor)
increased
the
accu-
mulation
of
calcein
in
the
cells.
Considering
that
both
phyllanthin
and
hypophyllanthin
could
increase
intracellu-
lar
calcein
accumulation
even
in
the
presence
of
the
active
MRP2,
their
inhibitory
effects
on
P-gp
activity
should
be
rather
profound.
It
is
worth
noting
that
the
data
generated
by
our
study
might
underestimate
the
effect
of
phyllanthin
and
hypophyllantin
on
P-gp
function.
This
argument
is
cor-
roborated
by
the
fact
that
both
lignans
could
increase
intracellular
accumulation
of
calcein,
even
in
the
presence
of
the
MRP2
activity.
Some
P-gp
moderators,
including
verapamil,
are
known
to
have
direct
interaction
with
P-gp
at
the
substrate
binding
2012
The
Authors.
JPP
2012
Royal
Pharmaceutical
Society
2013
Journal
of
Pharmacy
and
Pharmacology,
65,
pp.
292-299
297
P-gp
inhibition
by
lignans
of
P
amarus
Naphassamon
Sukhaphirom
et
al.
site.
[
"
-
"
1
The
inhibitory
action
of
the
two
lignans
against
P-gp
activity
might
well
result
from
such
direct
interaction.
We
applied
three
experimental
inhibitory
conditions,
in
which
the
existence
of
the
substrate
and
the
inhibitor
in
the
assay
system
varied,
to
elucidate
the
reversibility
of
the
inhi-
bition.
Both
phyllanthin
and
hypophyllanthin
clearly
elic-
ited
their
inhibitory
action
against
P-gp
in
the
standard
pre/
co-treatment
condition,
in
which
the
cells
were
pre-treated
with
the
test
substance
for
30
min
before
the
co-existence
with
the
substrate.
The
variation
in
the
data
increased
markedly
in
the
co-treatment
condition,
in
which
the
period
of
pre-treatment
with
the
lignans
was
excluded.
It
could
be
anticipated
that
the
differences
in
the
physico-
chemical
properties
of
calcein-AM
and
the
lignans
affected
their
cellular
encounter
and
the
ability
to
compete
at
the
P-gp
binding
sites.
The
pre-treatment
with
the
lignans
might
allow
their
steady
levels
at
the
binding
site
to
compete
readily
with
calcein-AM
during
the
uptake
assay.
This
pre-
treatment
condition
might
be
necessary,
especially
where
the
accessibility
of
the
test
compounds
to
P-gp
was
less
than
that
of
calcein-AM.
Furthermore,
the
inhibitory
action
of
both
lignans
and
verapamil
was
lost
in
the
condition
where
the
inhibitors
were
present
only
in
the
pre-treatment
period.
It
was
possible
that
the
binding
of
the
test
com-
pounds
to
P-gp
was
reversible.
Verapamil
is
a
known
P-gp
inhibitor
and
exerts
its
effect
through
competitive
inhibi-
tion
at
the
substrate
binding
sites.
[
"'
Hence,
due
to
their
similar
inhibitory
pattern
to
that
of
verapamil,
both
phyllanthin
and
hypophyllanthin
might
also
act
as
competi-
tive
inhibitors
of
P-gp
efflux
pump.
We
excluded
the
potential
effect
of
these
two
lignans
on
ATPase
activity.
This
notion
was
based
upon
the
fact
that
P-gp
and
MRP2
are
both
members
of
the
ABC
transporters,
which
need
ATP
hydrolysis
for
their
action.
In
this
study,
it
was
unlikely
that
ATPase
was
the
inhibitory
target
since
the
MRP2
could
still
function
normally
in
the
presence
of
the
test
lignans.
In
the
Caco-2
cells,
the
activity
of
P-gp
could
possibly
be
altered
through
changes
in
the
protein
expression
levels.m
In
addition
to
the
direct
inhibition
of
P-gp
activity,
pro-
longed
exposure
to
certain
compounds,
including
natural
substances,
could
affect
the
level
of
P-gp
function
and
expression.
Examples
of
such
compounds
are
capsaicin,
[191
piperine,
[20]
curcumin,
[341
cyclooxygenase
inhibitors,
[
"
]
astilbin
and
taxifolin.
[361
It
was
reported
that
the
loss
of
P-gp
function
in
the
Caco-2
cells
after
48
and
72
h
exposure
to
indometacin
heptyl
ester
was
due
to
the
reduction
of
P-gp
expression.
[351
In
contrast,
capsaicin
increased
P-gp
activity
through
an
upregulation
of
cellular
P-gp
protein
and
MDR1
mRNA
levels.
[191
In
this
study,
neither
phyllanthin
nor
hypo-
phyllanthin
(at
non-cytotoxic
concentrations)
affected
the
function
of
P-gp
in
the
Caco-2
cells
after
prolonged
expo-
sure
for
up
to
7
days.
It
was
likely
that
the
amount
of
func-
tional
P-gp
in
the
Caco-2
cell
monolayers
was
not
affected
by
these
two
lignans
under
our
experimental
condition.
However,
the
effect
of
the
lignans
on
P-gp
expression
could
not
be
ruled
out
exclusively.
It
was
still
possible
that
the
(over)expressed
protein
(if
any)
did
not
function
properly
as
the
membrane-bound
efflux
pump.
Further
study
on
the
protein
expression
is
needed
if
this
presumption
is
to
be
verified.
Conclusions
This
study
demonstrated
that
phyllanthin
and
hypophyllan-
thin,
the
major
lignans
of
P.
amarus,
could
directly
inhibit
P-gp
activity
without
interfering
with
MRP2
activity.
It
was
likely
that
both
phyllanthin
and
hypophyllanthin
reversibly
bound
to
the
substrate
binding
sites
on
P-gp.
Moreover,
these
two
lignans
had
no
influence
on
P-gp
function
after
prolonged
exposure
for
up
to
7
days.
Declarations
Conflict
of
interest
The
Author(s)
declare(s)
that
they
have
no
conflicts
of
interest
to
disclose.
Funding
This
work
was
supported
by
the
Graduate
Research
Fund
of
Chulalongkorn
University
and
the
Higher
Education
Research
Promotion
and
National
Research
University
Project
of
Thailand
(Project
AS
638A),
Office
of
the
Higher
Education
Commission.
Acknowledgements
We
thank
Chulalongkorn
University
Drugs
and
Health
Products
Innovation
Promotion
Center
for
their
facilities.
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rug
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M
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The
Authors.
JPP
2012
298
Royal
Pharmaceutical
Society
2013
Journal
of
Pharmacy
and
Pharmacology,
65,
pp.
292-299
Naphassamon
Sukhaphirom
et
a/.
P-gp
inhibition
by
lignans
of
P
amarus
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