Vol.
34,
No.
2
ANTIMICROBIAL
AGENTS
AND
CHEMOTHERAPY,
Feb.
1990,
p.
277-280
0066-4804/90/020277-04$02.00/0
Copy igh
C
1990,
Ame ican
Socie y
o
Mic obiology
Up ake
and
In acellula
Ac i i y
o
an
Op ically
Ac i e
O loxacin
Isome
in
Human
Neu ophils
and
Tissue
Cul u e
Cells
ALVARO
PASCUAL,*
ISABEL
GARCIA,
AND
EVELIO
J.
PEREA
Depa men
o
Mic obiology,
School
o
Medicine,
Uni e si y
o
Se ille,
Apdo.
914,
Se ille41080,
Spain
Recei ed
28
June
1989/Accep ed
31
Oc obe
1989
The
pene a ion
o
an
op ically
ac i e
o loxacin
isome
[(-)-o loxacin]
in o
human
neu ophils
and
di e en
issue
cul u e
cells
(HEp-2,
McCoy,
MDCK,
and
Ve o)
was
s udied
and
compa ed
wi h
ha
o
o loxacin
by
a
luo ome ic
assay.
The
cellula - o-ex acellula -concen a ion
a ios
(CIE)
o
(-)-o loxacin
we e
always
highe
han
6,
signi ican ly
g ea e
han
hose
o
o loxacin
a
ex acellula
concen a ions
o
5
and
10
mg/li e .
The
pene a ion
o
(-)-o loxacin
and
o loxacin
was
doubled
when
neu ophils
we e
s imula ed
by
pho bol
my is a e
ace a e
bu
no
a ec ed
a e
inges ion
o
opsonized
S aphylococcus
au eus.
The
CIE
a ios
o
(-)-o loxacin
and
o loxacin
o
di e en
issue
cul u e
epi helial
cells
and
ib oblas s
we e
lowe
han
hose
o
neu ophils
bu
s ill
highe
han
2.
Bo h
compounds
p oduced
a
signi ican
educ ion
in
iable
in aphagocy ic
S.
au eus
du ing
3
h
o
exposu e
o
an imic obial
agen s.
We
conclude
ha
(-)-o loxacin
appea s
o
each
highe
in acellula
concen a ions
han
o loxacin,
emaining
ac i e
inside
he
neu ophils.
O loxacin
(OFLX)
is
a
quinolone
compound
ha
shows
high
ac i i y
agains
a
a ie y
o
g am-posi i e
and
g am-
nega i e
bac e ia
(17).
By
using
high-pe o mance
liquid
ch oma og aphy,
wo
op ically
ac i e
OFLX
isome s
ha e
been
p epa ed:
(+)-OFLX
and
(-)-OFLX
(6).
The
la e
isome
inhibi ed
he
supe coiling
ac i i y
o
gy ase
om
Mic ococcus
lu eus
7.4
imes
mo e
e ec i ely
han
did
he
(+)-isome
(7).
Mo eo e ,
he
in
i o
ac i i y
o
(-)-OFLX
agains
a
a ie y
o
g am-posi i e
and
g am-nega i e
bac e ia
was
8
o
128
imes
highe
han
ha
o
(+)-OFLX
and
wice
as
high
as
ha
o
acemic
OFLX
(6).
The
in acellula
pene a ion
and
ac i i y
o
an imic obial
agen s
can
be
an
impo an
ac o
in
he
ea men
o
in ec ions
caused
by
in acellula
acul a i e
o
obliga e
pa hogens.
Mos
s udies
on
he
abili y
o
an imic obial
agen s
o
pene a e
in o
cells
ha e
u ilized
phagocy ic
cells,
and
only
a
ew
s udies
ha e
e alua ed
he
pene a ion
in o
o he
ypes
o
cells,
such
as
epi helial
cells
(1,
2).
The e
is,
howe e ,
s ong
e idence
ha
some
en e oin asi e
bac e ia
such
as
en e oin asi e
Esche ichia
coli
su i e
in
epi helial
cells
despi e
high
ex e nal
aminoglycoside
concen a ions
because
o
he
low
in acellula
pene a ion
a e
o
hese
an imic obial
agen s
(8).
We
ha e
p e iously
epo ed
ha
OFLX
pene a es
in o
human
polymo phonuclea
leukocy es
(PMNs)
by
an
ac i e
mechanism
and
emains
ac i e
in acellula ly
agains
S aph-
ylococcus
au eus
(13).
Since
he
an imic obial
ac i i y
o
OFLX
seems
o
be
media ed
mainly
by
(-)-OFLX,
i
would
be
impo an
o
s udy
whe he
his
isome
can
be
concen-
a ed
in o
phagocy es
and
o he
euca yo ic
cells.
The
pu pose
o
his
s udy
is
o
e alua e
he
in acellula
pene a ion
o
his
op ically
ac i e
OFLX
isome ,
(-)-
OFLX,
in o
human
PMNs
and
di e en
issue
cul u e
cells
compa ed
wi h
ha
o
OFLX.
The
in acellula
ac i i y
in
PMNs
o
bo h
compounds
agains
S.
au eus
is
also
assessed.
MATERIALS
AND
METHODS
Isola ion
o
PMNs.
PMNs
we e
eco e ed
om
hepa in-
ized
enous
blood
o
heal hy
dono s
and
we e
pu i ied
by
p e iously
desc ibed
me hods
(16).
PMN
p epa a ions
we e
*
Co esponding
au ho .
97%
pu e.
Final
cell
suspensions
we e
adjus ed
o
5
x
106
PMNs
pe
ml
in
Hanks
balanced
sal
solu ion.
PMNs
we e
95%
iable
by
ypan
blue
exclusion.
Tissue
cul u e
cells.
HEp-2,
McCoy,
MDCK,
and
Ve o
cells
(Flow
Labo a o ies,
I ine,
Uni ed
Kingdom)
we e
g own
in
minimal
essen ial
medium
(Flow)
supplemen ed
wi h
1
mM
HEPES
(N-2-hyd oxye hylpipe azine-N'-2-
e hanesul onic
acid;
Flow)
and
con aining
10%
e al
cal
se um
(Flow)
wi hou
an ibio ics.
Fo
each
expe imen
cells
we e
de ached
om
issue
cul u e
bo les
wi h
ypsin-
EDTA
(Flow)
and
suspended
i
Hanks
balanced
sal
solu-
ion
a
a
concen a ion
o
5
x
106
cells
pe
ml.
An imic obial
agen
up ake
by
cells.
A
p e iously
de-
sc ibed
luo ome ic
assay
o
measu e
quinolone
up ake
by
human
PMNs
was
used
(13).
The
op ically
ac i e
o loxacin
isome
(-)-OFLX
was
kindly
supplied
by
Daiichi
Seiyaku
Co.,
L d.
(Tokyo,
Japan),
and
OFLX
was
supplied
by
Hoesch
(Fede al
Republic
o
Ge many).
In
hese
expe i-
men s
PMNs
o
issue
cul u e
cells
we e
incuba ed
in
Hanks
balanced
sal
solu ion
con aining
di e en
concen a ions
o
he
an imic obial
agen
(2,
5,
and
10
mg/li e ).
A e
di e en
incuba ion
imes
(1.5,
20,
and
30
min)
a
37°C,
cells
we e
sepa a ed
om
ex acellula
solu ion
by
cen i uga ion
h ough
a
wa e -impe meable
silicone-oil
ba ie
in
a
mic o-
cen i uge
ube
(9).
The
en i e
cell
pelle ,
ob ained
by
cu ing
o
he
po ion
o
he
mic o uge
ube
con aining
he
pelle ,
was
placed
in
2
ml
o
0.1
M
glycine-HCl
bu e
(pH
3.0)
and
agi a ed
igo ously
in
a
o ex
shake .
Incuba ion
o
2
h
a
oom
empe a u e
was
su icien
o
elease
ully
in acellula
an imic obial
agen .
The
samples
we e
cen i uged
o
5
min
a
5,600
x
g,
and
he
amoun
o
an imic obial
agen
was
de e mined
by
luo escence
emission
o
he
supe na an s
wi h
a
Kon on
SFM
25
luo escence
spec opho ome e
(Kon on,
Swi ze land).
The
luo escence
exci a ion
and
emission
maxima
o
OFLX
and
(-)-OFLX
in
0.1
M
glycine-
HCl
(pH
3.0)
we e
292
and
496
nm,
espec i ely.
Con ols
wi hou
an imic obial
agen s
we e
always
used
o
de e mine
he
backg ound
luo escence.
The
in acellula
wa e
space
was
measu ed
by
using
i ia ed
wa e
and
he
ex acellula
ma ke
[14C]polye hy-
lene
glycol
(1.4
mCi/g;
New
England
Nuclea
Co p.,
Bos on,
Mass.).
The
cells
we e
incuba ed
wi h
hese
adiolabeled
277
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ANTIMICROB.
AGENTS
CHEMOTHER.
compounds
o
2
min
a
37°C,
and
hen
cells
we e
sepa a ed
om
ex acellula
luid
by
eloci y
g adien
cen i uga ion
as
desc ibed
abo e
and
coun ed
in
a
liquid
scin illa ion
coun e .
The
o al
wa e
con en
o
he
cell
pelle
was
co ec ed
o
apped
ex acellula
wa e ,
i.e.,
polye hylene
glycol
space,
o
ob ain
he
in acellula
wa e
space.
F om
he
alues
ob ained
om
his
p ocedu e,
cell-associa ed
an imic obial
agen
concen a ions
we e
calcula ed
and
exp essed
as
a
a io
o
he
cellula
concen a ion
o
ex acellula
concen a-
ion
(CIE)
(9).
In
a
se ies
o
expe imen s,
OFLX
and
(-)-OFLX
(ex a-
cellula
concen a ion,
5
mg/li e )
up ake
by
human
PMNs
was
measu ed
a e
s imula ion
o
he
cells
wi h
200
nM
ei he
pho bol
my is a e
ace a e
(PMA;
Sigma
Chemical
Co.,
S .
Louis,
Mo.)
o
S.
au eus
ATCC
25923
opsonized
in
5%
pooled
human
se um
(15
min,
37°C)
a
a
10/1
a io
o
bac e ia
o
PMNs.
PMA
o
opsonized
bac e ia
we e
added
o
he
PMN
suspensions
a
he
same
ime
as
he
an imic obial
agen s,
and
he
up ake
was
measu ed
as
desc ibed
abo e.
Since
he
measu emen
o
OFLX
and
(-)-OFLX
is
indi ec
and
dependen
upon
luo escence,
con ols
we e
always
pe o med
o
e alua e
he
e ec
o
s imuli
on
he
luo es-
cence
o
he
an imic obial
agen s
in
cell- ee
sys ems.
In acelula
ac i i y
o
an imic obial
agen s.
To
e alua e
he
in acellula
ac i i y
o
an imic obial
agen s,
a
p e iously
desc ibed
me hod
was
used
(15).
B ie ly,
0.1
ml
o
opsonized
bac e ial
suspension
(5
x
107
CFU/ml)
and
0.1
ml
o
PMNs
(5
x
106
PMNs
pe
ml)
we e
combined
in
a
se ies
o
polyp opylene
bio ials
(Beckman
Ins umen s,
Inc.,
Fulle -
on,
Cali .)
and
incuba ed
in
a
shake
(250
pm)
o
60
min
a
370C.
A e
incuba ion,
ex acellula
bac e ia
we e
emo ed
by
di e en ial
cen i uga ion.
Cells
we e
hen
suspended
in
0.2
ml
o
RPMI
medium
(GIBCO,
Uni ed
Kingdom).
A
his
ime
(designa ed
ime
ze o)
he
di e en
an imic obial
agen s
we e
added,
and
he
ials
we e
incuba ed
in
a
shake
(250
pm)
a
370C.
Vials
we e
emo ed
a
ime
ze o
(con ol)
and
a e
3
h
o
incuba ion
(con ol
and
samples
wi h
an imic o-
bial
agen s).
Cells
we e
lysed
in
dis illed
wa e ,
and
samples
we e
dilu ed
and
pou
pla ed
in
aga .
Colonies
we e
coun ed
a e
24
h
o
incuba ion
a
370C.
The
da a
we e
exp essed
as
pe cen ages
o
su i ing
s aphylococci
compa ed
wi h
con-
ols
(wi hou
an imic obial
agen s)
a
ime
ze o.
In
addi ion
10-
9-
8-
7-
0
~
6-
4
lx
5
w
'-%
4-
3-
2-
I-
2
5
10
Ex ocellula
concen a ion
(mg/I)
FIG.
1.
OFLX
(C1)
and
(-)-OFLX
(O)
up ake
by
human
PMNs
a
di e en
ex acellula
concen a ions
(n
=
6).
Incuba ions
we e
done
o
20
min.
Fo
he
da a
om
concen a ions
o
5
and
10
mg/li e ,
P
<
0.05.
10
8'
w
5
J4
3
2
I
5
10
20
TIME
(min)
FIG.
2.
OFLX
(0)
and
(-)-OFLX
(A)
up ake
by
human
PMNs
a
di e en
imes
(n
=
4).
The
ex acellula
concen a ion
was
5
mg/li e .
o
de e mining
bac e ial
su i al,
mo phologic
s udies
we e
also
ou inely
pe o med
a
ime
ze o
and
a e
3
h
o
incuba ion
o
e alua e
he
disposi ion
o
bac e ia
(cell
asso-
cia ed
o
ex acellula ).
Samples
o
50
,ul
we e
emo ed
om
bio ials
and
deposi ed
on
glass
slides.
A e
s aining
wi h
W igh
s ain,
samples
we e
examined
by
ligh
mic oscopy.
All
assays
we e
pe o med
in
duplica e
wi h
PMNs
om
i e
di e en
dono s.
S a is ical
analysis
o
da a.
Da a
we e
exp essed
as
he
means
s anda d
de ia ions.
Di e ences
among
g oups
we e
compa ed
by
analysis
o
a iance
wi h
he
Bon e oni
me hod
(18),
used
o
assess
s a is ical
signi icance
a
P
s
0.05.
RESULTS
Up ake
o
an imic obial
agen s
by
PMNS.
Figu e
1
shows
he
CIE
a ios
o
OFLX
and
(-)-OFLX
incuba ed
wi h
human
PMNs
a
di e en
ex acellula
concen a ions.
Bo h
OFLX
and
(-)-OFLX
showed
high
CIE
a ios,
being
signi -
ican ly
highe
o
he
la e
a
ex acellula
concen a ions
o
S
and
10
mg/li e .
The
pene a ion
o
cell
associa ion
o
OFLX
occu ed
sligh ly
mo e
apidly
han
did
ha
o
(-)-OFLX
(Fig.
2).
The
s imula ion
o
PMNs
by
a
memb ane
ac i a o
(PMA)
enhanced
he
in acellula
pene a ion
o
OFLX
and
(-)-
OFLX
2.4
and
1.5
imes,
espec i ely
(Table
1).
The
incu-
ba ion
o
PMNs
wi h
opsonized
S.
au eus
ATCC
25923,
howe e ,
did
no
modi y
he
up ake
o
ei he
compound.
TABLE
1.
E ec
o
he
incuba ion
o
human
PMNs
wi h
ei he
opsonized
S.
au eus
ATCC
25923
o
PMA
on
he
up ake
o
OFLX
and
(-)-OFLX
by
hese
cells
(n
=
4)1
CIE
a io
S imulus
OFLX
(-)-OFLX
None
(con ol)
6.0
±
1.3
8.3
±
1.7
Opsonized
S.
au eus
5.2
±
1.1
7.0
±
1.6
PMA
(200
nM)
14.4
±
2.3"
13.0
±
2.3"
a
Incuba ions
we e
done
o
20
min.
The
ex acellula
concen a ion
o
OFLX
o
(-)-OFLX
was
5
mg/li e .
b
p
<
0.05
compa ed
wi h
he
con ol.
278
PASCUAL
ET
AL.
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OFLOXACIN
ISOMER
UPTAKE
BY
HUMAN
NEUTROPHILS
TABLE
2.
OFLX
and
(-)-OFLX
up ake
by
human
PMNs
and
di e en
issue
cul u e
cells
(n
=4)
An imic o-
CIE
a io
bial
agen
PMN
HEp-2
McCoy
MDCK
Ve o
OFLX
5.8
±
1.2
2.1
±
0.2 3.9
±
1.1
2.1
±
0.9
3.4
±
1.2
(-)-OFLX
8.2
1.7b
2.8
±
0.6
4.7
±
1.3
1.9
±
0.9
2.4
±
0.6
a
Incuba ions
we e
done
o
20
min.
The
ex acellula
concen a ion
o
OFLX
o
(-)-OFLX
was
5
mg/li e .
b
p
<
0.05
compa ed
wi h
alue
o
OFLX.
Up ake
o
an imic obial
agen s
by
issue
cul u e
cells.
The
CIE
a ios
o
OFLX
and
(-)-OFLX
incuba ed
wi h
h ee
issue
cul u e
epi helial
cells
om
di e en
o igins
(HEp-2,
McCoy,
and
MDCK)
and
one
issue
cul u e
ib oblas
cell
line
(Ve o)
o
20
min
a
37°C
a e
shown
in
Table
2.
The
CIE
a ios
o
OFLX
and
(-)-OFLX
we e
2
o
3
imes
lowe
han
hose
obse ed
wi h
PMNs,
and
he e
we e
no
signi ican
di e ences
be ween
he
wo
compounds.
In acellula
ac i i ies
o
an imic obial
agen s.
The
in ac-
ellula
ac i i ies
o
di e en
concen a ions
o
OFLX
and
(-)-OFLX
agains
S.
au eus
ATCC
25923
we e
e alua ed
in
a
3-h
assay
(Table
3).
A
all
o
he
concen a ions
e alua ed,
(-)-OFLX
p oduced
a
signi ican
educ ion
o
he
su i al
o
S.
au eus
in
PMNs,
simila
o
ha
p oduced
by
OFLX.
DISCUSSION
(-)-OFLX
is
an
op ically
ac i e
OFLX
isome
ha
shows
highe
in
i o
ac i i y
han
(+)-OFLX
and
he
acemic
compound
OFLX
agains
di e en
mic oo ganisms
(6,
7).
The
pu pose
o
his
s udy
was
o
e alua e
whe he
his
inc eased
ac i i y
was
accompanied
by
a
highe
in acellula
pene a ion
and
ac i i y
han
hose
o
OFLX.
The
(-)-
OFLX
up ake
by
human
PMNs
was
signi ican ly
highe
han
ha
o
OFLX
a
ex acellula
concen a ions
o
5
and
10
mg/li e .
This
di e ence
was
no
obse ed
a
a
lowe
con-
cen a ion
(2
mg/li e ).
Ne e heless,
bo h
compounds
eached
high
in acellula
concen a ions
in
hese
phago-
cy es,
as
has
been
desc ibed
o
o he
quinolones
wi h
di e en
me hodology
(3,
10).
The
pene a ion
o
(-)-OFLX
and
OFLX
was
no
a ec ed
when
PMNs
we e
incuba ed
wi h
opsonized
S.
au eus.
This
has
been
desc ibed
o
o he
an imic obial
agen s
such
as
e y h omycin,
i ampin,
and
gen amicin
(5).
Howe e ,
he
TABLE
3.
E ec
o
OFLX
and
(-)-OFLX
on
he
su i al
o
S.
au eus
ATCC
25923
wi hin
human
PMNs
(n
=
5)
An imic obial
agen
and
%
Su i al
o
concn
(mg/li e )
S.
au eus
a
3
h
No
agen
45.5
±
5.2
OFLX
0.5
20.9
±
3.
la
2
17.6
±
3.0O
5
15.0
±
2.7a
10
5.8
±
1.4a
(-)-OFLX
0.5
17.8
±
3.2a
2
15.5
±
3.
la
5
12.2
±
2.6a
10
9.1
±
2.1la
a
P
<
0.05
compa ed
wi h
he
con ol.
pene a ion
o
o he
an imic obial
agen s,
such
as
clindamy-
cin,
has
been
shown
o
inc ease
a e
phagocy osis
o
S.
au eus
(5).
The
s imula ion
o
PMN
memb anes
by
PMA,
howe e ,
doubled
he
up ake
o
bo h
compounds
by
human
PMNs,
poin ing
ou
ha
he
pene a ion
o
(-)-OFLX
in o
PMNs
could
be
media ed
by
an
ac i e
mechanism,
as
has
been
demons a ed
o
o loxacin
(13),
since
PMA
ac i a es
he
NADPH
oxidase
sys em
ia
p o ein
kinase
C
(11).
To
s udy
he
pene a ion
o
hese
an imic obial
agen s
in o
o he
euca yo ic
cells,
we
used
a
issue
cul u e
cell
model.
The
pene a ion
o
bo h
compounds
in o
issue
cul u e
epi helial
cells
and
ib oblas s
was
signi ican ly
lowe
han
ha
ob-
se ed
in
PMNs
bu
s ill
eached
in acellula
concen a ions
ha
we e
a
leas
double
he
ex acellula
concen a ions.
The
use
o
his
model
o e s
an
in e es ing
insigh
in o
he
pene a ion
o
an ibac e ial
subs ances
in o
cells
and
may
ha e
ele ance
o
human
in ec ions
p oduced
by
mic oo -
ganisms
ha .
a e
able
o
su i e
and
e en
mul iply
wi hin
epi helial
cells.
Con as s
be ween
phagocy e
an imic obial
agen
up ake
and
in acellula
ac i i y
ha e
been
desc ibed
o
a
ew
an imic obial
agen s
(5,
15).
In
ou
s udy,
(-)-OFLX
showed
a
good
in acellula
pene a ion
in o
and
ac i i y
in
PMNs
agains
S.
au eus.
The
in acellula
an imic obial
ac i i y
o
his
isome
was
simila
o
ha
obse ed
o
he
acemic
compound
and
simila
o
hose
desc ibed
o
o he
quino-
lones
agains
di e en
bac e ia
(4,12,
14).
In
summa y,
(-)-OFLX,
an
op ically
ac i e
OFLX
iso-
me ,
pene a ed
in o
human
PMN,
eaching
concen a ions
highe
han
- hose
eached
by
OFLX
and
emaining
ac i e
in acellula ly.
This
compound
also
showed
a
high
pene a-
ion
in o
issue
cul u e
epi helial
cells
and
ib oblas s.
The
high
an imic obial
ac i i y
o
his
compound,
highe
e en
han
ha
o
he
acemic
compound
OFLX,
added
o
he
p ope ies
obse ed
in
his
s udy,
could
jus i y
addi ional
s udies
o
e alua e
he
possible
clinical
ele ance o
hese
indings.
ACKNOWLEDGMENTS
We
hank
M.
C.
Guzman
o
echnical
assis ance
and
C.
Acos a
o
yping
he
manusc ip .
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