Intracellular vs extracellular activity of antibiotics : PK – PD in action
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15-09-2009 ISAP 1 Cellular PK/PD of oxazolidinones: Cellular PK/PD of oxazolidinones: studies with radezolid (RX-1741; RDZ) and studies with radezolid (RX-1741; RDZ) and linezolid (LZD) linezolid (LZD) Sandrine Lemaire, Paul M. Tulkens, and Françoise Van Bambeke Unité de Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain
description
Cellular PK/PD of oxazolidinones: studies with radezolid (RX-1741; RDZ) and linezolid (LZD) Sandrine Lemaire, Paul M. Tulkens, and Françoise Van Bambeke Unité de Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain. - PowerPoint PPT Presentation
Transcript of Intracellular vs extracellular activity of antibiotics : PK – PD in action
15-09-2009 ISAP 1
Cellular PK/PD of oxazolidinones:Cellular PK/PD of oxazolidinones:
studies with radezolid (RX-1741; RDZ) and linezolid (LZD) studies with radezolid (RX-1741; RDZ) and linezolid (LZD)
Sandrine Lemaire, Paul M. Tulkens, and Françoise Van Bambeke
Unité de Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain
15-09-2009 ISAP 2
Intracellular vs extracellular activity of antibiotics :PK – PD in action
bacterialresponsiveness
physico-chemicalconditions
cooperation withhost defenses
accumulation and
bioavailability
metabolism binding
influx
efflux
Carryn et al., Infect Dis Clin North Am. (2003) 17:615-34
what about oxazolidinones ?
15-09-2009 ISAP 3
Heteroaryl substituant
Biaryl spacer
Linezolid vs. Radezolid
O
NO
HN
F
N
O
O
O
NO
HN
F
O
NH
HN
N
N
2 protonable functions
linezolid
radezolid
15-09-2009 ISAP 5
Physico-chemical properties
drug logP(Qikprop)
pKa1 pKa2
Linezolid 0.47 5.0
Radezolid 0.7 6.8 9.4
Azithromycin 2.98 8.1 8.6
Similar lipophilicity
O
NO
HN
F
N
O
O
O
NO
HN
F
O
NH
HN
N
N
linezolid radezolid
15-09-2009 ISAP 6
Physico-chemical properties
drug logP(Qikprop)
pKa1 pKa2
Linezolid 0.47 5.0
Radezolid 0.7 6.8 9.4
Azithromycin 2.98 8.1 8.6weak dibasic character
O
NO
HN
F
O
NH
HN
N
N
O
H3C
OH
OH
OH
CH3
H3CH2C O
O
CH3
CH3
H3C
O
O
OHO
(H3C)2N
CH3
OH
N
CH3
H3C
H3C
H3COCH3
radezolid azithromycin
15-09-2009 ISAP 7
Let’s start with pharmacokinetics !
Munich subway, ECCMID 2007
Yes, but CELLULARPK !
15-09-2009 ISAP 8
Aim of the study
to investigate the cellular pharmacokinetics of radezolid
to decipher the mechanisms of its cellular accumulation
in comparison with linezolid and azithromycin
Oxazolidinone - low accumulation - cellular conc. ~ extracell. conc.
Macrolide - dicationic amphiphile - high accumulation by
diffusion-segregation in acidic compartments
15-09-2009 ISAP 9
General methodology: cellular accumulation
cell prot. (Lowry)
drugmicrobiological assay (B. subtilis)
scintillation counting
accumulation calculated considering a cell volume of 5 µl/mg prot.
• cells incubated with linezolid or 14C-radezolid
• washed in PBS and collected by low-speed centrifugation
• resuspended in water
15-09-2009 ISAP 10
Kinetics of accumulation and efflux
phagocytic cells
0 30 60 90 1200.0
2.5
5.0
7.5
10.0
12.5
THP-1
PMNJ774
RDZLDZ
THP-1uptake
300
time (min)
ce
llula
r a
cc
um
ula
tio
n (
Cc
/Ce
)
0 10 20 30 40 50 60
release
0
25
50
75
100
time (min)
res
idu
al c
on
ce
ntra
tion
(% o
f ac
cu
mu
late
d a
mo
un
t)
t1/2 = 5.9 min (4.0-11.0) t1/2 = 8.7 min (7.0-11.6)plateau = 11.3-fold (10.5-12.1)
Accumulation and efflux rapid; RDZ >> LZD
15-09-2009 ISAP 11
Comparative accumulation level at equilibrium
phagocytic cells
Cell lines cellular accumulation
Human THP-1 macrophages 11.0 ± 0.4
J774 mouse macrophages 10.4 ± 0.5
Human PMNs 12.5 ± 0.7
Rat fibroblasts (primary culture) 10.9 ± 2.1
Human skin keratinocytes (primary cultures) 16.1 ± 2.0
Normal human osteoblasts (NHost) 9.7 ± 0.4
Human Umbilical Vein endothelial cells (HUVEC) 10.5 ± 0.1
Human lung epithelial cells (Calu-3) 8.3 ± 1.0
non phagocytic cells
15-09-2009 ISAP 12
Influence of binding to serum proteins
accumulation varies depending on serum content
phagocytic cells
0 5 10 15 200
5
10
15
20
25
30
THP-1J774
*
*
0
5
10
15
20
25
30
*
fetal calf serum (%)
ce
llula
r a
cc
um
ula
tio
n (
Cc
/Ce
)
15-09-2009 ISAP 13
Influence of binding to serum proteins
accumulation varies depending on serum content
phagocytic cells
0 5 10 15 200
5
10
15
20
25
30
THP-1J774
*
*
0
5
10
15
20
25
30
*
fetal calf serum (%)
ce
llula
r a
cc
um
ula
tio
n (
Cc
/Ce
)%
dru
g b
ou
nd
15-09-2009 ISAP 15
Importance of pH gradients
B
BH+
BB
BH+BH+
pH 7.4pH 7.0
pH 5.4
De Duve. Biochem Pharmacol. (1974) 23:2495-531
15-09-2009 ISAP 16
How to collapse pH gradients ?
B
BH+
BB
BH+BH+
pH 7.4pH 7.4
pH 7.4 H+
monensin
H+
15-09-2009 ISAP 17
How to collapse pH gradients ?
B
BH+
BB
BH+BH+
pH 7.4 pH 5.4pH 7.0
pH 5.4
15-09-2009 ISAP 18
Influence of pH gradients
accumulation reduced by 60-80 % for both oxazolidinoneswhen collapsing pH gradients
RDZ LZD AZI0
25
50
75
100
125 control+ monensin
**
*
antibiotics
ce
llula
r a
cc
um
ula
tio
n (
% o
f c
on
tro
l)
5.0 5.5 6.0 6.5 7.0 7.50
25
50
75
100
125 RDZ
AZILZD
**
*
**
*
*
*
*
*
extracellular pH
15-09-2009 ISAP 19
Where is the drug ?
ICAAC 2003
15-09-2009 ISAP 20
Subcellular localization of radezolid
solublefraction
extractnuclei / unbroken cells
low speed centrifugation
high speed centrifugation
sedimentablefraction
J774 mouse macrophages
lysosome
mitochondria
cytochrome C oxydase
(CytOx)
N-acetyl--glucosaminidase(NAB)lacticodehydrogenase
(LDH)
lysosome
mitochondria
cytochrome C oxydase
(CytOx)
N-acetyl--glucosaminidase(NAB)lacticodehydrogenase
(LDH)
15-09-2009 ISAP 21
Subcellular localization of radezolid
solublefraction
extractnuclei / unbroken cells
low speed centrifugation
high speed centrifugation
sedimentablefraction
Radezolid 58.2 % 41.8 %
NAB 10.6 % 89.4 %
CytOx 1.7 % 98.3 %
LDH 97.0 % 3.0 %
dual subcellular localization• cytosol• organelles
J774 mouse macrophages
lysosome
mitochondria
cytochrome C oxydase
(CytOx)
N-acetyl--glucosaminidase(NAB)lacticodehydrogenase
(LDH)
lysosome
mitochondria
cytochrome C oxydase
(CytOx)
N-acetyl--glucosaminidase(NAB)lacticodehydrogenase
(LDH)
15-09-2009 ISAP 22
Subcellular localization of radezolid
solublefraction
high speed centrifugation
on sucrose gradient
sedimentablefraction
dual subcellular localization• cytosol• lysosomes
1.12 1.14 1.16 1.18 1.200
10
20
30
NABCytOxRDZ
density
Q
/
lysosome
mitochondria
cytochrome C oxydase
(CytOx)
N-acetyl--glucosaminidase(NAB)lacticodehydrogenase
(LDH)
lysosome
mitochondria
cytochrome C oxydase
(CytOx)
N-acetyl--glucosaminidase(NAB)lacticodehydrogenase
(LDH)
15-09-2009 ISAP 26
Cellular pharmacodynamics
Does accumulation translate in activity ?
Madison, Craig’s symposium 2008
15-09-2009 ISAP 27
General methodology: intracellular activity
cell prot. (Lowry) plating and CFU counting
activity expressed as change from initial inoculum
• cells infected by pre-opsonized bacteria
• extracellular bacteria eliminated by short incubation with gentamicin
• incubation with increasing concentrations of antibiotics for 24/48 h
• washings
• resuspended in water
Barcia-Macay et al., Antimicrob. Ag. Chemother. (2006) 50:841-51
15-09-2009 ISAP 28
Activity on different bacteria
PHAGOSOMES LYSOSOMES
Listeria monocytogenes
CYTOSOL
PHAGOLYSOSOMES
Staphylococcus aureus
~ pH 7.0
~ pH 5.0 – 5.5
Legionella pneumophila
ENDOPLASMIC RETICULUM-DERIVEDVESICLES
~ pH 6.1
15-09-2009 ISAP 29
Activity on different bacteria
Listeria monocytogenes Staphylococcus aureusLegionella pneumophila
radezolid 10-fold more potent that linezolid in the 3 models
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Comparison of static concentrations
organism linezolid radezolid linezolid/radezolid
mg/L xMIC mg/L xMIC mg/L xMIC
L. monocytogenes 4 4 0.5 17 8 0.25
L. pneumophila 4 1 0.25 2 16 0.5
S. aureus 4 2 1 4 4 0.5
radezolid 10-fold more potent that linezolid in the 3 modelsbut similar potency at equivalent x MIC
15-09-2009 ISAP 31
Comparison of static concentrations
organism linezolid radezolid linezolid/radezolid
mg/L xMIC mg/L xMIC mg/L xMIC
L. monocytogenes 4 4 0.5 17 8 0.25
L. pneumophila 4 1 0.25 2 16 0.5
S. aureus 4 2 1 4 4 0.5
radezolid 10-fold more potent that linezolid in the 3 modelsbut similar potency at equivalent x MIC
any role for accumulation ?
15-09-2009 ISAP 32
Further analysis for S. aureus
strain phenotypeMIC pH 7.4 (mg/L) MIC pH 5.5 (mg/L)
linezolid radezolid linezolid radezolid
SA 238 LZD-S 2 1 4 16
SA 238L LZD-R 16 2 8 16
• at neutral pH, radezolid is ~equipotent against the LZD-R strain
15-09-2009 ISAP 33
Further analysis for S. aureus
strain phenotypeMIC pH 7.4 (mg/L) MIC pH 5.5 (mg/L)
linezolid radezolid linezolid radezolid
SA 238 LZD-S 2 1 4 16
SA 238L LZD-R 16 2 8 16
• at neutral pH, radezolid is ~equipotent against the LZD-R strain• at acidic pH, radezolid activity is reduced
15-09-2009 ISAP 34
Intracellular activity (clinically-relevant comparison)
drug SA 238 SA 238L
Cstatic Emax Cstatic Emax
linezolid 5.8 - 0.3 > 100 0.2
radezolid 0.5 - 0.6 0.9 -0.8
Radezolid is • more potent and effective than LZD• as potent and effective against both strains
SA 238
-2 -1 0 1 2-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
LNZRX-1741
lo
g C
FU
fro
m t
ime
0SA 238L
-2 -1 0 1 2
LNZRX-1741
Log extracell. conc. (mg/L)
15-09-2009 ISAP 35
Intracellular activity (equipotent concentrations)
drug SA 238 SA 238L
Cstatic Cstatic
linezolid 2.1 > 10
radezolid 1.1 0.5
• RDZ and LZD ~ equipotent against the LZD-S strain• RDZ ~ equipotent against both strains
SA 238
-3 -2 -1 0 1 2 3-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
LNZRX-1741
lo
g C
FU
fro
m t
ime
0SA 238L
-3 -2 -1 0 1 2
LNZRX-1741
Log extracell. conc. (x MIC [pH 7.4])
15-09-2009 ISAP 36
Intracellular activity (x MIC pH 5.5)SA 238
-3 -2 -1 0 1-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
LNZRX-1741
lo
g C
FU
fro
m t
ime
0SA 238L
-3 -2 -1 0 1
LNZRX-1741
Log extracell. conc. (x MIC [pH 5.5])
drug SA 238 SA 238L
Cstatic Cstatic
linezolid 1.1 > 10
radezolid 0.1 0.1
• Radezolid is active at lower multiples of the MIC assumed in the infected compartment
15-09-2009 ISAP 37
Intracellular activity (pharmacological comparison)
drug SA 238 SA 238L
Cstatic Cstatic
linezolid 1.9 > 10
radezolid 0.7 0.6
• cellular accumulation of RDZ compensates for the effect of acid pH on activity
SA 238
-3 -2 -1 0 1 2-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
LNZRX-1741
lo
g C
FU
fro
m t
ime
0SA 238L
-3 -2 -1 0 1 2
LNZRX-1741
Log extracell. conc. (x MIC [pH 5.5] x accumulation)
15-09-2009 ISAP 38
Comparison for strains of clinical interest (MRSA and VISA)
-3 -2 -1 0 1 2 3
-1
0
1
2
3ATCC 25923
ATCC 33591
NRS 384
NRS 18
SA 040
SA 238
Linezolid
-3 -2 -1 0 1 2 3
ATCC 25923
ATCC 33591
NRS 384
NRS 18
SA 040
SA 238
Radezolid-1
0
1
2
3
extracellular concentration (mg/L)
lo
g C
FU
fro
m t
ime
0
Cstatic : 2.75 mg/L ~ 1.4 x MIC
Cstatic : 0.37 mg/L ~ 0.7 x MIC
difference in potency maintained
15-09-2009 ISAP 39
Conclusions
oxazolidinones ~ macrolides regarding mechanism of accumulation/distribution accumulation level depends on basic character dual distribution cytosol/lysosomes for radezolid
ISA
P –
po
st I
CA
AC
20
07
& 2
00
8 accumulation may conpensate for defeating effect of intracellular milieu on activity
oxazolidinones show intracellular activity in all subcellular compartments examined lower MICs translate in improved potency
what is the cellular bioavailability of antibiotics ? how do antibiotics express their activity inside the cells ?
15-09-2009 ISAP 40
ConclusionsIS
AP
– p
ost
IC
AA
C 2
00
7 &
20
08
Still room for thinking …
15-09-2009 ISAP 41
Architectural cruse, Chicago, ICAAC 2007
