Transcript The novel oxazolidinone Radezolid (RX

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
15-09-2009
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Intracellular vs extracellular activity of antibiotics :
PK – PD in action
metabolism binding
cooperation with
host defenses
influx
physico-chemical
conditions
efflux
accumulation
and
bioavailability
bacterial
responsiveness
what about oxazolidinones ?
Carryn et al., Infect Dis Clin North Am. (2003) 17:615-34
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Linezolid vs. Radezolid
F
O
N
O
O
N
H
N
linezolid
2 protonable functions
O
H
N
F
N
H
N
N
O
radezolid
O
Heteroaryl substituant
N
H
N
O
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Biaryl spacer
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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
H
N
F
O
F
O
O
N
N
H
N
H
N
O
O
linezolid
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N
N
N
O
O
N
H
radezolid
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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
weak dibasic character
CH3
F
N
H3C
H
N
N
H
OH
N
N
CH3
OH
H3C
OH
CH3
O
(H3C)2N
H3C
H3CH2C
O
CH3
O
O
radezolid
azithromycin
H3CO
CH3
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CH3
O
H3C
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O
N
H
N
O
OHO
O
OH
6
Let’s start with
pharmacokinetics !
Yes, but
CELLULAR
PK !
Munich subway,
ECCMID 2007
15-09-2009
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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.
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Macrolide
- dicationic amphiphile
- high accumulation by
diffusion-segregation in
acidic compartments
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General methodology: cellular accumulation
• cells incubated with linezolid or 14C-radezolid
• washed in PBS and collected by low-speed centrifugation
• resuspended in water
scintillation counting
drug
cell prot.
(Lowry)
microbiological assay
(B. subtilis)
accumulation calculated considering a cell volume of 5 µl/mg prot.
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Kinetics of accumulation and efflux
 phagocytic cells
RDZ
uptake
THP-1
release
12.5
100
10.0
75
7.5
t1/2 = 8.7 min (7.0-11.6)
t1/2 = 5.9 min (4.0-11.0)
plateau = 11.3-fold (10.5-12.1)
5.0
50
25
2.5
0.0
residual concentration
(% of accumulated amount)
cellular accumulation (Cc/Ce)
LDZ
THP-1
J774
PMN
0
0
30
60
90
120
300
0
time (min)
10
20
30
40
50
60
time (min)
Accumulation and efflux rapid; RDZ >> LZD
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Comparative accumulation level at equilibrium
Cell lines
phagocytic
cells
non
phagocytic
cells
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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
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Influence of binding to serum proteins
cellular accumulation (Cc/Ce)
 phagocytic cells
*
30
30
25
25
*
20
20
*
15
15
10
10
5
THP-1
J774
0
5
0
0
5
10
15
20
fetal calf serum (%)
accumulation varies depending on serum content
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Influence of binding to serum proteins
*
30
30
25
25
*
20
20
*
15
15
10
10
5
THP-1
J774
0
% drug bound
cellular accumulation (Cc/Ce)
 phagocytic cells
5
0
0
5
10
15
20
fetal calf serum (%)
accumulation varies depending on serum content
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Importance of pH gradients
pH 7.4
pH 7.0
pH 5.4
B
B
B
+
BH
BH+
BH+
De Duve. Biochem Pharmacol. (1974) 23:2495-531
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How to collapse pH gradients ?
monensin
pH 7.4
pH 7.4
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H+
pH 7.4
H+
B
B
B
BH+
BH+
BH+
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How to collapse pH gradients ?
pH 7.4 pH 5.4
pH 7.0
pH 5.4
B
+
BH
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B
B
BH+
BH+
17
cellular accumulation (% of control)
Influence of pH gradients
125
125
control
+ monensin
100
100
75
75
50
50
*
25
RDZ
LZD
AZI
*
*
*
*
*
*
*
*
5.0
5.5
6.0
6.5
25
*
*
*
*
0
0
RDZ
LZD
AZI
7.0
7.5
extracellular pH
antibiotics
accumulation reduced by 60-80 % for both oxazolidinones
when collapsing pH gradients
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Where is the drug ?
ICAAC 2003
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Subcellular localization of radezolid
 J774 mouse macrophages
N-acetyl--glucosaminidase
lacticodehydrogenase
(NAB)
(LDH)
cytochrome C
oxydase
low speed
centrifugation
nuclei /
unbroken cells
soluble
fraction
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lysosome
(CytOx)
mitochondria
extract
high speed
centrifugation
sedimentable
fraction
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Subcellular localization of radezolid
 J774 mouse macrophages
N-acetyl--glucosaminidase
lacticodehydrogenase
(NAB)
(LDH)
cytochrome C
oxydase
low speed
centrifugation
(CytOx)
mitochondria
extract
nuclei /
unbroken cells
high speed
centrifugation
soluble
fraction
15-09-2009
lysosome
sedimentable
fraction
Radezolid
58.2 %
41.8 %
NAB
10.6 %
89.4 %
CytOx
1.7 %
98.3 %
LDH
97.0 %
3.0 %
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dual subcellular localization
• cytosol
• organelles
21
Subcellular localization of radezolid
N-acetyl--glucosaminidase
lacticodehydrogenase
(LDH)
sedimentable
fraction
soluble
fraction
cytochrome C
oxydase
high speed
centrifugation
on sucrose gradient
Q/
30
(NAB)
lysosome
(CytOx)
mitochondria
NAB
CytOx
RDZ
20
dual subcellular localization
• cytosol
• lysosomes
10
0
1.12
1.14
1.16
1.18
1.20
density
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Cellular pharmacodynamics
Does accumulation
translate in activity ?
Madison, Craig’s symposium 2008
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General methodology: intracellular activity
• 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
cell prot.
(Lowry)
plating and CFU counting
activity expressed as change from initial inoculum
Barcia-Macay et al., Antimicrob. Ag. Chemother. (2006) 50:841-51
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Activity on different bacteria
PHAGOSOMES
LYSOSOMES
Staphylococcus aureus
CYTOSOL
~ pH 7.0
ENDOPLASMIC
RETICULUMDERIVED
VESICLES
PHAGOLYSOSOMES
~ pH 5.0 – 5.5
Listeria monocytogenes
Legionella pneumophila
~ pH 6.1
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Activity on different bacteria
Listeria monocytogenes
Legionella pneumophila
Staphylococcus aureus
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 models
but similar potency at equivalent x MIC
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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 models
but similar potency at equivalent x MIC
any role for accumulation ?
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Further analysis for S. aureus
strain
phenotype
SA 238
SA 238L
MIC pH 7.4 (mg/L)
MIC pH 5.5 (mg/L)
linezolid
radezolid
linezolid
radezolid
LZD-S
2
1
4
16
LZD-R
16
2
8
16
• at neutral pH, radezolid is ~equipotent against the LZD-R strain
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Further analysis for S. aureus
strain
phenotype
SA 238
SA 238L
MIC pH 7.4 (mg/L)
MIC pH 5.5 (mg/L)
linezolid
radezolid
linezolid
radezolid
LZD-S
2
1
4
16
LZD-R
16
2
8
16
• at neutral pH, radezolid is ~equipotent against the LZD-R strain
• at acidic pH, radezolid activity is reduced
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Intracellular activity (clinically-relevant comparison)
SA 238
SA 238L
log CFU from time 0
3.0
LNZ
RX-1741
2.5
LNZ
RX-1741
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2
-1
0
1
2
-2
-1
0
1
2
Log extracell. conc. (mg/L)
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
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Radezolid is
• more potent and effective
than LZD
• as potent and effective
against both strains
34
Intracellular activity (equipotent concentrations)
SA 238
SA 238L
log CFU from time 0
3.0
LNZ
RX-1741
2.5
LNZ
RX-1741
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5
-3
-2
-1
0
1
2
3
-3
-2
-1
0
1
2
Log extracell. conc. (x MIC [pH 7.4])
drug
SA 238
SA 238L
Cstatic
Cstatic
linezolid
2.1
> 10
radezolid
1.1
0.5
15-09-2009
• RDZ and LZD ~ equipotent against
the LZD-S strain
• RDZ ~ equipotent against both strains
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Intracellular activity (x MIC pH 5.5)
SA 238
SA 238L
log CFU from time 0
3.0
LNZ
RX-1741
2.5
LNZ
RX-1741
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5
-3
-2
-1
0
1
-3
-2
-1
0
1
Log extracell. conc. (x MIC [pH 5.5])
drug
SA 238
SA 238L
Cstatic
Cstatic
linezolid
1.1
> 10
radezolid
0.1
0.1
15-09-2009
• Radezolid is active at lower
multiples of the MIC assumed
in the infected compartment
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Intracellular activity (pharmacological comparison)
SA 238
SA 238L
log CFU from time 0
3.0
LNZ
RX-1741
2.5
LNZ
RX-1741
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5
-3
-2
-1
0
1
2
-3
-2
-1
0
1
2
Log extracell. conc. (x MIC [pH 5.5] x accumulation)
drug
SA 238
SA 238L
Cstatic
Cstatic
linezolid
1.9
> 10
radezolid
0.7
0.6
15-09-2009
• cellular accumulation of RDZ
compensates for the effect
of acid pH on activity
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Comparison for strains of clinical interest
(MRSA and VISA)
log CFU from time 0
3
2
ATCC 25923
ATCC 25923
ATCC 33591
ATCC 33591
SA 040
SA 040
SA 238
SA 238
NRS 384
NRS 384
NRS 18
NRS 18
3
2
1
1
0
0
-1
-1
Radezolid
Linezolid
-3
-2
-1
0
1
2
3 -3
-2
-1
0
1
2
3
extracellular concentration (mg/L)
Cstatic : 2.75 mg/L
~ 1.4 x MIC
Cstatic : 0.37 mg/L
~ 0.7 x MIC
difference in potency maintained
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Conclusions
 oxazolidinones ~ macrolides regarding mechanism of
accumulation/distribution
 accumulation level depends on basic character
 dual distribution cytosol/lysosomes for radezolid
ISAP – post ICAAC 2007 & 2008
 oxazolidinones show intracellular activity in all
subcellular compartments examined
 lower MICs translate in improved potency
15-09-2009
 accumulation may conpensate for defeating effect
of intracellular milieu on activity
 what is the cellular bioavailability of antibiotics ?
 how do antibiotics express their activity inside the cells ?
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Conclusions
ISAP – post ICAAC 2007 & 2008
Still room for thinking …
15-09-2009
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Architectural cruse,
Chicago, ICAAC 2007
15-09-2009
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