ANTIVIRAL AGENTS FOR THE PREVENTION AND TREATMENT ON INFLUENZA
Competition between Ciprofloxacin and Antiviral Agents … · 2009-05-19 · and Antiviral Agents...
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16/05/2009 1 ECCMID 2009 O23 - Pharmacokinetics/Pharmacodynamics: Clinical relevance Competition between Ciprofloxacin and Antiviral Agents ( Adefovir, Saquinavir, Ritonavir) for Efflux Transporters in J774 Macrophages L. Garcia*, B. Marquez, S. Carryn, M.P. Mingeot-Leclercq, P.M. Tulkens, F. Van Bambeke Unité de Pharmacologie Cellulaire et Moléculaire Louvain Drug Research Institute Université Catholique de Louvain <www.facm.ucl.ac.be>
Transcript of Competition between Ciprofloxacin and Antiviral Agents … · 2009-05-19 · and Antiviral Agents...
16/05/2009 1ECCMID 2009
O23 - Pharmacokinetics/Pharmacodynamics: Clinical relevance
Competition between Ciprofloxacin and Antiviral Agents (Adefovir, Saquinavir, Ritonavir) for Efflux Transporters in J774 Macrophages
L. Garcia*, B. Marquez, S. Carryn, M.P. Mingeot-Leclercq, P.M. Tulkens, F. Van Bambeke
Unité de Pharmacologie Cellulaire et Moléculaire Louvain Drug Research Institute Université Catholique de Louvain <www.facm.ucl.ac.be>
16/05/2009 2ECCMID 2009
Active efflux is an ubiquitous process which protects cells against foreign substances
Cellular ConcentrationPharmacological Activity
Active Efflux
Linton KJ (2007) Physiology (Bethesda) 22: 122-130
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ATP-Binding Cassette transporters
ABCs transporters
ABCA ABCB ABCC ABCD ABCE ABCF ABCG
P-gp MRPs BCRP
Ciprofloxacin, Adefovir,
Saquinavir, Ritonavir
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Wild-Type Cells
CIP-Resistant Cells
Chronic Exposure to Ciprofloxacin
Michot et al. AAC (2006) 50:1689-1695
CT + Gem0
250
500
750
1000WTRS
cellu
lar c
once
ntra
tion
(ng/
mg
prot
)C
ellu
lar C
once
ntra
tion
(ng/
mg
of p
rote
ins)
Control + Gemfibrozil
Identification of Ciprofloxacin transporter
The CIPRO-resistant cellsoverexpress Mrp2 and Mrp4,
but Mrp4 predominates
Marquez et al. AAC (2009) [Epub ahead of print]
16/05/2009 5ECCMID 2009
Aim of our study
The aim of our work is to examine, in wild-type and Ciprofloxacin-resistant macrophages, the potential competition between Ciprofloxacin and preferential
substrates of Mrp2 and Mrp4 in order to better identify and further
characterize the Fluoroquinolone transporter.
16/05/2009 6ECCMID 2009
Adefovir and MRP4PMEA
9-(2-PhosphonylMethoxyEthyl)Adenine
Reid et al. Mol Pharmacol. 2003 63:1094-103Imaoka et al. Mol Pharmacol. 2007 71:619-27.
Saquinavir and MRP2
Ritonavir and MRP2
N
C
O
O-
O
HN
N
F
CIPROFLOXACIN
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Bis(POM)PMEA
Mono(POM)PMEA
PMEA
9-[2-(BisPivaloyolOxyMethyl)PhosphoMEthoxy]Adenine[Bis(POM)PMEA or Adefovir Dipivoxil]
Hydrolysis of Bis(POM)PMEA
16/05/2009 8ECCMID 2009
Measure of cellular content in CIP
CIP 50 µM
Bis(POM)PMEA300 µM
Wild-Type Cells CIP-Resistant Cells
Competition between CIP and Bis(POM)PMEA
Fluorimetry: λ
excitation = 275 nm; λ
emission = 450 nm
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Accumulation of Ciprofloxacin
The accumulation of CIP is increased in the presence of Gemfibrozil or Bis(POM)PMEA in both cell types
Competition between CIP and Bis(POM)PMEA
0
100
200
300
400
a*
b
cControl+ Bis(POM)PMEA+ GEM
0
100
200
300
400
A
B
CControl+ BIS(POM)PMEA+ GEM
CIP-Resistant CellsWild-Type Cells
Extr
acel
lula
r Con
cent
ratio
n of
CIP
RO
(%
of t
he c
ontr
ol in
WT)
16/05/2009 10ECCMID 2009
Measure of cellular content in PMEA
CIP 500 µM
Bis(POM)PMEA50 µM
HPLC: 4°C, flow rate 1 mL/min, volume injected 25 µL. Stationary phase: column Agilent C8 (4,6x250mm)
Mobile phase A : Acetonitrile Mobile phase B : a mixture of 900 mL of buffer [10 mM KH2 PO4 et 2 mM (But)4 N+ HSO4
-] and 50 mL Acetonitrile.
Wild-Type Cells CIP-Resistant Cells
Competition between Bis(POM)PMEA and CIP
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The accumulation of PMEA is increased in the presence of Gemfibrozil or CIP, especially in resistant cells
Competition between Bis(POM)PMEA and CIP
Accumulation of PMEA
0
50
100
150
200
250
AA A
Control+ CIPRO+ GEM
0
50
100
150
200
250
a*
b
c
Control+ CIPRO+ GEM
CIP-Resistant CellsWild-Type Cells
Extr
acel
lula
r Con
cent
ratio
n of
PM
EA(%
of t
he c
ontr
ol in
WT)
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The accumulation of CIPin CIPRO-resistant cells in the presence of Bis(POM)PMEA
The accumulation of PMEAin CIPRO-resistant cells in the presence of CIP
PMEA is substrate and inhibitor of the CIP transporter
Conclusions
16/05/2009 13ECCMID 2009
Huisman et al. AIDS. 2002 16:2295-301.Williams et al . AAC. 2002 46:3456-62.
Saquinavir and MRP2
N
C
O
O-
O
HN
N
F
CIPROFLOXACIN
Huisman et al. AIDS. 2002 16:2295-301.Williams et al . AAC. 2002 46:3456-62.
Ritonavir and MRP2
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Wild-Type Cells CIP-Resistant Cells
Fluorimetry: λ
excitation = 275 nm; λ
emission = 450 nm
SQV 100 µM
Competition between CIP and SQV
Measure of cellular content in CIP
CIP 50 µM
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Accumulation of Ciprofloxacin
Competition between CIP and SQV
0
100
200
300
400
500
600
A
BBControl+ SQV+ GEM
0
100
200
300
400
500
600
a*
b*b*
Control+ SQV+ GEM
CIP-Resistant CellsWild-Type Cells
Extr
acel
lula
r Con
cent
ratio
n of
CIP
RO
(%
of t
he c
ontr
ol in
WT)
The accumulation of CIP is increased in the presence of Gemfibrozil or SQV in both cell types
16/05/2009 16ECCMID 2009
Measure of cellular content in SQV
SQV 50 µM
HPLC: 25°C, gradient isocratique, flow rate 1 mL/min. Stationary phase : column Agilent C8 (4,6x250mm)
Mobile phase : 45% Acetonitrile / 55% of buffer [3,042 g KH2 PO4 and 0,6 mL Triethylamine /L, adjusted to pH 3,1]
Wild-Type Cells CIP-Resistant Cells
Competition between SQV and CIP
CIP 500 µM
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The accumulation of SQV is not affected by CIP or by Gemfibrozil in the wild-type and resistant cells
Accumulation of Saquinavir
Competition between SQV and CIP
0
50
100
150
200
AAA
Control+ CIPRO+ GEM
CIP-Resistant CellsWild-Type Cells
0
50
100
150
200
a aa
Control+ CIPRO+ GEM
Extr
acel
lula
r Con
cent
ratio
n of
SQ
V(%
of t
he c
ontr
ol in
WT)
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The accumulation of CIPin CIPRO-resistant cells in the presence of SQV
The accumulation of SQV is unchanged in CIPRO-resistant cells or in the presence of CIP
SQV is a potent inhibitor (but NOT substrate) of the CIP transporter
Conclusions
NO effect of RTV on the CIP accumulationNO effect of CIP on the RTV accumulation
RTV is neither an inhibitor nor a substrate of the CIP transporter
16/05/2009 19ECCMID 2009
SQV is a potent inhibitor (but NOT substrate) of the CIP transporter
PMEA is substrate and inhibitor of the CIP transporter
General Conclusions
Do PMEA and SQV act upon the same transporter?
Is there a competition between PMEA and SQV, as inhibitor?
16/05/2009 20ECCMID 2009
Measure of cellular content in PMEA
SQV 100 µM
Bis(POM)PMEA300 µM
HPLC: 4°C, flow rate 1 mL/min, volume injected 25 µL. Stationary phase: column Agilent C8 (4,6x250mm)
Mobile phase A : Acetonitrile Mobile phase B : a mixture of 900 mL of buffer [10 mM KH2 PO4 et 2 mM (But)4 N+ HSO4
-] and 50 mL Acetonitrile.
Wild-Type Cells CIP-Resistant Cells
Competition between Bis(POM)PMEA and SQV
16/05/2009 21ECCMID 2009
Competition between Bis(POM)PMEA and SQVAccumulation of PMEA
The accumulation of PMEA is increased in the presence of SQV, especially in resistant cells
CIP-Resistant CellsWild-Type Cells
0
50
100
150
200
250
a*
b
+ GEM
Control+ SQV c
Extr
acel
lula
r Con
cent
ratio
n of
PM
EA(%
of t
he c
ontr
ol in
WT)
0
50
100
150
200
250
AA
+ GEM
Control+ SQV
A
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This competition leads to a cross-modulation of their cellular concentration
There is a competition between Ciprofloxacin and Antiviral Agents
General Conclusions
This may alter their activity on intracellular bacteria or target viruses
CIP
PMEABis(POM)PMEA
SQV
N
C
O
OH
O
HN
N
F
N
C
O
OH
O
HN
N
F
