Transcript Tulkens

PK/PD in an Era of Drug Transporters
Paul M. Tulkens & Françoise Van Bambeke
Unité de pharmacologie cellulaire et moléculaire
Université catholique de Louvain
Brussels, Belgium
http://www.md.ucl.ac.be/facm
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Discovery of drug transporters
• anticancer drugs
• antibiotics
• antifungals
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Anticancer drugs transporters : discovery
•
Decreased retention of actinomycin D as the basis for crossresistance in anthracycline-resistant sublines of P388 leukemia.
Inaba M & Johnson RK Cancer-Res. 1977 Dec; 37(12): 4629-34
•
Active efflux of daunorubicin and adriamycin in sensitive and
resistant sublines of P388 leukemia.
Inaba M, Kobayashi H, Sakurai Y, Johnson RK, Cancer-Res. 1979 Jun;
39(6 Pt 1): 2200-3
There is an active outward transport mechanism for
anthracyclines in P388 leukemia cells and an
enhanced activity of this efflux process renders cells
highly resistant to the cytostatic and cytotoxic effects
of ADR and DAU.
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Anticancer drugs transporters :
inbition by chemically-unrelated
other anticancer drugs
• Alteration of methotrexate uptake in human leukemia cells by
other agents.
Bender RA, Bleyer WA, Frisby SA, Oliverio VT. Cancer-Res. 1975
May; 35: 1305-8
Vincristine sulfate enhanced MTX uptake by inhibiting
MTX efflux, thus increasing the level of intracellular drug
in excess of the tightly bound fraction. The potential
clinical implications of using MTX in commbination with
the aforementioned drugs for cancer chemotherapy are
discussed.
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Anticancer drug efflux inhibited by an antibiotic ...
The semi-synthetic
antibiotic tiamulin, …
overpasses the capacities
of reference products such
as verapamil and
cyclosporin-A in terms of in
vitro reversal effect [of
anthracycline resistance].
In Vitro and In Vivo Reversal of Cancer
Cell Multidrug Resistance by the Semisynthetic Antibiotic Tiamulin. L.G.
Baggetto, M. Dong, J. Bernaud, L.
Espinosa, D. Rigal, R., & E. Marthinet.
Biochem. Pharmacol (1998) 56 12191228.
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Antifungal drugs transporters : discovery
•
A leptomycin B resistance gene of Schizosaccharomyces pombe
encodes a protein similar to the mammalian P-glycoproteins. Nishi-K;
Yoshida-M; Nishimura-M; Nishikawa-M; Nishiyama-M; Horinouchi-S; Beppu-T.
Mol-Microbiol. 1992 Mar; 6(6): 761-9
•
Mechanisms of resistance to azole antifungal agents in Candida albicans
isolates from AIDS patients involve specific multidrug transporters.
Sanglard-D; Kuchler-K; Ischer-F; Pagani-JL; Monod-M; Bille-J. AntimicrobAgents-Chemother. 1995 Nov; 39(11): 2378-8
Fluconazole-resistant C. albicans isolates failed to accumulate 3Hlabelled fluconazole. This phenomenon was reversed in resistant cells
by inhibiting the cellular energy supply with azide, suggesting that
resistance could be mediated by energy-requiring efflux pumps such
as those described as ATP-binding cassette (ABC) multidrug
transporters.
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Antifungal drug efflux
Molecular mechanisms of azole resistance. Drugs enter the cell through an unknown
mechanism (passive diffusion ?). Two types of efflux pumps are expressed at low
levels.
In a "model" resistant cell, the drugs are less effective because … the azoles are
effluxed through overexpression of the CDR (ABCT) and MDR (MF) genes.
Adapted from White, T. C. 1997. Antifungal drug resistance in Candida albicans. ASM News 63:427-433
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Antibiotic transporters in bacteria : discovery
• Plasmid-mediated tetracycline resistance in Escherichia coli
involves increased efflux of the antibiotic. Ball-PR; Shales-SW;
Chopra-I. Biochem-Biophys-Res-Commun. 1980 Mar 13; 93(1): 74-81
• Active efflux of tetracycline encoded by four genetically different
tetracycline resistance determinants in Escherichia coli. McMurryL; Petrucci-RE Jr; Levy-SB. Proc-Natl-Acad-Sci-U-S-A. 1980 Jul;
77(7): 3974-7
The decrease in tretracycline intrabacterial accumulation was attributable
to an active efflux ... …
Active export of tetracycline is a common component of the mechanism
for tetracycline resistance encoded by different plasmid-borne
determinants in bacteria.
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Why drug transporters ?
• because cells like to build up specific
resistance mechanism ?
• because cells have been exposed to drugs
in their history ?
• because drugs have special properties ?
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There is much more than drugs ...
mid-90’s :
The genomic analysis of Saccharomyces
cerevisiae and of Pseudomonas
aeruginosa reveals a very large number of
potential “drug” transporters
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Inventory of transporters in
the complete genomes of S. cerevisiae and E. coli
Abundance of proteins
of differing predicted membrane topologies
nb TMS
nb proteins
Yeast
E. coli
% of total
Yeast
E. coli
0
4364
2861
70.8
66.8
soluble
proteins
1
937
655
15.3
15.3
signal
peptides
2-3
4-6
7-9
> 10
390
185
144
121
220
211
153
82
6.5
3.1
2.3
2.0
5.1
4.9
3.6
4.3
potential
transport
proteins
(14 %)
Paulsen et al FEBS Lett (1998) 430:116-125
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There is indeed much more than drugs ...
mid-90’s :
Proteomic and functional analyses reveals
that the potential transporters identified by
genomics are responsible for the efflux of
a very large variety of substances with a
common biophysical property:
amphiphilicity...
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Why amphiphilic compounds ?
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Because they are the only ones that can
easily pass across membrane bilayers ...
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Why should cells extrude amphiphilic
compounds ?
Efflux
Detoxifying
metabolism
toxicity
Concerted barrier against invasion ...
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But why drugs ?
Most if not all of our drugs are
amphiphilic ...
because we
selected or designed them
to be so….
in order to penetrate cells
and tissues ...
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• Oral
bioavailability
• tissue
distribution
• penetration in
difficult-toreach
compartments,
etc...
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Transporters - data bases
http://www-biology.ucsd.edu/~msaier/transport/titlepage.html
main
drug
transporters
Classification page
Transport analysis page
Phylogenetic page
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combination of phylogenetic and functional information
comparison of transporters in complete genomes
phylogenetic trees of transporters families
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Most frequent antibiotic-pumps in procaryotes (1/2)
• Resistance Nodulation Division (Gram - )
TOPOLOGY
MECHANISM
ANTIBIOTICS
tetracyclines
fluoroquinolones
erythromycin
rifampicin
H+
a
b
-lactams
fluoroquinolones
fusidic acid
c
H+
d
NH 2
chloramphenicol
COOH
proton antiport
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+
aminoglycosides
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Most frequent antibiotic-pumps in procaryotes (2/2)
• Major Facilitator Superfamily (Gram + and - )
TOPOLOGY
MECHANISM
ANTIBIOTICS
12 TMS
b
d2
c
g
tetracyclines
fluoroquinolones
macrolides
lincosamides
rifampicin
pristinamycin
H+
NH2 a
COOH
14 TMS
c
d1
chloramphenicol
H+
b
h
e
+
f
a
COOH
NH2
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aminoglycosides
proton antiport
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Most frequent antibiotic-pumps in eucaryotes (1/2)
• Multiple Drug Resistance (P-gp)
TOPOLOGY
MECHANISM
ANTIBIOTICS
tetracyclines
fluoroquinolones
erythromycin
lincosamides
rifampicin
?
COOH
NH2
ATP
ATP-driven efflux
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chloramphenicol
ADP + Pi
+
aminoglycosides
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Most frequent antibiotic-pumps in eucaryotes (2/2)
• Multidrug Resistance Proteins (MRP)
TOPOLOGY
MECHANISM
ANTIBIOTICS
fluoroquinolones
NH2
tetracyclines
macrolides
COOH
?
GSH
ATP
ADP + Pi
ATP-driven efflux
with GSH as co-factor
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Antibiotic classes recognized by efflux pumps
in different types of organisms
Antibiotic
class
bacteria
Gram (+)
Gram(-)
fungi
superior
eucaryotes
-lactams
fusidic acid
macrolides
streptogramins
tetracyclines
aminoglycosides
chloramphenicol
rifamycins
sulfamides
trimethoprim
fluoroquinolones
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Two probable mechanisms of drug
transport by eucaryotic efflux pumps ...
• “vacuum cleaner”
• “flippases”
• the drug never really
penetrates the cell
• all effects are on the influx
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Do efflux pumps act as vacuum cleaners ?
Structural evidence for P-glycoprotein:
Rosenberg et al, JBC (1997) 272:10685-94
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Two probable mechanisms of drug
transport by eucaryotic efflux pumps...
• “vacuum cleaner”
• “flippases”
• the drug gets in the cell, and
is sucked from the
cyplasmic domain
• most effects are on the
efflux
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Do efflux pumps act as flippases ?
Functional evidences for MDR & MRP:
Smith et al,
J Biol Chem (2000) 275:23530-9
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Translocation of phosphatidylcholine at the apical
(closed bars) and basolateral (open bars) membranes
of MDR-3 (left) and MRP-1 (right) transfected LLCPK1cells, in the absence of in the presence of
inhibitors of the corresponding pump.
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Drugs and physiological substrates for
transporters in eucaryotic cells
superfamily
transporter
ABC
MDR1
phospholipids
MRP1
phospholipids
leukotrienes
conjugates
conjugates
MRP2
MFS
OAT
NPT1
OATP1
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physiol.
antibiotics
substrates
phosphates
bile salts
steroids
fluoroquinolones
macrolides
-lactams
tetracyclines
streptogramins
fluroquinolones
macrolides
rifamycins
fluoroquinolones
-lactams
-lactams
-lactams
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Biological significance of antibiotic
transport ...
• at the level of the bacteria (procaryotes)
– multi / cross-resistance
– role in emergence of resistance through
exposure to subinbitory levels
• at the level of the host (eucaryotes)
– modulation of transport
– modulation of intracellular activity
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Efflux and multi / cross-resistance in
pathogenic bacteria
1 bacteria
several pumps
multiresistance
1 pump
several classes
of antibiotics
crossresistance
1 class
of antibiotics
several pumps
efficacy of
inhibitors ?
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Efflux and selection of resistance to FQ
A certain serum AUC and a certain serum peak
will determine the drug concentration at the
target level which may prevent the selection of
first and second mutation resistants
Gyrase/ Topoisomerase
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Efflux and selection of resistance to FQ
Get a AUC !
Get a peak !
Gyrase/ Topoisomerase
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Efflux and selection of resistance to FQ
Oh!
Efflux pumps will
reduce the
concentration
at the level of the
target
and thereby favor
the selection of
target mutants!
Crazy !!
Gyrase/ Topoisomerase
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Efflux and selection of resistance
Frequency of Levofloxacin-resistant mutants in
Pseudomonas aeruginosa with deletions of the efflux pump operons
Pump status
WT
 mexAB-oprM
 mexCD-oprJ
 mexEF-oprN
 mexAB-oprM; mexEF-oprN
 mexCD-oprJ;  mexEF-oprN
 mexAB-oprM;  mexCD-oprJ
 mexAB-oprM;  mexCD-oprJ;
 mexEF-oprN
Lomovskaya et al,
AAC (1999) 43:1340-1346
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LVX MIC
Frequency of LVXresistant mutants
0.25
0.015
0.25
0.25
0.015
0.25
0.015
0.015
2 × 107 - 4 × 107
2 × 107 - 4 × 107
2 × 107 - 4 × 107
2 × 107 - 4 × 107
2 × 107 - 107
2 × 106
1 × 109
<1 × 1011
Selection of mutants in FQ target
undetectable if ALL pumps are disrupted
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Biological significance of antibiotic
transport ...
• at the level of the bacteria (procaryotes)
• at the level of the host (eucaryotes)
– modulation of tissue pharmacokinetics
– modulation of cellular pharmacokinetics
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Tissue PK : combined action of drug transporters for
drug elimination / absorption
efflux / reuptake of -lactams in kidney
proximal tubular cell
urine
Mrp2
Npt-1
Mct1
Oat1/3
blood
PepT2
OctN2
Mrp1
distal tubular cell
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Tissue PK: combined action of drug transporters for
drug elimination / absorption
efflux / reuptake of -lactams in kidney
proximal tubular cell
Mrp2
cloxacilline Npt-1
urine
cephalexine
cephaloridine
Mct1
Oat1/3
PepT2
OctN2
cephaloridine
cephalexine
carbenicilline
bloodG
penicilline
...
probenecid
Mrp1
distal tubular cell
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Tissue PK: efflux cooperates
with other mechanisms of drug clearance
hepatocyte
CONJUGATION CYP
bile
GS-D’ D’
D’ D
Phase
II
Phase
I
blood
D
Mdr1
Mrp2
Phase
III
Ishikawa, Trends Biochem Sci (1992) 17:463-468.
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Tissue PK: Efflux may also contribute to
poor oral absorption
enterocyte
D
D
CYP
lumen
blood
D’ D
Mdr1
Mrp2
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Cellular PK: ciprofloxacin efflux in macrophages
1. Probenecid (PRB) increases ciprofloxacin
accumulation and decreases ciprofloxacin efflux
Michot et al., ICAAC 2000
Residual fraction (%)
Ce = 50 µM (17µg/ml)
Cc/Ce
10.0
7.5
5.0
2.5
0.0
Control 5 mM PRB
2h incubation at 37°C
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100
PRB 5 mM
PRB 0 mM
75
50
25
0
0
10
20
30
Time (min)
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Cellular PK: ciprofloxacin efflux in macrophages
2. ATP depletion also increases ciprofloxacin
accumulation and decreases ciprofloxacin efflux
Michot et al., ICCAC 2000
Ce = 50 µM (17µg/ml)
Residual fraction (%)
10.0
Cc/Ce
7.5
5.0
2.5
75
50
25
0
0.0
Control ATP depletion
2h incubation at 37°C
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ATP depletion
Control
100
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0
5
10
15
Time (min)
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Cellular PK : handling of macrolides macrophages
Influence of
P-gp inhibitors
on azithromycin
accumulation
(5µg/ml; 3 h)
Seral et al., unpublished
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Towards the future ...
Inhibitors of transporters
should help you to keep your stuff in ...
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They contributed to this review
and/or gave us unpublished data...
•
•
•
•
•
•
Elisabetha Balzi
Jean-Michel Michot
Cristina Seral
Hugues Chanteux
Marie-Paule Mingeot-Leclercq
and many others...
And thank you to Franco
Scaglione for such an
excellent meeting !!
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