Intracellular Pharmacodynamics: a Journey from Cell biology to the Design of
New Chemotherapeutic Agents
Paul M. Tulkens, MD, PhDCellular and Molecular Pharmacology Unit
Catholic University of Louvain, Brussels, Belgium&
International Society of Antiinfective Pharmacology
Advances in the Pharmacology of Antiinfective Therapy,
Bangalore, India, January 20-22, 2000
What is Intracellular Infection ?
The Cell
(black box)
antibiotic bacteria
These they are… OBLIGATORY OR MAINLY INTRACELLULAR:
respiratory infections (pneumopathies): Chlamydia pneumoniae: 10% in children
Legionella pneumophila: frequent if immunosuppression Mycobacterium spp.: frequent if immunosuppression
sexually transmitted diseases Chlamydia trachomatis: most common pathogen
CNS infections + other sites: Listeria monocytogenes: pregnant women;
immunosuppression
FACULTATIVE OR MAINLY EXTRACELLULAR: digestive tract infections
Salmonella spp., Shigella spp. respiratory, cutaneous, etc…tract infections
Streptococcus spp., Staphylococcus spp.
1. where are the bacteria ? 2. which antibiotics accumulate in cells ?3. where are antibiotics located ?4. what is the intracellular expression of activity ?5. what is the bacterial responsiveness ? 6. cooperation with the cell own defenses and cytokines?
You need to consider (at least)the following questions:
1st question (and answer) : where do intracellular bacteria sojourn and thrive ?
LegionellaChlamydia, ...
S. aureusSalmonella,M. leprae, ...
Listeria hly+,Shigella
phagosomesphagolysosomes
cytosol endosomes
Antibiotics and Intracellular Bacteria : a Bit of Theory
Eur. J. Microbiol Infect. Dis. 10: 100-106, 1991
2d question (and answer): which antibiotics do accumulate in cells ?
• beta-lactams: 1x• aminoglycosides: <1 to 2 x• ansamycins: 2-3 x• tetracyclines: 2-4 x• fluoroquinolones: 10-20 x• macrolides: 4 to > 100 x
endosomes?
phagosomes?
phagolysososomes• macrolides• aminoglycosides
cytosol• fluoroquinolones• beta-lactams• ansamycins
3d question (and answer) : where are intracellular antibiotics located ?
endosomes
phagosomes• macrolides: reduced• aminoglycosides: very reduced
• fluoroquinolones: yes• ansamycins: excellent• beta-lactams: yes
4th question: what are the antibiotic intracellular expressions of activity
pH = 7
pH < 6
in endosomes:?
in phagosomeslikely to excellent
5th question: what is the bacterial responsiveness to the antibiotics ?
pH = 7
pH < 6
in cytosol:excellent
in phagolysosomes:very variable
in endosomes:likely
in phagosomeslikely
6th question [1 of 2]: cooperation with the cell own defenses ?
pH = 7
pH < 6
in cytosol:unlikely
in phagolysosomes:very variable
O2. -
NO.
myeloperoxydaseacidity, cationic proteins...
6th question [2 of 2]: cooperation or antagonism with the actions of cytokines ?
The very black box
antibiotic
interferonsILxgrowth fact.TNF...
antibiotics:
• ampicillin• sparfloxacin• azithromycin
interferongamma
Listeria monocytogeneshly+ and hly-
INTRACELLULAR INFECTION CYCLE OF Listeria monocytogenes hly+
from Portnoy et al.
Bacteria are stained withfluorescein(FITC) [green]and cell actinwith phalloidinrhodamin[red];
hly+: virulenthly-: non virulent
Listeria and Interferon : confocal microscopy
A: phagocytosisB: escape from vacuoleC: surrounded by actin
D,E: cells pre-treated with interferon-gamma, L. m. hly+ remains in vacuoles.
F,G, H: L.m. hly- remains constantly in vacuoles
A = 1 h post infection; B, C, D, F, G = 3 h post infection E, H = 5 h post infection
Following the intracellular fateof Listeria m. in EM
control
IFN 100 U/ml
control
IFN 100 U/ml
IFN contains L. monocytogenes hly+ in phagosomes and impairs its growth.
A: dose-dependency; B: specifity
IFN 50 U/ml
IFN 100 U/m+ anti IFN IgG
The growth containment of L. monocytogenes caused by
IFN is due to NO and H202 production...
IFN and antibiotics...
control
+ IFN
+ IFN and
LMMA / catalase
Intracellular activity:
CFU @ 0 h CFU @ 5 hlog10
Bacterial growth
Bacterial kill
Listeria, ampicillin and Interferon gamma
1. Ampicillin is poorly active against intracellular Listeria;
2. Ampicillin activity is completely defeated when IFN is present (IFN makes the job !)
Why do you keep ampicillin ? extracellular bacteria get activity with very large doses
Listeria, fluoroquinolones and Interferon
-1
0
1
2
3
Intr
ac
ellu
lar
ac
tiv
ity
(lo
g(C
FU
0h
/5h
))
SPSP+
INF
CT
SP + INFSP + INF+ LMMA + + LMMA + catalasecatalase
sparfloxacin
(Ouadrhiri et al, 1999)
0
5
10
15
20
Ac
cu
mu
lati
on
fa
cto
r (C
c/C
e)
SP
SP + INFSP + INF+ LMMA + + LMMA + catalasecatalase
T = 5h
AccumulationActivity
SP+
INF
Listeria, fluoroquinolones and Interferon
1. Sparfloxacin appears highly active against intracellular Listeria;
2. Sparfloxacin activity is enhanced when IFN is present (IFN and Sp cooperate !)
Why don’t you use fluoroquinolones ? too low intrinsic activity ...
Where published ?
Scorneaux et al.,Antimicrob. Agents Chemother. 40:1225-30, 1996
Ouadrhiri et al., Antimicrob. Agents Chemother. 43:1242-51, 1999
Ouadrhiri et al., J. Infect. Dis. 180:1195-204, 1999
C NO
COOH
R
CHN
O
COOH
H2N
CH3
CH3
D-alanine beta-lactam
O
COOH
R
ALL beta-lactam have (and must have) a free carboxyl group
(or an equivalent acidic group [electron donor])
at appropriate distance from the C —— N bound
NC
phagolysososomespH 5-6
exatracellularpH 7.4
Why do beta-lactams not accumulate in cells (1 of 2) ?
cytosolpH 7.2
Why beta-lactams do not accumulate in cells (2 of 2) ?
AH
A-
AH
A-
AH
A-
H+ H+
H+
What if you are basic ?
B
BH+
B
BH+
B
BH+
H+ H+
H+
O
CO- R
R
To get accumulation, make a basic beta-lactam...
NC
- NH2
O
CO- NH-CH2-CH2-CH2-N (CH3)2
R
N-(3-dimethylaminopropyl)benzylpenicillamide...
NC
S
CH3
CH3
H
N-(3-dimethylaminopropyl)benzylpenicillamide…
get accumulated in cells (5 to 10-fold concentrates in lysosomes
but…
is inactive because the amide linkage is not splitted by lysosomal enzymes...
O
CO - O - CH2 - X - NH2
R
You need to make a chemically labile ester...
NC
S
CH3
CH3
H
What you can do with old pivampicillin...
N
S
COO
CH 3O
CH3
HNC
O
NH2
OO
H2CO
C C
O
CH 3
CH3
CH3
C
N
S
CH3O
CH 3
HNC
O
NH2
N
S
COOH
CH 3O
CH3
HNC
O
NH3
OO
H2CO
C C
O
CH 3
CH3
CH3
C
N
S
CH3O
CH 3
HNC
O
NH3
ampicillin
pivampicillin
pH 7 pH < 6+
+
-
0.0
0.5
1.0
Intr
ac
ellu
lar
ac
tiv
ity
(lo
g(C
FU
0h
/24
h))
0 1 2 3 4Time (h)
0
20
40
60
80
Ac
cu
mu
lati
on
fa
cto
r (
Cc
/Ce
)Old pivampicillin does bring ampicillin in cells...
free AMPIPIVAAMPI FROM PIVA
CT AMPI PIVA
T = 24h
(Fan et al, 1997)
Where published ?
Renard et al., J. Med. Chem. 29:1291-1293, 1986
Renard et al., Antimicrob. Agents Chemother. 31:410-416, 1987
Fan et al., Bioorg. Med. Chem. Letters 24:3107-3112, 1997
Thanks to the crew ...
Zubida Bumkhalla (beta-lactams [chemistry])
Marie-Béatrice Carlier (macrolides, fluoroquinolones)
Huajuan Fan (betlactams [chemistry] )
Christine Renard (beta-lactams)
Yousef Ouadrhiri (Listeria, interferon, IL6, fluoroquinolones)
Isabelle Paternotte (beta-lactams [chemistry], efflux mech.)
Bernard Scorneaux (Listeria, interferon, fluoroquinolones)
Etienne Sonveaux (beta-lactams [chemistry] )
Andrée Zenebergh (lincosaminides, macrolides)
But where do they work ?
Catholic University of LouvainFaculty of Medicine Brussels, Belgium
But, where is this university?
Cellular and Molecular Pharmacology UnitCatholic University of Louvain, Belgium
http://www.md.ucl.ac.be/facm
intracellularpharmacodynamics
AUIC
postantibiotic effect...
population pharmacokinetics
tissue concentrations
efficacy/toxicity ratios
http://www.isap.org
But don’t forget ISAP...
intracellularpharmacodynamics
AUIC
postantibiotic effect...
population pharmacokinetics
tissue concentrations
efficacy/toxicity ratios
And thank you to Bangalore...