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...