Inserm - Drepamag

Hydroxycarbamide:
Mode of action
Jacques Elion, MD, PhD
[email protected]
National Reference Centers for Sickle Cell Disease
Department of Medical Genetics
and Inserm UMR 665
Robert Debré University Hospital, 75019 Paris, France
Guadeloupe University Hospital, 97139 Les Abymes (French West Indies)
3èmes Journées de la Drépanocytose en Martinique
Trois Ilets
19-21 février 2013
HYDROXYCARBAMIDE (HC)
Hydroxyurea
is the only effective drug in SCD
FDA 1998 (adults)
DROXIA® Bristol-Myers Squibb
EMEA 2007 (adults and children)
SIKLOS® AddMedica
Initial expectation from HC:
increased HbF expression
deoxyHbS
polymer
decreased HbS polymerisation
HbF
a2g2
Glu
reduced vaso-occlusion
Glu
Heterotetramer
a2bSg
g
g
Inserm
UMR665
HYDROXYCARBAMIDE
& Sickle Cell Disease
 Oral HC : proven efficacy in clinical
trials (adults & children)
but
(Charache S et al, N Engl J Med 1995)
 Early clinical response
 Delayed and variable
Hb F increase
Ware RE, et al.
Blood. 2002;99:10-4
Inserm
UMR665
Induction of HbF by HC
French pediatric trial
Discordance
HbF (%) between clinical effect
and expected biological effect
40
35
30
25
What are the targets of HC?
20
15
10
5
What are the molecular mechanisms of
HC
action?
treatment
period (M)
0
0
3
6
9
12
15
18
De Montalembert M, et al, J Pediatr Hematol, 1997, 19:313-318
Maier-Redelsperger M, et al. Blood, 1998, 91:4472-4479
21
24
Inserm
UMR_S 665
In Sickle Cell Disease
abnormal cell adhesion to the endothelium plays a major in
targetting vaso-occlusion
Mohandas & Evans, Blood 1984
….but so does the vascular tone
Could HC affect cell adhesion?
from JP Cartron
SCADHESION project C Le Van Kim, PL Tharaux, J Elion
- expression of adhesion molecules on RBCs?
- endothelial cells?
- leukocytes?
Inserm
UMR665
Could HC affect cell adhesion?
from JP Cartron
SCADHESION project C Le Van Kim, PL Tharaux, J Elion
- expression of adhesion molecules on RBCs?
- endothelial cells?
- leukocytes?
Inserm
UMR665
Number of a4b1/ reticulocyte
% of reticulocytes with
overexpression of a4b1
SS reticulocytes express high levels of the
a4b1 integrin (and CD36)
Inserm
UMR665
40
a4b1
30
20
10
0
Controls
HC
VOC
NonVOC
(n=8)
(n=19)
(n=18)
(n=23)
160
120
80
40
0
Controls
HC
VOC
Non VOC
(n=20)
(n=20)
(n=18)
(n=23)
Odièvre et al, Haematologica, 2008
Number of a4b1/ reticulocyte
% of reticulocytes with
overexpression of a4b1
HC treatment reduces the expression of the
a4b1 integrin (and CD36) on SS reticulocytes
Inserm
UMR665
40
40
a4b1
30
30
20
20
10
10
0
Controls
HC
VOC
NonVOC
(n=8)
(n=19)
(n=18)
(n=23)
0
Before HC
On HC
Before HC
On HC
200
160
120
80
100
40
0
Controls
HC
VOC
Non VOC
(n=20)
(n=20)
(n=18)
(n=23)
0
Odièvre et al, Haematologica, 2008
in vivo results are mimicked in vitro on
erythroid progenitors in culture
(a4b1)
Number of VLA-4 /
GPA+ cell at J20
VLA-4
30000
20000
10000
0
No HC
Inserm
UMR665
With HC
Odièvre et al, Haematologica, 2008
Cellular targets of hydroxycarbamide in SCD
RBCs
- Hb F production
- adhesion molecules
- decreased production of a4b1 integrin, CD36…
Inserm
UMR 665
Mechanisms of sickle red blood cell adhesion:
impact of HC treatment
Epinephrine
β2-AR
HC
cAMP
Sickle Red
Blood Cell
PKA
Gauthier et al, J Biol Chem 2005
α4β1 integrin
Lu/BCAM
El Nemer et al, Blood 2007
Adhesion
Less adhesion
Lu/BCAM
Odièvre et al, Haematologica 2008
Bartolucci et al, Blood 2010
Abnormal cell-cell interactions in SCD are triggered
by signalling Laminin
eventsα5
HC diminishes cell adhesion by inhibiting Lu/BCAM phosphorylation,
Inserm
independently of its expression level
UMR665
Endothelial cells
Cellular targets of hydroxycarbamide in SCD
RBCs
- Hb F production
- adhesion molecules
- decreased production of a4b1 integrin, CD36…
- decreased phosphorylation of Lu/BCAM
Inserm
UMR 665
Could HC affect cell adhesion?
from JP Cartron
SCADHESION project C Le Van Kim, PL Tharaux, J Elion
- expression of adhesion molecules on RBCs?
- endothelial cells?
- leukocytes?
Inserm
UMR665
Microcinematic recording of RBCs
dynamics on treated or untreated ECs
Emmanuelle Verger
submitted results
Travelling distance (µm)
RBC trajectories
140
120
100
80
60
40
20
0
Time (s)
75 cells per experiment are
individually traced at 20 msec
intervals for each treatment condition
5 SS patients + 5 controls =
3.000 individual kinetics
Velocities of interacting (rolling?) SS-RBCs
are lower than those of AA-RBCs
mean advanced velocity (µm/s)
greater interaction
1200
HPMEC
1100
1000
900
800
700
600
***
***
500
400
NT
cyt
RBC type/treatment of EC
Emmanuelle Verger
unpublished results
Inserm
UMR665
HU treatment increases velocities of
interacting (rolling?) RBCs
mean advanced velocity (µm/s)
reduces transient interactions
1200
HPMEC
1100
1000
+ HC
900
800
***
***
700
600
***
***
500
400
NT
HC
cyt
cyt+HC
RBC type/treatment of EC
Emmanuelle Verger
unpublished results
Inserm
UMR665
adherent RBC number /mm²
AA-RBCs vs SS-RBCs firm adhesion on
EC+/- HU (1dyne/cm2 flow)
65
60
55
50
45
40
35
30
25
20
15
10
5
0
RBC type
*
basal
inflammatory
*
AARBC
NT
SSRBC
AARBC
SSRBC
NT
Inserm
Emmanuelle Verger, unpublished results
UMR665
adherent RBC number /mm²
AA-RBCs vs SS-RBCs firm adhesion on
EC+/- HU (1dyne/cm2 flow)
65
60
55
50
45
40
35
30
25
20
15
10
5
0
RBC type
*
basal
inflammatory
*
+ HC
AARBC
HC treatment
NT
SSRBC
HC
AARBC
NT
SSRBC
HC
After HC treatment of CE, adhesion potential of SS-RBCs is equivalent
to that of AA-RBCs
Inserm
Emmanuelle Verger, unpublished results
UMR665
Could HC affect cell adhesion?
from JP Cartron
SCADHESION project C Le Van Kim, PL Tharaux, J Elion
- expression of adhesion molecules on RBCs?
- endothelial cells: decreased adhesion
- leukocytes?
Inserm
UMR665
Mechanism of action of HC on endothelial cells
Sickle
reticulocyte
Laurance et al,
Pharmacogenet Genomics 2010
Haematologica 2011
HC
α4β1 integrin
TSP-1
vWF
PECAM-1
Less adhesion
Verger
et al, submitted
Adhesion
Lu/BCAM
Chaar et al, submitted
AMP
PDE
cAMP
PKA
AC
HC inhibits
the adhesive recruiting potential of endothelial cells by
ATP
Endothelialproteins
cells
modulating membrane
adhesion
and their activation state
Inserm
via phosphodiesterase activation
UMR665
Cellular targets of hydroxycarbamide in SCD
RBCs
- Hb F production
- adhesion molecules
Endothelial cells
- decreased adhesion
vascular tone?
Inserm
UMR 665
HC also decreases endothelin-1 production by
endothelial cells in culture
Brun et al, 2003, Pharmacogenomics J.
1200
100
ET-1(pg/ml)
% of expression
mRNA
*
*
60
20
Control HC Cyto + HC
Protein
* p<0.05 (n=5)
800
*
*
400
NT HC
Cyto Cyto + HC
ET-1(pg/ml)
1500
1000
HC
HC+Cyto
TrHBMEC cells
Incubation time : 48h
500
0
HUVEC cells: identical results
0
125
250
500 HC (M)
Inserm
UMR 665
In vivo HU decreases ET-1 plasmatic levels
Lapouméroulie et al, 2005, Hematologica
longitudinal follow up
(two 8 year old SS girls)
16 months
2
25
2
25
1,6
20
1,6
20
1,2
15
1,2
15
0,8
10
0,8
10
0,4
5
0,4
5
0
0
months
0
0
0
5
10
months
ET-1 (pg/ml)
ET-1 (pg/ml)
14 months
0
5
10
15
Decrease of plasmatic ET-1 does not correlate with
HbF induction
Inserm
UMR 665
Cellular targets of hydroxycarbamide in SCD
RBCs
- Hb F production
- adhesion molecules
Endothelial cells
- decreased adhesion
- decreased production of endothelin-1
vasoconstriction
Inserm
UMR 665
Could HC affect cell adhesion?
from JP Cartron
?
SCADHESION project C Le Van Kim, PL Tharaux, J Elion
- expression of adhesion molecules on RBCs?
- endothelial cells?
- leukocytes?
Inserm
UMR665
HC normalizes L-selectin expression and H202
production by PMNs of SCD patients in vivo
Benkerrou et al Blood, 2002
120
100
**
**
80
controls (n=11)
60
**
**
severe SS patients (n=17)
HU treated (n=9)
40
20
0
1
H202 production
2
L-selectin

by whole blood PMNs stimulated with TNFa and fMLP
 expressed at the surface of fMLP-stimulated PMNs
Inserm
UMR 665
Cellular targets of hydroxycarbamide in SCD
RBCs
- Hb F production
- adhesion molecules
Endothelial cells
- adhesion molecules
- endothelin-1
Polymorphonuclear neutrophils
- activation
Inserm
UMR 665
Conclusions
HC has a pleiotropic action in SCD
HC decreases deoxy HbS polymerisation
-  HbF expression -  RBC hydration
HC decreases cellular adhesion to endothelium
-  proadhesive RBCs
-  adhesion properties of ECs
-  PMN activation
HC decreases vasoconstrictive stimulus
-  ET-1
Paris
Guadeloupe
Pointe-à-Pitre
UMR 665
Institut national de la santé et
de la recherche médicale
Réunion