Hydroxycarbamide in Sickle Cell Disease : Mode of action

Hydroxycarbamide:Mode of action

Jacques Elion, MD, PhD Inserm UMR 665National Institute for Blood Transfusion, 75015 ParisRobert Debré Mother and Children University Hospital, 75019 ParisGuadeloupe University Hospital, 97139 Les Abymes (French West Indies)Laboratory of Excellence on the Red Blood CellNational Reference Centers for Sickle Cell Disease

Institut national de la santé et de la recherche médicale

4th REDAC Meeting, Yaoundé, Cameroun, June 13-15, 2013

S

S

S

S

S

S

S

S

Glu Glu

HbF22

deoxyHbSpolymer

85Phe 88

Leu

S

6Val

decreased HbS polymerisation

Initial expectation from HC: increased HbF expression

reduced vaso-occlusion

HYDROXYCARBAMIDE (HC)Hydroxyurea is the only effective drug in SCD

InsermUMR665

FDA 1998 (adults) DROXIA® Bristol-Myers SquibbEMEA 2007 (adults and children) SIKLOS® AddMedica

S

Glu

Heterotetramer2SHeterotetramer2S

Glu

Heterotetramer2S

Oral HC : proven efficacy in clinicaltrials (adults & children)(Charache S et al, N Engl J Med 1995)

Early clinical response

Delayed and variable Hb F increase

HYDROXYCARBAMIDE& Sickle Cell Disease

but

InsermUMR665

Ware RE, et al.Blood. 2002;99:10-4

HbF

0

5

10

15

20

25

30

35

40

0 3 6 9 12 15 18 21 24

treatment period (M)

HbF (%)

De Montalembert M, et al, J Pediatr Hematol, 1997, 19:313-318Maier-Redelsperger M, et al. Blood, 1998, 91:4472-4479 Inserm

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Induction of HbF by HCFrench pediatric trial

0

5

10

15

20

25

30

35

40

0 3 6 9 12 15 18 21 24


HbF (%)


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Discordance between clinical effectand expected biological effect

0

5

10

15

20

25

30

35

40

0 3 6 9 12 15 18 21 24


HbF (%)


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What are the targets of HC?

0

5

10

15

20

25

30

35

40

0 3 6 9 12 15 18 21 24


HbF (%)


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What are the molecular mechanismsof HC action?


What are the targets of HC?

Mohandas & Evans, Blood 1984

In Sickle Cell Diseaseabnormal cell adhesion to the endothelium plays a major in targetting vaso-occlusion

….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

SS reticulocytes express high levels of the 41 integrin (and CD36)

Controls(n=20)

HC(n=20)

VOC(n=18)

Non VOC(n=23)

0

40

80

120

160

Num

bero

f 4

1/ re

ticul

ocyt

e

Controls(n=8)

HC(n=19)

VOC(n=18)

NonVOC(n=23)

0

10

20

30

40

% o

f ret

icul

ocyt

es w

ithov

erex

pres

sion

of

41

41

Odièvre et al, Haematologica, 2008 Inserm

UMR665

HC treatment reduces the expression of the 41 integrin (and CD36) on SS reticulocytes

Controls(n=20)

HC(n=20)

VOC(n=18)

Non VOC(n=23)

0

40

80

120

160

Num

bero

f 4

1/ re

ticul

ocyt

e

Controls(n=8)

HC(n=19)

VOC(n=18)

NonVOC(n=23)

0

10

20

30

40

% o

f ret

icul

ocyt

es w

ithov

erex

pres

sion

of

41

41

Odièvre et al, Haematologica, 2008

Before HC On HC0

100

200

Before HC On HC0

10

20

30

40

InsermUMR665

VLA-4(41)

No HC With HC0

10000

20000

30000

Num

bero

f VLA

-4 /

GP

A+

cell

atJ2

0

in vivo results are mimicked in vitro on erythroid progenitors in culture

Odièvre et al, Haematologica, 2008 Inserm

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Cellular targets of hydroxycarbamide in SCD

RBCs- Hb F production- adhesion molecules

- decreased production of 41 integrin, CD36…

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InsermUMR665

β2-AR

Lu/BCAM

Sickle Red Blood Cell

Laminin α5

Epinephrine

cAMP

Mechanisms of sickle red blood cell adhesion:impact of HC treatment

PKA

InsermUMR665

β2-AR

Lu/BCAM


Adhesion

Gauthier et al, J Biol Chem 2005

Laminin α5

Epinephrine

cAMP


PKA

InsermUMR665

β2-AR

Lu/BCAM


Adhesion


Laminin α5

Epinephrine

cAMP

α4β1 integrinEl Nemer et al, Blood 2007

Lu/BCAM

Endothelial cells


PKA

InsermUMR665

β2-AR

Lu/BCAM



Laminin α5

Epinephrine

cAMP


Lu/BCAM

Endothelial cells


PKA

Odièvre et al, Haematologica 2008Bartolucci et al, Blood 2010

Less adhesion

HC

InsermUMR665

β2-AR

Lu/BCAM


Adhesion


Laminin α5

Epinephrine

cAMP


Lu/BCAM

Endothelial cells


PKA

Odièvre et al, Haematologica 2008Bartolucci et al, Blood 2010

Less adhesion

HC

Abnormal cell-cell interactions in SCD are triggered by signalling events

HC diminishes cell adhesion by inhibiting Lu/BCAM phosphorylation, independently of its expression level



- decreased production of 41 integrin, CD36…- decreased phosphorylation of Lu/BCAM

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- expression of adhesion molecules on RBCs?- endothelial cells?- leukocytes? Inserm

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Microcinematic recording of RBCs dynamics on treated or untreated ECs

Emmanuelle VergerClin Hemoheol 2013

RBC trajectories

020406080

100120140

0,02

0,06 0,1 0,14

0,18

0,22

0,26 0,3 0,34

0,38 Time (s)

Trav

ellin

g di

stan

ce (µ

m)

75 cells per experiment are individually traced at 20 msecintervals for each treatment condition

5 SS patients + 5 controls = 3.000 individual kinetics

AARBCSSRBC

AARBCSSRBC

400

500

600

700

800

900

1000

1100

1200

*** ***

RBC type/treatment of EC

mea

nad

vanc

edve

loci

ty(µ

m/s

)

NT cyt

Velocities of interacting (rolling?) SS-RBCs are lower than those of AA-RBCs

greater interaction

HPMEC

InsermUMR665Emmanuelle Verger

Clin Hemoheol Microcircul 2013

AARBCSSRBCAARBCSSRBCAARBCSSRBC AARBCSSRBC

400

500

600

700

800

900

1000

1100

1200

***

***

***

***

RBC type/treatment of EC

mea

nad

vanc

edve

loci

ty(µ

m/s

) HPMEC

NT HC cyt cyt+HC

HU treatment increases velocities of interacting (rolling?) RBCs

reduces transient interactions

+ HC



AA-RBCs vs SS-RBCs firm adhesion on EC+/- HU (1dyne/cm2 flow)

AARBC SSRBC AARBC SSRBC

05

101520253035404550556065

*

RBC type

adhe

rent

RB

C n

umbe

r/m

m²

NT NT

*basalinflammatory



AA-RBCs vs SS-RBCs firm adhesion on EC+/- HU (1dyne/cm2 flow)

basalinflammatory

After HC treatment of CE, adhesion potential of SS-RBCs is equivalentto that of AA-RBCs

AARBC SSRBC AARBC SSRBC

05

101520253035404550556065

*

RBC type

adhe

rent

RB

C n

umbe

r/m

m²

NT NTHC HCHC treatment

*

+ HC





- expression of adhesion molecules on RBCs?- endothelial cells: decreased adhesion- leukocytes? Inserm

UMR665

InsermUMR665

Adhesion

Mechanism of action of HC on endothelial cells

α4β1 integrin

Endothelial cells

Sickle reticulocyte

Lu/BCAMTSP-1 vWFPECAM-1

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α4β1 integrin

Endothelial cells

Sickle reticulocyte

Lu/BCAMVerger et al, Clin Hemoheol Microcircul 2013Chaar , Laurance et al, J Biol Chem 2014

Less adhesionLaurance et al, Pharmacogenet Genomics 2010Haematologica 2011

HC

TSP-1 vWFPECAM-1

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α4β1 integrin

Endothelial cells

Sickle reticulocyte



HC

AC

AMP

ATP

cAMP PKA

PDE

TSP-1 vWFPECAM-1

InsermUMR665

Adhesion


α4β1 integrin

Endothelial cells

Sickle reticulocyte



AC

AMP

ATP

cAMP PKA

PDE

HC

InsermUMR665


α4β1 integrin

Endothelial cells

Sickle reticulocyte



HC

AC

AMP

ATP

cAMP PKAPDE

TSP-1 vWFPECAM-1

InsermUMR665


α4β1 integrin

Endothelial cells

Sickle reticulocyte



HC

AC

AMP

ATP

cAMP PKAPDE

TSP-1 vWFPECAM-1

HC inhibits the adhesive recruiting potential of endothelial cells by modulating membrane adhesion proteins and their activation state

via phosphodiesterase activation


Endothelial cells- decreased adhesion

vascular tone?

InsermUMR 665


HCHC+Cyto

0 125 250 5000

500

1000

1500

HC (M)

ET-

1(pg

/ml)

ET-

1(pg

/ml)

NT Cyto + HC

* *400

800

1200

HC Cyto

Protein * p<0.05 (n=5)

HC also decreases endothelin-1 production by endothelial cells in culture

TrHBMEC cellsIncubation time : 48h

Brun et al, 2003, Pharmacogenomics J.

20

60

100

Control HC Cyto + HC

% o

f exp

ress

ion

mRNA

**

HUVEC cells: identical results

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In vivo HU decreases ET-1 plasmatic levels

0

0,4

0,8

1,2

1,6

2

0 5 100

5

10

15

20

25

ET-1

(pg/

ml)

HbF

(%)

months

14 months

0

0,4

0,8

1,2

1,6

2

0 5 10 150

5

10

15

20

25

ET-1

(pg

/ml)

HbF

(%)

months

16 months

Lapouméroulie et al, 2005, Hematologica

Decrease of plasmatic ET-1 does not correlate withHbF induction

longitudinal follow up (two 8 year old SS girls)

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Endothelial cells- decreased adhesion

- decreased production of endothelin-1

vasoconstriction


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- expression of adhesion molecules on RBCs?- endothelial cells?- leukocytes?

?

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HC normalizes L-selectin expression and H202production by PMNs of SCD patients in vivo

0

20

40

60

80

100

120

1 2

controls (n=11)severe SS patients (n=17)HU treated (n=9)

H202 production L-selectin

by whole blood PMNs stimulated with TNF and fMLP

expressed at the surface of fMLP-stimulated PMNs

**

**

**

**

Benkerrou et al Blood, 2002

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Endothelial cells- adhesion molecules- endothelin-1

Polymorphonuclear neutrophils- activation


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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

Conclusions

HC has a pleiotropic action in SCD

Paris

UMR 665Guadeloupe

Pointe-à-Pitre

Réunion

Institut national de la santé et de la recherche médicale

Hydroxycarbamide in Sickle Cell Disease : Mode of action

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