NeuroprotectionNeuroprotection

in preterm infants:in preterm infants:

hope or hope or utopyutopy? ?

Olivier BAUD, MD, PhD

INSERM U676

Children’s Hospital Robert Debré, Paris, France

The International Neonatology Conference, Kiev 6th march 2013

Brain damages neonatesBrain damages neonates

� Term

� HIE

� Stroke

� Preterm� Intraventricular/parenchymal haemorrhage↓� Cystic white matter damage ↓� Diffuse white matter damage ↑

Volpe, 2001, 2009

Non cystic PVL: Non cystic PVL: the the ““newnew”” white matter damagewhite matter damage

� Mostly observed in very preterm infant: MRI

features detected near term

� Risk factors: VLBW-ELBW, perinatal

infection/inflammation, postnatal steroids,

BPD

� Associated with myelination defect,

involvement of SP neurons leading to

reduction in cortical volumes Nosarti et al., 2002

Inder et al., 1999

Woodward et al., 2006

Functional consequences of PVLFunctional consequences of PVL

� Motor deficit (Little)

� Executive dyspraxia

� Visual-perceptal impairment (optic

radiations)

� Feeding and deglutition disorders

� Cognitive impairment

� Comportemental deficits (ADHD)

� Psychiatric disorders (autism) +++

- Axonal damage (association and projection

fibers)

- Quantitative decrease of Oligodendrocytes

WMD and WMD and brainbrain developmentdevelopment

20 25 32

Neuronal migrationNeuronal migration

astrocytogenesisastrocytogenesis

oligodendrogenesisoligodendrogenesis

AxonalAxonal and and dendriticdendritic growthgrowth

synaptogenesissynaptogenesis

ProgrammedProgrammed cellcell deathdeath

Post-conceptional weeks

PerinatalPerinatal brainbrain lesionslesions: a multiple : a multiple hit concepthit concept

Preterm labor

Inflammation Free radicals

Excitotoxicity

WMD

Repair

Main Main targetstargets for for neuroprotectionneuroprotection in in pretermpreterm

RepairRepair //plasticityplasticity

glutamate

inflammation Oxidative stress

NMDA AMPA

microglia

cytokines

X

X XMg2+

Melatonin

Epo

Xenon

Topiramate

Tianeptine

Quinolones

X

Melatonin

iNO

E2

Melatonin

iNO

Growth factors

Cell therapy

Tetracyclines

Steroids

X

ShortcomingsShortcomings in in neuroprotectionneuroprotection of the of the

pretermpreterm brainbrain

� False candidate molecules

� Not accurate design or preclinical model

� Experimental biases

� Wrong rationale/endpoints

� Confounding variables (sex, T°C, care, stress…)

� Methodological biases (power+++, variability…)

� Responders vs non-responders

� Sick vs healthy models

StrategiesStrategies for for perinatalperinatal neuroprotectionneuroprotection

1- Unexpected neuroprotective effect of well-known molecules-Magnesium sulfate

-Nitric Oxide

-Antibiotics

-Tianeptine

-Melatonin

-Erythropoietin

-Allopurinol

-NAC

2- New neuroprotective molecules-Growth factors

−β-estradiol

-A2A rec antagonist

-Xenon

3- New neuroprotective strategies-Exogenous stem cells

-Neural progenitors

MelatoninMelatonin and and circadian rhythmcircadian rhythm

Claustrat et al. Restorative Neurology 1998

No rhythmic secretion in newborn

A B

C D

* *

* *

Ctl RCIU

Me

lato

nin

PB

S

MelatoninMelatonin (ROS+(ROS+inflaminflam++excitoexcito))

Husson et al., 2002

Olivier et al., 2009

Perinatal oxidative stress excitotoxicity

Bouslama et al., 2007

Welin et al., 2007

Villapol et al., 2010

Mouse P5: excitotoxicity model

Rat P7 : hypoxia-ischemia model

Fetal sheep: ischemia model

NeuroprotectiveNeuroprotective effects of Melatonineffects of Melatonin

in animal modelsin animal models

Learning disorders

Periventricular white matter damage

Mechanisms

Direct free radical scavenging

OLs maturation

Excitotoxicity modulation

Neuroprotection

Melatonin in Preterm

3 animal models

of WM damage

MELIP

Melatonin deficiency

Pharmacokinetic studyOptimal regimen of administration

Therapeutic trial

WM damage, MRI 40 GA

MelatoninMelatonin as as neuroprotectantneuroprotectant for for pretermpreterm infants: infants:

a 3a 3--stepssteps proofproof--ofof--concept concept researchresearch programprogram

1

2

3

MagnesiumMagnesium sulfate sulfate (NMDA)(NMDA)

MgMg2+2+

-NMDA receptors blocker

-Preclinical prevention of developing WM

-Safe for neonates but potential side

effects in mothers

-Demonstrated benefits (gross motor

dysfunction and CP) in preterm infants…

… but NNT = 63 mothers to save one CP!

-No effect in asphyxiated term infants

Marret et al., 1995

Crowther et al., 2003

Doyle et al., 2009

Ctr

l

1 m

g/kg

10 m

g/kg

30 m

g/kg

Ctr

l

1 m

g/kg

10 m

g/kg

30 m

g/kg

0

250

500

750

1000

cortical plate white matter

*

**

*Lesi

on s

ize

(µm

)

TopiramateTopiramate (AMPA)(AMPA)

Follett et al., 2004

Sfaello et al., 2005

Kurul et al., 2009

excitotoxicity ischaemia

FluroquinolonesFluroquinolones (inflammation)(inflammation)

Ct CTX CIP0

5

10

15 *

****

ED-1

pos

itive

cel

ls /

0,06

5mm

2

Ct CTX CIP0

10

20

30

40

50

60

***

MB

P-po

sitiv

e fib

ers

(O.D

.)

E. Coli infectedLoron et al., 2011

Le Saché et al., 2011

ED1

MBP - Anti-inflammatory effect in situ

- OLs maturation

- Molecular determinant?

Potential new therapeutic agents

Schreiber et al., 2003

Van Meurs et al., 2005

Mestan et al., 2005

InhaledInhaled NO & NO & outcomeoutcome of of pretermpreterminfantsinfants

- iNO was finally associated with a

lower incidence of brain damage in

preterm infants

- 2y outcome revealed a better MDI

- No side effect

- These data must be confirmed

NO, NO, myelinationmyelination& & neuroprotectionneuroprotection

P7 P14

• Inhaled NO enhances angiogenesis and myelination

• Inhaled NO induces neuroprotection

Ctl iNO5

Olivier et al., 2010

Pansiot et al. , 2011

0

200

400

600

800

1000

1200

Cortical plate White matter

CtliNO20ppm

lesi

on s

ize

(µm

)Ibo NMDA Will Ibo NMDA Will

******

*

*

*

NO, NO, myelinationmyelination& & neuroprotectionneuroprotection

P7 P14 Pham et al., AJRCCM 2012

Ischemia

ACAs

BT

r.PCAl.PCA

r.MCAl.MCA

Azygos ACA

P Com

r.ICAl.ICA

**

InhaledInhaled NO & NO & neonatalneonatal strokestroke

Bonnin et al., 2012

Charriaut-Marlangue et al., 2012

iNOiNO isis neuroprotectiveneuroprotective……

……but not but not atat anyany time and not time and not atat anyany dose!dose!

iNO induces benefits on neonatal stroke only at

low dose and during ischemia

GrowthGrowth factorsfactors & & neuroprotectionneuroprotection

� Leptin

� VIP

� VEGF

� BDNF

� Epo

BDNF

Husson et al., 2005

Sizonenko et al., 2007

PBS BDNF

CerebralCerebral effectseffects of Epoof Epo

� Cellular effects

� ↓↓↓↓ apoptosis

� ↑↑↑↑ angiogenesis

� ↓↓↓↓ inflammation

� ↓↓↓↓ excitotoxicity

� ↓↓↓↓ oxidative stress

� Neuroprotective effects in human adults

� ↓↓↓↓ lesion size of adult stroke

� ↓↓↓↓ handicap

� ↓↓↓↓ paraplegia after medullarysection

� Epo and neuroprotection in human neonates

� ↑↑↑↑ MDI

� ↓↓↓↓ CP after HIE

� High doses safe in preterm Strunk et al., 2004

Zhu et al., Pediatrics, 2009

Brown et al., Pediatrics, 2009

Melatonine Epo NAC Epo

mimetics

Allopurinol Xenon Vit C&E Memantine Topiramate Adenosine

A2A rec antag

nNOS

inh

BH4

Easy of use 10 10 10 10 7 4 9 3 4 5 1 1

Loading

dose

7 7 7 7 8 6 6 5 4 5 1 1

SAE 10 8 10 8 8 8 6 6 5 8 1 1

Neurotox 10 10 10 7 10 8 8 10 9 2 1 1

Benefits 8 8 3 6 3 8 4 3 3 5 1 1

FDA oui oui oui non oui non oui oui oui non non oui

Score

/50

45 43 40 38 36 34 33 27 25 22 5 5

Ranking

% score

1 (90%) 2 (86%) 3 (80%) 4 (76%) 5 (72%) 6 (68%) 7 (66%) 8 (54%) 9 (50%) 10 (44%) 11 (10%)

Ranking of potential Ranking of potential

neuroprotectants in the newbornneuroprotectants in the newborn

From Robertson et al., 2012

Treatment Mechanism Study Primary outcome

Hypothermia multiple many Proven benefits and NNT 6-8

EPO growth factor Jull et al., 2008 High dose EPO safe in ELGA infants

iNO antioxidant Scheiber et al., 2006

Ballard et al., 2006

- Lower incidence of severe IVH/PVL in ELGA- Decreased neurodevelopmental disability in ELGA

Melatonin antioxidant Gitto et al., 2004, 2005, 2012

- Anti-inflammatory effects and improves clinical outcome (BPD)- Additional analgesic therapy during procedural pain in LGA

Allopurinol antioxidant Clancy et al., 2001

Benders et al., 2006

- Neurocardiac protection in HLHS infants- Postnatal treatment had no effect in HIE

Magnesium sulfate Anti-excitotoxic Doyle et al., cochrane 2009

Established neuroprotection in VLGA but NNT 63

HumanHuman StudiesStudies of of NeuroprotectantsNeuroprotectants

Gonzalez and Ferriero, 2009

Cell therapy and neural progenitorsCell therapy and neural progenitors

Stem cells:• embryonic, • umbilical cord blood,• mesenchymal, • neural…

Syková et al., 2007

Umbilical cord: stem cells from Umbilical cord: stem cells from

blood and Wharton jellyblood and Wharton jelly

CBMNCCBMNC UCMSCUCMSC

Umbilical cordUmbilical cord--derived stem cellsderived stem cells

Advantages

• rich source of various stem cells

• No potential ethical issues

• easily available with no harm to the infant or mother

• low immunogenicity

• international standards for sampling and storage

Limits

• Few and conflicting data on efficacy

• Major concerns in pre-clinical models (injection route, cell tracking,

variability…)

• Discrepancy between « emotional infatuation » and scientific reality

NeuroprotectiveNeuroprotective mechanismmechanism(s) of (s) of

UCdUCd stem stem cellscells

- Immuno-modulation of systemic and/or

local inflammation

- Enhancement of endogenous repair:-growth factor

-apoptotic cell death

-endogenous stem cells?

- Marginal engraftment and

transdifferenciation

Van Velthoven et al., 2012

Dalous et al., 2012

NeurogenesisNeurogenesis and and neuroprotectionneuroprotection

Burns et al., JCN 2009

Neural growth factorsEpo

ConclusionConclusion

� Neuroprotection of the developing brain is feasible

� Many pharmacological strategies based on better underst ood physiopathology

� Ongoing proof-of-concept studies :� Melatonin� EPO� iNO� …

� Many new promising strategies in the pipeline

AknowledgmentsAknowledgments

� U676:� C Charriaut-Marlangue� H Pham� J Pansiot� G Loron� J Dalous� P Olivier� J Gallego� P Gressens

� NICU & Robert Debré:� V Biran� C Alberti� L Maury

� PremUP foundation:� D Evain-Brion� L Bordarier� ML Hamon

� Supports:� INSERM� PremUP� IKARIA� Paris Diderot Univ� Mairie de Paris