Tamoxifen: a potential new drug for Duchenne
Urs Ruegg
University of Geneva, Geneva, Switzerland
Why tamoxifen in DMD?
A Treatment of mdx mice for 15 months
Performance
Diaphragm
Cardiac fibrosis
B Treatment of adult mdx mice (2 months at “mid-life”)
Dose – effect
Muscle contractibility
Membrane permeability
C Tamoxifen
Mechanism of action
Plasma levels of cytokines
Mdx cardiomyocytes
Summary of effects
Pro’s & Con’s
Action Duchenne 12th International Annual Conference “From the Labs to the Clinic”
Why evaluate tamoxifen?
A Physiology of estrogens
•
•
•
•
•
Female mdx mice have stronger muscles than male ones.
Force is increased when plasma estrogen levels are high.
Estrogens improve muscle resistance to fatigue.
Estrogens increase myofiber regeneration.
Estrogens increase muscle mass recovery from disuse atrophy.
tamoxifen
B Why tamoxifen?
•
•
•
Tamoxifen has estrogen-like activity.
Tamoxifen is an antagonist in the mammary gland – used to treat breast
cancer and an agonist in bone (and maybe in muscle).
Over 30 years of clinical experience with tamoxifen.
Note: Tamoxifen is a pro-drug: metabolized into 4-hydroxy amoxifen – 100 times more potent.
Major signalling events in the dystrophic pathogenesis
LACK of DYSTROPHIN
kinase
activity
Ca2+ influx
oxidative
stress
membrane
fragility
multiple dysfunctions of muscle cell homeostasis
protein function
metabolism
FORCE GENERATION
mitochondrial function
gene expression
ATP deficit
muscle cell death
regeneration
fibrosis/adiposis
muscle wasting
inflammation
DEATH
A. Treatment of mdx5Cv mice with tamoxifen for 15 months: Experimental design
Birth
normal
4-5 weeks
necrosis
6-8 weeks
regeneration
Death
16 months
cachexia
lowintensity, chronic disease
TAM, 10 mg/kg/day via food for 15 Mo
males
body weight, food consumption
Dys:
TAM:
wt:
FEM:
untreated mdx
TAMtreated mdx
untreated wildtype
untreated female mdx
wire test
force recording
The mdx mouse
postmortem analyses
Histology
Western blots
gene expression
(From SCIENCEphotoLIBRARY)
A. Treatment of mdx5Cv mice with tamoxifen for 15 months – Performance & CK
Performance at the wire test
3500
*** ***
††
***
3000
60
*
40
20
14
2500
2000
**
1500
1000
500
Dys TAM wt FEM
12
10
8
6
***
***
***
4
2
0
0
0
plasma CK activity (U/mL)
80
†
C
††
impulse (g.s)
B
time (s)
A
Creatine kinase levels
Dys TAM wt FEM
Dys TAM wt FEM
TAM treatment for 15 months increases motor performance and reduces CK blood levels
A. Treatment of mdx5Cv mice with tamoxifen for 15 months: Diaphragm morphology
A
- TAM increased myofibre number in dystrophic diaphragm
- TAM reduced necrosis and fibrosis in dystrophic diaphragm
- TAM normalized the size of the myofibres
- TAM increased the size of dystrophic diaphragm
H&E
Dys
B
TAM
wt
Fem
WGA
†††
40
< adiposis
fiber count
***
60
***
***
4000
**
***
20
***
2000
0
Dys TAM wt FEM
fiber diameter (m)
***
***
***
†
6000
< fibrosis
**
muscle surface (% total)
80
†††
†††
100
60
***
***
TAM
wt
40
20
< myofibres
0
0
Dys TAM wt FEM
Dys
FEM
TAM treatment for over a year makes diaphragm bigger, less fibrotic, and normalizes myofibre size
A.Treatment of mdx5Cv mice with tamoxifen for 15 months – Cardiac fibrosis
B
A
C
†††
fibrosis (% surface)
5
†
4
3
**
2
1
***
0
Dys TAM wt FEM
TAM treatment for 15 months reduces cardiac fibrosis
B. Treatment of adult mdx mice for 2 months : experimental design
AIm: effect on the low intensity stage of the disease – “mid-life”
TAM, 10 mg/kg/day for 15 Mo
Birth
normal
4-5 wks
necrosis
6-8 wks
regeneration
Death
16 months
cachexia
lowintensity, chronic disease
TAM (mg/kg/day)
6 Mo
8 Mo
The mdx mouse (muscular dystrophy, Xlinked)
0.1
1
10
raloxifene (RAL), fulvestrant (Faslodex)
weekly grid tests
locomotor activity
force recording
tissue collection
plasma CK
postmortem analyses
(From SCIENCEphotoLIBRARY)
B. Treatment of adult mdx mice for 2 months : dose - effect
Grid test
900
wt
1
10
time (sec)
700
TAM
500
0.1
RAL
Dys
300
100
0
1
2
3
4
5
6
7
8
1000
after treatment
before
***
time (sec)
800
600
***
400
***
**
***
***
**
***
200
reaches on top of the grid
treatment (week)
50
** **
*
** **
Dys 0.1 0.3
1
3
ns
**
40
30
20
10
0
0
Dys wt
Dys 0.1 0.3 1
TAM
3
10 RAL wt
10 RAL wt
TAM
TAM treatment of adult mice dose-dependently ameliorates motor performance
In vivo muscle contraction test
single twitches / phasic contractions
stimulator
intensity
duration
frequency
mouse
forcefrequency curve / tetanic contractions
force
transducer
data acquisition and analysis
muscle fatigue
1500
Dys
0 .1
1200
TAM
1
10
900
RAL
FEM
600
wt
300
0
0
20
40
60
80
100
125
p h a s ic t e n s io n ( % m a x .)
a b s o lu te p h a s ic t e n s io n (m N )
2
s p e c if ic p h a s ic t e n s io n (m N /m m )
B. Treatment of adult mdx mice for 2 months : Isometric muscle contractibility
100
75
50
25
0
100
20
§§§
§§§
§§§
§§§
*** *** ***
§§§
§
25
***
125
40
60
80
t im e to p e a k (m s )
100
75
50
0
*
§§§
§§§
§§§
*** *** ***
§
*
1
TAM
3
10
R A L F EM
wt
§
§§§
30
***
15
10
20
40
60
80
100
§
§§§
§§§
§§§
*** *** *** *** *** **
*
***
25
20
15
10
5
0
0
0.3
0
t im e ( m s )
5
25
0.1
20
100
20
Dys
40
t im e ( m s )
t im e f o r h a lf r e la x a tio n ( m s )
**
2
s p e c ific p h a s ic te n s io n (m N /m m )
§§§
60
0
0
t im e ( m s )
150
80
Dys
0.1
0.3
1
TAM
3
10
R A L F EM
wt
Dys
0.1
0.3
1
3
10
R A L F EM
TAM
TAM treatment of adult mice dose-dependently causes muscle to contact stronger and slower
wt
B. Treatment of adult mdx mice for 2 months: Membrane permeability
Evans blue dye uptake
Creatine kinase
diaphragm
10000
TAM
RAL
plasma CK activity (U/L)
Dys
§§§
§§
§
*** *** *** *** *** *** *** ***
8000
A mouse 18h post EBD injection
6000
4000
2000
0
Dys 0.1
0.3
1
3
10
RAL FEM wt
TAM
wt
20
15
CK activity (U/mL)
20
% EBD positive fibres
Poloxamer
***
***
***
10
5
**
**
**
***
5
0
Dys
Dys
**
10
*** ***
0
0.1
1
TAM
gastrocnemius
15
10
RAL P188 wt
Dys
0.1
1
10
RAL P188 wt
TAM
TAM
TAM treatment of adult mice dose-dependently stabilizes myofibre membranes
C. Mechanisms of action of tamoxifen on dystrophic muscle
Antagonism of TAM effects by pure antiestrogen fulvestrant / Faslodex
Faslodex
TAM
wt
wt + Fas
900
TAM
time (sec)
700
500
TAM + Fas
300
Dys
Dys + Fas
100
0
1
2
3
4
5
6
7
8
treatment (week)
Data suggest that
TAM acts, at least partly, via estrogen receptors:
TAM effects are antagonized by fulvestrant
TAM is potent: low nanomolar concentrations in plasma and tissues
TAM effect is antagonized by the pure antiestrogen fulvestrant
C. Tamoxifen alters plasma levels of cytokines
5
15
*
*
10
5
PDGF-BB (ng/ml)
T G F  ( n g /m l)
20
4
*
3
2
1
0
0
D ys
0 .1
1
10
R AL
Dys 0.1
wt
TAM
600
14
TAM
12
**
400
** **
***
***
300
200
100
IG F - I ( n g /m l)
OPN (ng/ml)
500
**
1
10
10
RAL
wt
***
**
*
8
**
6
4
2
0
0
Dys 0.1
1
TAM
10
RAL
wt
D ys
0 .1
1
10
R AL
wt
TAM
TAM  TGF, PDGF & osteopontin but strongly

IGF-1 levels
C. Tamoxifen reduces stress-induced calcium elevations in mdx cardiomyocytes
Results by
Emmanuel. Lauber,
Charlotte Lorin &
Ernst Niggli University of Berne
Evaluation of tamoxifen in dystrophic mice: summary of effects
Some signalling events in the dystrophic pathogenesis
LACK of DYSTROPHIN
kinase
activity
Ca2+ influx
oxidative
stress
membrane
fragility
multiple dysfunctions of muscle cell homeostasis
protein function
metabolism
FORCE GENERATION
mitochondrial function
gene expression
ATP deficit
muscle cell death
regeneration
fibrosis/adiposis
muscle wasting
inflammation
DEATH
Tamoxifen – Pro’s & Con’s
Pro’s
1. Mimics natural physiological pathway – females, estrogen action
2. Well known profile – 30 years of experience, incl. children
3. Exon-independent
4. Results validated in another lab
5. Cost – £ 1000 - 5000/year
Con’s
1.
2.
3.
4.
Not done according to GLP rules
Not a cure
Mechanism not yet fully clarified
No patent – difficulty in large funding
Action Duchenne 12th International Annual Conference “From the Labs to the Clinic”
Thank you for your attention
Special thanks to
Collaborators and contributors
Pharmacology (Geneva)
Olivier PETERMANN
Olivier DORCHIES
Royal Veterinary College (London, UK)
Hesham ISMAIL
Julie PATTEREUTENAUER
Dominic WELLS
Ophélie PATTHEYVUADENS
University of Bern
Emmanuel LAUBER
Ernst NIGGLI
Lausanne University Hospital (Lausanne)
Elyes DAHMANE
Laurent DECOSTERD
Swiss Federal Institute of Technology
(Lausanne)
Nicolas MERMOD
Funding
USA
France
Switzerland
Stefania PUTTINI
The Netherlands

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