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1. STATE-OF-THE-ART OF HMC, HUMAN AND
VETERINARY REALITY (PART 2)
Raffaele Coppini
University of Florence
Department NeuroFarBa, division of Pharmacology
Human HCM: state of the art and focus on drug therapy

2. 2
Human
HCM:
state-of-the-art
and
focus
on
therapy
Hypertrophic cardiomyopathy (HCM)
is a primary disorder of the myocardium,
characterized by the presence of
increased left ventricular (LV) wall thickness*
that is not solely explained by abnormal loading
conditions.
DEFINITION
European Society of Cardiology Guidelines Eur. H. Journal 2015
* >15mm or >12mm in patients
with pathogenic gene mutations

3. 3
Human
HCM:
state-of-the-art
and
focus
on
therapy
Mayo clinic website: “http://www.mayoclinic.com/health/medical/IM00586”
CARDIOMIOPATIA IPERTROFICA (HCM)
• Prevalenza: 1/500
• Prima causa di morte improvvisa
cardiaca <40aa
• Importante causa di scompenso
cardiaco a tutte le età
Cuore Normale HCM
No ostruzione Ostruzione
OSTRUZIONE DINAMICA

4. 4
Human
HCM:
state-of-the-art
and
focus
on
therapy
MORTE CARDIACA IMPROVVISA NEI
GIOVANI (<40aa)
• 1000 casi/anno in Italia
• HCM è la prima causa di MI negli
atleti
• Generalmente avviene a causa di
una aritmia letale (fibrillazione
ventricolare)

5. 5
Human
HCM:
state-of-the-art
and
focus
on
therapy
SCOMPENSO CARDIACO IN HCM
• 5-10% dei casi di HCM evolve verso lo
scompenso
• Lo scompenso è più frequente se le
mutazioni sono in specifici geni
(filamento sottile)
FORMA RESTRITTIVA FORMA DILATATIVA
Da Coppini et al. JACC 2014

6. 6
Human
HCM:
state-of-the-art
and
focus
on
therapy
• Malattia del sarcomero
• Mutazioni sarcomeriche in >65%
dei pazienti
• Anomalie funzionali dei
miofilamenti

7. 7
Human
HCM:
state-of-the-art
and
focus
on
therapy
MYBPC3- non E258K
MYBPC3-E258K
MYH7
MYL-2
Complex
TPM1
TNNT2
TNI
MYH7 (36%)
MYL-2
TPM1
Complex
TNNT2
TNNI2
MYBPC3 (57%)
Myofilament
Positive (62%)
Myofilament
Negative (38%)
Florence Cohort (>1000 pts.)

8. 8
Human
HCM:
state-of-the-art
and
focus
on
therapy

9. 9
Human
HCM:
state-of-the-art
and
focus
on
therapy
GENE TROPONINA T
TNNT2
GENE
A-ACTINA
ACTC
MIOSINA
Catene leggere regolatrici
MYL2
GENE A-TROPOMIOSINA
TPM1
GENE
PROTEINA C
MYBPC3
GENE Beta-MIOSINA
Catene pesanti
MYH7
MIOSINA
Catene leggere essenziali
MYL3
GENE TROPONINA I
TNNI3
TEST GENETICO TRADIZIONALE: 8 geni sarcomerici
“Richard, Circulation 2003”

10. 10
Human
HCM:
state-of-the-art
and
focus
on
therapy
THE ERA OF NEXT GENERATION SEQUENCING (>90 genes)
10
HCM
MYBPC3, MYH7, MYL2, MYL3,
TNNI3, TNNT2, TPM1, LAMP2, CSRP3,
PRKAG2, ACTA1,TCAP, TNNC1, ACTN2, JPH2,
TTN, ANKRD1, MYH6, MYOZ2, GLA,
ACTC1,CALM3, MYO6, OBSCN, NEXN,
FKTN, SMC1A, CALR3, COX15, MYLK2,
CASQ2, CAV3, DES, FXN, NDUFV2, PLN,
RAF1, SRI, VCL
DCM
MYBPC3, MYH7, TNNI3,
TNNT2, TPM1, CSRP3, TCAP,
TNNC1, ACTN2, TTN, ANKRD1,
MYH6, MYOZ2, ACTC1, DES,
PLN, VCL, LMNA, TAZ,
MYOZ1, FHL2, LDB3/ZASP,
SCN5A, EYA4, SGCD, BAG3,
TMPO, DMD, ABCC9,
SCO2,XIRP2, CMYA5, PDE4DIP,
XIRP1, UNC45B, MYO6, EMD,
PLEC, MLYCD, DNAJC19,
SLC25A3
ARVC
DSG2, DSP, PKP2,
DSC2,
TMEM43, JUP, RYR2, TGFB3,TTN
RCM
MYL2,MYL3,
PRKAG2,GLA, TNNI3
LVNC
CASQ2, MYBPC3,
MYH7,TNNI3,TNNT2,TP
M1,ACTC1,PLN,LMNA,
TAZ,LDB3/ZASP,SCN5A,
DTNA, DMPK FLNA
AKAP9, ANK2,
CACNA1C, CACNB2,
CASQ2, CAV3, GPD1L,
KCNE1,KCNE2,KCNE3,
KCNJ2,KCNQ1,RYR2,
SCN1B,SCN4B,SCN5A,
SNTA1
CHANNELLOPATHIES
Courtesy of Dr. Francesca Girolami, Careggi, Florence

11. 11
Human
HCM:
state-of-the-art
and
focus
on
therapy
Yacoub, M. H. (2014) Cardiomyopathy on the move
Nat. Rev. Cardiol. doi:10.1038/nrcardio.2014.157
GENES AND CARDIOMYOPATHIES

12. 12
Human
HCM:
state-of-the-art
and
focus
on
therapy
MYBPC3- non E258K
MYBPC3-E258K
MYH7
MYL-2
Complex
TPM1
TNNT2
TNI
MYH7 (36%)
MYL-2
TPM1
Complex
TNNT2
TNNI2
MYBPC3 (57%)
Myofilament
Positive (62%)
Myofilament
Negative (38%)
Florence Cohort (>1000 pts.)

13. 13
Human
HCM:
state-of-the-art
and
focus
on
therapy
MYBPC3- non E258K
MYBPC3-E258K
MYH7
MYL-2
Complex
TPM1
TNNT2
TNI
MYH7 (36%)
MYL-2
TPM1
Complex
TNNT2
TNNI2
MYBPC3 (57%)
Myofilament
Positive (62%)
Myofilament
Negative (38%)
Florence Cohort (>1000 pts.)
GENOTYPE-PHENOTYPE CORRELATIONS

14. 14
Human
HCM:
state-of-the-art
and
focus
on
therapy GENOTYPE-PHENOTYPE CORRELATIONS
Thin-filament HCM:
• Increased risk of sudden death
• Increased risk of heart failure
(systolic or diastolic)
Coppini et al. JACC 2014

15. 15
Human
HCM:
state-of-the-art
and
focus
on
therapy
Onde Q, ipertrofia e sovraccarico del VS (QRS ampi)
DIAGNOSI: ECG

16. 16
Human
HCM:
state-of-the-art
and
focus
on
therapy
Il Cuore Normale
DIAGNOSI: ECOCARDIOGRAFIA
ASSE LUNGO

17. 17
Human
HCM:
state-of-the-art
and
focus
on
therapy
Il Cuore Normale
DIAGNOSI: ECOCARDIOGRAFIA
APICALE 4-CAMERE

18. 18
Human
HCM:
state-of-the-art
and
focus
on
therapy
Cardiomiopatia Ipertrofica
DIAGNOSI: ECOCARDIOGRAFIA
ASSE LUNGO:
• Ipertrofia settale

19. 19
Human
HCM:
state-of-the-art
and
focus
on
therapy
Cardiomiopatia Ipertrofica
DIAGNOSI: ECOCARDIOGRAFIA
ASSE CORTO:
• Ipertrofia
spesso anche
della parete
libera

20. 20
Human
HCM:
state-of-the-art
and
focus
on
therapy
Cardiomiopatia Ipertrofica
DIAGNOSI: ECOCARDIOGRAFIA
APICALE 4-
CAMERE:
• Funzione
sistolica
supernormale
• Disfunzione
diastolica

21. 21
Human
HCM:
state-of-the-art
and
focus
on
therapy DIAGNOSI: ECOCARDIOGRAFIA
Doppler
Trans
mitrale
Doppler
Vene polm
Tissue-
doppler

22. 22
Human
HCM:
state-of-the-art
and
focus
on
therapy RISONANZA MAGNETICA

23. 23
Human
HCM:
state-of-the-art
and
focus
on
therapy IMAGING AVANZATO
Contrast MRI:
LGE (fibrosis)
PET (Ammonia):
Local blood flow
(microvascualar
dysfunction)

24. 24
Human
HCM:
state-of-the-art
and
focus
on
therapy Hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
STAGES OF HCM

25. 25
Human
HCM:
state-of-the-art
and
focus
on
therapy Hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
STAGES OF HCM 30-35%

26. 26
Human
HCM:
state-of-the-art
and
focus
on
therapy
Ostruzione al TEVS: SAM
ECOCARDIOGRAFIA
Insuff.
Mitr

27. 27
Human
HCM:
state-of-the-art
and
focus
on
therapy
Ostruzione al TEVS: Gradiente
ECOCARDIOGRAFIA
AO
LV
LA

28. 28
Human
HCM:
state-of-the-art
and
focus
on
therapy
Ostruzione: Terapia chirurgica
ECOCARDIOGRAFIA
Miectomia settale
transaortica PRE
POST
Scomparsa del SAM
Abbattimento del gradiente

29. 29
Human
HCM:
state-of-the-art
and
focus
on
therapy Hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
STAGES OF HCM

30. 30
Human
HCM:
state-of-the-art
and
focus
on
therapy
Ipocinetico-dilatativa

31. 31
Human
HCM:
state-of-the-art
and
focus
on
therapy
Restrittiva

32. 32
Human
HCM:
state-of-the-art
and
focus
on
therapy Hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
STAGES OF HCM

33. 33
Human
HCM:
state-of-the-art
and
focus
on
therapy Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
-β blockers
-Verapamil/
Diltiazem
-Disopyramide
-β blockers
-Diuretics
-Verapamil
-β blockers
-Diuretics
-ACEi – ARBs
-MRA
-------------- Current therapies according to
AHA 2013 and ESC 2014 HCM guidelines
-β blockers
TREATMENT OF HCM-RELATED SYMPTOMS

34. 34
Human
HCM:
state-of-the-art
and
focus
on
therapy Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
-β blockers
-Verapamil/
Diltiazem
-Disopyramide
-β blockers
-Diuretics
-Verapamil
-β blockers
-Diuretics
-ACEi – ARBs
-MRA
-------------- Current therapies according to
AHA 2013 and ESC 2014 HCM guidelines
-β blockers
-β blockers
-Amiodarone
-Sotalol
-Anti-
coagulants (AF)
TREATMENT OF HCM-RELATED SYMPTOMS

35. 35
Human
HCM:
state-of-the-art
and
focus
on
therapy Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
-β blockers (I B)
-Verapamil/
Diltiazem (I B)
-Disopyramide (I B)
-β blockers (II C)
-Diuretics (II C)
-ACEi – ARBs (II C)
-MRA (II C)
-------------- Current therapies according to
AHA 2013 and ESC 2014 HCM guidelines
(CLASS OF RECOMMENDATION)
-β blockers (II C)
-β blockers (I C)
-Amiodarone(I C)
-Sotalol (II C)
-Anti-
coagulants (AF)
(I B)
-β blockers (II C)
-Diuretics (II C)
-Verapamil (II C)
CLASS I B = Benefit greatly exceeds the risk; limited evidence
from a single randomized trial or multiple nonrandomized studies.
CLASS I C = Treatment should be administered; evidence only
from expert opinions/ case studies.
CLASS II C = Benefit exceeds
the risk and it is reasonable to treat;
evidence only from expert opinions/
case studies.

36. 36
Human
HCM:
state-of-the-art
and
focus
on
therapy
1. Disopyramide: new roles for an old drug
2. β- blockers: new mechanisms and
perspectives
3. Other experimental therapies for HCM
4. Late sodium current inhibition in
symptomatic HCM: from basic science to
randomized trials
5. Preventing phenotype development in
HCM mutation carriers
DRUG THERAPY IN HCM: OUTLINE

37. 37
Human
HCM:
state-of-the-art
and
focus
on
therapy
1. Disopyramide: new roles for an old drug
2. β- blockers: new mechanisms and
perspectives
3. Other experimental therapies for HCM
4. Late sodium current inhibition in
symptomatic HCM: from basic science to
randomized trials
5. Preventing phenotype development in
HCM mutation carriers
OUTLINE

38. 38
Human
HCM:
state-of-the-art
and
focus
on
therapy Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
-β blockers
-Verapamil/
Diltiazem
-Disopyramide
-β blockers
-Diuretics
-Verapamil
-β blockers
-Diuretics
-ACEi – ARBs
-MRA
-------------- Current therapies according to
AHA 2013 and ESC 2014 HCM guidelines
-β blockers
TREATMENT OF HCM-RELATED SYMPTOMS

39. 39
Human
HCM:
state-of-the-art
and
focus
on
therapy ROLE OF DISOPYRAMIDE
• First-line drug for LVOT
obstruction (Class I B
indication), alone or in
addition to β-blockers or
verapamil.
• Consistently reduces LVOT
gradient (negative inotropic
effect?)
• Improves survival in
obstructive patients
• Mechanisms poorly studied
38%
13%
50%
Disopyramide + β blocker
Disopyramide
β blocker
Disopyramide in the Florence cohort
of obstructive patients (>250)

40. 40
Human
HCM:
state-of-the-art
and
focus
on
therapy DISOPYRAMIDE IMPROVES SURVIVAL

41. 41
Human
HCM:
state-of-the-art
and
focus
on
therapy
HCM patients with Obstruction Septal myectomy Surgical Sample
1 cm
Diastole
Left
Atrium
Left
Ventricle
Aorta
CONTROLS: non-failing surgical patients: septal thickness <
14mm, ejection fraction >55%, aortic valve disease
Basal inter-ventricular septum
Turbulent blood flow in the LVOT
STUDY COHORT: HCM patients with symptomatic obstruction
Late Sodium Current in Hypertrophic Cardiomyopathy
Modified from: Coppini C.,
Mugelli A. et al. Late Sodium
Current Inhibition Reverses
Electromechanical
Dysfunction in Hypertrophic
Cardiomyopathy. Circulation
2013
HCM
Control
Calibration bar = 10 µm
Cardiomyocytes

42. 42
Human
HCM:
state-of-the-art
and
focus
on
therapy
Basal Dis
0
100
200
300
400
Time
to
peak
(ms)
Basal Dis
0
100
200
300
400
500
600
RT
50%
1E-6 1E-5 1E-4
0,0
0,5
1,0
Relative
tension
(%)
Disopyramide (M)
EC50: 5.29±1.55 µM
Therapeutic conc.: 5 µM
Basal
Dis 5uM
200 ms 200 ms
Normalized
1
mN/mm
2
# p<0.05, paired
Coppini et al. Unpublished Data
#
#
DISOPYRAMIDE IN HUMAN HCM MYOCARDIUM

43. 43
Human
HCM:
state-of-the-art
and
focus
on
therapy DISOPYRAMIDE IN HUMAN HCM CARDIOMYOCYTES
0,2 0,5 1
0,10
0,15
0,20
0,25
0,30
Diastolic
[Ca
2+
]
i
(A.
U.)
Pacing rate (Hz) Means±SEM from
13 cells/ 4 patients
• Disopyramide shortens APD
• Disopyramide reduces
diastolic Ca and the
amplitude of Ca transients
• Effects mediated by INaL
block?

44. 44
Human
HCM:
state-of-the-art
and
focus
on
therapy
1. Disopyramide: new roles for an old drug
2. β- blockers: new mechanisms and
perspectives
3. Other experimental therapies for HCM
4. Late sodium current inhibition in
symptomatic HCM: from basic science to
randomized trials
5. Preventing phenotype development in
HCM mutation carriers
OUTLINE

45. 45
Human
HCM:
state-of-the-art
and
focus
on
therapy BETA BLOCKERS IN HCM
• First-line drug for LVOT obstruction (Class I B indication).
• To be used in patients with ICD and recurrent arrhythmias.
Primary arrhythmia prevention?
• No apparent effects on survival in adult patients
• Improve survival in children with HCM (single study).
High-dose beta-blockers
Low-dose beta-blockers
No beta-blockers

46. 46
Human
HCM:
state-of-the-art
and
focus
on
therapy BETA BLOCKERS IN HCM
• Hypertrophic cardiomyopathy (HCM) presenting in
childhood has higher mortality (annual rate 4.6% to
5.8%) than in adult life (1% to 4%)
• HCM-related deaths during childhood are:
• Sudden (arrhythmic) 48%
• Heart failure 36%
• Surgical complications 16%
• Hypertrophic cardiomyopathy during childhood is
associated with increased activity of cardiac sympathetic
nerves (extreme sympathetic over-activity)

47. 47
Human
HCM:
state-of-the-art
and
focus
on
therapy
High-dose beta-blockers
Standard or no Therapy
-Standard Dose βB, n = 18
patients; 0.8 to 4 mg/kg/day of
propranolol)
-High-dose therapy (HDβB), n
= 26 patients; 5 to 23
mg/kg/day of propranolol, or
equivalent doses of metoprolol
or atenolol)
-No specific therapy, n=20
BETA BLOCKERS IN HCM

48. 48
Human
HCM:
state-of-the-art
and
focus
on
therapy
• Likely beneficial for symptomatic patients with no
obstruction (Class II B indication).
• Nadolol (80-120 mg/d) to be preferred in obstructive pts.
over propranolol or metoprolol (Class II C).
• Bisoprolol (5-10 mg/d) to be preferred in end-stage (II C).
• All studies with β-blockers in HCM are 20+ years old
• Beta-blockers reduce obstruction and improve diastolic
function, however the mechanisms are unclear
• No studies on human cardiomyocytes or samples
BETA BLOCKERS IN HCM: CONTROVERSIES

49. 49
Human
HCM:
state-of-the-art
and
focus
on
therapy
1. Disopyramide: new roles for an old drug
2. β- blockers: new mechanisms and
perspectives
3. Other experimental therapies for HCM
4. Late sodium current inhibition in
symptomatic HCM: from basic science to
randomized trials
5. Preventing phenotype development in
HCM mutation carriers
OUTLINE

50. 50
Human
HCM:
state-of-the-art
and
focus
on
therapy Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
-β blockers
-Verapamil/
Diltiazem
-Disopyramide
-β blockers
-Diuretics
-Verapamil
-β blockers
-Diuretics
-ACEi – ARBs
-MRA
-------------- Current therapies according to
AHA 2013 and ESC 2014 HCM guidelines
-β blockers
-β blockers
-Amiodarone
-Sotalol
-Anti-
coagulants (AF)
TREATMENT OF HCM-RELATED SYMPTOMS

51. 51
Human
HCM:
state-of-the-art
and
focus
on
therapy Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
-β blockers
-Verapamil/
Diltiazem
-Disopyramide
-INaL blockers
-β blockers
-Diuretics
-Verapamil
- INaL blockers
-Perhexiline
-β blockers
-Diuretics
-ACEi – ARBs
-MRA
-------------- Current therapies according to
AHA 2013 and ESC 2014 HCM guidelines
----------------- Promising Future Options
-β blockers
- INaL blockers
-Perhexiline
-β blockers
-Amiodarone
-Sotalol
-Anti-
coagulants (AF)
-INaL blockers
DISEASE-MODIFYING THERAPIES?
-Anti-oxydants
-ARBs
-Statins

52. 52
Human
HCM:
state-of-the-art
and
focus
on
therapy
Polakit Teekakirikul et al. J Cell Biol 2012;199:417-421
TARGETING CELLULAR AND EXTRACELLULAR REMODELLING

53. 53
Human
HCM:
state-of-the-art
and
focus
on
therapy EXPERIMENTAL THERAPIES: STATINS
Atorvastatin reduced hypertrophy in HCM rabbits
….but failed to reduce hypertrophy in a pilot study on 32 patients

54. 54
Human
HCM:
state-of-the-art
and
focus
on
therapy
Reduced fibrosis
EXPERIMENTAL THERAPIES: SARTANS
Baseline
1 y treatment
Reduced TGF-β
Disarray persists
Change in LGE%
Change in LV Mass
Losartan reduced fibrosis in transgenic rabbits and reduced
LGE and LV mass in patients after 1 year of treatment (pilot
study on 20 patients)

55. 55
Human
HCM:
state-of-the-art
and
focus
on
therapy EXPERIMENTAL THERAPIES: SARTANS
• Single center, randomized, placebo-controlled trial with
losartan in HCM
• Primary endpoint: change in LV mass at MR
• 133 total patients (50% per branch)
• No significant change in LV mass after 12 months of
treatment when compared with placebo
INHERIT TRIAL

56. 56
Human
HCM:
state-of-the-art
and
focus
on
therapy
1. Disopyramide: new roles for an old drug
2. β- blockers: new mechanisms and
perspectives
3. Other experimental therapies for HCM
4. Late sodium current inhibition in
symptomatic HCM: from basic science to
randomized trials
5. Preventing phenotype development in
HCM mutation carriers
OUTLINE

57. 57
Human
HCM:
state-of-the-art
and
focus
on
therapy Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
-β blockers
-Verapamil/
Diltiazem
-Disopyramide
-INaL blockers
-β blockers
-Diuretics
-Verapamil
- INaL blockers
-Perhexiline
-β blockers
-Diuretics
-ACEi – ARBs
-MRA
-------------- Current therapies according to
AHA 2013 and ESC 2014 HCM guidelines
----------------- Promising Future Options
-β blockers
- INaL blockers
-Perhexiline
-β blockers
-Amiodarone
-Sotalol
-Anti-
coagulants (AF)
-INaL blockers
DISEASE-MODIFYING THERAPIES?
-Anti-oxyd.
-ARBs
-Statins

58. 58
Human
HCM:
state-of-the-art
and
focus
on
therapy
0,5 1,0 1,5 2,0 2,5
-1,0
-0,5
0,0
Current
density
(pA/pF)
Time (s)
0
50
100
150
200
250
I
NaL
integral
(A*ms*F
-1
)
**
-120
-60
0
1s
Increased INaL
Modified from: Coppini C., Mugelli A. et al. Late Sodium
Current Inhibition Reverses Electromechanical
Dysfunction in Hypertrophic Cardiomyopathy.
Circulation 2013
-80
-40
0
40
Membrane
Voltage
(mV)
300 ms
Control HCM
-80
-40
0
40
Membrane
Voltage
(mV)
300 ms
Prolonged APD
Late Sodium Current in Hypertrophic Cardiomyopathy
2 s
40
mV
Stimuli
= Early After Depolarization, EAD
Cellular arrhythmias Delayed relaxation

59. 59
Human
HCM:
state-of-the-art
and
focus
on
therapy
ATP
ase
PLB
Sarcoplasmic
reticulum
Ca
Na
Ca
Na
K
Na
Myofilaments
Ca
RyR
SERCA
Sarcolemma
T-tubule
Ca
Mitochondria
ATP
produced
MCU
K
Na
H
Na Na
P
Ca
P
500 ms
150
nM
200 ms
5
mN/mm2
AP
Ca Transient
Force
From: Coppini R et al. Regulation of intracellular Na(+) in health and disease: pathophysiological mechanisms and implications for treatment. Glob Cardiol Sci Pract. 2013
Consequences of Increased INaL in cardiomyocytes
CONTROL CARDIOMYOCYTE

60. 60
Human
HCM:
state-of-the-art
and
focus
on
therapy
-80
-40
0
40
300 ms
ATP
ase
PLB
Ca
Na
Ca
Na
K
Myofilaments
Ca
RyR
SERCA
Sarcolemma
T-tubule
Ca
Mitochondria
ATP
produced
MCU
K
Na
H
Na Na
P
AP
Ca2+
Force
Ca
P
P
P
Sarcoplasmic
reticulum
Na
Na
From: Coppini R et al. Regulation of intracellular Na(+) in health and disease: pathophysiological mechanisms and implications for treatment. Glob Cardiol Sci Pract. 2013
Consequences of Increased INaL in HCM cardiomyocytes
HCM CARDIOMYOCYTE

61. 61
Human
HCM:
state-of-the-art
and
focus
on
therapy Effects of Late Sodium Current Inhibition.
 Direct effects (decreased net inward
current during AP plateau)
o Shortening of AP duration
o Suppression of Early after-
depolarizations (EADs)
o Increased repolarization stability
 Mediated by the reduction of [Na+]i
 ↓ [Ca2+]i (NCX)
 ↓ Delayed after-depolarizations
(DADs)
 Ameliorated Diastolic function and
reduction of myocardial ischemia
 Effects on long term remodelling
Beneficial effects of INaL block
Na+
Na+
Modified from: Antzelevitch C, Belardinelli L et al.: The Role of Late INa in Development of Cardiac Arrhythmias. Handb Exp Pharmacol. 2014

62. 62
Human
HCM:
state-of-the-art
and
focus
on
therapy Antiarrhythmic Drugs that inhibit INaL
Modified from: Antzelevitch C. et al. The Role of Late INa in
Development of Cardiac Arrhythmias Handb Exp Pharmacol. 2015.
DRUG INaL/INaT INaL/IKr
Amiodarone 13 ≤ 1.5
Flecainide 2.9–5 < 0.1–2
Ranolazine 9–38 1.5–2
GS-967 50-100 >100
Selectivity for INaL
(uM)
Late INa
Peak INa
Modified from: Belardinelli L. et al.
Cardiac late Na+ current: Proarrhythmic
effects, roles in long QT syndromes, and
pathological relationship to CaMKII and
oxidative stress. Heart Rhythm 2015

63. Ranolazine: Key Clinical Trials
ROLE
N=746
Chronic angina • Chaitman BR et al. J Am Coll Cardiol 2004;43:1375-82
• Chaitman BR, et al. JAMA. 2004;291:309-316
• Stone PH, et al. J Am Coll Cardiol. 2006;48:566-575
• Morrow DA, et al. JAMA. 2007;297:1775-1783
• Kosiborod M et al. J Am Coll Cardiol 2013;61:2038–45
• Koren MJ et al. J AM Coll Cardiol 2007;49:1027-34
CARISA
N=823
Chronic
angina
Ranolazine
vs placebo
on top of
standard therapy
ERICA
N=565
Chronic
angina
Ranolazine
vs placebo
on top of
amlodipine 10mg
MERLIN
TIMI-36
N=6560
Non-STE
ACS
Ranolazine
vs placebo
on top of
standard care
MARISA
N=191
Chronic
angina
Ranolazine
vs placebo
Total patients enrolled = 9,088
TERISA
N=949
Chronic
Angina/
DM2
Ranolazine
vs placebo
on top of
1 to 2 antianginals
• Approved for Chronic Stable Angina treatment
• Studies in pts with Diabetes, Neuropathic Pain, Heart Failure, HFpEF

64. 64
Human
HCM:
state-of-the-art
and
focus
on
therapy Effects of Late Sodium Current Inhibition.
 Direct effects (decreased net inward
current during AP plateau)
o Shortening of AP duration
o Suppression of Early after-
depolarizations (EADs)
o Increased repolarization stability
 Mediated by the reduction of [Na+]i
 ↓ [Ca2+]i (NCX)
 ↓ Delayed after-depolarizations
(DADs)
 Ameliorated Diastolic function and
reduction of myocardial ischemia
 Effects on long term remodelling
Beneficial effects of INaL block
Na+
Na+
Modified from: Antzelevitch C, Belardinelli L et al.: The Role of Late INa in Development of Cardiac Arrhythmias. Handb Exp Pharmacol. 2014

65. 65
Human
HCM:
state-of-the-art
and
focus
on
therapy
0.2 Hz
0.5 Hz
1 Hz
0.0
0.2
0.4
0.6
0.8
1.0
APD90%
(s)
-80
-40
0
40
80
Membrane
Voltage
(Vm)
200 ms
HCM Basal
HCM + Ran
500ms
50
pA
-120
0
1s
HCM Basal
HCM + Ran
HCM + TTX
B
a
s
a
l
R
a
n
0
100
200
I
Na-L
integral
(A*ms*F
-1
)
Transmembrane
Current
INaL Inhibition shortens APD and abolish EADs in human HCM
Ranolazine 10µM
Modified from: Coppini C., Mugelli A. et al. Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Hypertrophic Cardiomyopathy. Circulation 2013

66. 66
Human
HCM:
state-of-the-art
and
focus
on
therapy
500ms
50
pA
-120
0
1s
HCM Basal
HCM + Ran
HCM + TTX
B
a
s
a
l
R
a
n
0
100
200
I
Na-L
integral
(A*ms*F
-1
)
Transmembrane
Current
INaL Inhibition shortens APD and abolish EADs in human HCM
1 s
50
mV
= Early After Depolarization (EAD)
EAD
DAD
0
20
40
60
Occurrence
(%
of
cells)
Stimuli
Ranolazine 10µM
Modified from: Coppini C., Mugelli A. et al. Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Hypertrophic Cardiomyopathy. Circulation 2013

67. 67
Human
HCM:
state-of-the-art
and
focus
on
therapy
-80
-60
-40
-20
0
20
40
60
Membrane
potential
(mV)
Basal
GS-967 1M
Wash-out
500 ms
0.2 Hz
0.5 Hz
1 Hz
0,0
0,2
0,4
0,6
0,8
1,0
APD90%
(s)
Basal
GS-967 1M
Wash-out
0.2 Hz
##
##
##
## p<0.01, paired
Means±SEM from
25 cells/ 9 patients
INaL Inhibition shortens APD and abolish EADs in human HCM
GS-967 1µM
Coppini et al. Unpublished Data

68. 68
Human
HCM:
state-of-the-art
and
focus
on
therapy Effects of Late Sodium Current Inhibition.
 Direct effects (decreased net inward
current during AP plateau)
o Shortening of AP duration
o Suppression of Early after-
depolarizations (EADs)
o Increased repolarization stability
 Mediated by the reduction of [Na+]i
 ↓ [Ca2+]i (NCX)
 ↓ Delayed after-depolarizations
(DADs)
 Ameliorated Diastolic function and
reduction of myocardial ischemia
 Effects on long term remodelling
Beneficial effects of INaL block
Na+
Na+
Modified from: Antzelevitch C, Belardinelli L et al.: The Role of Late INa in Development of Cardiac Arrhythmias. Handb Exp Pharmacol. 2014

69. 69
Human
HCM:
state-of-the-art
and
focus
on
therapy
-80
-40
0
40
300 ms
ATP
ase
PLB
Ca
Na
Ca
Na
K
Myofilaments
Ca
RyR
SERCA
Sarcolemma
T-tubule
Ca
Mitochondria
ATP
produced
MCU
K
Na
H
Na Na
P
AP
Ca2+
Force
Ca
P
P
P
Sarcoplasmic
reticulum
Na
Na
From: Coppini R et al. Regulation of intracellular Na(+) in health and disease: pathophysiological mechanisms and implications for treatment. Glob Cardiol Sci Pract. 2013
HCM CARDIOMYOCYTE
Consequences of Increased INaL in cardiomyocytes

70. 70
Human
HCM:
state-of-the-art
and
focus
on
therapy
ATP
ase
PLB
Ca
Na
Ca
Na
K
Na
Myofilaments
Ca
RyR
SERCA
Sarcolemma
T-tubule
Ca
Mitochondria
ATP
produced
MCU
K
Na
H
Na Na
P
AP
Ca2+
Force
Ca
P
P
P
Sarcoplasmic
reticulum
Na
-80
-40
0
40
80
Membrane
Voltage
(Vm) 200 ms
From: Coppini R et al. Regulation of intracellular Na(+) in health and disease: pathophysiological mechanisms and implications for treatment. Glob Cardiol Sci Pract. 2013
HCM CARDIOMYOCYTE
Consequences of INaL inhibition in HCM cardiomyocytes

71. 71
Human
HCM:
state-of-the-art
and
focus
on
therapy
HCM Basal
HCM + Ran
0.2 0.5 1
0.30
0.35
0.40
0.45
0.50
Diastolic
[Na
+
]
(F/F
max
)
Pacing rate (Hz)
* * **
Ranolazine decreases [Na+]i
500 ms
200
nM
TP T50% T90%
0.0
0.4
0.8
1.2
Duration
(s)
**
* **
Ranolazine
hastens Ca
transient
kinetics
Modified from: Coppini C., Mugelli A. et al. Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Hypertrophic Cardiomyopathy. Circulation 2013
INaL inhibition ameliorates Ca homeostasis in HCM cardiomyocytes

72. 72
Human
HCM:
state-of-the-art
and
focus
on
therapy
0.2 0.5 1
100
150
200
250
Diastolic
[Ca
2+
]
i
(nM)
Pacing rate (Hz)
**
** **
HCM Basal
HCM + Ran
Control
** =p<0.01
Ranolazine reduces intracellular diastolic [Ca]
INaL inhibition ameliorates Ca homeostasis in HCM cardiomyocytes

73. 73
Human
HCM:
state-of-the-art
and
focus
on
therapy
0,5
1,0
1,5
2,0
2,5
3,0
3,5
Intracellular
Ca
2+
(arbitrary
units)
TP T50%T90%
0,0
0,4
0,8
1,2
1,6
2,0
2,4
Duration
(s)
0,2 0,5 1
0,10
0,15
0,20
0,25
0,30
Diastolic
[Ca
2+
]
i
(A.
U.)
Pacing rate (Hz)
0.2 Hz 0.5 Hz 1 Hz
##
##
#
##
##
##
# p<0.05, paired
## p<0.01, paired
Means±SEM from
15 cells/ 6 patients
INaL inhibition ameliorates Ca homeostasis in HCM cardiomyocytes
GS-967

74. 74
Human
HCM:
state-of-the-art
and
focus
on
therapy Effects of Late Sodium Current Inhibition.
 Direct effects (decreased net inward
current during AP plateau)
o Shortening of AP duration
o Suppression of Early after-
depolarizations (EADs)
o Increased repolarization stability
and reduced spatial APD dispersion
 Mediated by the reduction of [Na+]i
 ↓ [Ca2+]i (NCX)
 ↓ Delayed after-depolarizations
(DADs)
 Ameliorated Diastolic function and
reduction of myocardial ischemia
 Effects on long term remodelling
Beneficial effects of INaL block
Na+
Na+
Modified from: Antzelevitch C, Belardinelli L et al.: The Role of Late INa in Development of Cardiac Arrhythmias. Handb Exp Pharmacol. 2014

75. 75
Human
HCM:
state-of-the-art
and
focus
on
therapy
Ranolazine lowers diastolic tension and
accelerates twitch relaxation
1s Basal Ran Wash out
3mN/mm
2
HCM Basal
HCM+Ran
75
200 ms
Normalized
Amplitude
INaL inhibition ameliorates diastolic function in HCM myocardium
0,0 0,5 1,0 1,5 2,0 2,5 3,0
120
160
200
240
280
Peak
Time
(ms)
Frequency (Hz)
Basal
GS967
Wash-out
Basal
GS-967 1M
10
mN/mm
2
#
#
#
#
# p<0.05, paired
Means±SEM from 8 trabeculae / 7 patients
GS-967
Ranolazine

76. 76
Human
HCM:
state-of-the-art
and
focus
on
therapy
Basal Iso Iso+Ran
0
100
200
300
Force
%
Basal
Iso
Iso + Ran
200 ms
1
mN/mm
2
# #
Basal Iso Iso+Ran
0
100
200
300
400
500
600
RT50%
(ms)
Basal Iso Iso+Ran
0
100
200
300
400
Time
to
peak(ms)
# n.s. # n.s.
# p<0.05
RANOLAZINE CAN BE A DRUG FOR OBSTRUCTION

77. 77
Human
HCM:
state-of-the-art
and
focus
on
therapy
Basal Iso Iso+Ran
0
100
200
300
Force
%
Basal
Iso
Iso + Ran
200 ms
1
mN/mm
2
# #
Basal Iso Iso+Ran
0
100
200
300
400
500
600
RT50%
(ms)
Basal Iso Iso+Ran
0
100
200
300
400
Time
to
peak(ms)
# n.s. # n.s.
# p<0.05
RANOLAZINE CAN BE A DRUG FOR OBSTRUCTION
0
1
2
3
4
5
Tension
(mN/mm
2
)
0
1
2
3
4
5
Tension
(mN/mm
2
) #
Basal
Contraction
Diso
Ran

78. 78
Human
HCM:
state-of-the-art
and
focus
on
therapy Clinical Trials with Late Na Current Blockers in HCM
RESTYLE-HCM
• Recruitment: 80 non-obtructive
symptomatic patients with reduced exercise
capacity (VO2
max <75% of predicted)
• Double-blind, randomized, placebo-
controlled multicenter study (11 centers
across europe)
• Primary Objective: improved exercise capacity at CPET
• Secondary Objectives: improved diastolic function,
ameliorated symptomatic status, lowered proBNP, reduced
arrhythmic burden

79. 79
Human
HCM:
state-of-the-art
and
focus
on
therapy Clinical Trials with Late Na Current Blockers in HCM
RESTYLE-HCM
• Objective: demonstrate the efficacy
of ranolazine in improving exercise
capacity and reducing symptoms in
HCM patients
• Ranolazine or placebo added on
top of standard treatment
• Primary: objective not met (p≈0.10)
• Secondary: significant reduction of proBNP, reduced
arrhythmic burden at Holter
Placebo
V3
Treatment phase
Ranolazine
Time 0
V4 V6
3° month
5° month
V6
V4
3° month
V5
V5
1° month
1° month
V3
Placebo
Time 0
Treatment phase
V1
Ranolazine
500 mg bid
Time -14 d
V2
Ranolazine
750 mg bid
Time -7 d
Titratio
n
phase
Titratio
n
phase
Placebo
Time -14d
V1
Placebo
Time -7d
V2
5° month
Ranolazine
1000 mg bid
screening
Symptomatic
standard therapy
RESULTS (preliminary: final statistics analysis ongoing)
Problems: low number of pts., difficult recruitment

80. 80
Human
HCM:
state-of-the-art
and
focus
on
therapy
• 40 specialized centers in USA and Europe (Italy, Germany, UK) to enrol 180
symptomatic patients (including obstructive).
 PRIMARY: improvement of exercise capacity
(VO2
max at CPET)
 SECONDARY: improvement of: symptomatic status (questionnaire),
arrhythmias, diastolic function, degree of obstruction (gradient)
• Level 2/3, randomized, double-blind, placebo-controlled, with GS-6615
Objectives
TREATMENT: 30mg single dose, 3 mg/die for 12 weeks, then 6 mg/die for 12
weeks (total 24 weeks)
Clinical Trials with Late Na Current Blockers in HCM

81. 81
Human
HCM:
state-of-the-art
and
focus
on
therapy
Exercise
Capacity
Diastolic
Function
QoL
Arrhythmias
Zio Patch
Echo
MLHFQ
CPET (VO2)
Obstruction
(GS-6615)

82. 82
Human
HCM:
state-of-the-art
and
focus
on
therapy Clinical Trials with Late Na Current Blockers in HCM
• The first multicentric randomized trial in HCM with a novel drug (eleclazine)
• Aimed at registration with specific HCM indication
• Results expected beginning of 2017
• Based on solid preclinical data on human samples
-80
-60
-40
-20
0
20
40
60
Membrane
potential
(mV)
Basal
GS-967 1M
Wash-out
500 ms
0,5
1,0
1,5
2,0
2,5
3,0
3,5
Intracellular
Ca
2+
(arbitrary
units)

83. 83
Human
HCM:
state-of-the-art
and
focus
on
therapy
1. Late sodium current inhibition in
symptomatic HCM: from basic science to
randomized trials
2. Disopyramide: new roles for an old drug
3. β- blockers: new mechanisms and
perspectives
4. Other experimental therapies for HCM
5. Preventing phenotype development in
HCM mutation carriers
OUTLINE

84. 84
Human
HCM:
state-of-the-art
and
focus
on
therapy
Stages of hypertrophic cardiomyopathy (HCM)
Modified from Iacopo Olivotto et al. Circ Heart Fail. 2012;5:535-546
Non-Hypertrophic
Development
of LV
Hypertropy
No current therapies are able to
prevent cardiac phenotype
development in young carriers of
mutations associated with HCM
Non-Hypertrophic Non-Hypertrophic
Healthy
mutation
carrier
Prevention of HCM phenotype

85. 85
Human
HCM:
state-of-the-art
and
focus
on
therapy Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
No disease expression
-Diltiazem
-Ranolazine/
new INaL blockers
-Gene therapy
-β blockers
-Verapamil/
Diltiazem
-Disopyramide
-INaL blockers
-β blockers
-Diuretics
-Verapamil
- INaL blockers
-Perhexiline
-β blockers
-Diuretics
-ACEi – ARBs
-MRA
-------------- Current therapies according to
AHA 2013 and ESC 2014 HCM guidelines
----------------- Promising Future Options
-β blockers
- INaL blockers
-Perhexiline
-β blockers
-Amiodarone
-Sotalol
-Anti-
coagulants (AF)
-INaL blockers
PHENOTYPE PREVENTION
-Anti-oxydants
-ARBs
-Statins

86. 86
Human
HCM:
state-of-the-art
and
focus
on
therapy Prevention of HCM phenotype

87. 87
Human
HCM:
state-of-the-art
and
focus
on
therapy
Means ± S.E. from 38 patients: 18 Diltiazem and 20 Placebo Age, yrs: 14,1 ± 1,7 Diltiazem and 17,3 ± 2,1 Placebo
During treatment After treatment
Prevention of HCM phenotype

88. 88
Human
HCM:
state-of-the-art
and
focus
on
therapy
ATP
ase
PLB
Ca
Na
Ca
Na
K
Na
Myofilaments
Ca
RyR
SERCA
Sarcolemma
T-tubule
Ca
Mitochondria
ATP
produced
MCU
K
Na
H
Na Na
P
AP
Ca2+
Force
Ca
P
P
P
Sarcoplasmic
reticulum
Na
-80
-40
0
40
80
Membrane
Voltage
(Vm) 200 ms
From: Coppini R et al. Regulation of intracellular Na(+) in health and disease: pathophysiological mechanisms and implications for treatment. Glob Cardiol Sci Pract. 2013
HCM CARDIOMYOCYTE
INaL inhibition may exert long-term effects on myocardial remodeling

89. 89
Human
HCM:
state-of-the-art
and
focus
on
therapy
Genotyping
45
ranolazine-
treated
mice
90 newborn
(R92Q or WT)
siblings
45 vehicle-
treated mice
1 month 11-12 months
~ 22 mice
R92Q (R92Q-RAN)
WT (WT-RAN)
R92Q (R92Q-KET)
WT (WT-KET)
Chow
0.5% Ran.
+ 0.03%
Ketoc.
6 couples of
progenitors
(♀WT+♂R92
Q)
~ 22 mice
~ 22 mice
~ 22 mice
Chow
0.03%
Ketoc.
(vehicle)
-In vivo experiments (Echo, MRI)
-Ex vivo experiments
(cells, trabeculae,hystology, PCR)
Diast. Septum
0,5
1,0
Septal
Thickness
(mm)
0
2
4
6
8
Areas
(mm
2
)
Left Atrium
WT R92Q
-KET
R92Q
-RAN
LV
LA
LAA
LV
LA
LAA
LV
LA
LAA
WT R92Q-KET
E
A
E A
R92Q-RAN
E
A
E/A
0,0
0,4
0,8
1,2
1,6
E/A
Ratio
WT
R92Q-KET
R92Q-RAN
**
*
*
**
NS
**
WT
R92Q
-KET
R92Q
-RAN
INaL inhibition exerts beneficial effects on myocardial remodeling in HCM
R92Q-TnT transgenic mice were
treated with Ran lifelong
Ran lifelong treatment prevented the development
of LV hypertrophy and LA dilatation in mutants
Ran lifelong treatment prevented the development
of diastolic dysfunction in mutants **
NS
**
Bar=1mm
Bar=1mm
* =p<0.05
** =p<0.01
Coppini et al. Unpublished

90. 90
Human
HCM:
state-of-the-art
and
focus
on
therapy
1 minute after Gd injection 30 minutes after Gd injection
WT
R92Q-KET
R92Q-RAN
RV
LV
RV
LV
RV
LV
END-DIASTOLE END-SYSTOLE
Extracellular Space
0.0
0.2
0.4
0.6
LGE
Fraction
**
NS
**
LV Mass
60
70
80
90
100
mg
**
NS
*
**
NS
*
LV diastole
0
20
40
60
80
Volume
(L)
LV EF
40
50
60
70
80
Fraction
(%)
**
NS
*
Cardiac Magnetic Resonance
Fibrosis
0
1
2
3
4
5
6
Pico
Sirius
Red
Fraction
**
**
**
WT
R92Q-KET
R92Q-RAN
0.0
0.5
1.0
1.5
2.0
2.5
mRNA
expression
of
TGF
**
*
*
R92Q-KET
WT
R92Q-
RAN
Histology
INaL inhibition prevents structural myocardial remodeling in HCM

91. 91
Human
HCM:
state-of-the-art
and
focus
on
therapy
• Administration of ranolazine in HCM mice prevents LV
hypertrophy and hypercontractility, ameliorates diastolic
function and reduces extracellular fibrosis
• Ranolazine-treatment normalizes cellular dysfunction
• Ranolazine or novel late Na current blockers may be employed
in young carriers of HCM-mutations to prevent phenotype
onset and disease progression
Prevention of HCM phenotype

92. 92
Human
HCM:
state-of-the-art
and
focus
on
therapy GENE THERAPY
In homozygous Mybpc3 mutants,
reintroduction of WT Mybpc3 gene via
AAV-mediated cardiac-specific gene-
transfer prevents hypertrophy and
functional deterioration (single
injection at 1 week of age).

93. 93
Human
HCM:
state-of-the-art
and
focus
on
therapy THE BRIGHT FUTURE: HCM AS A TREATABLE DISEASE
Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012
No disease expression
-Diltiazem
-Ranolazine/
new INaL blockers
-Gene therapy
-β blockers
-Verapamil/
Diltiazem
-Disopyramide
-INaL blockers
-β blockers
-Diuretics
-Verapamil
- INaL blockers
-Perhexiline
-β blockers
-Diuretics
-ACEi – ARBs
-MRA
-------------- Current therapies according to
AHA 2013 and ESC 2014 HCM guidelines
----------------- Promising Future Options
-β blockers
- INaL blockers
-Perhexiline
-β blockers
-Amiodarone
-Sotalol
-Anti-
coagulants (AF)
-INaL blockers
PHENOTYPE PREVENTION
-Anti-oxydants
-ARBs
-Statins

94. 94
Human
HCM:
state-of-the-art
and
focus
on
therapy
Dept. Physiology
University of Florence
N. Piroddi
B. Scellini
J.M. Pioner
F. Gentile
B. Tosi
C. Tesi
C. Ferrantini
C. Poggesi
Referral Center for
Cardiomyopathies
AOU Careggi – Firenze
I. Olivotto
F. Cecchi
B. Tomberli
K. Baldini
Dept. Pharmacology
University of Florence
L. Sartiani
V. Spinelli
M. del Lungo
L. Diolaiuti
L. Dini
S. Blescia
L. Mazzoni
E. Cerbai
Genetics AOU Careggi
F. Girolami
S. Bardi
F. Torricelli
Cardiac Surgery Units
AOU Careggi: M.Yacoub,
A.Rossi, S.Stefano
Villa Beatrice: G. Popov
LENS: C. Crocini
M. Scardigli
L. Sacconi
F. Pavone
Thanks
Tucson: J. Tardiff
Gilead Sciences
L. Belardinelli
S. Rajamani
Fundings: