Hemoglobin F Enhancers by Suthat Fucharoen MD, Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Thailand

1. Hemoglobin F Enhancers
Suthat Fucharoen M.D.
Thalassemia Research Center
Th l i R hC
Institute of Molecular Biosciences
Mahidol University, Salaya Nakornpathom Thailand
University Salaya, Nakornpathom,
grsfc@mahidol.ac.th

2. Hb switching
-globin cluster
g
-globin cluster

 
HbF HbA

3. Fetal Hemoglobin Augmentation
in -Thalassemia
Increase -globin to bind excess -globin
HbF= 2 2
•Ameliorate the imbalance in globin chain
synthesis
    •Decreased ineffective erythropoiesis
•Reduced anemia
 
 
Genetic evidence:
Normal - Thalassemia higher F level -> milder disease

4. Genetic modifers of fetal globin expression: 3 QTL provide
50% of variability (Thein, Chui,Uda, Lettre 2008-9)
• Bcl-11A • Repressor of  globin
• p
-158 CT G promoter • Increases G globin
• 6p22.3-23.2 QTL, myb • Decreases F-cell
• Xp22 QTL
p number
• PDE 7B • Increases F-cells
• MAPK5 • Reduces  globin
• PEX7 • Increases  globin thru far
upstream site
• TOX
• Binds factor that binds a
negative regulator
• N td t
Not determined
i d

5. Proof‐of‐Concept with first generation inducers:
Hematologic (total Hgb) responses of 1‐4 gms/dl
• 5‐Azacytidine: 2.5 g/dl (1.5‐4)
(Ley & Neinhuis, Dunbar, Lowrey)
• H d
Hydroxyurea: mean 0.6‐1.5 g/dl
0 6 1 5 /dl
(Zeng, Hajjar, Fuchareon, Singer)
• A Butyrate +/‐ EPO: Mean 2 7 g/dl (1 ‐ 5 g/dl)
A. Butyrate +/ EPO: Mean 2.7 g/dl (1 5 g/dl)
Perrine et al
• Sodium Phenylbutyrate: 2 g, (1‐2.5)
(Collins, Giardina, Dover, 1994)
• EPO, Darbopoietin: 1.6‐2 g/dl
(Nisli, Rachmilevitz, Bourantas, Singer)
(Nisli Rachmilevitz Bourantas Singer)
• Isobutyramide: HbF increased in 4‐8 wks, Tx reduced
( p
(Capellini, Reich, 2000)
, , )

6. Mechanism of Action
• Erythropoietin: Stimulation of erythropoiesis
Optimize
O ti i erythroid survival, i hibit apoptosis, prevent maturation
th id i l inhibit t i t t ti
block
• Hydroxyurea, cytotoxic agents:
Induce proliferation of erythroid precursors, retain high
g
-globin synthesis
y
Less alpha chain excess, better survival, less ineffective erythropoiesis
5 Azacytidine,
5-Azacytidine, Butyrates :
Increase accessibility to -globin gene promoters by
altering chromatin structure (Hypomethylation, Histone
g ( yp y ,
deacetylase inhibition)
Specific activation of -globin gene

7. Limitations of first candidates
• Butyrate, PB require IV infusion or large
doses, intermittent Rx to avoid cell growth
suppression
• 5-Azacytidine  mutagenic /genotoxic,
injection required
• HU  cytostasis, maturation block
• EPO  requires injection, expensive
New candidate SCFADs:
ST20, ST7 - in preclinical development
ST20, ST7
Boosalis M et al, 2001; Pace, 2002; Castaneda S, 2005.
2001; 2002; 2005.
Cao H, Stamatoyannopoulos, G. 2005.
2005.

8. USE OF HYDROXYUREA IN THALASSEMIA
To reduce extramedullary erythropoietic masses;
To decompress spinal cord
● Konstantopoulos et al, Haematologica, 77 : 352-4, 1992
● Saxon et al., Brit.J. Haematol, 101:416-419, 1998
● Rigano et al, Hemoglobin 21 : 219-226, 1997
To treat patients
● Hajjar and Pearson J. Pediatrics, 125 : 490-492, 1994
● Huang SZ et al., Sci.Chin Bulletin, 37 : 1350-1359, 1994
● Zeng et al Brit J Haematol 90 : 557 563 1995
al, Haematol, 557-563,
● Fucharoen et al (in HbE/β-thal) Blood 87 : 887-892, 1996
● Olivieri et al, Lancet 350 : 491-492, 1997 (with Na-butyrate)
( y )
● Saxon et al, Ann NY Acad Sci, 850 : 459-460, 1998
● Styles et al, Ann NY Acad Sci, 850 : 461-462, 1998
● Loukopoulos et al, Ann NY Acad Sci, 850 : 120-128, 1998
● Tsiarta et al, The Bangkok Thalassemia Conference, 1999

9. Patient characteristics
N=
N 20 patients, male/female = 11/9
ti t l /f l 11/
Age: 19-55 (30 + 10) years
19- 10)
14 patients were splenectomized.
Transfusion requirement:
occasional 3/20,
20, none 17/20
17/
-thalassemia mutation:
17 8 4bp 8
IVSII-
IVSII-654 1 IVSI-
IVSI-5 1
71/
71/72 1 Capsite 1
(The other chromosome is / E )

10. Patient characteristics
Genotype of XmnI polymorphism
XmnI
• DNA polymorphism at -158 nt 5’ to the G -globin
gene was determined by PCR and XmnI digestion
XmnI
• 13 patients had XmnI -/+, HbF 30.22 + 8.77 %
mnI 30.
• 7 patients had XmnI -/- , HbF 13.86 + 9.16 %
mnI 13.

11. Hematologic Effects of Hydroxyurea
Baseline 36 months
Hb ( /dL)
(g/dL) 6.15  0.9 6.71  0.93**
93**
MCV (fL) 62.3  8.2
62. 68.3  8.2*
68.
MCH (pg) 17.25  
17.  **
 **
Reticulocyte (x106)
(x10 0.11  0.24 0.14  31**
31**
Hb E (%)
( ) 67.6  16.1
67. 16. 62.6  16.5**
62. 16.
Hb F (%) 24.2  11.9
24. 11. 28.8  16.7**
28. 16.
Absolute Hb F (g/dL) 1.88    **
 **
G:A ratio
i 1.18  0.18 1.65  0.79*
79*
Serum ferritin (ng/ml) 3,778    *
 *
Tf receptor (g/ml)
( 45.3  19
45. 37.4  15.1**
37. 15.
* = P-value < 0.001, ** = P-value < .05, N=17
P- 001, P- .05, N=17

12. Effect of hydroxyurea on HbF levels (%)
50
40
( )
HbF (%
30
20
10
0 3 6 9 12 15 18 21 24 27 30 33 36 mo

13. Quality of life in patients after treatment
with hydroxyurea
Data collection instruments: biodermographic data,
g p ,
QOL tool and treatment satisfaction tool
Improvement of QOL was measured by
1. Physical domain
2. Psychological domain
3. Social domain
4. Treatment related well-being domain

14. Quality of life after treatment with hydroxyurea
Domain Correlation P-value
Physical Before & After .522 .022
Psychological
P h l i l Before & After
B f Aft .804 < .001
Social Before & After .954 < .001
Others Before & After .723 < .001

15. Conclusion
• significant increase in HbF level(absolute
HbF, HbF) MCV
HbF % HbF), MCV, MCH
• HbF levels peaked at 6-12 months and
remain stable
• significant decrease in Hb E levels, serum
transferrin receptor
• slightly increase in Hb level, G : A ratio
• i
improvement on quality of life with minimal
t lit f lif ith i i l
toxicity

16. HYDROXYUREA (HU) AND THALASSEMIA
1.
1 WHAT PATIENTS YOU WILL PUT HIM/HER ON HU?
2. HOW DO YOU KNOW THAT HE/SHE IS A
RESPONDER OR NONRESPONDER?
3. AT WHAT DOSES/REGIMENS AND WHEN WILL
YOU CHANGE THE DOSES?
4. HOW DO YOU ASSESS THE LONG TERM
EFFECT OF HU?
5. WHAT IS THE LONG TERM TOXICITY OF HU?
IS IT SAVE TO HAVE BABY WHILE ON HU?
6.
6 WILL THERE ANY BETTER HB F ENHANCING
AGENTS AVAILABLE SOON?

17. 2 New Candidates / regimens which preserve
cellular proliferation
cellular proliferation
• Low dose SQ Decitabine in BTI ‐
Low‐dose, SQ, Decitabine in BTI
Y. Saunthararajah, J. DeSimone, TCRN
• Sodium 2,2 Dimethylbutyrate
– HemaQuest Pharmaceuticals

18. Low dose sub‐cutaneous
decitabine in  thalassemia intermedia
decitabine in ‐thalassemia intermedia
• Patient population: thalassemia intermedia (5 evaluable patients)
• Treatment: Decitabine 0.2 mg/kg SC 2X/week for 12 weeks
• Non‐cytotoxic mechanism of action: DNMT‐1 inhibition
• Dose limiting event: increase in platelet counts
• Safety: No increase in erythrocyte micronucleus or VDJ
y y y
recombination events
• Results: total Hgb baseline 7.88 ±0.88 g/dL, peak 9.04±0.77 g/dL
(p=0.004) (>1.5 g/dl increase in 2 patients)
‐ HbF 3.64±1.13 g/dL to 4.29 ±1.13 g/dL (p=0.003)
‐ significant favorable changes were noted in indices of red blood
cell densitometry and hemolysis

19. Decitabine Rx for 12 weeks in BTI
Decitabine Rx for 12 weeks in BTI

20. SDMB: Patient‐friendly,
SDMB: Patient friendly
2 tablets, once/day

21. Novel oral therapeutic SDMB: a gently
SDMB:
erythropoietic fetal globin inducer
• Targeted inducer, activates the  globin gene promoter
inducer,
without global epigenetic effects
• Favorable cellular actions- prolongs Bcl-xL
actions- Bcl-
• Active in transgenic and primate animal models
• Favorable PK (t1/2 =11 hrs) and safety profile in normal
subjects
• Phase I/II safety dose-ranging trials performed in beta
dose-
thalassemia intermedia, show HbF induction
• HbF induction 4-25%, mean 9% 8 of 9 patients
responded at 20 mg/kg dose (6 BTI, 3 HbE beta thal)
• L
Longer trials to determine effects on total Hb levels
ti l t d t i ff t t t l l l

22. Thalassemias t i l 2 representative sites for
Th l i trial: t ti it f
common mutations & QTLs

23. HbF with SDMB i B t Th l
ith in Beta Thalassemia i a d
i in dose
ranging trial: active dose induces HbF
Change from Baseline to End of Therapy
p = 0.0032
25
10
ange
5
Hb (%) Cha
0
bF
-5
-10
kg
kg
kg
kg
o
eb
g/
g/
g/
g/
ac
m
m
m
m
Pl
10
20
30
40

24. HbF Responses to SDMB in 6/6 BTI Lebanese Patients
(20 mg/kg to Day 41)
(20 /k t D 41)
% HbF in 003
% HbF in 001 % HbF in 002
64.0 70.0 63
63.0 62
% HbF
68.0 61
62.0 60
66.0 59
61.0 58
60.0 64.0 0 6 13 27 41
59.0
62.0
58.0 0 6 27 41 55
0 6 27 41 55 % HbF in 006
% HbF in 005
% HbF in 005 94.0
94 0
% HbF in 004 35.0 93.0
30.0
30.0 92.0
28.0
26.0 91.0
24.0 25.0
22.0 90.0
20.0 0 6 27 41 55
20.0
20 0
0 6 27 41 55
0 6 27 41 55
% HbF in Placebo
30.0
*PI: Dr. Adlette
28.0
28 0
26.0
Inati 0 6 27 41 55

25. Hematologic responses in Lebanese TI patients:
1.5 g/dl rise in total Hgb in 6 weeks
*Quantitative HbF, g/dl, not %

26. Ph I/II Dosing Trial: Total Hgb increased by 1.1 g/dl in
8 weeks in 4/6 Lebanese BTI, (range 0.6‐1.6)
9.5
9
8.5
g/dL)
8
l Hgb (g
7.5
7
Total
6.5
6 2001
2002
5.5 2003
2006
5
0 10 20 30 40 50 60 70 80 90 100 110 120
Day of Study

27. SUMMARY: Fetal globin gene inducers should benefit
l l b h l b f
many thalassemia patients, globally
• Proof‐of‐concept is established
• Novel oral, safe, tolerable agents now in trials – SDMB & DEC
• ‐ SDMB improves both cellular & globin deficiencies
– Decitabine has high activity, oral agent nearing clinical trial
• Novel highly‐potent, oral inducers identified
• New strategy: combine inducers with complimentary
molecular mechanisms
• Future approaches: designer Rx for genotypes, QTLs
• International collaborations are essential for thalassemia
trials
ti l