GTx011, A Novel Agent That Improves Rheological Properties Of Sickle Cell Blood By Increasing
Oxygen Affinity For Hemoglobin.
Mira Patel, Donna Oksenberg, Abel Silva, Andreas Betz, Brian Metcalf and Uma Sinha
Global Blood Therapeutics, Inc., South San Francisco, CA 94080
Oxygen Affinity Studies
Polymerization and Rheological Studies
Hb modified by GTx011 maintains the oxyHb state in a low O2 environment
Hb modified by GTx011 recapitulates HbA at comparable fractions
1.2
1
1
0.8
0.6
0.6
0.4
0.2
0.2
0
0
350
390
430
470
510
350
550
590
630
λ
(nm)
670
A. %oxyHb at various concentrations
100
-x
-x
-x GTx011-HbS
released to tissues
time
(min)
0 .0
0 .0
0
Materials and Methods
Determination of oxygen equilibrium curves (OECs) in purified Hb, washed RBCs, and blood
Purified Hb samples (25µM) were incubated for 45 min at 37°C. Washed RBCs and blood
samples (Hematocrit, Hct, adjusted to 20%) were incubated for 1 hour at 37°C in the absence
or presence of compound. 100 µl of RBCs or blood was added to 5 mL of Hemox buffer at
37°C. Oxygen equilibrium curves (OECs) were then collected with a Hemox analyzer (8).
40
G Tx011 @ 4M
G Tx011 @ 2M
G Tx011 @ 1M
H b c o n tr o l
0
In vitro HbS Polymerization
HbS polymerization was evaluated in an adapted version of the assay described in ref. 9. HbS
was purified from SS RBC lysates through gel filtration and DE-52 anion exchange
chromatography. Purified HbS was mixed with either HbA or GTx011-HbS (final [Hb]=50µM) in
1.8 M potassium phosphate for 1 hour at 37oC. The reaction mixture was then passively deoxygenated (99.5 % N2/ 0.5% O2) at 4°C for 90 min and polymerization was induced via a
temperature jump from 4°C to 37°C. Polymerization was quantitated by measuring turbidity of
the HbS solution at 700 nm under continued hypoxia.
Viscosity
The viscosity of treated and control blood was measured in a cone plate viscometer (10).
Blood Hct was adjusted to 30%, and then incubated for 30 min at room temperature with
GTx011 or DMSO. The reaction mixture was deoxygenated for 2 hours in a gas permeable 24well plate placed within a humidified hypoxic chamber (97.6% N2/2.4% O2). Room
temperature equilibrated blood samples were run as oxygenated controls. Data was collected
at sheer rates ranging from 60 s-1 to 415 s-1.
40
50
20
E C 50
G T x 0 1 1 2 .4  M
A. GTx011 improves rheology in SS blood
-6
100
-4
10
80
EC50 of GTx011 in SS patient blood
400μM (n=3)
80
[G T x 0 1 1 ]
300M
100M
30M
25M
20M
10M
3M
1M
c o n tr o l
40
20
200 µM G Tx011
C o n tro l
60
Washed RBC
OEC % Δp50
cmp 1mM 3mM
5HMF
30
65
GTx006 59
78
GTx007 69
78
GTx011 76
83
40
20
0
Whole Blood
OEC % Δp50
1mM 3mM
10
47
49
71
63
>80
80
>80
0
10
20
30
40
40
50
B. GTx011 dose dependently decreases
viscosity in SS blood
2
0
50
[GTx011] Viscosity (cP)
deoxy SS cells
6.33
0.6 mM
5.78
0.8 mM
4.52
1.2 mM
4.02
1.4 mM
4.13
Oxy SS cells
3.69
S S c e lls
0 .6 m M
1 .2 m M
1 .4 m M
o x y S S c e lls
0
S
S
A
A
11
S
S
A
A
0
y
xy
xy
xy
Tx
o
o
O
Ox
G
De
De
M
S)
S
m
y
1 .6
ox
e
(D
100
200
300
400
S h e a r R a te
(s
-1
)
Figure 5. (A) Blood viscosity was determined for patient and donor samples under oxygenated and deoxygenated conditions. Hct was
adjusted to 30%. GTx011 reduced the viscosity of the deoxygenated SS blood (n=15) when compared to untreated deoxygenated SS blood.
(B) The viscosity of deoxygenated SS blood improves with increasing GTx011 concentrations; at higher doses GTx011 has a viscosity profile
similar to oxygenated SS blood. (cP=centiPoise)
Conclusions
B. Whole blood
100
4
2
0
A. Purified Hb
30
6
-2
GTx011 modulates O2 affinity in purified Hb, washed RBCs and SS Blood
60
4
G Tx006 38 M
5H M F
1 .6 m M
0
80
20
T im e ( m in )
(c P )
40
G T x 0 0 7 9 .0  M
60
10
6
20
40
0
Figure 4. (A) HbA mixed at various ratios with HbS (final [Hb]=50 μM) in 1.8 M potassium phosphate buffer. The increasing concentrations
of HbA delays the onset of polymerization. (B) GTx011 incubated with HbS was added to unmodified HbS at varying ratios. As the ratio of
GTx011-HbS to HbS increases a delay in polymerization can be observed, similar to the delay observed with HbA.
8
c m p lo g [ M ]
Figure 2. (A) %oxyHb at various time points during a 2 hour passive deoxygenation of Hb. GTx011 dose dependently delays the progression
to a deoxy state ([Hb] = 3 μM). The %oxyHb is calculated based upon the AUC of the sample spectra compared to the AUC of deoxyHb and
oxyHb spectra. (B) Dose response of GTx006, GTx007, GTx011 and 5HMF in the presence of 3µM Hb. GTx011 is the most potent compound
in the Oxygen Dissociation Assay (ODA). The %oxyHb is calculated at 108 minutes after deoxygenation. Δ%oxyHb calculated by subtracting
the hemoglobin control from the samples values.
% O 2 S a t u r a t io n
Oxygen Dissociation Assay
Determines the ability of GTx compounds to maintain the oxygenated state of hemoglobin
(oxyHb) under deoxygenated conditions. Purified Hb (3 µM) was incubated for 1 hour at 37oC
in the presence or absence of compound in 50 mM potassium phosphate buffer, pH=7.4 in 96well half-area plates. Plates were then placed in a SPECTROstar Nano plate reader (BMG
Labtech) at 37oC and N2 (flow rate = 20 L/min) was blown over the plates for 2 hours. Visible
spectra measurements were obtained every 6 min for 2 hours.
30
GTx011 reduces the hyperviscosity observed in deoxygenated SS blood
t im e ( m in )
100
20
8
0
Preserved RBC
morphology
10
100% H bS
1 0 % G T x 0 1 1 -H b S
2 0 % G T x 0 1 1 -H b S
3 0 % G T x 0 1 1 -H b S
4 0 % G T x 0 1 1 -H b S
T im e ( m in )
(c P )
60
-x
©2013 Global Blood Therapeutics
670
0 .4
0 .2
60
20
Fewer HbS polymers
630
0 .6
0 .2
B. EC50s of various GTx compounds
80
of GTx011
-x
O2
590
V is c o s it y
% oxyH b
Altered RBC
morphology
-x
-x
550
λ
(nm)
 % oxyHb
released to tissues
510
0 .4
GTx compounds dose dependently maintain the oxyHb state
% O 2 S a t u r a t io n
HbS
470
time
(min)
80
Extensive HbSpolymers formed
430
Figure 1. (A) Spectra of 3 µM Hb that is passively deoxygenated with N2 in a 96-well plate reader over a 2 hour time period. The Soret
bands (400-450nm) shows the Hb transitioning from an oxyHb state to a deoxyHb state. The same transition can be seen in the Q-band
region (500-600nm). (B) Spectra of 3 μM Hb incubated with 4 μM GTx011 shows that GTx011-Hb maintains the oxyHb state after 2 hours
of passive deoxygenation.
Under physiologic conditions, HbS will form polymers upon deoxygenation. The formation
of extensive polymers leads to sickling in the microcirculation. In vitro experiments show
that GTx011-treated HbS delays polymerization and thereby should delay formation of
sickled cells, allowing them to exit the microcirculation and get reoxygenated in the lungs.
O2
390
0 .6
B. GTx011-HbS + HbS mixing experiment
0 .8
100% H bS
10% H bA
20% H bA
30% H bA
40% H bA
V is c o s it y
Mechanism of Action
0.8
0.4
A. HbA + HbS mixing experiment
1 .0
0 .8
O D (7 0 0 n m )
1.2
OD
1.4
1 .0
B. GTx011 modified Hb
1.6
1.4
Though the childhood death rate of individuals with SCD has
drastically fallen due to disease management, transfusion therapy and hydroxyurea, there
is no standard treatment for SCD (5). It has been shown that modifying O2 affinity can
alter the polymerization dynamics in a manner that may be beneficial in SCD (6, 7).
However, to date, none of these modifiers are approved for use in patients.
In order to fulfill this unmet need, Global Blood Therapeutics has
developed a novel series of antisickling compounds that increase the O2 affinity of Hb,
delay polymerization and improve the rheological properties of SS blood.
A. Unmodified Hb
1.6
OD
Sickle cell disorder (SCD) is an inherited hemoglobinopathy
affecting a substantial portion of the African population and more than 90,000 AfricanAmericans (1, 2). It is a genetic disorder characterized by the presence of sickled red
blood cells (RBCs) that are the result of a single point mutation in Hemoglobin (Hb) in the
β globin at the 6th position. The hydrophilic glutamic acid is replaced by a hydrophobic
valine. This mutation does not alter the oxygen affinity or change the structure of Hb in
the R (relaxed) state. However, in the T (tense) state, Hb from SS patients (HbS) forms
polymers in low oxygen environments. When polymers within erythrocytes grow large
enough, they can lead to the development of non-deformable RBCs. If the RBCs are
further deoxygenated, they develop into the quintessential sickle shape. The nondeformable sickled cells increase the blood viscosity leading to a diminished flow through
capillary beds. These non-deformable cells lead to the physiologic effects of SCD: anemia,
ulcers, and major organ damage, including renal insufficiency and non-functional spleens
(3,4).
O D (7 0 0 n m )
Introduction
100
GTx011, an antisickling compound, has been shown to
• Left-shift the OEC curve, indicating a higher oxygen affinity than control Hb, RBCs and Blood.
• Maintain the oxyHb state under hypoxic conditions.
• Delay polymerization similar to HbA.
• Reduce the viscosity of deoxygenated SS blood.
GTx011 is anticipated to improve blood flow by increasing RBC deformability and reducing in vivo sickling events.
References
1. http://www.cdc.gov/ncbddd/sicklecell/data.html
3. Ashley-Koch, et al., Am. J. Epidemiol. 2000; 151: 839
5. Quinn, et al., Blood 2010; 115: 3447
7. Abdulmalik et al., British Journal of Haematology 2005; 128: 552
9. Adachi et al., Blood Cells 1982; 8:213
2. http://www.sicklecelldisease.org/index.cfm?page=scd-global
4. Nash, et al., Blood 1986; 67: 110
6. Nnamani et al., Chem. Biodivers. 2008; 5:1762
8. Guarnone et al., Haematologica 1995, 80:426
10. Wells/ Brookfield (Lubin et al., PNAS 1975; 72:43)
150
m m H g (p O 2 )
m m H g (p O 2 )
Figure 3. (A) Oxygen Equilibrium Curves (OEC) of 25 µM purified Hb incubated with various concentrations of GTx011 indicates that
modified Hb has a higher oxygen affinity when compared to unmodified Hb. (B) Representative OEC of 200 μM GTx011 incubated in SS
blood at 20% Hct displays a left shifted curve when compared to unmodified SS blood. Table summarizes the OECs of various GTx and
control compounds in washed RBCs and in whole blood.
Acknowledgements
Blood from SCD patients were kindly provided by:
UNC Comprehensive Sickle Cell Program, Chapel Hill
Children Hospital and Research Center Oakland
Kobe Dufu for polymerization data
Global Blood Therapeutics Project Team
For more information on sickling and polymerization results of GTx011 please visit our Oral presentation tomorrow (#316)