A Novel Selectin Antagonist, GMI1070, Prevents VasoOcclusion in Sickle Cell Mice by Inhibiting Leuko

1
A Novel Selectin Antagonist, GMI-1070, Prevents
Vaso-Occlusion in Sickle Cell Mice by Inhibiting
Leukocyte Adhesion and Activation Jungshan
Chang, John Patton, Arun Sarkar, John L.
Magnani, Paul S. FrenetteMount Sinai School of
Medicine, New York, NY, USA GlycoMimetics Inc.,
Gaithersburg, MD, USA
Results
Abstract
Summary
Figure 4. Effect of selectin inhibitors, GMI-1070
and GMI-1077, on leukocyte behavior and RBC
capture in TNF-a treated sickle mice.
Previous studies using intravital microscopy
in a sickle cell disease mouse model suggest that
adherent leukocytes play a key role in
vaso-occlusion by capturing circulating
erythrocytes in cremasteric venules1. In
addition, mice deficient in both P-and
E-selectins are protected from vaso-occlusion
(VOC) induced by surgical trauma and TNF-a
stimulation1, suggesting that targeting selectins
or their ligands represents a potentially useful
strategy. Selectins bind to specific sialylated
and fucosylated carbohydrate structures presented
by glycoprotein or glycolipid ligands. Here, we
tested the effect of novel small glycomimetic
selectin inhibitors, GMI-1070 and GMI-1077, on
leukocyte behavior and sickle cell VOC. Fully
engrafted male SCD mice were treated with TNF-a
and prepared for intravital microscopy
examination of the cremaster muscle 90 min later.
GMI-1070, GMI-1077, or vehicle (PBS) were
administered immediately prior to cytokine
stimulation (t 0 min), and an additional dose
was given at t 70 min. Another group of mice was
injected with antibodies against P-and
E-selectins (1 mg/kg) as positive control.
Several post-capillary and collecting venules
were examined between t 90min and t 150 min.
Antibody blockade of endothelial selectins
completely ablated leukocyte rolling, whereas
GMI-1070 and GMI-1077 significantly increased the
rolling flux fractions. Furthermore GMI-1070 and
GMI-1077 significantly reduced the recruitment of
adherent leukocytes compared to sickle mice
injected with PBS control. Although the reduction
in leukocyte adhesion was not as marked as with
anti-P and E-selectins, GMI-1070, in particular,
dramatically inhibited the capture of sickle RBCs
by adherent leukocytes and markedly improved the
blood flow in venules to levels observed in
non-sickle mice. The increased leukocyte rolling
fluxes by these glycomimetics suggest that they
inhibit E-selectin gt P-selectin. Since the
hallmark of E-selectin-mediated adhesion is the
slow leukocyte rolling, we analyzed leukocyte
rolling velocities in the various group and
indeed found a near 2-fold increase in rolling
velocities in sickle mice treated with GMI-1070
compared to PBS control. These studies suggest
that E-selectin-mediated adhesion/signaling may
play a more important role than previously
appreciated in the pathophysiology of SCD, and
suggest that GMI-1070 may be beneficial for the
treatment of sickle cell vaso-occlusion.
Figure 1. Schema of intravital microscopy
protocol

• GMI-1070 markedly improves blood flow rates in
  sickle cell mice challenged with surgical
  trauma and TNF-?.
• GMI-1070, but not GMI-1077, dramatically
  inhibited the ability of adherent leukocytes to
  capture circulating sickle RBCs.
• Both selectin antagonists, GMI-1070 and GMI-1077
  can increase rolling flux fraction and reduce the
  recruitment of adherent leukocytes in cremasteric
  venules in sickle cell mice.
• Leukocytes of mice treated with GMI-1070 rolled
  significantly faster than controls on cremasteric
  endothelium. In contrast, GMI-1077 had a
  marginal effect on rolling velocities.

A.
B.
C.




Table 1. Hemodynamic parameters in TNF-a primed
sickle cell mice treated with selectin
antagonists, antibodies and PBS.


12
1.5
2500

10
1.2
2000
8
0.9
1500
RBC Interactions/WBC/min
Rolling Flux Fraction()
Number WBCs / mm2
6
0.6
1000
4
0.3
500
2
Data are presented as mean SEM.P lt 0.05
compared to PBS - control or GMI-1076.
0
0.0
Experimental sickle cell mice weight ranged
from 22 to 29 g. During observation under
brightfield intravital microscopy, venular size
from 17 to 25 µm were recorded for future
analyses. The mean centerline velocity (Vrbc) in
sickle mice treated with GMI-1077 was slightly
higher than PBS-treated animals but the
difference was not significant. Vrbc in mice
treated with GMI-1070 or antibodies was gt 2-fold
increased of compared to PBS control. p lt 0.05.
0
PBS
PBS
PBS
anti-Psel anti-Esel
anti-Psel anti-Esel
anti-Psel anti-Esel
GMI-1070
GMI-1070
GMI-1077
GMI-1077
GMI-1070
GMI-1077
(A) Both GMI-1076 and GMI-1070 dramatically
increased leukocyte rolling flux fraction by
nearly 2-fold. (B) Average number of leukocytes
adherent to endothelium was significantly reduced
in sickle cell mice treated with either GMI-1077
or GMI-1070. (C) Both small molecule inhibitors
reduced the capture rates of erythrocytes per
adherent leukocytes, but only GMI-1070 displayed
a statistically significant inhibitory effect.
plt0.05 plt0.01 and plt0.001
Material Methods
Discussion
Figure 2. Mean calculated blood flow rate

Here, we demonstrate that the administration
of a pan-selectin antagonist GMI-1070 can
profoundly alter the course of acute
vaso-occlusive episodes in sickle cell mice. We
show that GMI-1070 dramatically improves flow
rates in microvessels of sickle cell mice that
have been challenged with a lethal crisis. This
increase in flow may prolong survival of sickle
cell animals since a strong positive correlation
between blood flow rate and survival has been
demonstrated in sickle cell mice2. GMI-1070
exhibits the capability to increase leukocyte
rolling flux fraction and leukocyte rolling
velocities, resulting in reduced numbers of
adherent leukocytes. These attributes suggest
that GMI-1070 effectively interrupted
E-selectin-mediated rolling machinery since slow
rolling is controlled by E-selectin. Furthermore,
GMI-1070 markedly affected the capture of
circulating erythrocytes by adherents leukocytes,
suggesting an association between RBC-WBC
interactions and signals emerging from E-selectin
ligands. These in vivo studies thus suggest
that GMI-1070 or similar compounds may be
beneficial in the treatment or prevention of
sickle cell disease manifestations. Although
further studies are needed to understand further
its mechanisms and evaluate the conditions that
would benefit from selectin inhibition, these
data indicate that GMI-1070 should be assessed in
a clinical trial to treat acute painful crises.
Figure 5. Leukocyte rolling velocity histograms

1000

A.
750
25
1
Blood flow rate (nL/s)
PBS control (n193)
The blood flow rate in GMI-1070- and P-/E-sel
antibodies-treated mice was significantly higher
than in PBS control or GMI-1077-treated animals.
This difference was not due to venular size as
the average venular diameter was nearly identical
(21µm) among the three groups. plt0.01 and
plt0.001
20
500
0.8
15
Vmean20.91.1 mm/s
0.6
250
Cumulated frequency
10
0.4
0
0.2
5
PBS
anti-Psel anti-Esel
GMI-1070
GMI-1077
0
0
B.
0
20
40
60
80
100
120
140
160
180
200
220
Leukocyte rolling velocity ( mm/s)
25
Figure 3 Representative images of venules from
sickle mice treated with PBS, GMI-1070, GMI-1077,
or anti- P and E selectin antibodies.
GMI-1077 (n467)
20
The velocity of leukocyte rolling was evaluated
from (A) 193 leukocytes in 42 venules of
PBS-treated sickle cell mice (n10). Leukocytes
in sickle cell mouse venules treated with PBS
rolled at average velocity of 20.9 1.1 µm/s,
ranging from 0.3 to 90 µm /s. (B) Leukocyte
rolling velocities in GMI-1077-treated sickle
mouse venules were 25.5 1.1 µm/s, ranging from
2.5 to 250 µm/s (analyses derived from 467
leukocytes in 51 venules of 4 GMI-1077-treated
mice). (C) In contrast, leukocyte rolling
velocity in GMI-1070-treated mice were
significantly greater than PBS-treated controls
and GMI-1077 treated animals, with an average
rolling velocity of 37.8 1.2µm/s, ranging from
2.5 to 250 µm/s (data from 481 leukocytes in 58
venules of 5 GMI-1070-treated mice) plt0.001.
(D) A cumulative frequency histogram for these
three groups demonstrated that GMI-1070 shifted
leukocyte rolling from slower to faster with
2-fold higher median rolling velocities than PBS.
15
frequency ()
Vmean25.51.1 mm/s
References
10
5
0
C.

• Turhan A, Weiss LA, Mohandas N, Coller BS,
  Frenette PS. Primary role for adherent leukocytes
  in sickle cell vascular occlusion a new
  paradigm. Proc Natl Acad Sci U S A.
  2002993047-3051.
• Jungshan Chang, Patricia A. Shi, Elaine Y Chiang,
  and Paul S FrenetteIntravenous immunoglobulins
  reverse acute vaso-occlusive crises in sickle
  cell mice through rapid inhibition of neutrophil
  adhesion. Blood, Oct 2007 doi10.1182/blood-2007-
  04-084061.

PBS
10 m
GMI-1077
25
GMI-1070 (n481)
20
15
Vmean37.81.2 mm/s
10
5
Each still frame was taken at the 30 min
timepoint after TNF-a injection. Both small
molecule selectin antagonists and anti-P/E
selectin antibodies significantly reduced the
number of adherent leukocytes (yellow circles),
and RBCs interacting with adherent leukocytes
(yellow arrows). The white arrows indicate the
direction of blood flow.
0
10
20
30
40
50
60
70
80
90
100
300
0
Leukocyte rolling velocity ( mm/s)
.