Results

1. 1st dose (GMI-1077,GMI-1070, anti-P-/E-selantibodies or PBS i.c. ) Carotid artery cannulation TNF- a 0.5 mg i.p. 2nd dose -60 -50 -20 0 70 90 150 X Recording Time of death Tracheotomy Cremaster surgery Animals Berkeley SCD mouse bone marrow was transplanted into lethally irradiated C57BL/6 animals to generate age- and gender-matched genetically identical cohorts of SCD mice. Fully chimeric male sickle cell mice (expressing >97% human globin, including betaS) were subjected to intravital microscopy 3-5 months after bone marrow transplantation1. Intravital experimental protocol We used the protocol shown on Fig. 1 in which sickle cell mice were anesthetized by i.p. injection of a mixture of 2% chloralose and 10% α-urethane in PBS (6 mL/kg). A polyethylene tube was inserted into the trachea in order to facilitate spontaneous respiration and the right carotid artery was cannulated for administration of selectin antagonists (GMI-1070 and GMI-1077), mixture of antibodies against P- and E- selectins, or vehicle PBS control. The cremaster muscle was gently exteriorized and then continuously superfused throughout the experiment with warmed (37°C) bicarbonate-buffered (pH 7.4) saline aerated with a mixture of 95% N2 and 5% CO2. Each experimental mouse received 2 doses of each antagonist, PBS saline containing antibodies or equal volume of PBS control. The first injection was performed immediately after TNF–a injection (T0) and the second injection was made at 70 minutes (T70) after TNF-a administration. Twenty minutes after exposure to antagonists, PBS or the antibody cocktail, 8-12 venules of each mouse were videotaped over a period of 60 min, with each venule recorded continuously for at least 2 min. Venules were visualized with a custom-designed intravital microscope (MM-40, Nikon), using a 60X water immersion objective (Nikon). Images were recorded using a charge-coupled device video camera (Hamamatsu, Bridgewater, NJ) and video recorder (Sony SVHS, SVO-9500). Venular diameter was measured with a video caliper. Centerline red cell velocity (VRBC) was measured for each venule in real time using an optical Doppler velocitometer (Texas A&M, College Station, TX). Wall shear rate (g) was calculated based on Poiseuille’s law for a Newtonian fluid, g = 2.12 (8Vmean) / Dv, where Dv is the venule diameter, Vmean is estimated as VRBC / 1.6, and 2.12 is a median empirical correction factor obtained from actual velocity profiles measured in microvessels in vivo. Blood flow rate (Q) was calculated as Q= Vmeanπd2/4, where d is venule diameter, and is expressed as nL/s. Blood samples taken immediately after recording, through a cardiac puncture, were used to determine periphery white blood cell counts by using Hemocytometer methods. Image analyses for brightfield intravital microscopy All analyses were made using playback assessment of videotapes as previously described. Briefly, rolling WBCs were defined as those moving at a velocity less than that of free-flowing erythrocytes in a given vessel and counted over 1-min intervals per venule. Adherent WBCs were defined as those remaining stationary for at least 30 s over a 100 µm venular segment. RBCs were identified by their size and shape (discoid and sickle-shaped cells). An interaction between RBCs and adherent WBCs was defined as the arrest of an RBC on an adherent WBC for more than 2 video frames (> 0.07 s). This time interval corresponds to a readily discernable adhesion event when the videotape is played in real time. Leukocyte rolling velocity (Vwbc) was calculated by dividing the traveled distance by the tracking time or as the average translation over 2 seconds for 10 WBCs per venule, and expressed as μm / s. Leukocyte transit time was calculated as 100 μm / Vwbc. The flux fraction (F) of rolling leukocytes, corresponding to the percentage of leukocytes that are rolling per min, was calculated by F = WBCr / (0.25πd2 Vrbc60 [WBC]), where WBCr r is the number of leukocytes rolling past a fixed reference point in the venule per minute, d is venule diameter, Vrbc is centerline velocity, and [WBC] is the systemic leukocyte count. Statistical analyses All data are displayed as mean ± SEM. Parametric data were analyzed using ANOVA. Statistical significance for non-parametric distributions (RBC-WBC interactions) was assessed using the Mann-Whitney test. A value of p less than 0.05 was deemed significant. PBS Vmedian=16 mm/s GMI-1077 Vmedian=20 mm/s GMI-1070 Vmedian=32.5 mm/s Anti-P/Esel GMI-1070 A Novel Selectin Antagonist, GMI-1070, Prevents Vaso-Occlusion in Sickle Cell Mice by Inhibiting Leukocyte Adhesion and ActivationJungshan 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-α 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 > 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-α 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 < 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 > 2-fold increased of compared to PBS control. *p < 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-1070 GMI-1077 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. *p<0.05;**p<0.01 and ***p<0.001 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 (n=193) 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. **p<0.01 and ***p<0.001 20 500 0.8 15 Vmean=20.9±1.1mm/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 (n=467) 20 The velocity of leukocyte rolling was evaluated from (A) 193 leukocytes in 42 venules of PBS-treated sickle cell mice (n=10). 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) *** p<0.001. (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 (%) Vmean=25.5±1.1mm/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. 2002;99:3047-3051. • 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; doi:10.1182/blood-2007-04-084061. 10 m PBS GMI-1077 25 GMI-1070 (n=481) 20 15 Vmean=37.8±1.2mm/s*** 10 5 Each still frame was taken at the 30 min timepoint after TNF-α 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) .