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Polymers bearing sLe^x-mimetics are superior inhibitors of E-selectin-dependent leukocyte rolling in vivo¹

MAJID ALI^*, ANNE E. R. HICKS^*, PAUL G. HELLEWELL^*, GEBHARD THOMA and KEITH E. NORMAN^*,2

^* Cardiovascular Research Group, Division of Clinical Sciences (North), University of Sheffield, Sheffield, UK; and
Novartis Pharma AG, Basel 4002, Switzerland

²Correspondence: Cardiovascular Research Group, Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7AU, UK. E-mail: k.norman{at}shef.ac.uk

SPECIFIC AIMS

To investigate whether multivalent presentation of sLe^x-mimetics on a polylysine backbone enhances their activity against selectin-dependent rolling in post capillary venules.

PRINCIPAL FINDINGS

1. CGP77175 A and polymer 4 inhibit E-selectin-dependent leukocyte rolling
Mono- and multivalent selectin antagonists (Fig. 1 ) were tested against E-selectin-dependent leukocyte rolling in vivo. E-selectin-dependent leukocyte rolling was induced in the cremaster muscle of P-selectin-deficient mice using TNF- (500 ng, intrascrotal for 2 h). After observation of control rolling, different doses of CGP77175A were injected i.v. and changes in leukocyte rolling determined. Substantial inhibition was seen in mice treated with 3mg/kg CGP77175A (Fig. 2 A). A higher dose of CGP77175A gave no further inhibition. Polymer 4 (1200 lysines with 50% CGP77175A loading) also dose-dependently inhibited E-selectin-dependent leukocyte rolling and was effective at doses as low as 0.1 mg/kg (Fig. 2A) . E-selectin blocking antibody (10E6) produced a similar effect to Polymer 4 (Fig. 2A) at 0.3 mg/kg but not at 0.1 mg/kg.

View larger version (31K): [in this window] [in a new window]   Figure 1. Structures of tested inhibitors.

View larger version (26K): [in this window] [in a new window]   Figure 2. Effect of inhibitors on E- and P-selectin-dependent leukocyte rolling. A) Inhibition of E-selectin-dependent leukocyte rolling by CGP77175A and polymer 4. Baseline leukocyte rolling was determined from a 1 min intravital microscopy observation recorded 2 h 30 min after TNF--stimulation of a P-selectin –/– mouse cremaster. Inhibitors were injected iv at 2 h 31 min and changes in leukocyte rolling determined between 2 h 32 and 2 h 33 min. E-selectin blocking antibody was injected at the end of some experiments as a positive control. Data are presented as mean ± SEM of n = 4 experiments. Asterisks indicate significant difference from baseline rolling (*P<0.05). B) Duration of action of CGP77175A and polymer 4. Mice treated as above were monitored for up to 1 h after inhibitor injection. Data are typical of n = 4 experiments. All plots originate at rolling ratio = 1 (baseline) and t = 0 (immediately prior to inhibitor treatment). Subsequent measurements were taken at 1 min and every 10 min until rolling ratio returned to or exceeded baseline. C) Effect of polymer 4 on P-selectin-dependent leukocyte rolling. Baseline leukocyte rolling was determined from a 1 min recording collected 30 min after preparation of a C57BL/6 mouse cremaster for intravital microscopy. P-selectin blocking antibody (RB40.34) was injected at the end of some experiments as a positive control. Inhibitor was injected at 31 min and changes in leukocyte rolling determined between 32 and 33 min. Data are presented as mean ± SEM of n = 4 experiments. Asterisks indicate significant difference from baseline rolling (*P<0.05).

2. Effects of other polymers against E-selectin-dependent leukocyte rolling
Polymer 3 (1200 lysines, 20% CGP77175A) was also a good E-selectin inhibitor (>90% inhibition at 0.1 mg/kg) whereas other polymers were less effective. Polymer 1 (1200 lysines, 0% CGP77175A) had no measurable effect on leukocyte rolling. Polymers 2 and 5 (1200 lysines, 5 and 100% loading, respectively) increased rolling velocity, but did not reduce the percentage of leukocytes rolling even when given at 10 mg/kg.

3. Extended duration of action of polymer 4
Duration of action of CGP77175A and polymer 4 were compared by monitoring leukocyte rolling for up to 1 h after single bolus injection (Fig. 2B) . Treatment with 3 mg/kg CGP77175A reduced rolling for up to 10 min, whereas the effect of 10 mg/kg was maintained for more than 20 min. Effects of polymer 4 were more sustained. When injected at 0.1 mg/kg, polymer 4 kept rolling below 50% of control for more than 40 min whereas 0.3 mg/kg was active for more than 1 h.

4. Polymer 4 does not inhibit P- or L-selectin-dependent leukocyte rolling
We also tested polymer 4 against P- and L-selectin-dependent leukocyte rolling (Fig. 2, C and D) . For P-selectin-dependent rolling, wild type C57BL/6 mice were surgically stimulated and P-selectin-dependent leukocyte rolling was observed in the cremaster muscle. For L-selectin-dependent rolling, E-selectin –/– mice were treated for 6 h with TNF-, P-selectin blocking antibody and heparin. Cremasters were then prepared for intravital microscopy and L-selectin-dependent rolling observed. Polymer 4 did not affect P- or L-selectin-dependent leukocyte rolling, even at doses 100 times higher than those required to inhibit E-selectin-dependent leukocyte rolling. P- and L-selectin blocking antibodies virtually abolished leukocyte rolling in the appropriate experiments.

CONCLUSIONS AND SIGNIFICANCE

Interaction between the selectins and their ligands mediates initial attachment and rolling of leukocytes on inflamed endothelium. Leukocyte rolling requires new bond formation at the front of a cell coupled with detachment at the rear. The constant need for new bond formation in this system suggests that it may be susceptible to pharmacological intervention. As leukocyte rolling is the first observable event in the recruitment of leukocytes from the systemic circulation, it may be a good target for anti-inflammatory drugs.

Several mechanisms combine to permit rolling under the challenging conditions in the circulation. Selectin/selectin ligand bonds form rapidly and are relatively strong. Rolling leukocytes may actively disperse forces exerted on adhesion molecules by deforming to maximize contact area or by stretching microvilli at points of attachment. Finally, the avidity of interactions between rolling leukocytes and endothelium may be increased by clustering of molecules at points of contact allowing multivalent molecular interactions. These factors may all make leukocyte rolling a more difficult target than initially appreciated.

Although carbohydrates (sLe^x and variants thereof) integral to all selectin ligands provided lead structures for rational development of competitive selectin inhibitors, direct activity of such inhibitors against rolling in vivo is limited. In an earlier investigation we described CGP69669A (an sLe^x mimetic) as a selective E-selectin inhibitor, but found that high doses (30 mg/kg) were required to reduce established leukocyte rolling. In the present study, we show that CGP77175A, a derivative of CGP69669A, has clearly improved activity against E-selectin-dependent rolling compared with the parent compound.

Comparing CGP77175A with polymer 4 suggests that multivalent presentation gives an 30-fold improvement in activity in vivo. This is somewhat lower than the 700-fold improvement measured previously for these compounds using an in vitro flow chamber assay, but is impressive nonetheless. Polymers 3 and 4 were both able to inhibit almost all E-selectin-dependent rolling at lower doses than E-selectin blocking antibody and the effect of polymer 4 was maintained for up to 1 h. In spite of greatly enhanced activity against E-selectin, polymers carrying CGP77175A fail to influence P- or L-selectin-dependent rolling even at doses 100-fold higher than those blocking E-selectin.

We propose that rapid on/off kinetics and clearance from the circulation limit the ability of sLe^x-based selectin antagonists to block E-selectin on inflamed endothelium (Fig. 3 , upper panel). A large excess of monovalent inhibitors is therefore required to inhibit interactions between endothelial cell E-selectin and sLe^x-bearing ligands on leukocytes (Fig. 3 , middle panel). Presenting an E-selectin antagonist such as CGP77175A on a polymer backbone improves its competitive position dramatically. Interaction of one CGP77175A epitope with E-selectin will promote the likelihood of interactions at other positions on the polymer backbone as structures are held in close proximity to vascular endothelium (Fig. 3 , lower panel). Interactions with E-selectin along the polymer backbone may then form, break and reform at different times, ultimately keeping polymers in contact with E-selectin on the endothelium for longer. Sustained blockade of E-selectin, using a multivalent antagonist, results in a level of inhibition that is comparable to that seen with E-selectin blocking antibody.

View larger version (22K): [in this window] [in a new window]   Figure 3. Proposed advantage of multivalent selectin antagonists. A) Leukocyte rolling is critically dependent on constant bond formation and rupture and can be inhibited by decoy ligands. B) Monovalent inhibitors bind transiently and are subject to rapid clearance, limiting their efficacy and duration of action. C) Mulivalent inhibitors may form more stable interactions with E-selectin as bonds break and reform along the length of the polymer chain. Stable interaction with the endothelium will also limit clearance of multivalent inhibitors.

Ours is the first demonstration that multivalent presentation can dramatically increase the in vivo activity of a selectin antagonist against leukocyte rolling in vivo.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0346fje

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