Carbohydrates in an Acidic Multivalent Assembly:  Nanomolar P-Selectin Inhibitors (1996)

Neutrophil recruitment to tissues is initiated by an adhesion cascade.1 Through this process, cells roll and eventually attach firmly to the endothelium.2-9 The factors that contribute to the high binding strength of this interaction are not well understood. Here we report that multivalent carbohydrate assemblies can mimic neutrophils with respect to adhesion. However, the multiplicity of sugar copies in itself is not sufficient to provide tight binding. The chemical constituents making up the underlying backbone play an important, if not greater, role in this interaction. This is demonstrated by the nanomolar inhibition of adhesion by a simple sugar (lactose) presented on the surface of an acidic, polymerized liposome. Since various diseases such as ARDS (adult respiratory distress syndrome), rheumatoid arthritis, septic shock, and reperfusion injury can result from neutrophil adhesion, polymerized liposomes presenting carbohydrates (glycoliposomes) may form the basis for a new therapeutic agent. A physiological ligand for the adhesion protein Pselectin has been recently identified as a mucin-like structure designated as PSGL-1 (P-selectin glycoprotein ligand 1).10,11 Some sialylated or sulfated fucooligosaccharides, for example sialyl Lewisx (sLex, 1) (Figure 1), exhibit weak binding to P-selectin.4,12-14 Although much evidence suggests that the PSGL-1 carbohydrate epitope is an sLex-type structure, it is apparent that other factors are required for the high, in vivo affinity of P-selectin.2,15-22 We have designed macromolecular structures capable of potent inhibitory activity toward P-selectin binding. Polymerizable, carbohydrate-terminated lipid monomers are readily assembled into liposomes.23 These structures are made stable by polymerization with UV light. Nanomolar P-selectin inhibition is observed when liposomes are prepared from a bicomponent mixture of sLex-like glycolipids and acidic lipids. The polymerizable glycolipid 2b was synthesized by coupling the N-glycoside of compound 2a to commercially available 10,12-pentacosadiynoic acid (PDA, 5) (Figure 1).24 Compound 5 (PDA) or 6 is employed as the “matrix” lipid in the liposome preparations. The glycolipids are mixed with the matrix lipids and are formed into glycoliposomes by the probe sonication method25 and polymerized by UV irradiation (254 nm).26 Compounds 5 and 6 give the polymer scaffold an acidic (5) or neutral (6) character and separate the large carbohydrate headgroups on the bilayer surface (Figure 2). Glycoliposomes were prepared with acidic matrix lipid 5 and varying percentages of glycolipid 2b in the range 0-50%. Activity was determined by assaying for inhibition of P-selectin IgG chimera binding to HL-60 cells.12 The glycoliposomes showed a dose dependent inhibition (shown in Figure 3A). The inhibition is highly dependent on the percentage of glycolipid in the glycoliposomes. Concentrations of approximatly 5 molar % glycolipid 2b gave the best inhibition (IC50 ) 2 nM) that diminished with higher or lower surface concentrations (shown in Figure 3B). Glycoliposomes prepared from lactose or maltose glycolipids (3b or 4b, with matrix lipid 5) also showed binding inhibition (Figure 3A). The IC50 values for lactose glycoliposomes (3b) is 7 times weaker, and maltose glycoliposomes (4b) is 2 orders of magnitude weaker, than the sLex analog (2b) (Figure 3C). The same trend with respect to surface percentages vs activity is observed for all glycoliposomes tested (data not shown). These IC50 values are in stark contrast to the monomeric carbohydrate 2a, 3a, or 4a. The monomeric IC50 value for 2a is greater than 5 mM4 and lactose or maltose have no observed binding. In contrast, glycoliposome preparations with 5% 2b in neutral matrix lipid 6 showed no inhibitory activity at the highest concentration tested, 500 nM (Figure 3A). Therefore, the acidic groups of matrix lipid 5 are essential for strong binding activity. This result correlates with the apparent requirement for anionic groups in P-selectin antagonists. For example, the known inhibitors heparin, inositol hexakisphosphate, * To whom correspondence should be addressed. † Lawrence Berkeley National Laboratories. ‡ Glycomed Inc. § New address: Terrapin Technologies Inc. South San Francisco, CA 94080. ⊥ New address: Ranbaxy Research Laboratories, A1 Phase-1 Okhla Industrial Area, New Delhi: 110020, India. Figure 1. The sLex tetrasaccharide (1) recognized by Pselectin. Carbohydrates 2a (sLex analog), 3a (lactose), and 4a (maltose) are used for synthesizing the polymerizable glycolipids 2b, 3b, and 4b, respectively. Compound 5 or 6 is used as the matrix lipid in liposome preparation. 1018 J. Med. Chem. 1996, 39, 1018-1020