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Monomeric and Multimeric Blockers of Selectins: Comparison of in vitro and in vivo Activity

N. A. Ushakova1, M. E. Preobrazhenskaya1, M. I. Bird2, R. Priest2, A. V. Semenov3, A. V. Mazurov3, N. E. Nifantiev4, T. V. Pochechueva5, O. E. Galanina5, and N. V. Bovin5*

1Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, ul. Pogodinsksya 10, 119121 Moscow, Russia; E-mail: natalia.ushakova@ibmc.msk.ru

2Discovery Research, GlaxoSmithKline Ltd, Stevenage, UK; E-mail: mike.i.bird@gsk.com

3Institute of Experimental Cardiology, Cardiology Research Center, Russian Ministry of Health, ul. 3-ya Cherepkovskaya 15A, Moscow, Russia

4Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky pr. 47, 117913 Moscow, Russia; E-mail: nen@ioc.ac.ru

5Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (7-095) 330-5592; E-mail: bovin@carbohydrate.ru

* To whom correspondence should be addressed.

Received December 16, 2003; Revision received May 6, 2004
The potency of the oligosaccharides SiaLex, SiaLea, HSO3Lex, and HSO3Lea, their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC50 values for the neoglycoconjugate SiaLea-PAA were 6, 40, and 85 µM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO3Lea-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLex is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO3Lea-PAA-sTyr.
KEY WORDS: fucoidan, HSO3Lex, HSO3Lea, inflammation, neoglycoconjugates, platelets, polyacrylamide, selectins, SiaLex, SiaLea, tyrosine-O-sulfate