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

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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. Bovin^5*

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

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The potency of the oligosaccharides SiaLe^x, SiaLe^a, HSO₃Le^x, and HSO₃Le^a, 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 SiaLe^a-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 HSO₃Le^a-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 SiaLe^x 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 HSO₃Le^a-PAA-sTyr.

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KEY WORDS: fucoidan, HSO₃Le^x, HSO₃Le^a, inflammation, neoglycoconjugates, platelets, polyacrylamide, selectins, SiaLe^x, SiaLe^a, tyrosine-O-sulfate

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