Normal recruitment of circulating white blood cells (leukocytes) into tissues is important in inflammation, lymphocyte homing, and immunological responses. The mechanism of recruitment involves receptors termed selectins that recognize their counter receptors on leukocytes. Selectin mediated recruitment of leukocytes, however, also plays a role in many diseases, including atherosclerosis, ischemia-reperfusion injuries (e.g. transplant, stroke, myocardial infarct), inflammatory diseases (e.g. Sickle cell anemia, rheumatoid arthritis, thrombosis), and metastatic spreading of some cancers. The laboratories of Drs. R.P. McEver and R.D. Cummings, the scientific founders of Selexis Corporation, discovered that a key early adhesion event in the inflammatory response is the specific interaction of P-selectin, a glycoprotein receptor on activated platelets and endothelial cells, with its counterreceptor, the P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. Subsequently, they found that the critical molecular interactions involved only a small glycosulfopeptide (GSP) region within PSGL-1. Selexis has now licensed a proprietary chemoenzymatic approach to synthesize GSP (termed Glysopep), which is a small, homogeneous compound of 3,734.3 Da. Glysopep has the specificity and affinity of the native, intact PSGL-1 from human leukocytes and is being developed by Selexis as a new drug to treat inflammatory diseases. Selexis researchers have now shown that (i) Glysopep can be easily synthesized; (ii) in animal models Glysopep inhibits P-select independent leukocyte rolling; and (iii) Glysopep is highly stable and derivatizable. Here we propose to prepare polyethylene glycol (PEG)-modified forms of Glysopep to increase its circulating half-life and enhance its overall pharmacokinetic/pharmacodynamic (PK/PD) profile. There are three specific aims to this proposal. Aim 1 - We will generate PEG-Glysopep with PEG derivatives ranging from ~4 kD to ~40 kDa and test for their ability to inhibit P-selectin-dependent adhesion in vitro; Aim 2 - We will radioiodinate PEG-Glysopep derivatives to measure the effect of PEGylation on its PK; and Aim 3 - We will test the optimal PEG-Glysopep formulations in vivo to assess sustained inhibition and reversal of leukocyte rolling. This work will lead to a SBIR Phase II application to use PEG-Glysopep in animal models of disease in preparation for filing an Investigational New Drug application with the Food and Drug Administration.
Thesaurus Terms: antiinflammatory agent, cell adhesion, drug design /synthesis /production, glycoprotein, membrane protein, polyethylene glycol, selectin chemical stability, iodination, leukocyte, method development, pharmacokinetics, protein binding, protein structure function, receptor expression, synthetic peptide high performance liquid chromatography, laboratory mouse, laboratory rat, matrix assisted laser desorption ionization
