Ghrelin is a 28-amino-acid peptide that binds to growth hormone secretagogue receptor (GHS-R) and in so doing stimulates growth hormone release in a manner distinct from growth hormone regulation by the hypothalamic growth hormone-releasing hormone (GHRH) (Kojima et al., 1999). In addition to growth hormone release, it has also been linked to energy balance (Tschop et al., 2000) and parenteral administration of ghrelin led to increased food consumption in humans and caused obesity in rats (Wren et al., 2001a,b). Helmling et al. (2004) exploited a spiegelmer-based SELEX strategy to isolate a spiegelmer-based aptamer against ghrelin. Using biotinylated ghrelin bound to streptavidin or neutravidin beads, 17 rounds of selection were performed and binding and analysis identified a truncated 47-mer, L-NOX-B11 that bound to ghrelin with a Kd of 35 nmol/L. To measure inhibition of ghrelin binding to its receptor, CHO cells expressing human GHS-R1a were used and addition of L-NOX-B11 in ghre-

lin-stimulated cells inhibited binding with an ED50 of5 nmol/L. Ghrelin binding to its receptor GHS-R1a releases intracellular Ca2+. Aptamer L-NOX-B11 inhibited Ca2+ release with an IC50 of 5 nmol/L. To test the functional activity of L-NOX-B11, a 5'-PEG conjugated form of the spiegelmer was injected, and at doses of 15 and 30 nmol (in 5-10 excess of ghrelin challenge) no increase in growth hormone was seen in plasma when ghrelin was administered (Helmling et al., 2004).

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