The use of polyamidoamine (PAMAM) dendrimers as a method of drug delivery for ligands to G protein-coupled receptors (GPCRS)

This dissertation reports the first research showing that it is feasible to enhance the pharmacological profile of a G protein-coupled receptor (GPCR) ligand (specifically adenosine receptor [AR] ligand by conjugating the ligand to a dendrimer, a multi-branching polymer. First, it was determined that the 32 terminal amino groups on the generation 3 (G3) polyamidoamine (PAMAM) dendrimer caused ∼70% cell death to Chinese Hamster Ovary (CHO) cells at a 10 muM concentration. Therefore, a series of PAMAM G3-Acetyl (Ac) and PAMAM G3-poly(ethyleneglycol) (PEG) dendrimer conjugates was synthesized. To increase cell survival at a 10 muM concentration, a minimum of 20Ac or 7PEG550 groups was needed, allowing these PAMAM conjugates to serve as universal scaffolds for drug delivery. Next, nucleoside agonists of the A2A AR, an anti-aggregatory GPCR, were covalently attached to a PAMAM G3 dendrimers, and their binding to the human A2A AR was evaluated. Three drug conjugates were synthesized and characterized: 32, a conjugate with 5 nucleoside units and 20Ac (A2A K i 152 nM); 35, a dendrimer with 31 nucleoside moieties (A2A Ki 96 nM); and 34, a control dendrimer with 20Ac and no nucleoside (no A2A AR affinity). 32 and 35 significantly decreased platelet aggregation whereas 34 had no effect on platelet aggregation. Therefore, dendrimer-ligand conjugates are still able to bind and activate the A2A AR. Finally, synthesis and binding using derivatives of ADAC, an A1 agonist with anti-arrhythmic properties, were completed. N-(2-aminoethyl)-ADAC, was synthesized and found to be equipotent at the A1 AR and the anti-inflammatory A3 AR. This derivative was coupled by an amide bond to a fluorescently-labeled PAMAM G2.5 dendrimer. In binding and functional cAMP inhibition assays, this conjugate had a Ki of 2.4 nM and 100-fold selectivity for the A 3 AR. This is the first example showing that it is feasible to enhance the pharmacological profile of a GPCR ligand based on conjugation to a nanocarrier and the precise structure of the linking group. This dissertation proves that PAMAM dendrimers can serve as general scaffolds to deliver ligands to GPCRs.