Coumarinic acid-based cyclic prodrugs of opioid peptides that exhibit metabolic stability to peptidases and excellent cellular permeability

Purpose. To evaluate the cellular permeation characteristics and the chemic al and enzymatic stability of coumarinic acid-based cyclic prodrugs (1) und er bar and (2) under bar of the opioid peptides [Leu(5)]-enkephalin (H-Tyr- Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. Methods. The rates of conversion of the cyclic prodrugs (1) under bar and ( 2) under bar to [Leu(5)]-enkephalin and DADLE, respectively, in HBSS, pH 7. 4 (Caco-2 cell transport buffer) and in various biological media having mea surable esterase activity were determined by HPLC. The cell permeation char acteristics of [Leu(5)]-enkephalin, DADLE and cyclic prodrugs (1) under bar and (2) under bar were measured using Caco-2 cell monolayers grown onto mi croporus membranes and monitored by HPLC. Results. In HBSS, pH 7.4, cyclic prodrugs (1) under bar and (2) under bar d egraded chemically to intermediates that further degraded to [Leu(5)]-enkep halin and DADLE, respectively, in stoichiometric amounts. In 90% human plas ma and rat liver homogenate, the disappearance of cyclic prodrugs (1) under bar and (2) under bar was significantly faster than in HBSS, pH 7.4. The h alf-lives in 90% human plasma and in rat liver homogenate were substantiall y longer after pretreatment with paraoxon, a known inhibitor of serine-depe ndent esterases. When applied to the AP side of a Caco-2 cell monolayer, cy clic prodrug (1) under bar exhibited significantly greater stability agains t peptidase metabolism than did [Leu(5)]-enkephalin, Cyclic prodrug (2) und er bar and DADLE exhibited similar stability when applied to the AP side of the Caco-2 cell monolayer. Prodrug (1) under bar was 665-fold more able to permeate the Caco-2 cell monolayers than was [Leu(5)]-enkephalin, in part because of its increased enzymatic stability. Prodrug (2) under bar was sho wn to be approximately 31 fold more able to permeate a Caco-2 cell monolaye r than was DADLE. Conclusions. Cyclic prodrugs (1) under bar and (2) under bar, prepared with the coumarinic acid promoiety, were substantially more able to permeate Ca co-2 cell monolayers than were the corresponding opioid peptides. Prodrug ( 1) under bar exhibited increased stability to peptidase metabolism compared to [Leu(5)]-enkephalin. In various biological media, the opioid peptides w ere released from the prodrugs by an esterase-catalyzed reaction, which is sensitive to paraoxon inhibition.