Phenylpropionic acid-based cyclic prodrugs of opioid peptides that exhibitmetabolic stability to peptidases and excellent cellular permeation

Purpose, To evaluate the cellular permeation characteristics and the chemic al and enzymatic stability of phenylpropionic acid-based cyclic prodrugs (1 ) under bar and (2) under bar of 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 cyclic prodrugs (1) under bar and (2) u nder bar to [Leu(5)]-enkephalin and DADLE, respectively, in HBSS, pH 7.4 (C aco-2 cell transport buffer) and in various biological media having measura ble esterase activity were determined by HPLC. The cell permeation characte ristics of [Leu(5)]-enkephalin, DADLE, and cyclic prodrugs (1) under bar an d (2) under bar were measured using Caco-2 cell monolayers grown onto micro porus membranes and monitored by HPLC. Results. In HBSS. pH 7.4, cyclic prodrugs (1) under bar and (2) under bar d egraded to [Leu(5)]-enkephalin and DADLE, respectively, in stoichiometric a mounts. In 90% human plasma, the rates of disappearance of cyclic prodrugs (1) under bar and 2 Were slightly faster than in HBSS, pH 7.4. These accele rated rates of disappearance in 90% human plasma could be reduced to the ra tes observed in HBSS. pH 7.4. by pretreatment of the plasma with paraoxon, a known inhibitor of serine-dependent esterases. In homogenates nates of Ca co-2 cells and rat liver, accelerated rates of disappearance of cyclic prod rugs (1) under bar and (2) under bar were not observed. When applied to the AP side of a Caco-2 cell monolayer, cyclic prodrug (1) under bar exhibited significantly greater stability against peptidase metabolism than did [Leu (5)]-enkephalin. Cyclic prodrug (2) under bar and DADLE exhibited stability similar to prodrug (1) under bar when applied to the AP side of the Caco-2 cell monolayers. Prodrug (1) under bar was 1680 fold more able to permeate the Caco-2 cell monolayers than was [Leu5]-enkephalin, in part because of its increased enzymatic stability. Prodrug (2) under bar was shown to be ap proximately 77 fold more able to permeate a Caco-2 cell monolayer than was DADLE. Conclusions. Cyclic prodrugs (1) under bar and (2) under bar, prepared with the phenylpropionic acid promoiety, were substantially more able to permea te Caco-2 cell monolayers than were the corresponding opioid peptides. Prod rug (1) under bar exhibited increased stability to peptidase metabolism com pared to [Leu(5)]-enkephalin. In 90% human plasma but not in Caco-2 cell an d rat liver homogenates, the opioid peptides were released from the cyclic prodrugs by an esterase-catalyzed reaction that is sensitive to paraoxon in hibition. However, the rate of this bioconversion appears to be extremely s low.