Acyloxyalkoxy-based cyclic prodrugs of opioid peptides: Evaluation of the chemical and enzymatic stability as well as their transport properties across Caco-2 cell monolayers

Purpose. To evaluate the chemical and enzymatic stability, as well as the c ellular permeation characteristics, of the acyloxyalkoxy-based cyclic prodr ugs (1) under bar and (2) under bar of the opioid peptides [Leu(5)]-enkepha lin (H-Tyr-Cly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), re spectively. Methods. The rates of conversion of (1) under bar and (2) under bar to [Leu (5)]-enkephalin and DADLE, respectively, were measured by HPLC in HBSS, pH = 7.4, and in various biological media (e.g., human plasma and Caco-2 cell and rat liver homogenates) having measurable esterase activity. The cellula r permeation and metabolism characteristics of [Leu(5)]-enkephalin, DADLE a nd the cyclic prodrugs (1) under bar and (2) under bar were measured using Caco-2 cell monolayers grown onto microporous membranes and monitored by HP LC. Results. Cyclic prodrugs (1) under bar and (2) under bar degraded slowly bu t stoichiometrically to [Leu(5)]-enkephalin and DADLE, respectively, in HBS , pH = 7.4. In homogenates of Caco-2 cells and rat liver, as well as 90% hu man plasma, the rates of disappearance of the cyclic prodrugs were signific antly faster than in HBSS. The stabilities of the cyclic prodrugs (1) under bar and (2) under bar were increased significantly in 90% human plasma and Caco-2 cell homogenates when paraoxon, a potent inhibitor of serine-depend ent esterases, was included in the incubation mixtures. A similar stabilizi ng effect of paraoxon was not observed in 50% rat liver homogenates, but wa s observed in 10% homogenates of rat liver. When applied to the AP side of a Caco-2 cell monolayer, DADLE and cyclic prodrugs (1) under bar and (2) un der bar exhibited significantly greater stability than [Leu(5)]-enkephalin. Based on their physicochemical properties (i.e., lipophilicity), cyclic pr odrugs (1) under bar and (2) under bar should have exhibited high permeatio n across Caco-2 cell monolayers. Surprisingly, the AP-to-BL apparent permea bility coefficients (P-App) for cyclic prodrugs (1) under bar and (2) under bar across Caco-2 cell monolayers were significantly lower than the P-App value determined for the metabolically stable opioid peptide DADLE. When th e P-App values for cyclic prodrugs (1) under bar and (2) under bar crossing Caco-2, cell monolayers in the BL-to-AP direction were determined, they we re shown to be 36 and 52 times greater, respectively, than the AP-to-BL val ues. Conclusions. Cyclic prodrugs (1) under bar and (2) under bar prepared with an acyloxyalkoxy promoiety, were shown to degrade in biological media (e.g, , 90% human plasma) via an esterase-catalyzed pathway. The degradation of c yclic prodrug (1) under bar, which contained an ester formed with an L-amin o acid, degraded more rapidly in esterase-containing media than did prodrug (2) under bar, which contained an ester formed with a D-amino acid. Cyclic prodrugs (1) under bar and (2) under bar showed very low AP-to-BL Caco-2 c ell permeability, which did not correlate with their lipophilicities. These low AP-to-BL permeabilities result because of their substrate activity for apically polarized efflux systems.