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

Abstract

Purpose. To evaluate the chemical and enzymatic stability, as well as the cellular permeation characteristics, of the acyloxyalkoxy-based cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ of the opioid peptides [Leu⁵]-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 $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ to [Leu⁵]-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 cellular permeation and metabolism characteristics of [Leu⁵]-enkephalin, DADLE and the cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ were measured using Caco-2 cell monolayers grown onto microporous membranes and monitored by HPLC.

Results. Cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ degraded slowly but stoichiometrically to [Leu⁵]-enkephalin and DADLE, respectively, in HBSS, pH = 7.4. In homogenates of Caco-2 cells and rat liver, as well as 90% human plasma, the rates of disappearance of the cyclic prodrugs were significantly faster than in HBSS. The stabilities of the cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ were increased significantly in 90% human plasma and Caco-2 cell homogenates when paraoxon, a potent inhibitor of serine-dependent esterases, was included in the incubation mixtures. A similar stabilizing effect of paraoxon was not observed in 50% rat liver homogenates, but was observed in 10% homogenates of rat liver. When applied to the AP side of a Caco-2 cell monolayer, DADLE and cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ exhibited significantly greater stability than [Leu⁵]-enkephalin. Based on their physicochemical properties (i.e., lipophilicity), cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ should have exhibited high permeation across Caco-2 cell monolayers. Surprisingly, the AP-to-BL apparent permeability coefficients (P_App) for cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ across Caco-2 cell monolayers were significantly lower than the P_App value determined for the metabolically stable opioid peptide DADLE. When the P_App values for cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ crossing Caco-2 cell monolayers in the BL-to-AP direction were determined, they were shown to be 36 and 52 times greater, respectively, than the AP-to-BL values.

Conclusions. Cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ , 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 cyclic prodrug $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ , which contained an ester formed with an L-amino acid, degraded more rapidly in esterase-containing media than did prodrug $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ , which contained an ester formed with a D-amino acid. Cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ showed very low AP-to-BL Caco-2 cell 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.