Phenylpropionic Acid-Based Cyclic Prodrugs of Opioid Peptides that Exhibit Metabolic Stability to Peptidases and Excellent Cellular Permeation

Abstract

Purpose. To evaluate the cellular permeation characteristics and the chemical and enzymatic stability of phenylpropionic acid-based cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ of 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 cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ to [Leu⁵]-enkephalin and DADLE, respectively, in HBSS, pH 7.4 (Caco-2 cell transport buffer) and in various biological media having measurable esterase activity were determined by HPLC. The cell permeation characteristics of [Leu⁵]-enkephalin, DADLE, and 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 microporus membranes and monitored by HPLC.

Results. In HBSS, pH 7.4, cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ degraded to [Leu⁵]-enkephalin and DADLE, respectively, in stoichiometric amounts. In 90% human plasma, the rates of disappearance of cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ were slightly faster than in HBSS, pH 7.4. These accelerated rates of disappearance in 90% human plasma could be reduced to the rates observed in HBSS, pH 7.4, by pretreatment of the plasma with paraoxon, a known inhibitor of serine-dependent esterases. In homogenates of Caco-2 cells and rat liver, accelerated rates of disappearance of cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ were not observed. When applied to the AP side of a Caco-2 cell monolayer, cyclic prodrug $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ exhibited significantly greater stability against peptidase metabolism than did [Leu⁵]-enkepha-lin. Cyclic prodrug $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ and DADLE exhibited stability similar to prodrug $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ when applied to the AP side of the Caco-2 cell monolayers. Prodrug $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ 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 $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ was shown to be approximately 77 fold more able to permeate a Caco-2 cell monolayer than was DADLE.

Conclusions. Cyclic prodrugs $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ and $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2}$ , prepared with the phenylpropionic acid promoiety, were substantially more able to permeate Caco-2 cell monolayers than were the corresponding opioid peptides. Prodrug $\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1}$ exhibited increased stability to peptidase metabolism compared to [Leu⁵]-enkephalin. In 90% human plasma but not in Caco-2 cell and rat liver homogenates, the opioid peptides were released from the cyclic prodrugs by an esterase-catalyzed reaction that is sensitive to paraoxon inhibition. However, the rate of this bioconversion appears to be extremely slow.