ALBERT

Notes: Abstract Purpose. To evaluate the cellular permeation characteristics and the chemical and enzymatic stability of coumarinic acid-based cyclic pro-drugs $$\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1} $$ 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 the 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 chemically to intermediates that further degraded to [Leu5]-enkephalin and DADLE, respectively, in stoichiometric amounts. In 90% human plasma and rat liver homogenate, the disappearance of cyclic prodrugs $$\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1} $$ and $$\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2} $$ was significantly faster than in HBSS, pH 7.4. The half-lives in 90% human plasma and in rat liver homogenate were substantially longer after pretreatment with paraoxon, a known inhibitor of serine-dependent esterases. 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]-enkephalin. Cyclic pro-drug $$\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{2} $$ and DADLE exhibited similar stability when applied to the AP side of the Caco-2 cell monolayer. Prodrug $$\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{1} $$ was 665-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 31 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 coumarinic 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 various biological media, the opioid peptides were released from the prodrugs by an esterase-catalyzed reaction, which is sensitive to paraoxon inhibition.

Notes: Abstract Purpose. To compare the bioconversion rates in various biological media and the Caco-2 cell permeation characteristics of coumarin-based cyclic prodrugs (3a, 3b) and methylester-based linear prodrugs (1b, 2b) of two RGD peptidomimetics (la, 2a). Methods. Bioconversion rates of the prodrugs to the RGD peptidomimetics were determined in Hank balanced salt solution (HBSS), pH 7.4, at 37°C and in various biological media (human blood plasma, rat liver homogenate, Caco-2 cell homogenate) known to have esterase activity. Transport rates of the prodrugs and the RGD peptidomimetics were determined using Caco-2 cell monolayers, an in vitrocell culture model of the intestinal mucosa. Results. In HBSS, pH 7.4, the coumarin-based cyclic prodrugs 3a and 3b degraded slowly and quantitatively to the RGD peptidomimetics la and 2a, respectively (3a, t1/2= 630 ± 14 min; 3b, t1/2= 301 ± 12 min). The methylester-based linear prodrugs 1b and 2b were more stable to chemical hydrolysis (1b and 2b, t1/2〉 2000 min). Both the coumarin-based cyclic prodrugs and the methylester-based linear prodrugs degraded more rapidly in biological media containing esterase activity (e.g., 90% human blood plasma: 1b, t1/2〈 5 min; 2b, t1/2〈 5 min; 3a, t1/2〈 91 ± 1 min; 3b, t1/2〈 57 ± 2 min). When the apical (AP)-to-basolateral (BL) permeation characteristics were determined using Caco-2 cell monolayers, it was found that the methylester pro-drugs Ib and 2b underwent esterase bioconversion (〉80%) to the RGD peptidomimetics 1a and 2a, respectively. In contrast, the cyclic prodrugs 3a and 3b permeated the cell monolayers intact. Considering the appearance of both the prodrug and the RGD peptidomimetic on the BL side, the methylester prodrugs 1b and 2b were approximately 12-fold more able to permeate than were the RGD peptidomimetics la and 2a. When a similar analysis of the transport data for the coumarin prodrugs 3a and 3b was performed, they were shown to be approximately 6-fold and 5-fold more able to permeate than were the RGD peptidomimetics la and 2a, respectively. Conclusions. The coumarin-based cyclic prodrugs 3a and 3b were chemically less stable, but metabolically more stable, than the methylester-based linear prodrugs. The esterase stability of the cyclic prodrugs 3a and 3b means that they are transported intact across the Caco-2 cell monolayer in contrast to the methylester prodrugs 1b and 2b, which undergo facile bioconversion during their transport to the RGD peptidomimetics. However, both prodrug systems successfully delivered more (5-12-fold) of the RGD peptidomimetic and/or the precursor (prodrug) than did the RGD peptidomimetics themselves.