Pharmaceutical Research

, Volume 16, Issue 1, pp 24–29 | Cite as

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

  • Annette Bak
  • Olafur S. Gudmundsson
  • Gitte J. Friis
  • Teruna J. Siahaan
  • Ronald T. Borchardt

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 (PApp) 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 PApp value determined for the metabolically stable opioid peptide DADLE. When the PApp 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.

esterase-sensitive prodrugs peptide delivery opioid peptides Caco-2 cells chemical and enzymatic stability of peptides 

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Copyright information

© Plenum Publishing Corporation 1999

Authors and Affiliations

  • Annette Bak
    • 1
  • Olafur S. Gudmundsson
    • 2
  • Gitte J. Friis
    • 1
  • Teruna J. Siahaan
    • 2
  • Ronald T. Borchardt
    • 2
  1. 1.Department of Analytical and Pharmaceutical ChemistryThe Royal Danish School of PharmacyCopenhagenDenmark
  2. 2.Department of Pharmaceutical ChemistryThe University of KansasLawrence
  3. 3.Exploratory Biopharmaceutics and Drug DeliveryBristol-Myers Squibb Pharmaceutical Research InstituteNew Brunswick

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