Chirally Pure Prodrugs and Their Converting Enzymes Lead to High Supersaturation and Rapid Transcellular Permeation of Benzodiazepines

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Water-soluble prodrugs can be rapidly converted by enzymes to hydrophobic drugs, whose aqueous thermodynamic solubilities are low, but are maintained in aqueous solution at supersaturated concentrations due to slow precipitation kinetics. Recently, we investigated avizafone (AVF) in combination with Aspergillus oryzae protease as a prodrug/enzyme system intended to produce supersaturated diazepam (DZP). Several fold enhancement of permeation of supersaturated DZP across Madin-Darby canine kidney II-wild type (MDCKII-wt) monolayers was observed, compared to saturated DZP solutions. However, prodrug conversion was incomplete, putatively due to partial racemization of AVF and stereoselectivity of A oryzae protease. Here we report synthesis of chirally pure AVF, and demonstrate complete conversion to supersaturated DZP followed by complete DZP permeation at enhanced rates across MDCKII-wt cell monolayers. We also synthesized, for the first time, a chirally pure prodrug of midazolam (MDZ-pro) and carried out the same sequence of studies. A oryzae protease was identified as a benign and efficient activating enzyme for MDZ-pro. The MDZ-pro/A oryzae protease system showed greater than 25-fold increase in absorption rate of MDZ across MDCKII-wt monolayers, compared to saturated MDZ. Such chirally pure prodrug/enzyme systems are promising candidates for efficient intranasal delivery of benzodiazepine drugs used in the treatment of seizure emergencies.

Original languageEnglish (US)
Pages (from-to)2365-2371
Number of pages7
JournalJournal of Pharmaceutical Sciences
Issue number8
StatePublished - Aug 1 2016

Bibliographical note

Funding Information:
We thank the AHC Faculty Research Development Program at the University of Minnesota, the American Epilepsy Society, the Epilepsy Foundation, and the Lyle and Sharon Bighley Drug Development Fund, for research funding. The assistance of Heelim Lee, Patricia Maglalang, Rafael Castro Marin, Carolyn Rath, and Usha Mishra is gratefully acknowledged. We thank Profs William Elmquist and Karunya Kandimalla for providing cell culture facilities and HPLC equipment, respectively. Discussions with Prof. Edward Patterson are gratefully acknowledged.

Publisher Copyright:
© 2016 American Pharmacists Association ®


  • MDCK cells
  • chirality
  • enzymes
  • membrane transport
  • mucosal drug delivery
  • nasal drug delivery
  • prodrugs
  • supersaturation


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