The AAPS Journal

, Volume 16, Issue 3, pp 577–585 | Cite as

Rapid Delivery of Diazepam from Supersaturated Solutions Prepared Using Prodrug/Enzyme Mixtures: Toward Intranasal Treatment of Seizure Emergencies

  • Mamta Kapoor
  • Tate Winter
  • Lev Lis
  • Gunda I. Georg
  • Ronald A. Siegel
Research Article


Current treatments for seizure emergencies, such as status epilepticus, include intravenous or rectal administration of benzodiazepines. While intranasal delivery of these drugs is desirable, the small volume of the nasal cavity and low drug solubility pose significant difficulties. Here, we prepared supersaturated diazepam solutions under physiological conditions and without precipitation, using a prodrug/enzyme system. Avizafone, a peptide prodrug of diazepam, was delivered with—Aspergillus oryzae (A.O.) protease, an enzyme identified from a pool of hydrolytic enzymes in assay buffer, pH 7.4 at 32°C. This enzyme converted avizafone to diazepam at supersaturated concentrations. In vitro permeability studies were performed at various prodrug/enzyme ratios using Madin-Darby canine kidney II-wild type (MDCKII-wt) monolayers, a representative model of the nasal epithelium. Monolayer integrity was examined using TEER measurement and the lucifer yellow permeability assay. Prodrug/drug concentrations were measured using HPLC. Enzyme kinetics with avizafone-protease mixtures revealed K M = 1,501 ± 232 μM and V max = 1,369 ± 94 μM/s. Prodrug-protease mixtures, when co-delivered apically onto MDCKII-wt monolayers, showed 2–17.6-fold greater diazepam flux (S = 1.3–15.3) compared to near-saturated diazepam (S = 0.7). Data for prodrug conversion upstream (apical side) and drug permeability downstream (basolateral side) fitted reasonably well to a previously developed in vitro two compartment pharmacokinetic model. Avizafone-protease mixtures resulted in supersaturated diazepam in less than 5 min, with the rate and extent of supersaturation determined by the prodrug/enzyme ratio. Together, these results suggest that an intranasal avizafone-protease system may provide a rapid and alternative means of diazepam delivery.


avizafone enzyme activation diazepam delivery hydrophobic drugs MDCK monolayers rapid absorption seizure emergencies supersaturation 



We thank the AHC Faculty Research Development Program at the University of Minnesota for research funding (grant#1803-11406-21287-3672675, PI G. Georg). We thank Dr. Subhashree Francis for providing data for Supplemental 1 (LC-MS). We also thank Prof. William Elmquist for providing lab facilities to perform cell studies, and Prof. Karunya Kandimalla for providing the HPLC equipment. Discussions with Profs. James Cloyd, Edward Patterson and Patrick E. Hanna are gratefully acknowledged.

Supplementary material

12248_2014_9596_MOESM1_ESM.doc (244 kb)
ESM 1 (DOC 244 kb)


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Copyright information

© American Association of Pharmaceutical Scientists 2014

Authors and Affiliations

  • Mamta Kapoor
    • 1
  • Tate Winter
    • 1
  • Lev Lis
    • 2
    • 4
  • Gunda I. Georg
    • 2
    • 4
  • Ronald A. Siegel
    • 1
    • 3
  1. 1.Department of Pharmaceutics, College of PharmacyUniversity of MinnesotaMinneapolisUSA
  2. 2.Department of Medicinal ChemistryUniversity of MinnesotaMinneapolisUSA
  3. 3.Department of Biomedical EngineeringUniversity of MinnesotaMinneapolisUSA
  4. 4.Institute for Therapeutics Discovery and DevelopmentUniversity of MinnesotaMinneapolisUSA

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