title: Chirally Pure Prodrugs and Their Converting Enzymes Lead to High Supersaturation and Rapid Transcellular Permeation of Benzodiazepines.
abstract: 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.
authors.affiliation: Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota 55414.
authors.forename: Mamta
authors.initials: M
authors.lastname: Kapoor
authors.affiliation: Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55414; Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota 55414.
authors.forename: Narsihmulu
authors.initials: N
authors.lastname: Cheryala
authors.affiliation: Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota 55414.
authors.forename: Davin
authors.initials: D
authors.lastname: Rautiola
authors.affiliation: Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55414; Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota 55414.
authors.forename: Gunda I
authors.initials: GI
authors.lastname: Georg
authors.affiliation: Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, Minnesota 55414; Center for Orphan Drug Research, University of Minnesota, Minneapolis, Minnesota 55414.
authors.forename: James C
authors.initials: JC
authors.lastname: Cloyd
authors.affiliation: Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota 55414; Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55414. Electronic address: siege017@umn.edu.
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Siegel
mesh_terms: D000281:Administration, Intranasal; D000818:Animals; D001236:Aspergillus oryzae / Q000201:enzymology; D001569:Benzodiazepines / Q000737:chemistry / Q000378:metabolism*; D002463:Cell Membrane Permeability; D004151:Dipeptides / Q000378:metabolism; D004285:Dogs; D057927:Hydrophobic and Hydrophilic Interactions; D061985:Madin Darby Canine Kidney Cells; D008874:Midazolam / Q000378:metabolism; D009297:Nasal Mucosa / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry*; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D012996:Solutions
chemical_list: D004151:Dipeptides; D011355:Prodrugs; D012996:Solutions; D001569:Benzodiazepines; C065249:pro-diazepam; D010447:Peptide Hydrolases; D008874:Midazolam
keywords: MDCK cells; chirality; enzymes; membrane transport; mucosal drug delivery; nasal drug delivery; prodrugs; supersaturation