Mamta Kapoor,1 Tate Winter,1 Lev Lis,2,4 Gunda I. Georg,2,4 and Ronald A. Siegel1,3*

Rapid Delivery of Diazepamfrom Supersaturated Solutions Prepared Using Prodrug-Enzyme Mixtures: Toward Intranasal Treatment of Seizure Emergencies

Mamta Kapoor,1 Tate Winter,1 Lev Lis,2,4 Gunda I. Georg,2,4 and Ronald A. Siegel1,3*

Departments of 1Pharmaceutics, 2Medicinal Chemistryand 3Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA

4Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, MN 55414, USA

Electronic Supplementary Materials

*Corresponding Author:

Ronald A Siegel, Sc.D.

Professor, Department of Pharmaceutics, College of Pharmacy

University of Minnesota

Minneapolis, MN 55455

Ph: (612) 624 6164, Fax: (612) 626 2125

Email:

Supplemental 1.LC-MS data for AVF-DZP-TLB mixture in acidic mobile phase (pH 2-3). MS spectra of the 4thPeak (3.59 min) revealed the structure to be 2-(N-methylamino)-5-chlorobenzophenone (MW: 302.5) or open-ring DZP. Other peaks represent AVF (3.24 min), tolbulamide (TLB, 4.37 min), and DZP (4.6 min). MS is shown only for the unknown (X) peak. For this spectrum, uHPLC with 2.1 x 50 mm (1.7 µm) C18 BEH column with DAD, ELSD and ZQ MS detector. Mobile phase composition A: water with 0.1% formic acid, B: acetonitrile with 0.1% formic acid. Flow rate of 0.25 mL/min with gradient elution: 100% A for 1 min, 100% to 5% A for 4.5 min, 5% to 95% A for 0.5 min, 95% A for 0.5 min.

Supplemental 2.Permeability of Avizafone (AVF) across MDCKII-wt monolayer at various

initial prodrug concentrations (115.6-1992.7 µM), without enzyme. Mean+SD, n=4

Supplemental 3.Effect of proteaseenzyme on monolayer integrity: (a) % TEER and, (b) %

inulin permeability across MDCKII-wt monolayers when incubated with protease at different

concentrations (U/mL) for 2 h at 32°C with mild shaking.

Supplemental 4.Effect of enzyme (protease) on DZP (DZP at S = 0.7, cenz = 4 U/mL)

permeation. Apparent permeability of DZP in the presence and absence of enzyme was not

significantly different (average around Papp; 2.4 X 10-5 cm3/s) as seen from overlapping flux in

both cases. DZPE: DZP with protease.

Supplemental 5.AVF-protease reactions performed at various prodrug/enzyme ratios: (a) %

amount of AVF and DZP when AVF (S=7.5) was incubated with protease at different concentrations (1-16 U/mL) (b) % amount of AVF remaining in solution when AVF (S=0.8, 4.2 or 6.7) was incubated with protease at different enzyme concentrations (4 - 256 U/mL). These reactions were performed in assay buffer pH 7.4 in glass vials placed at 32°C on a shaker. ‘S’ represents AVF molar equivalent to supersaturated DZP.

Supplemental 6. Circular dichroism (CD) spectra of (A) starting material AVF (1 mg/mL AVF, no enzyme) and (B) unreacted AVF (remaining in prodrug-enzyme mixture: 1 mg/mL AVF + cenz = 8 U/mL, 1.5 h incubation at 32°C followed by methanol addition for enzyme precipitation), measured using J815, JASCO CD spectrometer (USA) in a 1mm quartz cuvette. Results are an average of 5 accumulations. CD spectra of DZP showed no optical activity whereas opposite signals were observed for (A) starting material AVF and (B) unreacted AVF in prodrug-enzyme mixture. Various combinations of A and B showed intermediate signals. HPLC analysis of this prodrug-enzyme mixture revealed 20% unreacted AVF and 80% DZP (data not shown).

Methods

TEER measurements. Intactness of the monolayer was examined by measuring its trans-epithelial electrical resistance (TEER) using the EVOM epithelial volt-ohm meter with STX-2 electrode (World Precision Instruments, Sarasota, Florida). The cell monolayer cultured in transwells was washed twice with pre-warmed assay buffer and then equilibrated with fresh assay buffer at 32°C for 30 min. TEER was measured using the chopstick electrode carefully placed across the transwell without disturbing the monolayer. Only monolayers with TEER values > 60 ohms cm2 were considered for the experiment. % TEER was obtained by normalizing the TEER value of treated cells by the value of untreated cells (cells alone).

Inulin permeability (for Fig. S3).Radiolabeled inulin (14C-inulin) was used as a marker for paracellular transport to determine any ‘leak’ in the tight junctions. A solution of 0.2 µCi/mL inulin was prepared (50 µCi stock in DMSO) in assay buffer and applied to the apical side of the transwells. Aliquots were withdrawn at 0 and 120 min from the apical chamber and at time 0, 15, 30, 60, 120 min, from the basal chamber. These aliquots were diluted with 4 mL scintillation cocktail and radiolabelling measurements were obtained using a liquid scintillation counter (Beckman LS 5000 TD, BeckmanInstruments, Fullerton, California).

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