Anxiety disorders (AD) are the most common mental conditions affecting an estimated 40 million adults in the United States. Amiloride, a diuretic agent, has shown efficacy in reducing anxious responses in preclinical models by inhibiting the acid-sensing ion channels (ASIC). By delivering amiloride via nasal route, rapid onset of action can be achieved due to direct "nose-to-brain" access. Therefore, this study reports the formulation, physical, chemical, and microbiological stability of an extemporaneously prepared amiloride 2 mg/mL nasal spray. The amiloride nasal spray was prepared by adding 100 mg of amiloride hydrochloride to 50 mL of sterile water for injection in a sterile reagent bottle. A stability-indicating high-performance liquid chromatography (HPLC) method was developed and validated. Forceddegradation studies were performed to confirm the ability of the HPLC method to identify the degradation products from amiloride distinctively. The physical stability of the amiloride nasal spray was assessed by pH, clarity, and viscosity assessments. For chemical stability studies, samples of nasal sprays stored at room temperature were collected at time-points 0, 3 hr., 24 hr., and 7 days and were assayed in triplicate using the stability-indicating HPLC method. Microbiological stability of the nasal spray solution was evaluated for up to 7 days based on the sterility test outlined in United States Pharmacopoeia (USP) chapter 71. The stability-indicating HPLC method identified the degradation products of amiloride without interference from amiloride. All tested solutions retained over 90% of the initial amiloride concentration for the 7-day study period. There were no changes in color, pH, and viscosity in any sample. The nasal spray solutions were sterile for up to 7 days in all samples tested. An extemporaneously prepared nasal spray solution of amiloride hydrochloride (2 mg/mL) was physically, chemically, and microbiologically stable for 7 days when stored at room temperature.

Purpose The main objectives of this study were 1) to evaluate the effects of phenytoin (antiepileptic drug) and losartan (TGF‐beta inhibitor) on cytotoxicity, ATP, and TGF‐beta in human gingival fibroblasts (HGF‐1) cells, 2) to identify whether losartan inhibits gingival overgrowth caused by phenytoin and 3) to establish HGF‐1 cell culture as a model to study druginduced gingival hyperplasia. Methods HGF‐1 cells obtained from ATCC were grown to 90% confluency in DMEM media supplemented with PenStrep, and bovine serum (FBS 10%). Cells were incubated in 5% CO2, 37°C and 95% relative humidity. For cytotoxicity, LDH leakage in the medium was measured using the CytoTox 96 Non‐radioactive Cytotoxicity Assay Kit, and for ATP, total % ATP was measured using CellTiter‐Glo Luminescent Cell Viability Assay Kit. For ATP and LDH measurements, cells were treated with various concentrations of phenytoin (0, 10, 25, 50, 100 and 200 μM) and losartan (0, 25, 50, 100, 250 and 500 μM) dissolved in DMSO, and incubated for 24h (30,000 cells/well in 96 well plates), 5% CO2, 37°C and 95% relative humidity. Final DMSO concentration was 0.1%. To identify effects on TGF‐beta, cells were treated with DMSO (0.1% final concentration), Phenytoin (100 μM), Losartan (100 μM), and Phenytoin (100 μM) + Losartan (100 μM). After 24h treatment of cells, TGF beta was measured as per supplier’s protocol using ELISA assay kit (Thermo Fisher, Grand Island, NY). All experiments were carried out as triplicate measurements and mean of triplicates were considered as one experiment (n). Minimum of three repetitions (n = 3) were carried out unless otherwise noted in results. Results Preliminary results show that none of the drug concentrations had a cytotoxic effect on HGF‐1 cells. No increase in ATP levels were observed for losartan. However, phenytoin showed greater increase in percent ATP levels when compared to vehicle control. Percent ATP levels for losartan and phenytoin were in the range of 101 ± 4 to 113.5 ± 8.2% and 135 ± 11.1 to 138.1 ± 16.5% respectively. Data from one TGF‐beta experiment showed no increase in TGF‐beta levels with phenytoin treatment, however losartan, and losartan plus phenytoin treatment showed 1.3 and 2‐fold increase in TGF beta levels over untreated cells respectively. Conclusion Our study suggests that phenytoin causes cell proliferation as noted from increased ATP levels. Preliminary results form TGF‐beta experiments were inconclusive and further experiments are needed to ascertain effects of losartan and phenytoin on TGF‐beta in HGF‐1 cells and identify whether HGF‐1 cells can be used as a model to study drug‐induced gingival hyperplasia. Support or Funding Information This project is supported by College of Pharmacy, Roseman University of Health Sciences, South Jordan, Utah 84095

20Anxiety disorders (AD) are the most common mental illnesses affecting an estimated 40 21 million adults in the United States. Amiloride, a diuretic agent, has shown efficacy in 22 treating AD in preclinical models by inhibiting the acid-sensing ion channels (ASIC). By 23 delivering amiloride via nasal route, rapid onset of action can be achieved due to direct 24 "nose-to-brain" access. Therefore, this study reports the formulation, physical, chemical, 25 and microbiological stability of an extemporaneously prepared amiloride 2 mg/mL nasal 26 spray. The amiloride nasal spray was prepared by adding 100 mg of amiloride 27 hydrochloride to 50 mL of sterile water for injection in a sterile reagent bottle. A stability-28 indicating high-performance liquid chromatography (HPLC) method was developed and 29 validated. Forced-degradation studies were performed to confirm the ability of the HPLC 30 method to identify the degradation products from amiloride distinctively. The physical 31 stability of the amiloride nasal spray was assessed by pH, clarity, and viscosity 32 assessments. For chemical stability studies, samples of nasal sprays stored at room 33 temperature were collected at time-points 0, 3 hr., 24 hr., and 7 days and were assayed 34 in triplicate using the stability-indicating HPLC method. Microbiological stability of the 35 nasal spray solution was evaluated for up to 7 days based on the sterility test outlined in 36 United States Pharmacopoeia (USP) chapter 71. The stability-indicting HPLC method 37 identified the degradation products of amiloride without interference from amiloride. All 38 tested solutions retained over 90% of the initial amiloride concentration for the 7-day 39 study period. There were no changes in color, pH, and viscosity in any sample. The 40 nasal spray solutions were sterile for up to 7 days in all samples tested. An 41 extemporaneously prepared nasal spray solution of amiloride hydrochloride (2 mg/mL) 3 42 was physically, chemically, and microbiologically stable for 7 days when stored at room 43 temperature.