Aerosol drug delivery devices
In this study, we compared the performance of the ModiFlow (MF) to an idealized Standard Spacer (SS). For standardization purposes, a hollow cylindrical tube of identical length and inner diameter represented both devices, with the only difference being that MF had specifically designed inner septal structures, which SS didn’t.
Since most, if not all spacer devices on the market have a cylindrical shape, and no internal structures, our Standard Spacer model was considered to be a fair representation of spacers on the market. In addition, several studies have demonstrated that with the exception of volume, all other structural modifications to the spacers, such as valves and masks, have not made a significant difference in their performance. Regardless, our goal was to minimize any structural variability between the tubes for the purpose of controlling all parameters, and leaving only one main difference – the presence of inner septal structures in MF, and the absence thereof in SS.
In accordance with the above, for this study the parameters for ModiFlow were selected as follows: Total length – 100mm, Inner Diameter – 38mm, Septi – 62mm, 34mm, 24mm. The calculated Inner Surface Area (ISA) was 164.9 sq. cm, of which 45.6 sq. cm is accounted for Septi only, and 119.3 sq. cm for inner walls. As a SS, a hollow cylindrical tube of the same Length (100mm) and Inner Diameter (38mm) as in MF was used, with the calculated ISA of 119.3 sq. cm.
To compare the efficiencies of aerosol delivery for ModiFlow vs a Standard Spacer, 4 different Test Tubes (TT) have been devised, all of which had a similar structure as depicted in Figure 1. Two of them had a length of 15cm, and two – 30cm. All four TT’s were of cylindrical shape, and of the same inner diameter (38 mm). In the TT of each length, either ModiFlow (Figure 1-5) or a Standard Spacer (Figure1-1) were inserted flush with the proximal end of the TT. The proximal end of TT’s was furnished with an adapter (4) for Medication Pump (5), and the distal end – with a receptacle (1) for tissue culture. This set the distances from the exit of the spacer (MF or SS) to the distal end of TT (where the tissue culture was placed) to be either 15-10=5 (cm) or 30-10=20 (cm), thus creating four different designs of TT: MF-5, MF-20, SS-5, SS-20. This distances from the spacer exit to the tissue culture were selected to be either “within” (5 cm) or “outside” (20 cm) of the average range of adult oro-pharyngeal cavity. This would allow for four pairs of comparisons to be made: MF-5 to SS-5, MF-5 to MF-20, MF-20 to SS-20, and SS-5 to SS-20. The total ISA for each Test Tube was respectively MF-5 – 224.6 sq. cm, MF-20 – 403.5 sq. cm, SS-5 – 179 sq. cm, SS-20 – 358 sq. cm, excluding the surface area of tissue cultures and receptacles.
Aerosolized medication
Fluticasone Propionate Metered Dose Inhaler (220 µg per spray) was used in all test tubes.
3D Oral Epidermal tissues
To better approximate physiological conditions of drug delivery, we measured aerosolized drug deposition on a SkinAxis model of the oral mucosal tissues. Normal human Gingival keratinocytes (SkinAxis) were cultured on specially prepared cell culture inserts using serum-free medium and differentiated in vitro using proprietary SkinAxis’ cell culture technology to form multilayered, highly differentiated models of the human gingival phenotypes (Figure 3, and www.skinaxis.com). SkinAxis oral epidermal tissue models are highly reproducible and exhibit in vivo-like morphological and growth characteristics. The differentiated tissue was inserted at the end of the spacer, as described above and to quantify drug deposition tissues were processed for Mass Spectrometry.
Fluticasone extraction from oral tissues and quantification.
Each tissue sample was processed by: adding 50 ml 0.1% formic acid and 200ml of methanol to a culture plate, scrapping with pipette tips, and transferring to an Eppendorf tube. The plate was washed sequentially with 200ml 0.2% formic acid and 100ml methanol and the washes combined with the initial extract. Extracts were sonicated for 1 min and centrifuged for 5 min at 25000 x g. Supernatants were diluted 10-fold using 50% methanol/0.1% formic acid before analysis by LC-MS.
HPLC–MS experiments were performed using a ThermoFisher Velos LTQ Orbitrap Pro mass spectrometer interfaced with a Dionex U3000 chromatography system. Samples (5 µL) were injected in microliter pick up mode and separated on a reverse-phase column (Discovery BIO Wide Pore C18, 5cm x 2.1mm, Supelco Analytical). Chromatography was conducted at a flow rate of 200µl/min using a gradient formed with an aqueous solution of 0.2 % acetic acid (solvent A) and methanol (solvent B) as follows: 60% B (1 min), 60-90% B (linear increase in 3 min), 90% B for 1 min, 90- 60% B (linear decrease in 0.1 min), and equilibration at 60% B (3 min). The column temperature was maintained at 45°C. MS acquisition parameters were as follows: the electrospray ion source was operated in positive ion mode (ESI+). The positively charged fluticasone (m/z= 501.3) was isolated in the ion trap with an isolation window of 3 m/z and fragmented with CID with a relative collision energy of 25% and activation time of 10 milliseconds. Fragments were detected using the ion trap and the 303.15 m/z fragment used for quantification.
A standard curve consisting of dilutions of fluticasone in methanol (0.01 ng/ml to 100mg/ml) was analyzed in parallel with samples. Peak areas of the 501-301 transitions were measured using XCalibur software. Concentrations of fluticasone in samples were determined with respect to standard curves by non-linear regression using a four-parameter sigmoidal fit, weighted by 1/y^2.
Statistical analysis
For each of the two spacers, independent t-tests were used to test the effects of varying drug deposition in different experimental settings. P-value < 0.05 was considered statistically significant.