An in vitro skin penetration model was used to optimize three formulations (A, B, and C) containing the compound SB-275833. In addition, during method development, three different approaches were used to evaluate the extraction efficiency of the molecule from skin tissues (epidermis and dermis): LE for 15 hours, LE for 48 hours, and homogenization using Precellys 24 homogenizer and customized tubes with 2.8 mm stainless steel beads (Precellys lysing kit MK28-R). These tubes were developed to be used with a Biocap® decapper (Biosero) thus minimizing the time spent with manual tube handling.
The skin permeation study was set up using custom-made Bronaugh diffusion cells and dermatomed human abdominal skin (approximately 500 µm thickness) from plastic surgeries. The flow of PBS was set at 60 µL/min, with the time points collected hourly for six hours, for posterior analysis by LC-MS/MS.
After six hours, the skin samples were split into epidermis and dermis by placing them in an oven set at 60°C for two minutes. Each skin layer was placed in individual homogenization tubes and 500 µL of extraction solvent (1:1 methanol:water + 1% formic acid) was added. The Precellys 24 protocols for each layer were the following: epidermis—one cycle of 30 seconds at 6,500 rpm and dermis—two cycles of 60 seconds each at 6,500 rpm, with a 30-second interval in between.
Next, 1,000 µL of methanol was added to each tube, followed by a 10 second vortexing, sonication for 10 minutes, and finally a centrifugation step for five minutes at 10,000 g and 5°C. The tubes were placed in a rack and samples were transferred and diluted in a 96-well plate using an EVO® 200 liquid handler (Tecan).
The samples (epidermis and dermis) used for LE were placed in 10 mL glass vials and 8 mL of a mixture of 1:1 methanol:water + 1% formic acid was added. The vials were incubated for 15 or 48 hrs at 5°C on a horizontal shaker. After this extraction step, the samples were sonicated for 10 minutes and then organized in a rack before being transferred and diluted in a 96-well plate using the liquid handler.
The compound was analyzed by LC-MS/MS using a Xevo TQ-MS (Waters) interface and multiple reaction monitoring in positive-ion mode for the transitions (m/z) 518.6>124.0 and 518.6>216.0. The chromatographic column was an UPLC BEH C18 50 × 2.1 mm, 1.7 μm particle size (Waters) with a mobile phase A containing 0.1% formic acid in water and a mobile phase B containing 0.1% formic acid in acetonitrile.
The flow rate was set at 0.4 mL/min with the following gradient 90:10 A:B from 0 to 0.4 min; 100:0 A:B from 0.6 to 1.3 min; and 90:10 A:B from 1.4 to 2 min. The injection volume was 5 µL, and the compound retention time was 1.1 minutes. The lower limit of quantification (LLOQ) was 20 pg/mL and the limit of detection, 5 pg/mL. The calibration curves were prepared over the range of 20 to 10,500 pg/mL, adequate for the detection and quantification of SB-275833 in the receiving fluid samples, dermis, and epidermis samples.