(M1130-10-64) Effect of Amounts of DMSO and Incubation Time on BBB-PAMPA Permeability of Drugs with High and Low BBB Permeability

Purpose: One of major challenges hampering the success of central nervous system (CNS) drugs is the difficulty in penetrating Blood Brain Barrier (BBB). During BBB- parallel artificial membrane permeability assay (PAMPA), which is useful for evaluating how well drugs can penetrate the BBB, dimethyl sulfoxide (DMSO) is often added to increase drug solubility in donor and acceptor solutions, as CSN drugs tends to be insoluble but needs to be dissolved in the solutions. However, DMSO is known to create pores in lipid membranes, thereby enhancing membrane penetration. In this study, the effect of DMSO and incubation time on BBB permeability of drugs with high and low permeability was investigated. The goal was to investigate the effect of DMSO concentration and incubation time on BBB-PAMPA permeability of drug with low and high permeability.

Methods: The parallel Artificial Membrane Permeability Assay (PAMPA) was conducted using five drugs including Caffeine, Donepezil-HCl, Donepezil freebase, Piroxicam, Verapamil-HCl. Each compound was dissolved in DMSO at 10mM and diluted with different ratios of DMSO/PBS solution to obtain donor solutions at a final concentration of 100 μM. Porcine polar brain lipid extract (PBLE) was dissolved in a hexane/dodecane solution and 4 μL of the resulting solution was coated to the donor plate.In order to evaluate the effect of DMSO on donor and acceptor plates, donor and acceptor plates were filled with 100 μL and 200 μL of solutions containing different proportion of DMSO to the total solution concentration (1%, 15%, 20%, 33.3%, 50%, and 66.7%), respectively. Incubation time was 4 hours. In order to evaluate the effect of incubation time, permeability was measured by varying incubation time of 4, 8, 12, and 16 hours at 37°C. Donor and acceptor plates were filled with 100 μL and 200 μL of solutions containing 1% DMSO.

Results: To confirm the effect of DMSO on BBB permeability of drugs, two variables were set under various conditions for experimentation. [Figure1] shows the permeability trend of five drugs in BBB-PAMPA when the incubation time such as 4, 8, 12, and 16 hours, varies. With the incubation times of 4 and 8 hours, the BBB permeability ranked in a descending order, as follows - Donepezil Freebase > Donepezil HCl > Verapamil HCl > Piroxicam > Caffeine while Donepezil Freebase > Donepezil HCl ≒ Piroxicam > Verapamil HCl ≒ Caffeine with the incubation times of 12 and 16 hours [Figure1]. The logPe values of Donepezil freebase, Piroxicam, and Caffeine tend to increase as time increases, while Donepezil HCl and Verapamil HCl exhibit relatively constant logPe values regardless of incubation time. BBB-PAMPA permeability was evaluated using Donepezil HCl with varying DMSO concentration ratios (1%-66.7%). [Figure3] showed that below 15%-20% concentration, the logPe value increased as the concentration of DMSO decreased. However, at higher concentrations, the logPe values tend to decrease. According to the experiment conducted on caffeine with different DMSO concentration ratios (1%-66.7%), shown in [Figure3], there was no significant change in logPe value from 1% to 33.3% DMSO concentration. However, a significant decrease in logPe value was observed at 50% concentration, and an opposite trend was observed as the concentration increased to 66.7%, showing a significant increase in permeability. Same donor concentration experiment with different acceptor conditions, using PBS only or PBS with 66.7% DMSO, showed significantly higher permeability in the latter condition.

Conclusion: Dimethyl sulfoxide is well-known for increasing lipid permeability. Using the same concentration of DMSO, we conducted PAMPA experiments at different time points to observe changes in logPe values for each drug and obtained the results shown in Figures 1. For the three freebase drugs, the logPe values increased as time increased. This can be interpreted as a result of increased exposure to DMSO and increased time for the drug to penetrate. In contrast, VerapamilHCl and Donepezil HCl, which contain salt, showed regions where logPe values did not increase significantly with increasing incubation time. This demonstrates that even though there was increased exposure to DMSO over time, permeability did not increase significantly. Therefore, it can be concluded that DMSO does not always increase lipid permeability, but its effects differ depending on the drug properties. Figure 3, which showed the permeability based on the ratio of DMSO, also demonstrated that logPe changes did not correspond proportionally to the ratio of DMSO. This also indicates that DMSO does not unconditionally increase BBB-PAMPA permeability at any ratio and that the tendency can vary for each drug. The study will provide a guide to understand the BBB-PAMPA permeability data and accelerate CNS drug development process.

Acknowledgements: supervisor: professor Eun Hee Lee

[Figure1. BBB permeability of control compunds in different incubation time with 10% v/v PBLE]

[Figure2. BBB permeability of caffeine in different incubation time with 5% v/v PBLE]

[Figure3. BBB permeability of DonepezilHCl and Caffeine in different concentration of DMSO]