0.4 %w/v control 0.4 %w/v SLN's
43
Rabbit No Basal
1 2 3 Avg
87 Right (G) 21 21 19 20.3
Left (N) 20 19 18 19.0
91 Right (G) 19 20 21 20.0
Left (N) 19 19 20 19.3
Results of basal IOP readings Table 3
After the basal IOP readings were recorded, both animals received instillations of the control formulation in the right eye. A total of 50 μL of 0.4% w/v control formulation was administered, with each dose containing 0.2 mg of WIN 55, 212. The study took place over a 120-minute duration, and IOP readings were taken at 30-minute time intervals. A total of three readings were taken of the right eye at each interval, and the average of the three was recorded, shown in Table 4.
44 Time Interval
Average IOP Reading (mmHg) of Rabbit Number: 87
Results of IOP readings after installation of control formulation Table 4
After the study was complete, the IOP levels were recorded as a function of the percent of baseline IOP. This allows the determination of when the maximum IOP reduction was for each animal. Both animals saw a maximum IOP reduction at the 30-minute time interval. Rabbit 87 experienced a 37.6% maximum IOP reduction rabbit 91 experienced a 29.2% maximum IOP reduction, shown in Table 5.
% Change in IOP from baseline with control formulation Table 5
45
After the control formulations had been tested and the results were quantified, the three remaining rabbits in the study population were given instillations of the WIN SLN formulations.
As with the control formulations, the basal IOP readings were taken after a two-week stabilization period. The results can be seen in Table 6.
Rabbit No Basal IOP Reading
1 2 3 Average
85 Right (G) 27 29 30 28.7
Left (N) 16 14 17 15.7
88 Right (G) 26 26 27 26.3
Left (N) 16 15 17 16.0
89 Right (G) 35 36 33 34.7
Left (N) 19 20 19 19.3
Results of basal IOP readings of rabbits receiving WIN-SLN Table 6
After the baseline IOP levels were recorded, each rabbit received 50 μL of the 0.4% w/v WIN-SLN formulation containing a total of 0.2 mg of WIN 55, 212. The study was terminated when the IOP levels of the glaucomatous eyes had reached 90% of the baseline IOP levels, which was 180 minutes. The IOP readings were taken every 30 minutes. The results can be seen in Table 7.
46
Results of IOP readings after instillation of WIN-SLN Table 7
The results of the WIN-SLN and control formulation studies were then compared to determine the effectiveness of the solid lipid nanoparticle. The duration of effect of the 0.4% w/v WIN-SLN formulation was found to be four times that of the control formulation, which can be seen in Figure 11.
47
Effectiveness of control formulation versus WIN-SLN Figure 11
Immediately following the conclusion of the WIN 55, 212 SLN study, the rabbits were euthanized by a lethal dose of pentobarbital. given through the marginal ear vein. After being euthanized, the right eye of each rabbit was enucleated and washed. The ocular tissues were then separated, weighed, and preserved at -80°C until they were shipped to Excalibur Pathology to assess them for any damages or changes when compared a control. The corneal cross-sections were stained with hematoxylin-eosin. The top cross-sections were the controls that were exposed to phosphate-buffered saline. The bottom cross-sections were the corneas exposed to the SLN formulation. The results show that corneas exposed to the SLN did not have any significant changes when compared to the control. Figure 12 shows the results of the histology testing, with
50
WIN 55 (0.4 % w/v) in Tocrisolve
WIN 55 SLN (0.4 %w/v) Formulation
48
(1) representing the control corneas that were exposed to a phosphate-buffered saline and (2) showing the corneas exposed to the SLN formulation.
Results of the histology testing Figure 12
49 Conclusion
When used correctly, current marketed glaucoma treatments have proven effective in lowering intraocular pressure levels. However, the search continues for a drug that could
successfully provide an increased duration of action and protection of the optic nerve. Topically administered eye drops for glaucoma are not able to permeate the posterior segment where the optic nerves are found, and are confined to the anterior segment where they exert their action by modifying aqueous humor production and outflow. Drug delivery systems utilizing
nanotechnology have been speculated as a viable option to provide what other glaucoma drugs cannot, the ability to reach the posterior segment and to sustain a drug’s length of action. Solid lipid nanoparticles serve as a vehicle for drugs with poor physiochemical properties, and we explored the effectiveness of these formulation systems in our research.
After the preparation of the solid lipid nanoparticle is complete, we had to confirm that the formulation possessed favorable physiochemical characteristics that would allow it to be successful in subsequent in vitro and in vivo studies. Our WIN 55, 212 SLN showed favorable physiochemical characteristics, prompting us to move forward with the in vitro studies. When compared to the control formulation, the WIN 55, 212 SLN showed a two-fold increase in rate, flux, and transpermeability. These results confirm the solid lipid nanoparticle delivery systems ability to enhance the permeability of the drug, and is indicative that higher bioavailability will be found during in vivo studies.
When tested in vivo, the WIN 55, 212 SLN formulation sustained a drop in intraocular pressure that was four times as long as the control formulation. These results confirm the solid lipid nanoparticle’s ability to extend the duration of action of WIN 55, 212. The results of this project provide evidence that solid lipid nanoparticles are a drug delivery system that can provide
50
increased bioavailability and can lengthen the duration of action. This delivery system was able to effectively deliver the cannabinoid WIN 55, 212 into the ocular tissues, and it can be
extrapolated that other topically administered ocular drugs may benefit from this delivery system or other forms of nanotechnology.
Overall, the WIN 55, 212 formulation exhibited desirable physiochemical characteristics and was successful in the in vitro and in vivo studies. Although the SLN formulation extended WIN 55 212’s ability to lower intraocular pressure, it did not outlast Pilocarpine eye drops, one of the many glaucoma treatments available on the market. Further studies are required to optimize the SLN formulation to increase its efficacy when compared to current FDA marketed glaucoma drugs.
51
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