Purpose: Consumption of alcohol is a common social incident in the western world. Excessive consumption can lead to major adverse events. Although there are treatments available to treat addiction to alcohol or alcoholism, unpleasant side effects such as anxiety, headache, dizziness, pain and abdominal cramps are reported to be resulting in lower patient compliance. β-caryophyllene, a sesquiterpene which is a strong anti-oxidant found to be useful in the treatment of pain, inflammation, atherosclerosis and osteoporosis, has currently been studied for its agonist activity on the CB2 cannabinoid receptors and has shown promise for the treatment of alcoholism. The aim of this study was to develop and validate a HPLC method for β-caryophyllene analysis and to evaluate its transdermal delivery across porcine ear skin.
Methods: A simple and rapid high performance liquid chromatography method coupled with an ultraviolet detector was developed and validated for the analysis of β-Caryophyllene. The analysis was carried out using a reverse phase isocratic technique. A C8 column; Agilent, Zorbax Eclipse XDB-C8, (3×150 mm, 5 µm) was used as the stationary phase and the mobile phase was composed of acetonitrile: 0.1% trifluoroacetic acid (TFA) solution in deionized water (75:25). The detection wavelength was set at 210 nm and the total run time was 10 min. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were determined following International Conference on Harmonization (Q2B) Guidelines and the repeatability of the results was evaluated by determining the intra-day and inter-day precision and accuracy. In vitro permeation studies were performed using vertical Franz diffusion cells to investigate the delivery of β-caryophyllene across dermatomed porcine ear skin. Being a hydrophobic drug (LogP=4.5), several compositions were tested as the receptor solution, to maintain sink conditions. Solubility of the drug was tested in different ratios of ethanol and phosphate buffered saline (PBS, pH 7.4, 10 Mm), PEG 400 and PBS, and hydroxypropyl-β-cyclodextrin PBS, Brij® O20 in PBS, and Captisol® in PBS. The donors of the Franz Cells contained 150µL or 135.15mg of β- caryophyllene. The sampling time points for the study were 0, 2, 4, 8, 22, 24 and 26 h. Following the permeation study, the skin was cleaned and tape stripped to remove any residual drug on the surface and was further analyzed for drug absorbed into the skin.
Results: The selectivity of the analysis method was established since there were no interfering peaks at the drug’s retention time (4.67 min). The linearity was within the range of 0.5 to 100 µg/mL (r-squared>0.999). The LOD and LOQ values were determined to be 0.07 µg/mL and 0.22 µg/mL, respectively. The inter-day accuracy and precision were determined to be 98.64-112.98% and 0.89-4.62%, respectively. For the intra-day accuracy and precision study the responses were determined to be 98.24-109.98% and 0.26-1.86% respectively. β-caryophyllene showed very low solubility in the solutions tested. However, a solubility of 6.23 mg/mL was observed in PEG 400 which was thus selected as the receptor solution for in vitro permeation testing. The average cumulative amount of drug permeated through the skin was found to be 41.44±14.68 µg/sq.cm (Fig.1) at the end of the in vitro permeation study. However, a lag time of 17.83 h was observed and the amount of drug retained in the skin was found to be 183.96±46.18 µg/sq.cm.
Conclusion: In this study, transdermal delivery of β-caryophyllene was found to be possible as drug was found in the receptor. A simple and rapid HPLC method was successfully developed and validated to analyze β-caryophyllene. Permeation of the drug into the receptor compartment as well as skin was successful but a slow permeation profile was obtained, with a high lag time.