Purpose: Anandamide is an endogenous potent cannabinoid CB1 receptor agonist effective in the management of severe inflammatory and neuropathic pain by inhibiting the signal initiation and transmission to brain. Anandamide is metabolized in biological tissues by the enzyme fatty acid amide hydrolase (FAAH). The approach to enhance the level of anandamide is either by inhibiting the breakdown of these endocannabinoids or by administration of exogenous anandamide at the site of action. Anandamide topical delivery co-administered with FAAH inhibitors would be beneficial in the management of peripheral neuropathic pain. The present study was aimed to determine the anandamide metabolism in invitro skin models (HaCaT cells) and to identify potential FAAH inhibitors to enhance anandamide therapeutic effect peripherally.
Methods: The human epidermal keratinocytes (HaCaT) cells were used to study in vitro skin metabolism of anandamide. The In-vitro skin clearance studies were performed by incubating anandamide at 10 µM individually and along with natural products FFI-1, FFI-2, FFI-3, FFI-4, FFI-5, FFI-6 and FFI-7 at different 20, 10, 5, 2.5 and 1 µM concentrations in 20,000 HACAT cells per well in growth medium at 37 °C with 5% CO2. The cells were treated with acidified ice-cold acetonitrile at 0, 1, 2, 4 and 6 h intervals to terminate the reaction. The anandamide was extracted from the cells and analyzed by LC-MS/MS.
Results: The substrate depletion method was used to determine intrinsic skin clearance of anandamide. The anandamide percentage metabolized at 6 h, intrinsic skin clearance and t1/2 of anandamide was 91.0 ± 7.45%, 825 ± 37.1 µL/hr/106 cells and 1.68 ± 0.34 h, respectively. The anandamide percentage metabolized at 6 h in presence of 10 µM FFI-2, FFI-4 and FFI-6 was 72.3 ± 3.14%, 23.9 ± 1.24% and 59.1 ± 3.28%, respectively. The anandamide intrinsic skin clearance in presence of 10 µM FFI-2, FFI-4 and FFI-6 was 466 ± 51.4, 84.8 ± 3.10 and 315 ± 14.8 µL/hr/106 cells, respectively. The t1/2 of anandamide intrinsic skin clearance in presence of FFI-2, FFI-4 and FFI-6 was 2.67 ± 0.13, 16.3 ± 1.10 and 5.91 ± 0.48 h, respectively.
Conclusion: The results illustrate FFI-4 and FFI-6 are potent inhibitors of FAAH, which upon incubation with anandamide in cells increases the t1/2 of anandamide in human skin model. This study illustrates that anandamide when co-administered with FFI-4/FFI-6 topically, would prevent breakdown of anandamide in skin and increase duration of analgesia.