Menthol-derived isoxazoles reduce capsaicin-induced allodynia in mice when applied as cutaneous gel

TRPM8 is widely believed to be the putative receptor for menthol [1-2]. Recently, it has been reported that TRPM8 is the major sensor of peripheral innocuous cool temperatures and is involved in cold allodynia after nerve injury [3]. Thus we have synthesized several isoxazole derivative in order to evaluate the efficacy of those compounds both in vitro and in vivo. Synthesis was carried out by one step procedure in which the appropriate amino-isoxazole was reacted with an aldehyde in acidic media. The intermediate thus obtained, was then reduced by adding sodium triacetoxyborohydride. Crude products were purified by column-chromatography and final products characterized by mass and NMR spectroscopy. In vitro data were collected by single-cell Ca2+ imaging experiments with FURA-2AM in F11 cells, a clonal cell line obtained by the fusion of embryonic rat dorsal root ganglion (DRG) neurons with mouse neuroblastoma cells [4]. F11 cells express typical nociceptive markers, such as delta-opioid, prostaglandin, and bradykinin receptors, and are a widely used cellular model for nociceptive molecular mechanisms [5]. Furthermore, RT-PCR and quantitative Real-Time PCR experiments revealed the expression of transcripts encoding for TRPM8 receptors in this cellular model. Although, among the compounds tested, menthol appeared as the most effective in eliciting Ca2+ responses in F11 cells, several menthol derivatives provided of up to 100-fold greater potency when compared to menthol were also identified. On the basis of these results, the most potent menthol derivatives were formulated as gels for cutaneous application, in order to increase their cutaneous permeability and thus their efficacy. These formulations were used for the following experiments using an in vivo model in mice. Mice were treated with 0.5 g/paw of gel containing 10% of the isoxazole compound under trial. 15 min after then 0.8 g capsaicin/paw were injected in the paw plantar surface and pressure allodynia was measured 30 min after capsaicin injection, using licking and shacking as allodynic parameter. Here we reported that the selected isoxazole derivatives were more effective than the parent compound menthol in inhibiting capsaicin-induced allodynia. Permeation enhancement was evaluated by in vitro models, using Franz-type diffusion cells. Formulated gels were compared with solution containing the selected compounds at the same concentration. In vivo experiments on formulated gels confirmed that the increase in cutaneous absorption lead to reduction in capsaicin-induced allodynia. We conclude that these compounds could be very useful in the pharmacotherapy of allodynia following neuropatic pain.