TRPV3 in Drug Development

Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP (Transient Receptor Potential) super-family. It is a relatively underexplored member of the thermo-TRP sub-family (Figure 1), however, genetic mutations and use of gene knock-outs and selective pharmacological tools are helping to provide insights into its role and therapeutic potential. TRPV3 is highly expressed in skin, where it is implicated in skin physiology and pathophysiology, thermo-sensing and nociception. Gain of function TRPV3 mutations in rodent and man have enabled the role of TRPV3 in skin health and disease to be particularly well defined. Pre-clinical studies provide some rationale to support development of TRPV3 antagonists for therapeutic application for the treatment of inflammatory skin conditions, itch and pain. However, to date, only one compound directed towards block of the TRPV3 receptor (GRC15300) has progressed into clinical trials. Currently, there are no known clinical trials in progress employing a TRPV3 antagonist.


ATP and GM-CSF Release from Mouse Keratinocytes
On the day of assay growth media was removed from cells plated in 96-well plates, before addition of 50 μl Hanks' Balanced Salt Solution; HBSS (composition,in mM,138 NaCl,5 KCl,0.5 CaCl2,0.9 MgCl2,5.6 glucose,20 HEPES,pH 7.4 adjusted with 5M NaOH) in the presence or absence of antagonists.
For ATP release plates were incubated with antagonists for 10 min at 37 °C. Agonists were then added (50 µ L/well) and the plates returned to the incubator for a further 10 min at 37 °C. Finally, the plates were removed from the incubator and 50 µ L from each well transferred to a 96-well white Optiplate. ATP release was measured using the ATP Assay System Bioluminescence Detection Kit for ATP Measurement (Promega, Cat No. FF2021, Madison, WI, USA) according to the manufacturer's instructions using a Viewlux (Perkin-Elmer) to quantify the resulting luminescence.
For GM-CSF release plates were incubated with antagonists for 10 min at 37 °C. Histamine (100 μM final concentration) +/− antagonists was then added (50 µ L/well) and the plates returned to the incubator for a further 48 h at 37 °C. Finally, the plates were removed from the incubator and 100 µ L from each well transferred to a GM-CSF ELISA Kit (Abcam-Cat. No. 46078, Cambridge, UK. The ELISA was run according to the manufacturer's instructions using a Spectramax plate reader (Molecular Devices) to quantify the resulting absorbance at 450 nm.

Animals
Male Sprague Dawley (SD) rats (body weight range 150 to 200 g) or male CD-1 mice (body weight range 25-28 g) were received from Harlan Laboratories, (Indianapolis, IN, USA). The animals were allowed to acclimate 3 days prior to performing any experimental procedures and were housed 3-4 per cage and allowed standard chow and house water ad libitum. The animals were maintained in a controlled environment with 12 h light cycles (6:00 a.m. to 6:00 p.m.). All experiments were carried out according to protocols approved by the Eli Lilly Institutional Animal Care and Use Committee.

Mouse Histamine-Induced Itch Model
Harlan CD-1 mice, 4-5 weeks old were acclimated to testing room for 1 h. FTP-THQ was administered at 30, 100, or 200 mg/kg i.p., 1 h prior to histamine, while diphenhydramine was administered at 20 mg/kg, 30 min prior to histamine. Histamine dihydrochloride; (Fluka 53300-1G) was prepared as 2 mg/mL (base) in saline and diluted to 10 μg/25 μL for intradermal injections between the shoulder blades. Animals were then placed inside a clear plexiglass chamber and the number of scratching bouts was scored for 20 min. Data were collected via Abacus software; one-way ANOVA with post-hoc Dunnett's was used for analysis. * p < 0.05. Data are expressed as means + SEM, with 8 animals per treatment group unless otherwise specified.
The method used for these studies is a modification of that used by Jett et al., 1996. Thirty minutes prior to the administration of formalin, rats were individually placed in cylinders (i.d. 8.5 cm; length 16 cm) on the recording equipment for acclimation. After acclimation in the cylinders, the animals were removed, administered formalin (50 μL of a 5% solution in saline) subcutaneously into the plantar surface of the right hindpaw using a 27½ gauge needle, and immediately placed back into the cylinders. The cylinders were positioned inside of automated test chambers for detecting movement by means of an accelerometer (SR-Lab Startle Response System, San Diego Instruments, San Diego, CA, USA). Rats were monitored continuously for 50 min in 1 s bins. The number of events was defined as the number of 1-s bins with a change in dynamic force that exceeded an empirically determined threshold value (a value of arbitrary load units, which corresponds visually with animals quietly breathing or sniffing). The formalin-induced movements detected by the system included licking and flinching of the affected paw, as well as hopping and turning. The number of "agitation" events was collected and summarized according to the typical biphasic response pattern (Early and Late Phases) in 5-min intervals. After the 50 min test period, animals were sacrificed with CO2 followed by cervical dislocation. Data was analyzed using One-way ANOVA, and post-hoc analysis of treatment groups compared to the vehicle group using Dunnett's test using JMP statistical software (SAS Institute Inc, Cary, NC, USA). Data are expressed as means ± SEM, with 8 animals per treatment group unless otherwise specified.