Activity of FAAH-inhibitor JZP327A in an experimental rat model of migraine

This study aimed to explore the pharmacological activity of JZP327A in the modulation of spontaneous and nocifensive behavior in the animal model of migraine based on nitroglycerin (NTG) administration. The inhibitor JZP327A (0.5mg/kg, i.p.) or vehicle was administered to male rats 3h after NTG (10mg/kg, i.p.) or vehicle injection. Rats were then exposed to the open field test and the orofacial formalin test 1 h later. We also evaluated the effects of JZP327A on the expression of some pain and inflammatory mediators in cranial tissues and in the serum and on the levels of endocannabinoids and lipid-related substances in the medulla-pons and trigeminal ganglia.

The in vivo and ex vivo assessments were:

-Evaluation of trigeminal hyperalgesia in the orofacial formalin test: the face rubbing was measured counting the seconds the animal spent grooming the injected area (upper lip, lateral to the nose) with the ipsilateral forepaw or hindpaw 0–3 min (Phase I) or 12–45 min (Phase II) after formalin injection (50 µl, s.c.). The observation time was divided into 15 blocks of 3 min each.

- in the open field test were evaluated: time spent (expressed in seconds) in the center of the apparatus as a measure of anxiety; distance (expressed in meters) travelled in the apparatus as a measure of motor behaviour; number of rearings as a measure of exploratory behavior; time spent in grooming behavior (expressed in seconds) used as nociception index.

-Gene expression analysis: mRNA expression levels of, CGRP, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL) 6, were evaluated brain areas mRNA levels were measured by rt-PCR. All samples were assayed in triplicate and gene expression levels were calculated according to 2−∆∆Ct = 2− (∆Ct gene − ∆Ct housekeeping gene) formula by using Ct (cycle threshold) values.

- CGRP serum levels: For CGRP serum levels, the blood samples were collected in a clot activator with gel separator serum tubes and centrifuged for 15 min at 1000× g at 2–8 °C. Serum levels of CGRP measured using a commercial ELISA kit (-CGRP: Elabsciences).

-lipids levels in cranial areas: lipids were quantified in rat brain tissue using a previously proven online solid phase extraction (SPE) high-performance liquid chromatography (HPLC) method coupled with tandem mass spectrometry (MS/MS).

Results – The findings show that JZP327A did not affect NTG-induced changes in the spontaneous behavior of rats, while inhibited NTG-induced hyperalgesia at the formalin test. Furthermore, JZP327A dramatically decreased the gene expression of CGRP, TNF-alpha, and IL-6 in the TG and medulla-pons, while it did not change CGRP serum levels, nor endocannabinoids or lipids levels in the cranial tissues. These findings suggest an anti-hyperalgesic role for JZP327A in the NTG model, which is mediated by the inhibition of the inflammatory cascade of events. This activity does not seem mediated by a change in levels of endocannabinoids and lipid