Antiallodynic effect of PhAR‐DBH‐Me involves cannabinoid and TRPV1 receptors

Abstract The antiallodynic effect of PhAR‐DBH‐Me was evaluated on two models of neuropathic pain, and the potential roles of CB1, CB2, and TRPV1 receptors as molecular targets of PhAR‐DBH‐Me were studied. Female Wistar rats were submitted to L5/L6 spinal nerve ligation (SNL) or repeated doses of cisplatin (0.1 mg/kg, i.p.) to induce experimental neuropathy. Then, tactile allodynia was determined, and animals were treated with logarithmic doses of PhAR‐DBH‐Me (3.2‐100 mg/kg, i.p.). To evaluate the mechanism of action of PhAR‐DBH‐Me, in silico studies using crystallized structures of CB1, CB2, and TRPV1 receptors were performed. To corroborate the computational insights, animals were intraperitoneally administrated with antagonists for CB1 (AM‐251, 3 mg/kg), CB2 (AM‐630, 1 mg/kg), and TRPV1 receptors (capsazepine, 3 mg/kg), 15 min before to PhAR‐DBH‐Me (100 mg/kg) administration. Vagal stimulation evoked on striated muscle contraction in esophagus, was used to elicited pharmacological response of PhAR‐DBH‐ME on nervous tissue. Systemic administration of PhAR‐DBH‐Me reduced the SNL‐ and cisplatin‐induced allodynia. Docking studies suggested that PhAR‐DBH‐Me acts as an agonist for CB1, CB2, and TRPV1 receptors, with similar affinity to the endogenous ligand anandamide. Moreover antiallodynic effect of PhAR‐DBH‐Me was partially prevented by administration of AM‐251 and AM‐630, and completely prevented by capsazepine. Finally, PhAR‐DBH‐Me decreased the vagally evoked electrical response in esophagus rat. Taken together, results indicate that PhAR‐DBH‐Me induces an antiallodynic effect through partial activation of CB1 and CB2 receptors, as well as desensitization of TRPV1 receptors. Data also shed light on the novel vanilloid nature of the synthetic compound PhAR‐DBH‐Me.


| INTRODUC TI ON
Neuropathic pain is defined as a pain caused by a lesion or disease of the somatosensory nervous system 1 , which produces functional disabling symptoms and anxiety disorders, addressing on the quality of life of those who suffer this condition 2 . Furthermore, pharmacological treatment of neuropathic pain has limited efficacy and provides an unsatisfactory relief, most often accompanied by side effects which complete an aberrant process that impairs the management and prognosis of the patients [3][4][5] . In a recent report, the Canadian Pain Society published a consensus statement for pharmacological management of chronic neuropathic pain, which includes cannabinoids as a third-line of treatment 6 . Thus, current trends in pharmacological research for neuropathic pain management consider the development of cannabinoid-like drugs and the subsequent characterization of its molecular mechanism of action 7 .
Cannabinoid drugs exert their anti-neuropathic effects through activation of CB1 and CB2 receptors at peripheral 8,9 , central 10,11 and supraspinal levels 12 . Interestingly, alternative targets have also been linked for the treatment of neuropathic pain. Some studies demonstrate that TRPV1 has a special role on antineuropathic-like behavior induced by anandamide 10,13 .
From a historical perspective, cannabinoid-based therapy has been used to treat pain on many cultures. In a previous study, we However, further studies on the nature of this compound such as its antiallodynic effect or its participation on CB2 and TRPV1 receptors have not been performed yet.
As anandamide produces antineuropathic effects through activation of CB1, CB2, and TRPV1 receptors and has structural similarity with PhAR-DBH-Me, in this work, it was hypothesized that PhAR-DBH-Me has antiallodynic effect on SNL-and cisplatin-induced neuropathy and that such effect may be also linked to the activation of the CB1, CB2, and TRPV1 receptors.

| Animals
Female Wistar rats (160-180 g) were used to induce experimental allodynia. Animals were acquired from Centro UNAM-Envigo (Envigo México, SA de CV) and maintained at controlled room temperature in a 12 h light/dark cycle with food and water ad libitum. In this study, the number of animals used per group was the minimum to obtain statistical significance. Animals were euthanized at the end of experiment. The animals used in this work were handled following the

| Drugs
PhAR-DBH-Me was synthetized at Facultad de Estudios Superiores de Zaragoza -UNAM, as was described by 14 .
Capsazepine was acquired from Tocris Bioscience (Ellisville, MO, USA), whereas AM-251 and AM-630 were acquired from Sigma Aldrich (San Luis, MO, USA). Cisplatin was used from a commercial presentation (PISA Laboratories, Mexico), at a concentration of 1 g/ml. Capsaicin was obtained from Sigma-Aldrich (St. Louis Mo. USA). Sodium pentobarbital was acquired from PISA laboratories.

| L5/L6 Spinal nerve ligationinduced neuropathy
Allodynia was induced in the rats by Kim and Chung surgery 16 .
Animals were anesthetized with a mixture of ketamine/xylazine (45/12 mg/kg, i.p.). After surgical preparation of the dorsal vertebral column, the left L5 and L6 spinal nerves were exposed and tightly ligated with 6-0 silk suture distal to the dorsal root ganglion. For sham-operated rats, the nerves were exposed but not ligated. The incisions were sutured, and the animals were observed until their recovery. Fourteen days after surgery, allodynia was evaluated.

| Cisplatin induced-neuropathy
In order to induce neuropathy, animals were exposed to repeated intraperitoneal injections of cisplatin (0.1 mg/kg) for 15 days, every third day. To prevent nephrotoxicity, the cisplatin-treated rats received a second intraperitoneal injection containing saline solution at 0.9% (2 ml/kg). Allodynia was evaluated 15 days after the first administration of cisplatin 17,18 .

| Allodynia assessment
Tactile allodynia was determined by the up-down method 19 . Rats exhibiting motor deficiency were discarded from tactile allodynia evaluation. Tactile allodynia was determined measuring paw withdrawal in response to probing with a series of calibrated fine filaments (von Frey filaments) ranging from 0.4 to 15 g. The stimulus intensity required to produce a response in 50% of the applications for each animal was defined as the 50% withdrawal threshold. All rats were verified for allodynia before experiment (responding to a stimulus of less than 4 g). The 50% of withdrawal threshold of the paw rat, was evaluated in a temporal course of 8 h. For all experiments a blind design was used, drugs were administrated for other personal, avoiding that experimenter associate the nociceptive behavior to the employed treatment. The area under the curve (AUC) was constructed from the temporal course, using the trapezoidal method. Then, from the AUC of the groups, we calculated the percentage of maximum possible effect (%MPE), using the following equation:

| Vagal nerve stimulation induced esophagus contractions: isolated organ assay
For isolated organ bioassay, a Krebs-Henseleit modified solution (KHS, mM: NaCl 136.9, KCl 2.7, CaCl 2 1.8, MgCl 2 2.1, NaH 2 PO 4 0.4, NaHCO 3 1.9 y glucosa 5.5) was freshly prepared before experiment. Firstly, animals were administrated with an overdose of sodium pentobarbital injection (150 mg/kg, i.p.) and then, a segment of the middle thoracic esophagus (a 1 cm-long) was dissected and immediately placed into the Krebs-Henseleit solution at room temperature. Quickly, the two enclosed vagal nerves were identified and separated from the esophagus striated muscle. In order to record the mechanical activity on esophagus muscle, an organ bath (capacity 60 ml) filled with Krebs solution (pH 7.4) in a continuous bubbling with carbogen gas (95% O 2 and 5% CO 2 ) and constant temperature (35°C) was used. Later, esophagus segment was placed between two rings of nichrome, which were connected as follow, one ring was tied to the bottom of chamber, and the other ring to an isometric force transducer (Grass FT 03E), both rings were suspended and tied using a silk thread. Isometric response was filtered and amplified through an amplifier and recorded in the Acqknowledge software, MP100 version 3.5.3 (Biopack systems, inc). A initial tension of 1.0 g was used to perform the assay, the isolated organ preparation was equilibrated and stabilized during 20 min. After this time, the vagus nerve segment was situated on the top of the electrode located on the isolated organ bath and, an electrical stimulation was applied as square-wave pulses in intensity of 80 V in a duration of 0.5 ms at intervals of 1 s. The contractile responses were registered during 10 min and, they were taken as control. In order to test the effect of the capsazepine, capsaicin or PhAR-DBH-ME, alone and combined, were added 10 µl of the capsazepine (10 mM), capsaicin (0.1 mM) and/or PhAR-DBH-Me (100 mM) drugs on the same nerve segment. Each vagal nerve was used only one time for each experiment.
Finally, data were processed from the area under the curve (AUC) of the temporal course of contractile response, which was calculated as follows: Were, contractile effect was measured as grams force (gf), observed as the spike height in the polygraph. Once this characterization was finished, in vivo assays were carried out using a series of pharmacological tools. Rats were administrated with the antagonist of TRPV1 capsazepine at 3 mg/kg, i.p. 27 , the selective antagonist of CB1 receptor AM-251 at 3 mg/ kg, i.p. 27,28 or the selective antagonist of CB2 receptor AM-630 at 1 mg/kg, i.p) 29 . All antagonists were administrated 15 minutes before systemic administration of PhAR-DBH-Me (100 mg/kg, i.p.).

| Statistical analysis
In vivo assays show the mean ± standard error of the mean (SEM) of 6 animals, on independent groups for each experiment. The temporal courses were constructed plotting the 50% withdrawal threshold vs time. The dose-response curve was constructed using the area under the curve obtained through the trapezoidal method.
Data were expressed as the percentage of maximum possible effect (%MPE). Statistical differences were determined by one-way analysis of variance (ANOVA), followed by Tukey's test with a P ≤ .05.

| Systemic administration of PhAR-DBH-Me produces an antiallodynic effect on SNL-and cisplatin-induced neuropathy in rats
The L5/L6 SNL surgery, but not the false ligature, produced allodynia ( Figure 1A). Likewise, the repeated intraperitoneal injection of cisplatin (0.1 mg/kg, every third day) produced a drastic reduction in the 50% withdrawal threshold, compared with animals administrated only with the vehicle ( Figure 1C). In both models, allodynia was In both experimental models of neuropathy, PhAR-DBH-Me administration produced a long-lasting antiallodynic effect, being observed since the first hour from administration to the eighth hour.
Thus, data suggest that the synthetic compound PhAR-DBH-Me has an antiallodynic effect on experimental neuropathy induced by either SNL or cisplatin in rats. where it can be seen hindering the movement of residues F200/ W356, important for agonist effects ( Figure 2B). The docking score of AM251 is slightly larger than the one for PhAR-DBH-Me (−9.9 kcal/mol).

| PhAR
On the CB2 receptor, Figure 3A  Overall, computational studies strongly suggest that PhAR-DBH-Me is a likely binder for the three analyzed targets, also indicating an agonist character for the two GPCRs and an activator for the ion channel.

| Capsaicin and PhAR-DBH-Me induces desensitization on vagal stimulation evoked esophagus contractions in rats
To test the pharmacological response of PhAR-DBH-Me on nervous tissue, we applied the electrical stimulation at vagus

| D ISCUSS I ON
In this work, the intraperitoneal administration of PhAR-DBH-Me For a more comprehensive work on the topology of TRPV1 ion channels, the reader is referred 33 .
In particular, when bound to a phosphatidylinositol (PTI) mole-  We also studied the participation of TRPV1, which is involved in nociception 54 , and more recently it has been suggested its modulation by cannabinoids 55  Taken together, our results indicate that antiallodynic effect of PhAR-DBH-ME is mediated by activation of cannabinoid and vanilloid receptors, suggesting that the nature of this compound acts as an analogue of the endocannabinoid/endovallinoid system for control of neuropathic pain.

D I SCLOS U R E
None of the authors have any conflict to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Please contact the corresponding author for additional data requests.