Behavioral Pharmacology

Adult male C57BL/6J and CD-1 mice housed five to a cage (8–12 weeks of age) were used. C57BL/6J mice were used for evoked locomotor rotarod and formalin assays.^35,60,61 Antinociception was confirmed with the use of CD-1 mice in the CCI nerve assay. The CD-1 strain has been well-validated for antinociceptive⁶² and mechanical anti-allodynic testing.^63,64 All test compounds were administered using the intraperitoneal (i.p.) route. All animal studies reported herein adhere to ARRIVE guidelines.⁶⁵ Animals were randomly assigned, and researchers were blinded to group treatments. Animals were housed on a 12:12 h light/dark cycle (lights off at 7:00 pm) with ad libitum access to food and water except during experimental sessions. All procedures were preapproved by the Institutional Animal Care and Use Committee (University of Florida) and conducted in accordance with the 2011 NIH Guide for the Care and Use of Laboratory Animals.

The efficacy of compound 15 to ameliorate inflammatory nociception was achieved with the use of C57BL/6J mice in the formalin assay as previously described.⁵³ After a 10 min pretreatment (i.p.) of vehicle control (saline), CM304 (3–30 mg/kg, i.p.), or compound 15 (3–30 mg/kg, i.p.), an intraplantar (i.pl.) injection of 5% formalin (2.5 μg in 15 μL) was administered into the right hind paw. Time spent licking the right hind paw was recorded in 5 min intervals for 60 min following injection. The last 55 min of assessment was used to determine the inflammatory response stimulus. Data were analyzed as the summed duration of licking hind paw.

CCI was introduced in CD-1 mice that were first anesthetized with isoflurane as described by Hoot et al.⁶⁶ and Cirino et al.³⁵ to induce mechanical allodynia.⁵¹⁻⁵⁴ After anesthetization, mice were subjected to surgery where an incision was made along the surface of the biceps femoris of the right hind paw.⁶⁶ Blunt forceps were used to split the muscle and expose the right sciatic nerve. The tips of two 0.1–10 μL pipet tips facing opposite directions were passed under the sciatic nerve to allow for easy passing of two sutures under the nerve, 1 mm apart. The sutures were tied loosely around the nerve and knotted twice, and the skin was closed with 29 mm skin staples. Mice were given a 7 day recovery period prior to the baseline von Frey testing as described below to confirm the induction of hyperalgesia in each mouse. Animals demonstrating allodynia or a response to lower pressure were deemed to have neuropathic pain. Allodynic mice were then administered (i.p.) either vehicle (saline), morphine (10 mg/kg, i.p.), gabapentin (50 mg/kg, i.p.), CM304 (45 mg/kg, i.p.), or compound 15 (10–60 mg/kg, i.p.). Note that gabapentin was tested 1 h postinjection to circumvent known sedative effects that may confound the assay.⁶⁷ Each mouse was then tested for the modulation of tactile allodynia every 20 min up until 80 min post-injection with the use of von Frey testing. The assessment of mechanical allodynia was performed to measure compound 15’s efficacy against CCI-induced allodynia as described.⁵¹⁻⁵⁴ Mice were habituated on a mesh platform for 1 h prior to testing. Filaments of increasing pressure (0.4–6 g) were applied and then held to the plantar surface of both the injured and uninjured hind paws of mice for approximately 1–2 s prior to drug administration to record baseline responses to a peripheral stimulus. The filaments were applied with increasing strengths, and threshold responses were defined as two hind paw responses per trial of the same filament strength.

Control or test compounds were administered (i.p.), and paw-withdrawal thresholds were again recorded from 20 to 80 min postinjection. Each hind paw was tested in a counterbalanced manner. Each time was measured in triplicate and then averaged. Responsiveness was a clear withdrawal, shaking, or licking of the paw. To account for variability between mice, data are presented as the percent of baseline paw withdrawal thresholds following filament stimulation of the ipsilateral hind paw. The following equation was used: % anti-allodynia = 100 × ([mean paw withdrawal force {g} in control group – paw withdrawal force {g} of each mouse]/mean paw withdrawal force [g] in control group).

The rotarod coordination assay was used to assess effects on evoked locomotor activity in C57BL/6J mice administered vehicle (saline, i.p.), morphine (10 mg/kg, i.p.), U50,488 (10 mg/kg, i.p.), CM304 (45 mg/kg, i.p.), or compound 15 (30–60 mg/kg, i.p.) using methods described previously.^68,69 Seven habituation trials were performed where the last habituation trial was used as an initial baseline of performance. The mice were administered (i.p.) test agents and then evaluated every 10 min in accelerated speed trials (180 s max latency at 0–20 rpm) over a 60 min period. The latency to fall was measured in seconds. Data are reported as the mean percent change from each mouse’s initial baseline latency to fall. Decreased latencies to fall in the rotarod test indicate impaired motor coordination or sedation

All data are presented as mean ± SEM. Significance is indicated as *p < 0.05 and was analyzed using two-way ANOVA with Tukey’s post hoc analysis. Statistical analysis was performed with the use of GraphPad Prism 9.0 software. Dose response lines were analyzed by linear or nonlinear regression modeling and ED₅₀ values (dose yielding 50% effect) along with 95% confidence limits using each individual data points. The rotarod data are expressed as the % change from baseline performance for each animal’s baseline response.