Resatorvid

Formulation

The table below describes the formulation of resatorvid and the controls morphine sulfate and gabapentin for the following in vivo experiments.

Experiment(s)	Compound	Dose(s) mg/kg	Correction factor	Dose volume mL/kg	Route	Vehicle	Suspension / Solution
Pharmacokinetics	Resatorvid	3, 30	1.00	10	SC	DMSO /	Solution
Pharmacokinetics	Resatorvid	3, 30	1.00	10	SC	Tween 80	Solution
Irwin, Rotarod	Resatorvid	3, 10, 30, 100	1.00	10	SC	DMSO /	Solution
Irwin, Rotarod	Resatorvid	3, 10, 30, 100	1.00	10	SC	Tween 80	Solution
Plantar incision,	Resatorvid	1, 3, 10, 30	1.00	10	SC	DMSO /	Solution
L5/L6 SNL	Resatorvid	1, 3, 10, 30	1.00	10	SC	Tween 80	Solution
Plantar incision	Morphine	6	1.33	1	SC	Saline	Solution
L5/L6 SNL	Gabapentin	60	1.00	1	PO	Saline	Solution

L5/L6 SNL = L5/L6 spinal nerve ligation; PO = per os; SC = subcutaneous; DMSO/Tween 80 = 10% dimethylsulfoxide (DMSO), 5% Tween 80, and 85% sterile water

Results

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Method: Pharmacokinetics

Animals were randomly assigned to treatment groups.

Experimenters collecting samples were masked to treatment.

Resatorvid concentrations were measured in serially collected plasma samples and terminally collected brain samples (shown on two graphs) from males and females treated with 3 or 30 mg/kg resatorvid (SC) in a vehicle of DMSO/Tween 80. There were 3-4 rats/group.

Method: Pharmacokinetics

A table shows pharmacokinetics parameters calculated for resatorvid using non-compartmental analysis in male and female rats treated with resatorvid (3 or 30 mg/kg, SC, vehicle of DMSO/Tween 80). The means for the 4 groups (males and females treated with 3 mg/kg resatorvid and males and females treated with 30 mg/kg resatorvid, respectively) are as follows. The highest concentration (Cmax) was 1.64, 1.4, 8.41, and 6.6 µM; the time at the maximum concentration (Tmax) was 0.75, 0.88, 0.75, and 1.13 hours; the half-life (T1/2) was 3.72, 2.69, 2.72, and 2.61 hours; area under the curve for time 0 to 24 hours was 206, 191, 1749, and 1674 minutes*µM; area under the curve for time 0 to infinity was 210, 193, 1751, and 1676 minutes*µM; the brain-to-plasma ratio at 2 hours was 0.17, 0.23, 0.08, and 0.06; the brain-to-plasma ratio at 24 hours could not be calculated because all samples were below the lower limit of quantification.

Method: Modified Irwin functional observational battery

Animals were balanced across treatment groups based on body weight.

Experimenters assessing behavior were masked to treatment.

Group size was determined using power analysis.

Two reviewers observed and scored 39 behaviors in a modified Irwin test. For each animal, the scores for behavioral observations from each reviewer are summed then averaged for male and female rats (shown on two graphs). Responses are shown at the following time points: baseline (before treatment) and at 1, 2, 4, and 6 hours after treatment with vehicle (10% DMSO and 5% Tween 80 in water, delivered PO) or resatorvid (3, 10, 30, or 100 mg/kg, delivered PO). There were 4 rats per group. Kruskal-Wallis tests were performed at each timepoint for each sex, and no significant effects were observed. Data were analyzed using GraphPad Prism version 10.0.2

Method: Modified Irwin functional observational battery

A heat map depicts the severity of the behaviors observed by two reviewers in a modified Irwin test at baseline and at 1, 2, 4, and 6 hours following the administration of vehicle (10% DMSO and 5% Tween 80 in water, delivered PO) or resatorvid (3, 10, 30, or 100 mg/kg, delivered PO). There were 8 rats per group (4 male and 4 female). Severity score was calculated as the sum of scores given by the two reviewers for all animals at that dose and timepoint divided by the maximum possible score, transformed into a percentage. In summary, resatorvid administration was well tolerated at all doses

Method: Modified Irwin functional observational battery

Two graphs show the body temperature of male or female rats during the modified Irwin test. Responses are shown at the following timepoints: baseline (before treatment) and at 1 and 6 hours after treatment with vehicle (10% DMSO and 5% Tween 80 in water, delivered PO) or resatorvid (3, 10, 30, or 100 mg/kg, delivered PO). There were 4 rats per group. A significant effect of time was found in a two-way repeated measures ANOVA for each sex. Dunnett’s tests found a significant decrease in body temperature after 1 hour relative to baseline for male rats (p<0.05). Dunnett’s tests found no significant comparisons between timepoints for females. Data were analyzed using GraphPad Prism version 10.0.2

Method: Rat Rotarod

Animals that achieved a latency of 40 seconds during the baseline trial were included in the study.

Two graphs show the latency for male or female rats to fall off a rotarod apparatus. Responses are shown at the following time points: baseline (before treatment) and at 1, 2, and 4 hours after treatment with vehicle (10% DMSO and 5% Tween 80 in water, delivered PO) or resatorvid (3, 10, 30, or 100 mg/kg, delivered PO). There were 10 rats per group. Neither males nor females had a significant interaction of time x treatment in a two-way repeated measures ANOVA.

Model: Plantar Incision Model in Rat (Brennan Model)

Animals were balanced across treatment groups based on body weight and post-surgery withdrawal thresholds.

Animals with ipsilateral post-surgery withdrawal thresholds ≤3.0g were included in the study.

Endpoint: Rat Hind Paw Mechanical Allodynia Behavior (von Frey Filaments) Method

Two graphs show ipsilateral hind paw mechanical allodynia behavior assessed with von Frey filaments in male or female rats that have undergone plantar incision surgery. Responses are shown at the following time points: baseline (before surgery), prior to treatment at 1 day after surgery, and at 1, 2, 4, and 6 hours after treatment at 1 day after surgery. The treatments are vehicle (DMSO/Tween 80, delivered SC), resatorvid (1, 3, 10, or 30 mg/kg, delivered SC), or the positive control morphine (6 mg/kg, delivered SC). There were 10 rats per group. A significant interaction of time x treatment was found in a two-way repeated measures ANOVA for each sex. Dunnett’s tests found significantly higher withdrawal thresholds relative to vehicle in males at 1, 2, and 4 hours post-treatment with morphine (p<0.0001, 0.001, 0.01, respectively) and in females at 1 and 2 hours post-treatment with morphine (p<0.0001 for both comparisons).

Model: Plantar Incision Model in Rat (Brennan Model)

Animals were balanced across treatment groups based on body weight and post-surgery guarding scores.

Animals with ipsilateral post-surgery guarding scores ≥10 were included in the study.

Endpoint: Guarding Behavior

Two graphs show ipsilateral hind paw guarding behavior evaluated in male or female rats that have undergone plantar incision surgery. Responses are shown at the following time points: baseline (before surgery), prior to treatment at 1 day after surgery, and at 1, 2, 4, and 6 hours after treatment at 1 day after surgery. The treatments are vehicle (DMSO/Tween 80, delivered SC), resatorvid (1, 3, 10, or 30 mg/kg, delivered SC), or the positive control morphine (6 mg/kg, delivered SC). There were 10 rats per group. A significant interaction of time x treatment was found in a two-way repeated measures ANOVA for each sex. Dunnett’s tests found significantly lower guarding scores relative to vehicle in males at 1, 2, and 4 hours post-treatment with morphine (p<0.0001, 0.0001, 0.01, respectively) and in females at 1, 2, and 4 hours post-treatment with morphine (p<0.0001, 0.0001, 0.01, respectively).

Model: L5/L6 Spinal Nerve Ligation Model

Endpoint: Rat Hind Paw Mechanical Allodynia Behavior (von Frey Filaments) Method

Model: L5/L6 Spinal Nerve Ligation Model

Animals were balanced across treatment groups based on body weight and post-surgery positive responses to acetone trials.

Animals with ipsilateral post-surgery number of positive responses to acetone trials ≥2 were included in the study.

Endpoint: Rat Hind Paw Cold Allodynia (Acetone Evaporation) Test

Two graphs show ipsilateral hind paw cold allodynia behavior assessed with 5 trials of the acetone evaporation test in male or female rats that have undergone L5/L6 spinal nerve ligation surgery. Responses are shown at the following time points: baseline (before surgery), prior to treatment at 3 weeks after surgery, and at 1 and 3 hours after treatment at 3 weeks after surgery. The treatments are vehicle (DMSO/Tween 80, delivered SC), resatorvid (1, 3, 10, or 30 mg/kg, delivered SC), or the positive control gabapentin (60 mg/kg, delivered PO). There were 10 rats per group. A significant interaction of time x treatment was found in a two-way repeated measures ANOVA for each sex. Dunnett’s tests found significantly lower acetone responses relative to vehicle at 1 hour post-treatment with gabapentin in males (p<0.05) and in females (p<0.05).

This work was conducted by PsychoGenics Inc. (Paramus, NJ) in collaboration with PSPP, NINDS, NIH under contract # 75N95019D00026. Prescribing information for clinically used controls can be found at labels.fda.gov. Information for icons representing experimental design details can be found through the NINDS Office of Research Quality https://go.nih.gov/Yw2tHGI.

Resatorvid

Formulation

Results

Pharmacokinetics: Concentration in Plasma and Brain

Pharmacokinetics: Calculated Parameters

Irwin: Total Observations

Irwin: Behavioral Severity Scores

Irwin: Body Temperature

Rotarod

Plantar Incision: von Frey

Plantar Incision: Guarding Behavior

L5/L6 Spinal Nerve Ligation: von Frey

L5/L6 Spinal Nerve Ligation: Acetone