DIAZEPAM

Formulation

The table below describes the formulation of diazepam 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	Diazepam	3, 10	1.00	1	PO	TPS	Suspension
Irwin, Rotarod	Diazepam	1, 3, 10, 30	1.00	5	PO	TPS	Suspension
Plantar incision,	Diazepam	1, 3, 10	1.00	5	PO	TPS	Suspension
L5/L6 SNL	Diazepam	1, 3, 10	1.00	5	PO	TPS	Suspension
CPP	Diazepam	0.3, 1, 3	1.00	1	IP	DTS	Suspension
CPP	Diazepam	1, 3, 10	1.00	5	PO	TPS	Suspension
Plantar incision	Morphine	6	1.33	1	SC	Saline	Solution
L5/L6 SNL	Gabapentin	60	1.00	1	PO	Saline	Solution
CPP	Oxycodone	3	1.12	1	IP	Saline	Solution
CPP	Oxycodone	3	1.12	1	PO	Saline	Solution

L5/L6 SNL = L5/L6 spinal nerve ligation; TPS = 5% Tween 80, 5% polyethylene glycol (PEG) 300, and 90% saline; DTS = 3% DMSO, 1% Tween 80, 96% saline; CPP = conditioned place preference; PO = per os; SC = subcutaneous; IP = intraperitoneal

Results

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Method: Protein binding

A table shows the results of rapid equilibrium dialysis to assess protein binding and percent recovery of diazepam. In rat plasma, diazepam was 86.6% bound to protein and 13.4% unbound and had a recovery of 93.1%. In rat brain homogenate, diazepam was 62.6% bound to protein and 37.4% unbound and had a recovery of 126.6%. Acebutolol, quinidine, and warfarin were included in the study as controls and returned values consistent with literature values.

Method: Pharmacokinetics

Animals were randomly assigned to treatment groups.

Experimenters collecting samples were masked to treatment.

Diazepam 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 10 mg/kg diazepam (PO) in a vehicle of TPS. There were 4 rats/group for plasma and 3-4 rats/group for brain.

Method: Pharmacokinetics

A table shows pharmacokinetics parameters calculated for diazepam using non-compartmental analysis in male and female rats treated with diazepam (3 or 10 mg/kg, PO, vehicle of TPS). The means for the 4 groups (males and females treated with 3 mg/kg diazepam and males and females treated with 10 mg/kg diazepam, respectively) are as follows. The highest concentration (Cmax) was 0.2, 0.58, 0.85, and 2.13 µM; the time at the maximum concentration (Tmax) was 0.81, 0.25, 1.38, and 0.56 hours; the half-life (T1/2) was 1.67, 1.78, 0.44, and 2.8 hours; area under the curve for time 0 to 8 hours was 25, 65, 157, and 481 minutes*µM; area under the curve for time 0 to infinity was 27, 69, 172, and 550 minutes*µM; the brain-to-plasma ratio at 0.5 hour was 0.66, 0.7, 0.75, and 0.77; the brain-to-plasma ratio at 8 hours was 0.78, 0.63, 0.57, and 0.5.

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 (5% Tween 80 and 5% PEG 300 in saline, delivered PO) or diazepam (1, 3, 10, or 30 mg/kg, delivered PO). There were 4 rats per group. Kruskal-Wallis tests were performed at each timepoint for each sex, followed by Dunn’s tests when a significant effect was observed. Dunn’s tests found an increase in the number of observations relative to vehicle in males at 4 hours post-treatment with 30 mg/kg diazepam (p<0.01) and at 6 hours post-treatment with 3, 10, and 30 mg/kg diazepam (p<0.05 for all comparisons). Dunn’s tests found an increase in the number of observations relative to vehicle in females at 1 hour post-treatment with 10 mg/kg diazepam (p<0.05) and 30 mg/kg diazepam (p<0.01) and at 2 hours post-treatment with 30 mg/kg diazepam (p<0.01). 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 (5% Tween 80 and 5% PEG 300 in saline, delivered PO) or diazepam (1, 3, 10, or 30 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, diazepam administration led to decreased body position, decreased locomotor activity, sedation, ataxia, decreased pupil size, decreased visual placement, and decreased grip strength in a dose-dependent manner.

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 (5% Tween 80 and 5% PEG 300 in saline, delivered PO) or diazepam (1, 3, 10, or 30 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 female rats. Dunnett’s tests found that body temperature in females was significantly decreased at 1 and 6 hours post-treatment relative to baseline (p<0.001 for both comparisons). Data were analyzed using GraphPad Prism version 10.0.2.

Method: 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, 4, and 6 hours after treatment with vehicle (5% Tween 80 and 5% PEG 300 in saline, delivered PO) or diazepam (1, 3, 10, or 30 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. Bonferroni’s tests found a significant decrease in latency to fall relative to the vehicle group in males at 1 hour post-treatment with 30 mg/kg diazepam (p<0.01). Bonferroni’s tests found significant decreases in latency to fall relative to the vehicle group in females at 1 hour post-treatment with 10 mg/kg diazepam (p<0.05) and 30 mg/kg diazepam (p<0.0001) and at 2 hours post-treatment with 30 mg/kg diazepam (p<0.05).

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 (5% Tween 80, 5% PEG 300, 90% saline, delivered PO), diazepam (1, 3, or 10 mg/kg, delivered PO), 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. Dunnett’s tests found significant differences from vehicle in males at 1 hour post-treatment with 3 mg/kg diazepam (p<0.05), 10 mg/kg diazepam (p<0.05), and morphine (p<0.0001); at 2 hours post-treatment with 10 mg/kg diazepam (p<0.01) and morphine (p<0.001); and at 4 hours post-treatment with 10 mg/kg diazepam (p<0.05) and morphine (p<0.01). Dunnett’s tests found significant differences from vehicle in females at 1 hour post-treatment with 1 mg/kg diazepam (p<0.05), 10 mg/kg diazepam (p<0.001), and morphine (p<0.0001); at 2 hours post-treatment with 1 mg/kg diazepam (p<0.05), 10 mg/kg diazepam (p<0.001), and morphine (p<0.001); and at 4 hours post-treatment with 10 mg/kg diazepam (p<0.05).

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 (5% Tween 80, 5% PEG 300, 90% saline, delivered PO), diazepam (1, 3, or 10 mg/kg, delivered PO), 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. Dunnett’s tests found significant differences from vehicle in males at 1 hour and 2 hours post-treatment with morphine (p<0.0001 for both comparisons). Dunnett’s tests found significant differences from vehicle in females at 1 hour post-treatment with 10 mg/kg diazepam (p<0.01) and morphine (p<0.0001) and at 2 hours post-treatment with 10 mg/kg diazepam (p<0.01) and morphine (p<0.0001).

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 2 hours after treatment at 3 weeks after surgery. The treatments are vehicle (5% Tween 80, 5% PEG 300, 90% saline, delivered PO), diazepam (1, 3, or 10 mg/kg, delivered PO), 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. Dunnett’s tests found significant differences from vehicle in males at 1 hour and 2 hours post-treatment with gabapentin (p<0.0001 and 0.001, respectively). Dunnett’s tests found significant differences from vehicle in females at 1 hour and 2 hours post-treatment with gabapentin (p<0.001 and 0.001, respectively).

Method: Conditioned place preference

Animals were balanced across treatment groups such that Day 1 preference values were equivalent with averages all close to the chance level.

Animals with a strong bias to one compartment (>75%) on Day 1 were removed during the rebalance procedure.

Two graphs show percent of time spent in the treatment-paired chamber on Day 10 after conditioning with diazepam (0.3, 1, 3 mg/kg, IP), the diazepam vehicle (DTS, IP), oxycodone (3 mg/kg, IP), or the oxycodone vehicle (saline, IP) in male and female rats (n=15-16 rats/group) via intraperitoneal (IP) administration. During conditioning, rats were placed in chambers immediately after treatment. A significant effect of treatment was found in a one-way ANOVA for each sex. Dunnett’s tests found significant increases in the percent of time spent in the treatment-paired chamber relative to the vehicle-paired chamber in males and females treated with oxycodone (p<0.05). Data were analyzed with GraphPad Prism version 10.5.0

Method: Conditioned place preference

Two graphs show the change in preference scores (calculated as the different in time in seconds spent in the treatment-paired chamber from Day 1 to Day 10) after treatment and conditioning with diazepam (IP), the diazepam vehicle (DTS, IP), oxycodone (IP), or the oxycodone vehicle (saline, IP) in male and female rats (n=15-16 rats/group) via intraperitoneal (IP) administration. During conditioning, rats were placed in chambers immediately after treatment. A significant effect of treatment was found in a one-way ANOVA for each sex. Dunnett’s tests found significant increases in the percent of time spent in the treatment-paired chamber relative to the vehicle-paired chamber in males and females treated with oxycodone (p<.05). Data were analyzed with GraphPad Prism version 10.5.0

Method: Conditioned place preference

Two graphs show percent of time spent in the treatment-paired chamber on Day 10 after conditioning with diazepam (1, 3, or 10 mg/kg, PO), the diazepam vehicle (TPS, PO), oxycodone (3 mg/kg, PO), or the oxycodone vehicle (saline, PO) in male and female rats (n=15-16 rats/group) via oral (PO) administration. During conditioning, rats were placed in chambers 30-minutes after treatment. A significant effect of treatment was found in a one-way ANOVA for each sex. Dunnett’s tests found significant increases in the percent of time spent in the treatment-paired chamber relative to the vehicle-paired chamber in males and females treated with oxycodone (p<0.05). Data were analyzed with GraphPad Prism version 10.5.0

Method: Conditioned place preference

This work was conducted by PsychoGenics Inc. (Paramus, NJ) in collaboration with PSPP, NINDS, NIH under contract # 75N95019D00026 and 75N95024D00038. 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.

DIAZEPAM

Formulation

Results

Protein Binding

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

Conditioned Place Preference: Bias Test (0.3-3 mg/kg IP)

Conditioned Place Preference: Preference Score (0.3-3 mg/kg IP)

Conditioned Place Preference: Bias Test (1-10 mg/kg PO)

Conditioned Place Preference: Preference Score (1-10 mg/kg PO)