BUPRENORPHINE

Formulation

The table below describes the formulation of buprenorphine hydrochloride 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	Buprenorphine	0.01, 0.1	1.08	1	SC	Saline	Solution
Irwin, Rotarod	Buprenorphine	0.01, 0.03, 0.1, 0.3	1.08	1	SC	Saline	Solution
Plantar incision,	Buprenorphine	0.003, 0.01, 0.03, 0.1	1.08	1	SC	Saline	Solution
L5/L6 SNL	Buprenorphine	0.003, 0.01, 0.03, 0.1	1.08	1			Solution
Plantar incision	Morphine	6	1.33	1	SC	Saline	Solution
L5/L6 SNL		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

Results

Expand and Collapse accordion content

Method: Protein binding

A table shows the results of rapid equilibrium dialysis to assess protein binding and percent recovery of buprenorphine. In rat plasma, buprenorphine was 99.5% bound to protein and 0.5% unbound and had a recovery of 120.1%. In rat brain homogenate, buprenorphine was 80.9% bound to protein and 19.1% unbound and had a recovery of 131.4%. 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.

Buprenorphine concentrations were measured in serially collected plasma samples and terminally collected brain samples (shown on two graphs) from males and females treated with 0.01 or 0.1 mg/kg buprenorphine (SC) in a vehicle of saline. There were 4 rats/group for plasma and 3-4 rats/group for brain.

Method: Pharmacokinetics

A table shows pharmacokinetics parameters calculated for buprenorphine using non-compartmental analysis in male and female rats treated with buprenorphine (0.01 or 0.1 mg/kg, SC, vehicle of saline). The means for the 4 groups (males and females treated with 0.01 mg/kg buprenorphine and males and females treated with 0.1 mg/kg buprenorphine, respectively) are as follows. The highest concentration (Cmax) was 0.005, 0.004, 0.093, and 0.089 µM; the time at the maximum concentration (Tmax) was 0.25 hours for all groups; the half-life (T1/2) was 1.60, 2.35, 1.29, and 2.21 hours; area under the curve for time 0 to 6 hours was 0.53, 0.60, 7.24, and 7.79 minutes*µM; area under the curve for time 0 to infinity was 0.57, 0.73, 7.44, and 8.83 minutes*µM; the brain-to-plasma ratio at 0.25 hour was 1.90, 1.66, 1.11, and 1.47; the brain-to-plasma ratio at 6 hours was 23.29, 20.13, 10.12, and 4.96.

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 (saline, delivered SC) or buprenorphine (0.01, 0.03, 0.1, or 0.3 mg/kg, delivered SC). 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 1 hour post-treatment with 0.3 mg/kg buprenorphine (p<0.01), at 2 hours post-treatment with 0.3 mg/kg buprenorphine (p<0.01), at 4 hours post-treatment with 0.3 mg/kg buprenorphine (p<0.01), and at 6 hours post-treatment with 0.3 mg/kg buprenorphine (p<0.05). Dunn’s tests found an increase in the number of observations relative to vehicle in females at 1 hours post-treatment with 0.1 mg/kg buprenorphine (p<0.01) and 0.3 mg/kg buprenorphine (p<0.05), at 2 hours post-treatment with 0.1 mg/kg buprenorphine (p<0.01) and 0.3 mg/kg buprenorphine (p<0.05), and at 4 hours post-treatment with 0.1 mg/kg buprenorphine (p<0.01). Data were analyzed using GraphPad Prism version 10.5.0.

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 (saline, delivered SC) or buprenorphine (0.01, 0.03, 0.1, or 0.3 mg/kg, delivered SC). 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, buprenorphine administration produced effects including decreased body position, decreased locomotor activity, sedation, and exophthalmos in a dose- and time-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 (saline, delivered SC) or buprenorphine (0.01, 0.03, 0.1, or 0.3 mg/kg, delivered SC). 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 at 1 hour post-treatment in females (p<0.001) relative to baseline of the same sex. Data were analyzed using GraphPad Prism version 10.5.0.

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 0.5, 1, and 2 hours after treatment with vehicle (saline, delivered SC) or buprenorphine (0.01, 0.03, 0.1, or 0.3 mg/kg, delivered SC). 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 (saline, delivered SC), buprenorphine (0.003, 0.01, 0.03, or 0.1 mg/kg, delivered SC), or 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 significant increases in withdrawal thresholds relative to vehicle in males at 1 hour post-treatment with .03 and 0.1 mg/kg buprenorphine (p<0.001, 0.0001, respectively) and morphine (p<0.0001); at 2 hours post-treatment with 0.03 and 0.1 mg/kg buprenorphine (p<0.001, 0.0001, respectively) and morphine (p<0.0001); and at 4 hours post-treatment with 0.1 mg/kg buprenorphine (p<0.001) and morphine (p<0.05). Dunnett’s tests found significant increases in withdrawal thresholds relative to vehicle in females at 1 hour post-treatment with 0.03 and 0.1 mg/kg buprenorphine (p<0.05, 0.0001, respectively) and morphine (p<0.0001); at 2 hours post-treatment with 0.03 and 0.1 mg/kg buprenorphine (p<0.05, 0.001, respectively) and morphine (p<0.0001); and at 4 hours post-treatment with 0.1 mg/kg buprenorphine (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 (saline, delivered SC), buprenorphine (0.003, 0.01, 0.03, or 0.1 mg/kg, delivered SC), or 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 significant decreases in guarding scores relative to vehicle in males at 1 hour post-treatment with 0.03 and 0.1 mg/kg buprenorphine (p<0.0001 for both comparisons) and morphine (p<0.0001); at 2 hours post-treatment with 0.03 mg/kg buprenorphine (p<0.0001), 0.1 mg/kg buprenorphine (p<0.01), and morphine (p<0.0001); at 4 hours post-treatment with 0.03 mg/kg buprenorphine (p<0.0001) and 0.1 mg/kg buprenorphine (p<0.0001); and at 6 hours post-treatment with 0.1 mg/kg buprenorphine (p<0.05). Dunnett’s tests found significant decreases in guarding scores relative to vehicle in females at 1 hour post-treatment with 0.03 mg/kg buprenorphine (p<0.01), 0.1 mg/kg buprenorphine (p<0.0001), and morphine (p<0.0001); at 2 hours post-treatment with 0.01 mg/kg buprenorphine (p<0.01), 0.03 mg/kg buprenorphine (p<0.0001), 0.1 mg/kg buprenorphine (p<0.0001), and morphine (p<0.0001); at 4 hours post-treatment with 0.03 mg/kg buprenorphine (p<0.001) and 0.01 mg/kg buprenorphine (p<0.0001); and at 6 hours post-treatment with 0.1 mg/kg buprenorphine (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 3 hours after treatment at 3 weeks after surgery. The treatments are vehicle (saline, delivered SC), buprenorphine (0.003, 0.01, 0.03, or 0.1 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 significant decreases in acetone responses relative to vehicle in males at 1 hour post-treatment with all doses of buprenorphine (p<0.05, 0.01, 0.0001, 0.0001, respectively) and morphine (p<0.0001) and at 3 hours post-treatment with 0.01, 0.03, and 0.1 mg/kg buprenorphine (p<0.05, .0.1, 0.0001, respectively) and morphine (p<0.0001). Dunnett’s tests found significant decreases in acetone responses relative to vehicle in females at 1 hour post-treatment with 0.01 mg/kg buprenorphine (p<0.001) and morphine (p<0.0001).

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.

BUPRENORPHINE

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