OXYCODONE

Formulation

The table below describes the formulation of oxycodone hydrochloride and the controls cocaine hydrochloride, duloxetine hydrochloride, and morphine sulfate for the following in vivo experiments.

Experiment(s)	Compound	Dose(s) mg/kg	Correction factor	Dose volume mL/kg	Route	Vehicle	Suspension / Solution
Pharmacokinetics	Oxycodone	0.1, 1	1.12	1	IV	Saline	Solution
Irwin, Rotarod	Oxycodone	0.1, 0.3, 1, 3	1.12	1	IV	Saline	Solution
CPP	Oxycodone	1, 3, 5	1.12	1	IP	Saline	Solution
IVSA	Oxycodone	0.01, 0.03, 0.06, 0.1	1.12	0.088 mL/infusion	IV	Saline	Solution
IVSA	Oxycodone	mg/kg/infusion	1.12	0.088 mL/infusion	IV	Saline	Solution
CPP	Cocaine	15	1.12	1	IP	Saline	Solution
CPP	Duloxetine	30	1.12	5	PO	Water	Suspension
IVSA	Morphine	0.6	1.33	0.088 mL/infusion	IV	Saline	Solution
IVSA	Morphine	mg/kg/infusion	1.33	0.088 mL/infusion	IV	Saline	Solution

L5/L6 SNL = L5/L6 spinal nerve ligation; CPP = conditioned place preference, IVSA = intravenous self-administration, IV = intravenous, IP = intraperitoneal, PO = per os

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 oxycodone. In rat plasma, oxycodone was 42% bound to protein and 58% unbound and had a recovery of 131.9%. In rat brain homogenate, oxycodone was 23.1% bound to protein and 76.9% unbound and had a recovery of 114%. 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.

Oxycodone concentrations were measured in serially collected plasma samples and terminally collected brain samples (shown on two graphs) from males and females treated with 0.1 or 1 mg/kg oxycodone (IV) in a vehicle of saline. There were 2-4 rats/group for plasma and 3 rats/group for brain.

Method: Pharmacokinetics

A table shows pharmacokinetics parameters calculated for oxycodone using non-compartmental analysis in male and female rats treated with oxycodone (0.1 or 1 mg/kg, IV, vehicle of saline). The means for the 4 groups (males and females treated with 0.1 mg/kg oxycodone and males and females treated with 1 mg/kg oxycodone, respectively) are as follows. The highest concentration (Cmax) was 0.05, 0.04, 0.69, and 0.61 µM; the time at the maximum concentration (Tmax) was 0.25, 0.25, 0.31, and 0.25 hours; the half-life (T1/2) was 0.84, 0.58, 0.7, and 0.96 hours; area under the curve for time 0 to 6 hours was 3, 2, 39, and 39 minutes*µM; area under the curve for time 0 to infinity was 3, 2, 39, and 40 minutes*µM; the brain-to-plasma ratio at 0.5 hours was 4.71, 4.6, 4.95, and 5.4; the brain-to-plasma ratio at 6 hours could not be calculated for the 0.1 mg/kg groups and was 6.51 and 4.66 for males and females in the 1 mg/kg groups, respectively.

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 IV) or oxycodone (0.1, 0.3, 1, or 3 mg/kg, delivered IV). There were 3-4 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 3 mg/kg oxycodone (p<0.05), at 2 hours post-treatment with 1 mg/kg oxycodone (p<0.05) and 3 mg/kg oxycodone (p<0.05), and at 4 hours post-treatment with 1 mg/kg oxycodone (p<0.01). Dunn’s tests found an increase in the number of observations relative to vehicle in females at 1 hour post-treatment with 3 mg/kg oxycodone (p<0.01). Data were analyzed using GraphPad Prism.

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 IV) or oxycodone (0.1, 0.3, 1, or 3 mg/kg, delivered IV). There were 8 rats per group (4 male and 4 female), except n=4 male and 3 female rats in the 3 mg/kg group at 2, 4, and 6 hours. 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, administration of 3 mg/kg oxycodone produced time-dependent decreased locomotion, sedation, and increased pupil size.

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 vehicle (saline, delivered IV) or oxycodone (0.1, 0.3, 1, or 3 mg/kg, delivered IV). There were 4 rats per group, except n=3 females in the 3 mg/kg group at 2, 4, and 6 h. A significant effect of time was found in a two-way repeated measures ANOVA for males. Dunnett’s tests found a significant increase in body temperature at 1 hour post-treatment (p<0.05) and a significant decrease in body temperature at 6 hours post-treatment (p<0.001) relative to baseline in males. Data were analyzed using GraphPad Prism.

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 30, 60, or 120 minutes after treatment with vehicle (saline, delivered IV) or oxycodone (0.1, 0.3, 1, or 3 mg/kg, delivered IV). There were 9-10 rats per group. A significant interaction of time x treatment was found in a mixed effects analysis for males and in a two-way repeated measures ANOVA for females. Dunnett’s tests found a significant decrease in latency to fall compared to vehicle in males at 30 minutes post-treatment with 3 mg/kg oxycodone (p<0.05). Bonferroni’s tests found a significant decrease in latency to fall compared to vehicle in females at 30 minutes post-treatment with 3 mg/kg oxycodone (p<0.05). Data were analyzed using GraphPad Prism.

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 vehicle (saline, IP), oxycodone (1, 3, or 5 mg/kg, IP), cocaine (15 mg/kg, IP), or duloxetine (30 mg/kg, PO) in male and female rats (n=14-16 rats/group). During conditioning, rats were placed in chambers immediately after treatment with vehicle, oxycodone, or cocaine or 60 minutes after treatment with duloxetine. 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 treated with 1 mg/kg oxycodone (p<0.001), 3 mg/kg oxycodone (p<0.01), 5 mg/kg oxycodone (p<0.001), or cocaine (p<0.05). Dunnett’s tests found significant increases in the percent of time spent in the treatment-paired chamber relative to the vehicle-paired chamber in females treated with 1 mg/kg oxycodone (p<0.001), 3 mg/kg oxycodone (p<0.0001), 5 mg/kg oxycodone (p<0.0001), or cocaine (p<0.05). Data were analyzed with GraphPad Prism.

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 vehicle (saline, IP), oxycodone (1, 3, or 5 mg/kg, IP), cocaine (15 mg/kg, IP), or duloxetine (30 mg/kg, PO) in male and female rats (n=15-16 rats/group). During conditioning, rats were placed in chambers immediately after treatment with vehicle, oxycodone, or cocaine or 60 minutes after treatment with duloxetine. A significant effect of treatment was found in a one-way ANOVA for each sex. Dunnett’s tests found significant increases in the change in preference score relative to vehicle in males treated with 1 mg/kg oxycodone (p<0.01), 3 mg/kg oxycodone (p<0.05), or 5 mg/kg oxycodone (p<0.05). Dunnett’s tests found significant increases in the change in preference score relative to vehicle in females treated with 1 mg/kg oxycodone (p<0.001), 3 mg/kg oxycodone (p<0.0001), 5 mg/kg oxycodone (p<0.0001), or cocaine (p<0.01). Data were analyzed with GraphPad Prism.

Method: Intravenous self-administration

Within each group on each day, data points that exceeded ± two standard deviations from the mean were removed from the analysis.

Two graphs show the number of infusions delivered in a fixed ratio of 3 lever presses for 1 intravenous infusion of vehicle (saline), oxycodone (0.01, 0.03, 0.06, or 0.1 mg/kg/infusion), or morphine (0.6 mg/kg/infusion) after food training in male and female rats (n=10-13/group). Dunnett’s tests were performed after finding a significant interaction of time x treatment in a two-way repeated measures ANOVA performed on the data for Days 5-20 for each sex. Significant increases in the number of infusions were found with each treatment relative to vehicle on several days in male and female rats. Data were analyzed with GraphPad Prism.

Method: Intravenous self-administration

Two graphs show the breakpoint in a progressive ratio schedule following acquisition training in male and female rats treated with vehicle (saline), oxycodone (0.01, 0.03, 0.06, or 0.1 mg/kg/infusion), or morphine (0.6 mg/kg/infusion) (n=9-13 rats/group). A significant effect of treatment was found in a one-way ANOVA for each sex. Dunnett’s tests found significant increases in the breakpoint relative to vehicle in males treated with 0.03 mg/kg oxycodone (p<0.0001), 0.06 mg/kg oxycodone (p<0.0001), and 0.1 mg/kg oxycodone (p<0.01). Dunnett’s tests found significant increases in the breakpoint relative to vehicle in females treated with 0.06 mg/kg oxycodone (p<0.05) and 0.1 mg/kg oxycodone (p<0.001). Data were analyzed with GraphPad Prism.

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.

OXYCODONE

Formulation

Results

Protein Binding

Pharmacokinetics: Concentration in Plasma and Brain

Pharmacokinetics: Calculated Parameters

Irwin: Total Observations

Irwin: Behavioral Severity Scores

Irwin: Body Temperature

Rotarod

Conditioned Place Preference: Bias Test

Conditioned Place Preference: Preference Score

Intravenous Self-Administration: Acquisition Training

Intravenous Self-Administration: Progressive Ratio