MORPHINE

Formulation

The table below describes the formulation of morphine sulfate and the controls pregabalin, ketoprofen, and gabapentin for the following in vivo experiments. For MIA, paclitaxel CIPN, and oxaliplatin CIPN studies, morphine showed efficacy in all endpoints and is used as the positive control when testing other compounds in these models; therefore, no additional positive control is used in the MIA, paclitaxel CIPN, and oxaliplatin CIPN morphine dose response studies.

Experiment(s)	Compound	Dose(s) mg/kg	Correction factor	Dose volume mL/kg	Route	Vehicle	Suspension / Solution
Pharmacokinetics	Morphine	0.3, 3, 6	1.33	1	SC	Saline	Solution
Irwin, Rotarod	Morphine	1, 3, 6, 10	1.33	1	SC	Saline	Solution
Plantar incision	Morphine	1, 3, 6	1.33	1	SC	Saline	Solution
L5/L6 SNL, MIA,	Morphine	0.3, 1, 3, 6	1.33	1	SC	Saline	Solution
Oxaliplatin CIPN
(Acetone)
Paclitaxel CIPN,	Morphine	0.3, 1, 3	1.33	1	SC	Saline	Solution
Oxaliplatin CIPN
(von Frey)
CPP	Morphine	1, 3, 10	1.33	1	SC	Saline	Solution
Plantar incision	Pregabalin	30	1.00	1	PO	Saline	Solution
Plantar incision	Ketoprofen	6	1.00	1	PO	Saline	Suspension
L5/L6 SNL	Gabapentin	60	1.00	1	PO	Saline	Solution
CPP	Oxycodone	3	1.12	1	IP	Saline	Solution

L5/L6 SNL = L5/L6 spinal nerve ligation; MIA = monosodium iodoacetate; CIPN = chemotherapy-induced peripheral neuropathy; 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 morphine. In rat plasma, morphine was 48.6% bound to protein and 51.4% unbound and had a recovery of 117.2%. In rat brain homogenate, morphine was 7.9% bound to protein and 92.1% unbound and had a recovery of 132.2%. 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.

Morphine concentrations were measured in serially collected plasma samples and terminally collected brain samples (shown on two graphs) from males and females treated with 0.3 or 3 mg/kg morphine (SC) in a vehicle of saline. There were 4-5 rats/group for plasma and 1-4 rats/group for brain.

Method: Pharmacokinetics

A table shows pharmacokinetics parameters calculated for morphine using non-compartmental analysis in male and female rats treated with morphine (0.3 or 3 mg/kg, SC, vehicle of saline). The means for the 4 groups (males and females treated with 0.3 mg/kg morphine and males and females treated with 3 mg/kg morphine, respectively) are as follows. The highest concentration (Cmax) was 0.21, 0.23, 2.45, and 2.45 µM; the time at the maximum concentration (Tmax) was 0.25, 0.25, 0.25, and 0.31 hours; the half-life (T1/2) was 0.91, 0.91, 1.5, and 0.97 hours; area under the curve for time 0 to 6 hours was 16, 17, 159, and 172 minutes*µM; area under the curve for time 0 to infinity was 17, 18, 164, and 175 minutes*µM; the brain-to-plasma ratio at 0.25 hours was 0.13, 0.16, 0.12, and 0.12; the brain-to-plasma ratio at 8 hours was 0.86, unable to calculate, 0.4, and 0.45.

Method: Pharmacokinetics

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 morphine (1, 3, 6, or 10 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, 2 hours, and 4 hours post-treatment with 10 mg/kg morphine (p<0.001, 0.01, 0.05, respectively). 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 morphine (p<0.001) and at 2 hours post-treatment with 10 mg/kg morphine (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 (saline, delivered SC) or morphine (1, 3, 6, or 10 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, morphine administration produced effects including decreased body position, decreased locomotor activity, sedation, exophthalmos, unusual behavior (chewing bedding), and increased pupil size 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 morphine (1, 3, 6, or 10 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 6 hours post-treatment in males (p<0.001) and females (p<0.01) relative to baseline of the same sex. 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 30, 60, and 90 minutes after treatment with vehicle (saline, delivered SC) or morphine (1, 3, 6, or 10 mg/kg, delivered SC). There were 10 rats per group. Both sexes had a significant interaction of time x treatment in a two-way repeated measures ANOVA. Dunnett’s tests found a significant decrease in latency to fall relative to the vehicle group at 60 minutes post-treatment with 10 mg/kg morphine (p<0.05). In females, the latency to fall was slightly decreased relative to the vehicle group at 30 minutes post-treatment with 10 mg/kg morphine, but this difference was not significant.

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, 6, and 24 hours after treatment at 1 day after surgery. The treatments are vehicle (saline, delivered SC), morphine (1, 3, or 6 mg/kg, delivered SC), or one of two positive controls: pregabalin (30 mg/kg, delivered PO) or ketoprofen (6 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 post-treatment with 1, 3, and 6 mg/kg morphine (p<0.001, 0.0001, 0.0001, respectively), pregabalin (p<0.0001), and ketoprofen (p<0.001); at 2 hours post-treatment with 1, 3, and 6 mg/kg morphine (p<0.01, 0.001, 0.0001, respectively), pregabalin (p<0.0001), and ketoprofen (p<0.01); and at 4 hours post-treatment with 6 mg/kg morphine (p<0.05), pregabalin (p<0.0001), and ketoprofen (p<0.05). Dunnett’s tests found significant differences from vehicle in females at 1 hour post-treatment with 1, 3, and 6 mg/kg morphine (p<0.0001 for all comparisons), pregabalin (p<0.0001), and ketoprofen (p<0.001); at 2 hours post-treatment with 6 mg/kg morphine (p<0.01), pregabalin (p<0.0001), and ketoprofen (p<0.0001); at 4 hours post-treatment with pregabalin (p<0.0001) and ketoprofen (p<0.05); and at 6 hours post-treatment with pregabalin (p<0.01).

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, 6, and 24 hours after treatment at 1 day after surgery. The treatments are vehicle (saline, delivered SC), morphine (1, 3, or 6 mg/kg, delivered SC), or one of two positive controls: pregabalin (30 mg/kg, delivered PO) or ketoprofen (6 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 post-treatment with 1, 3, and 6 mg/kg morphine (p<0.001, 0.0001, 0.0001, respectively), pregabalin (p<0.001), and ketoprofen (p<0.01); at 2 hours post-treatment with 1, 3, and 6 mg/kg morphine, pregabalin, and ketoprofen (p<0.0001 for all comparisons); at 4 hours post-treatment with 1, 3, and 6 mg/kg morphine (p<0.05, 0.01, 0.05, respectively), pregabalin (p<0.001), and ketoprofen (p<0.001); at 6 hours post-treatment with 1, 3, and 6 mg/kg morphine (p<0.05, 0.05, 0.0001, respectively), pregabalin (p<0.0001), and ketoprofen (p<0.001); and at 24 hours post-treatment with pregabalin (p<0.01). Dunnett’s tests found significant differences from vehicle in females at 1 hour post-treatment with 1, 3, and 6 mg/kg morphine (p<0.0001 for all comparisons), pregabalin (p<0.01), and ketoprofen (p<0.0001); at 2 hours post-treatment with 1, 3, and 6 mg/kg morphine (p<0.0001, 0.01, 0.0001, respectively), pregabalin (p<0.001), and ketoprofen (p<0.0001); at 4 hours post-treatment with 1, 3, and 6 mg/kg morphine (p<0.01 for all comparisons), pregabalin (p<0.0001), and ketoprofen (p<0.001); and at 6 hours post-treatment with pregabalin (p<0.001) and ketoprofen (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 4 hours after treatment at 3 weeks after surgery. The treatments are vehicle (saline, delivered SC), morphine (0.3, 1, 3, or 6 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. Dunnett’s tests found significant differences from vehicle in males at 1 hour post-treatment with 1, 3, and 6 mg/kg morphine (p<0.01, 0.0001, 0.0001, respectively) and gabapentin (p<0.0001) and at 4 hours post-treatment with 6 mg/kg morphine (p<0.01) and gabapentin (p<0.0001). Dunnett’s tests found significant differences from vehicle in females at 1 hour post-treatment with 1, 3, and 6 mg/kg morphine (p<0.001, 0.0001, 0.0001, respectively) and gabapentin (p<0.001) and at 4 hours post-treatment with gabapentin (p<0.01).

Method: Monosodium iodoacetate (MIA) model

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

Animals with ipsilateral pre-induction withdrawal responses >8.0g and post-induction withdrawal thresholds ≤4.0g were included in the study.

Endpoint: Rat hind paw mechanical allodynia behavior (von Frey filaments) method

Method: Monosodium iodoacetate (MIA) model

Animals were balanced across treatment groups based on body weight and post-induction weight bearing.

Animals with ipsilateral post-induction weight bearing of 10-40% of total hind paw weight bearing were included in the study.

Endpoint: Dynamic weight bearing measurement method

Two graphs show ipsilateral hind paw dynamic weight bearing behavior in male or female rats that have undergone injection of monosodium iodoacetate (MIA, 3 mg/50 µL) in the left hind limb knee joint. Responses are shown at the following time points: baseline (before MIA injection), Day 4 after MIA injection, and at 1 hour after treatment on Day 5. The treatments are vehicle (saline, delivered PO), pregabalin (10, 30, or 60 mg/kg, delivered PO), or the positive control morphine (6 mg/kg, delivered SC). There were 8-9 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 hind paw dynamic weight bearing relative to vehicle in males at 1 hour post-treatment with 1 mg/kg morphine (p<0.05), 3 mg/kg morphine (p<0.05), and 6 mg/kg morphine (p<0.01). Dunnett’s tests found significant increases in hind paw dynamic weight bearing relative to vehicle in females at 1 hour post-treatment with 6 mg/kg morphine (p<0.01).

Model: Paclitaxel chemotherapy-induced peripheral neuropathy (CIPN) model

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

Animals with average post-paclitaxel withdrawal thresholds ≤5.0g were included in the study.

Endpoint: Rat hind paw mechanical allodynia behavior (von Frey filaments) method

Two graphs show hind paw mechanical allodynia behavior assessed with von Frey filaments in male or female rats that have undergone paclitaxel administration to model chemotherapy-induced peripheral neuropathy. Responses are shown at the following time points: baseline (before paclitaxel administration), prior to treatment at 6 weeks after the start of paclitaxel administration, and at 1 hour after treatment at 6 weeks after paclitaxel administration. The treatments are vehicle (saline, delivered SC) or morphine (0.3, 1, or 3 mg/kg, delivered SC). Data are represented as the lowest response threshold (left or right hind paw) for each rat. There were 8-9 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 1 mg/kg morphine (p<0.01) and 3 mg/kg morphine (p<0.001). Dunnett’s tests found significant increases in withdrawal thresholds relative to vehicle in females at 1 hour post-treatment with 0.3, 1, and 3 mg/kg morphine (p<0.05, 0.01, 0.01, respectively).

Model: Paclitaxel chemotherapy-induced peripheral neuropathy (CIPN) model

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

Animals with post-paclitaxel 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 hind paw cold allodynia behavior assessed with 10 trials of the acetone evaporation test (5 per hind paw) in male or female rats that have undergone paclitaxel administration to model chemotherapy-induced peripheral neuropathy. Responses are shown at the following time points: baseline (before paclitaxel), prior to treatment at 5 weeks after the first paclitaxel administration, and at 1 hour after treatment at 5 weeks after paclitaxel administration. The treatments are vehicle (saline, delivered SC) or morphine (0.3, 1, or 3 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 and females at 1 hour post-treatment with 3 mg/kg morphine (p<0.0001 for each comparison).

Model: Oxaliplatin chemotherapy-induced peripheral neuropathy (CIPN) model

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

Animals with average post-oxaliplatin withdrawal thresholds ≤5.0g were included in the study.

Endpoint: Rat hind paw mechanical allodynia behavior (von Frey filaments) method

Two graphs show hind paw mechanical allodynia behavior assessed with von Frey filaments in male or female rats that have undergone oxaliplatin administration to model chemotherapy-induced peripheral neuropathy. Responses are shown at the following time points: baseline (before oxaliplatin administration), prior to treatment at 7 weeks after the start of oxaliplatin administration, and at 1 hour after treatment at 7 weeks after oxaliplatin administration. The treatments are vehicle (saline, delivered SC) or morphine (0.3, 1, or 3 mg/kg, delivered SC). Data are represented as the lowest response threshold (left or right hind paw) for each rat. There were 7-8 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 1 mg/kg and 3 mg/kg morphine (p<0.05, 0.001, respectively). Dunnett’s tests found significant increases in withdrawal thresholds relative to vehicle in females at 1 hour post-treatment with 0.3, 1, and 3 morphine (p<0.05, 0.05, 0.001, respectively).

Model: Oxaliplatin chemotherapy-induced peripheral neuropathy (CIPN) model

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

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

Endpoint: Rat hind paw cold allodynia (acetone evaporation) test

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, SC), morphine (1, 3, or 10 mg/kg, SC), or oxycodone (3 mg/kg, IP) in male and female rats (n=15-16 rats/group). During conditioning, rats were placed in chambers immediately after treatment with oxycodone or 15 minutes after treatment with vehicle or morphine. 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 morphine (p<0.01), 3 mg/kg morphine (p<0.001), 10 mg/kg morphine (p<0.001), or oxycodone (p<0.0001). 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 morphine (p<0.001), 3 mg/kg morphine (p<0.05), 10 mg/kg morphine (p<0.01), or 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 vehicle (saline, SC), morphine (1, 3, or 10 mg/kg, SC), or oxycodone (3 mg/kg, IP) in male and female rats (n=15-16 rats/group). During conditioning, rats were placed in chambers immediately after treatment with oxycodone or 15 minutes after treatment with vehicle or morphine. 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 morphine (p<0.01), 3 mg/kg morphine (p<0.01), 10 mg/kg morphine (p<0.01), or oxycodone (p<0.01). 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 morphine (p<0.0001), 3 mg/kg morphine (p<0.05), 10 mg/kg morphine (p<0.01), or oxycodone (p<0.05). Data were analyzed with GraphPad Prism version 10.5.0

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.

MORPHINE

Formulation

Results

Protein Binding

Pharmacokinetics: Concentration in Plasma and Brain (0.3, 3 mg/kg)

Pharmacokinetics: Calculated Parameters (0.3, 3 mg/kg)

Pharmacokinetics: Concentration in Plasma and Brain (6 mg/kg)

Pharmacokinetics: Calculated Parameters (6 mg/kg)

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

Monosodium Iodoacetate (MIA): von Frey (Single Administration)

Monosodium Iodoacetate (MIA): Dynamic Weight Bearing (Single Administration)

Paclitaxel Chemotherapy-Induced Peripheral Neuropathy: von Frey

Paclitaxel Chemotherapy-Induced Peripheral Neuropathy: Acetone

Oxaliplatin Chemotherapy-Induced Peripheral Neuropathy: von Frey

Oxaliplatin Chemotherapy-Induced Peripheral Neuropathy: Acetone

Conditioned Place Preference: Bias Test

Conditioned Place Preference: Preference Score