Morphine

Formulation

The table below describes the formulation of morphine sulfate and the controls pregabalin, ketoprofen, and gabapentin for the experiments shown on this page. For MIA studies, morphine showed efficacy in all endpoints tested and is used as the positive control when testing other compounds in this model; therefore, no additional positive control is used in the MIA morphine dose response studies.

Experiment(s)	Compound	Dose(s) mg/kg	Correction factor	Dose volume mL/kg	Route	Vehicle	Suspension / Solution
Pharmacokinetics	Morphine	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
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

L5/L6 SNL = L5/L6 spinal nerve ligation; MIA = monosodium iodoacetate; PO = per os; SC = subcutaneous

Results

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Method: Pharmacokinetics

Morphine concentrations were measured in serially collected plasma samples and terminally collected brain samples (shown on two graphs) from males and females treated with 6 mg/kg morphine (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 morphine using non-compartmental analysis in male and female rats treated with morphine (6 mg/kg, SC, vehicle of saline). The means for the 2 groups (males and females treated with 6 mg/kg morphine, respectively) are as follows. The highest concentration (Cmax) was 6.93 and 7.67 µM; the time at the maximum concentration (Tmax) was 0.25 and 0.31 hours; the half-life (T1/2) was 1.1 and 1.11 hours; area under the curve for time 0 to 8 hours was 477 and 573 minutes*µM; area under the curve for time 0 to infinity was 482 and 579 minutes*µM; the brain-to-plasma ratio at 0.25 hours was 0.19 and 0.15; the brain-to-plasma ratio at 8 hours was 0.73 and 0.49

Method: Modified Irwin functional observational battery

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: Rat Rotarod

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

Method: Monosodium Iodoacetate (MIA) Model
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).

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.

Morphine

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

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

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