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Research ArticleBehavioral Pharmacology

Behavioral Battery for Testing Candidate Analgesics in Mice. II. Effects of Endocannabinoid Catabolic Enzyme Inhibitors and ∆9-Tetrahydrocannabinol

C. M. Diester, A. H. Lichtman and S. S. Negus
Journal of Pharmacology and Experimental Therapeutics May 2021, 377 (2) 242-253; DOI: https://doi.org/10.1124/jpet.121.000497
C. M. Diester
Department of Pharmacology and Toxicology (C.M.D., A.H.L., S.S.N.), School of Pharmacy (A.H.L.), Virginia Commonwealth University, Richmond, Virginia
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A. H. Lichtman
Department of Pharmacology and Toxicology (C.M.D., A.H.L., S.S.N.), School of Pharmacy (A.H.L.), Virginia Commonwealth University, Richmond, Virginia
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S. S. Negus
Department of Pharmacology and Toxicology (C.M.D., A.H.L., S.S.N.), School of Pharmacy (A.H.L.), Virginia Commonwealth University, Richmond, Virginia
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  • Fig. 1.
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    Fig. 1.

    Selectivity of test compounds for the main endocannabinoid catabolic enzymes MAGL and FAAH based on competitive substrate binding and activity-based protein profiling assays. Data were obtained from the literature as cited below, with competitive substrate binding used for calculating selectivity if available and activity-based protein profiling used if no substrate binding data were available. All data are from assays using mouse brain tissue. No data for MAGL binding could be found for either endpoint for PF3845, which is considered to be a highly selective FAAH inhibitor. As a result, no selectivity ratio could be calculated for this inhibitor. PF3845: Ahn, 2009 (main paper denoting FAAH selectivity); URB597: Kathuria et al., 2003; SA57: Niphakis et al., 2012; JZL195: Long et al., 2009c; JZL184: Long et al., 2009a; MJN110: Niphakis et al., 2013.

  • Fig. 2.
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    Fig. 2.

    Effects of THC on IP acid–induced stretching, facial grimace, rearing, and nesting behaviors in male and female mice. Abscissae: dose of THC delivered subcutaneously in milligrams per kilogram (log scale). Ordinates: number of stretches (A), grimace score (B), number of rears (C), and nesting expressed as percent maximum nestlet consolidation (D). Each point shows ±S.E.M. for 12 mice (six male, six female). Filled symbols indicate a significant difference from vehicle (Veh) as determined by repeated-measures one-way ANOVA and Dunnett’s post hoc test for parametric data (A, C, and D) or by Friedman’s and Dunn’s post hoc test for nonparametric data (B), P < 0.05. Results of ANOVA and power analysis data for each panel are shown in Table 1.

  • Fig. 3.
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    Fig. 3.

    Effects of MAGL-selective inhibitors MJN110 and JZL184 on IP acid–induced stretching, facial grimace, rearing, and nesting behaviors in male and female mice. Abscissae: doses of MJN110 or JZL184 delivered subcutaneously in milligrams per kilogram (log scale). Ordinates: number of stretches (A), grimace score (B), number of rears (C), and nesting expressed as percent maximum nestlet consolidation (D). Other details as in Fig. 2.

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    Fig. 4.

    Effects of dual MAGL and FAAH inhibitors JZL195 and SA57 on IP acid–induced stretching, facial grimace, rearing, and nesting behaviors in male and female mice. Abscissae: doses of JZL195 or SA57 delivered subcutaneously in milligrams per kilogram (log scale). Ordinates: number of stretches (A), grimace score (B), number of rears (C), and nesting expressed as percent maximum nestlet consolidation (D). Other details as in Fig. 2.

  • Fig. 5.
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    Fig. 5.

    Effects of FAAH-selective inhibitors URB597 and PF3845 on IP acid–induced stretching, facial grimace, rearing and nesting behaviors in male and female mice. Abscissae: doses of URB597 and PF3845 delivered subcutaneously in milligrams per kilogram (log scale). Ordinates: number of stretches (A), grimace score (B), number of rears (C), and nesting expressed as percent maximum nestlet consolidation (D). Other details as in Fig. 2.

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    Fig. 6.

    Effects of endocannabinoid catabolic enzyme inhibitors and THC on nesting and locomotor behaviors in male and female mice in the absence of the IP acid noxious stimulus. Abscissae: doses of endocannabinoid catabolic inhibitors or THC in milligrams per kilogram. Doses of endocannabinoid catabolic enzyme inhibitors for locomotor studies were the highest tested in antinociception assays, whereas THC [inset in (B)] was tested across a range of doses. Ordinates: nesting expressed as percent maximum nestlet consolidation (A), and locomotor counts (B). Each point shows ±S.E.M. for 12 mice (six male, six female). Filled symbols in (A) and the asterisk in (B) inset indicate a significant difference from vehicle (Veh) as determined by repeated-measures one-way ANOVA and Dunnett’s post hoc test, P < 0.05. Results of ANOVA and power analysis data for each panel are shown in Table 1.

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    Fig. 7.

    Drug profiles for comparison of potency to produce antinociceptive effects on IP acid–stimulated and IP acid–depressed behaviors vs. general behavioral disruption. Abscissae: drug dose in milligrams per kilogram (log scale). Boxed letters (S, G, R, N) denote the dose range over which each drug significantly attenuated IP acid–induced stimulation of stretching (S) or facial grimace (G) or IP acid–induced depression of rearing (R) or nesting (N). In each panel, boxes for pain-stimulated behaviors (PSB) and pain-depressed behaviors (PDB) are shown above and below the dose axis, respectively. The gray zone at the right edge of panels for THC and PF3845 shows doses that produced motor disruption in assays of nesting and/or locomotion in the absence of the IP acid noxious stimulus. For the other drugs, no tested dose altered nesting or locomotion in the absence of the noxious stimulus, and no gray zone is indicated. The test compounds for inhibition of the two main endocannabinoid degradative enzymes, MAGL and FAAH, are organized by their selectivity, going from MAGL-selective to FAAH-selective, as indicated by the selectivity arrows on the right.

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    Fig. 8.

    Effects of time, cannabinoid receptor antagonism, and sex on MJN110 attenuation of IP acid–induced depression of nesting in male and female mice. Abscissae: (A) pretreatment time of 1.0 mg/kg MJN110 before administration of 0.32% IP acid, (B) treatments for assessment of the CB1R-selective antagonist rimonabant (3 mg/kg) and the CB2R-selective antagonist SR144528 (3 mg/kg), and (C) effect of sex on 1.0 mg/kg MJN110 antinociception, all delivered subcutaneously in a volume of 10 ml/kg. Ordinates: nestlet consolidation expressed as percent maximal nestlet consolidation. An asterisk (*) indicates a significant difference from vehicle (Veh) + Acid (A and C) or from Antagonist Veh:MJN110 (B), as determined by repeated-measures one-way ANOVA and Dunnett’s post hoc test (A and B) or two-way ANOVA and Holm-Šídák post hoc test (C), P < 0.05. Statistical results are as follows: (A) significant main effect of treatment [F(2.738, 30.12) = 7.367; P = 0.001]; (B) significant main effect of treatment [F(3.931, 43.24) = 7.84; P < 0.0001]. (C) The # indicates that there was a significant main effect of treatment [F(1, 34) = 53.41; P < 0.0001] and sex [F(1, 34) = 7.661; P = 0.0091] but no treatment × sex interaction [F(1, 34) = 3.605; P = 0.0661].

Tables

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    TABLE 1

    Summary of power analysis results from pooled one-way ANOVA data from Figs. 1–5

    Male and FemaleF Statistic, P ValueCurrent Effect Size (Cohen’s f)Current PowerSample Size: Power ≥ 0.8Friedman Statistic; P Value
    △9-Tetrahydrocannabinol
     Stretch + acidF(2.290, 25.19) = 11.83; P = 0.0001*1.0400.9947—a
     Rear + acidF(1.433, 15.77) = 0.362; P = 0.63250.7530.949—
     Grimace————F = 13.09; P = 0.0044*
     Nesting + acidF(2.469, 27.16) = 2.083; P = 0.13520.4350.43125—
     NestingF(2.351, 25.86) = 45.27; P < 0.0001*0.440.42725—
     LocomotorF(2.352, 25.87) = 11.64; P = 0.00011.0280.9947—
    MJN110
     Stretch + acidF(2.955, 32.51) = 6.231; P = 0.0019*0.7530.949—
     Rear + acidF(2.045, 22.49) = 0.9844; P = 0.39080.2990.20156—
     Grimace————F = 10.65; P = 0.0308*
     Nesting + acidF(2.528, 27.80) = 6.5; P = 0.0028*0.76815—
     nestingF(1.927, 21.19) = 2.789; P = 0.08570.5030.40727—
     LocomotorF(1.453, 15.98) = 2.166; P = 0.15560.4450.3334—
    JZL184
     Stretch + acidF(3.580, 35.80) = 2.726; P = 0.04960.5220.66214—
     Rear + acidF(1.992, 19.92) = 0.4745; P = 0.62830.2170.116>100—
     Grimace————F = 16.66; P = 0.0023*
     Nesting + acidF(2.777, 30.55) = 5.316; P = 0.0054*0.6950.8811—
     NestingF(2.072, 22.79) = 0.6424; P = 0.54060.2410.14584—
     LocomotorF(1.453, 15.98) = 2.166; P = 0.15560.4450.3334—
    JZL195
     Stretch + acidF(2.440, 26.84) = 5.007; P = 0.0102*0.6750.82112—
     Rear + acidF(2.413, 26.55) = 0.2673; P = 0.80660.1570.092>100—
     Grimace————F = 1.664; P = 0.645
     Nesting + acidF(2.252, 24.78) = 3.782; P = 0.0325*0.5870.66816—
     NestingF(1.696, 18.65) = 1.157; P = 0.32790.2990.18464—
     LocomotorF(1.516, 16.68) = 2.768; P = 0.10240.5020.41626—
    SA57
     Stretch + acidF(2.256, 24.82) = 4.219; P = 0.0228*0.6190.71814—
     Rear + acidF(2.278, 25.06) = 0.3463; P = 0.73770.1790.103>100—
     Grimace————F = 3.058; P = 0.3828
     Nesting + acidF(1.972, 21.7) = 4.391; P = 0.0255*0.6310.69115—
     NestingF(1.109, 12.20) = 5.032; P = 0.0413*0.6770.56519—
     LocomotorF(1.516, 16.68) = 2.768; P = 0.10240.5020.41626—
    URB597
     Stretch + acidF(2.643, 29.07) = 1.256; P = 0.3060.3370.28238—
     Rear + acidF(1.346, 14.81) = 1.129; P = 0.32580.320.18565—
     Grimace————F = 6.200; P = 0.1023
     Nesting + acidF(2.321, 23.21) = 4.102; P = 0.0251*0.6410.7113—
     NestingF(1.631, 17.94) = 0.5785; P = 0.53730.2290.125>100—
     LocomotorF(1.938, 21.31) = 3.418; P = 0.05290.5570.5719—
    PF3845
     Stretch + acidF(2.541, 27.95) = 0.5712; P = 0.61110.2270.14482—
     Rear + acidF(2.051, 22.56) = 0.252; P = 0.78470.150.084>100—
     Grimace————F = 0.0826; P = 0.9938
     Nesting + acidF(1.769, 19.46) = 2.045; P = 0.1600.4320.3531—
     NestingF(2.350, 25.85) = 17.43; P < 0.0001*1.2590.9995—
     LocomotorF(1.938, 21.31) = 3.418; P = 0.05290.5570.5719—
    • a Dashes indicate statistical analyses that were not conducted in accordance with parametric or nonparametric data.

Additional Files

  • Figures
  • Tables
  • Data Supplement

    • Supplemental Data -

      Supplemental Figure 1. Endpoints showing a main effect of sex in studies with JZL184 (A,B,C) and MJN110 (D).

      Supplemental Figure 2. Sex differences with the dual MAGL and FAAH eCB catabolic enzyme inhibitors JZL195 and SA57 on IP acid-stimulated stretching and locomotion in the absence of IP acid.

      Supplemental Tables 1-9. Summary of power analysis results from ANOVA data from Figures 2-6.

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Journal of Pharmacology and Experimental Therapeutics: 377 (2)
Journal of Pharmacology and Experimental Therapeutics
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1 May 2021
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Research ArticleBehavioral Pharmacology

Behavioral Battery for Testing Candidate Analgesics II

C. M. Diester, A. H. Lichtman and S. S. Negus
Journal of Pharmacology and Experimental Therapeutics May 1, 2021, 377 (2) 242-253; DOI: https://doi.org/10.1124/jpet.121.000497

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Research ArticleBehavioral Pharmacology

Behavioral Battery for Testing Candidate Analgesics II

C. M. Diester, A. H. Lichtman and S. S. Negus
Journal of Pharmacology and Experimental Therapeutics May 1, 2021, 377 (2) 242-253; DOI: https://doi.org/10.1124/jpet.121.000497
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