Research ArticlePain

A bifunctional nociceptin and mu opioid receptor agonist is analgesic without opioid side effects in nonhuman primates

See allHide authors and affiliations

Science Translational Medicine  29 Aug 2018:
Vol. 10, Issue 456, eaar3483
DOI: 10.1126/scitranslmed.aar3483
  • Fig. 1 Structure-based design and optimization of bifunctional NOP-MOP ligands.

    (A) Top: Docking and binding interactions of compound 2 (shown in green stick representation) into the active-state NOP homology model (38), shown in cartoon representation (pink). Amino acids in a 4 Å radius around the ligand are shown as pink sticks and are labeled. Polar interactions of the 2-aminoethyl moiety of 2 with Glu194 and Glu199 in the EL2 of the receptor are shown as green dotted lines. Bottom: Structures of the isoquinolinone-based bifunctional ligands and their binding affinities at NOP and MOP. (B) Docking and binding interactions of compound 5 (AT-121) (shown in cyan) in the active-state NOP structure (pink). Note the interactions of the nitrogens of the sulfamide group with the Glu194 and Glu199 of the EL2 loop of NOP. Gln280, the nonconserved amino acid between NOP and the opioid receptors [His297 in MOP, shown as green sticks in (C)], does not interact with the ligand. (C) Docking and binding interactions of bifunctional compound 5 in the MOP active-state crystal structure (73), shown in cyan cartoon representation. The interacting amino acids are shown as cyan stick representations. The lipophilic isopropylcyclohexyl group binds to a nonpolar pocket lined with Met131, His297 (green sticks), and Trp293 in the MOP binding pocket.

  • Fig. 2 Effects of systemic administration of AT-121 on modulating sensory processing in nonhuman primates.

    (A) Effect of AT-121 administration on acute noxious stimulus, 50°C water. (B) Bar graph showing the effect of AT-121 on antihypersensitivity against capsaicin-induced allodynia in 46°C water. (C) Effects of NOP receptor antagonist J-113397 (0.1 mg/kg) and MOP receptor antagonist naltrexone (0.03 mg/kg) on AT-121–induced antinociception. (D) Comparison of antinociceptive potency of AT-121 and morphine. (E) Comparison of itch-scratching responses elicited by AT-121 (0.03 mg/kg) and morphine (1 mg/kg) at antinociceptive doses. Each data point represents mean ± SEM (n = 4). All compounds were delivered subcutaneously. Data were analyzed by two-way analysis of variance (ANOVA) with repeated measures (A) or one-way ANOVA with repeated measures (B and E), followed by Bonferroni’s multiple comparisons test. *P < 0.05, significantly different from vehicle condition from the first time point to the corresponding time point.

  • Fig. 3 Effects of AT-121 on reinforcing effects in nonhuman primates.

    (A and B) Number of injections received as a function of dose in monkeys responding to cocaine (C; 0.03 mg/kg per injection), remifentanil (R; 0.3 μg/kg per injection), saline (S; ~0.14 ml/kg per injection), AT-121 (0.3 to 10 μg/kg per injection), or oxycodone (0.3 to 10 μg/kg per injection) under a progressive-ratio schedule of reinforcement. (C) Effects of the vehicle (0.1 ml/kg), AT-121 (0.03 mg/kg), buprenorphine (0.1 mg/kg), or naltrexone (0.01 mg/kg) on the reinforcing effects of oxycodone (3 μg/kg per injection). Each compound was administered intramuscularly 30 min before starting the progressive-ratio schedule of oxycodone. (D) Effect of the vehicle (0.1 ml/kg) or AT-121 (0.03 mg/kg) on the reinforcing effects of food pellets. AT-121 or its vehicle was administered intramuscularly 30 min before starting the fixed-ratio schedule of food pellets. Each data point represents mean ± SEM (n = 4). Data were analyzed by one-way ANOVA with repeated measures, followed by Bonferroni’s multiple comparisons test. *P < 0.05, a significant difference from saline or vehicle.

  • Fig. 4 Acute effects of systemic administration of AT-121 on physiologic functions of freely moving nonhuman primates.

    (A) Respiration rate before and after AT-121 administration. (B) Respiration rate before and after heroin (1 mg/kg) and naltrexone (0.01 mg/kg) administration. (C to F) Minute volume (C), heart rate (D), mean arterial pressure (E), and body temperature (F) before and after different doses of AT-121 administration. Each data point represents mean ± SEM (n = 4 to 7) from each individual data averaged from a 1-min time block. All compounds were delivered intramuscularly. Open symbols represent baselines of different dosing conditions for the same monkeys before administration.

  • Fig. 5 Development of physical dependence on morphine or AT-121 in nonhuman primates after short-term repeated administration.

    (A to D) Effects of naltrexone on respiration rate (A), minute volume (B) heart rate (C), and mean arterial pressure (D) in morphine- or vehicle-treated monkeys. (E to H) Effects of J-113397 (0.03 mg/kg) and naltrexone (0.01 mg/kg) on respiration rate (E), minute volume (F), heart rate (G), and mean arterial pressure (H) in AT-121– or vehicle-treated monkeys. Data are shown as changes from the baselines (before antagonist treatment). Each data point represents mean ± SEM (n = 4) from each individual data averaged from a 15-min time block. All compounds were delivered intramuscularly. Data were analyzed by two-way ANOVA with repeated measures, followed by Bonferroni’s multiple comparisons test. *P < 0.05, significantly different from vehicle from 15 to 30 min to the corresponding time point.

  • Fig. 6 Development of opioid-induced hyperalgesia and tolerance in nonhuman primates after repeated administration of morphine or AT-121.

    (A and B) Tail-withdrawal latencies in 46°C water after topical capsaicin (0.4 mg/ml × 0.3 ml) in vehicle-treated (A and B), morphine (1.8 mg/kg)–treated (A), and AT-121 (0.03 mg/kg)–treated (B) animals (n = 6). (C and D) Tail-withdrawal latencies in 50°C before (BL) and after (day 30) repeated administration of morphine (1.8 mg/kg, n = 4) (C) or AT-121 (0.03 mg/kg, n = 5) (D). Each data point represents mean ± SEM. Data were analyzed by two-way ANOVA with repeated measures, followed by Bonferroni’s multiple comparisons test. *P < 0.05, significantly different from the vehicle at 15 and 30 min. #P < 0.05, significantly different from BL values.

  • Table 1 In vitro pharmacological profile of NOP/MOP bifunctional agonists in binding and functional assays at the opioid receptors.

    Details of the experimental procedures are given in Supplementary Materials and Methods. Values are means ± SEM of three independent experiments run in triplicate. NT, not tested (compounds with binding affinity Ki >100 nM were not tested in the functional assay); DPDPE, (d-penicillamine2,d-penicillamine5)-enkephalin.

    Receptor binding Ki (nM)NOP functional assays*MOP functional assays*KOP functional assays*
    [35S]GTPγS NOP[35S]GTPγS MOP[35S]GTPγS KOP
    NOPMOPKOPDOPEC50 (nM)% StimEC50 (nM)% StimEC50 (nM)% Stim
    N/OFQ0.08 ± 0.03133 ± 30247 ± 3.42846 ± 5124.0 ± 0.1100>10,000>10,000
    DAMGO2.96 ± 0.5432.6 ± 4.06100
    DPDPE1.11 ± 0.07
    U69,5931.05 ± 0.0260.14 ± 7.45100
    111.7 ± 3.75130.3 ± 0.7500.3 ± 28331.5 ± 1.6181.7 ± 4.428.2 ± 0.6>10,000NT
    20.99 ± 0.0467.24 ± 2.5260.3 ± 221603 ± 63186 ± 4542.1 ± 1.0>10,000NT
    30.84 ± 0.0630.8 ± 2.568.9 ± 3.2834 ± 74229 ± 2234.1 ± 0.4128 ± 139.3 ± 2.236.8 ± 11.721.35 ± 3.4
    48.61 ± 0.5628.08 ± 2.769.7 ± 2243.09 ± 18.6123.5 ± 5027.5 ± 3.096 ± 38.78.2 ± 2.2>10,000
    5 (AT-121)3.67 ± 1.1016.49 ± 2.1301.3 ± 35.4145.6 ± 25.534.7 ± 6.2941.1 ± 0.319.6 ± 6.914.2 ± 0.40NT
    66.44 ± 0.4453.94 ± 2.0455.5 ± 63184.7 ± 63173.4 ± 2245.1 ± 12115.6 ± 206.7 ± 0>10,000
    Morphine>10,0001.1 ± 0.146.9 ± 14.5140.0 ± 1.5>10,0005.4 ± 115.6 ± 0.593 ± 9576 ± 81.525.0 ± 1.95
    Buprenorphine140 ± 11.20.15 ± 0.132.50 ± 1.26.13 ± 0.4>10,00012.6 ± 67.2 ± 3.527.0 ± 4.8>10,000

    *Functional activity was determined by stimulation of [35S]GTPγS binding to cell membranes, and % stimulation was obtained as a percentage of stimulation of the standard full agonists, N/OFQ (for NOP), DAMGO (for MOP), and U69,593 (for KOP), which showed at least two- to five-fold stimulation over basal, indicative of a robust assay. The stimulation by the standard full agonists was taken as 100% when comparing stimulation by the test compound.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/456/eaar3483/DC1

      Materials and Methods

      Fig. S1. Chemical synthesis scheme for compounds 1 to 6.

      Fig. S2. AT-121 plasma and brain concentrations after a single subcutaneous administration of 3 mg/kg to male Sprague-Dawley rats.

      Fig. S3. In vitro plasma stability assessment of AT-121 in nonhuman primate plasma.

      Fig. S4. Effects of systemic administration of AT-121 on physiologic functions of freely moving nonhuman primates implanted with telemetric probes.

      Table S1. Brain sample records for AT-121 brain penetration after subcutaneous administration of 3 mg/kg to male Sprague-Dawley rats.

      Table S2. Pharmacokinetic parameters (plasma and brain) for AT-121 after subcutaneous administration in male Sprague-Dawley rats.

      Table S3. Plasma stability assessment of AT-121 in nonhuman primate plasma.

      Table S4. Baseline values for the nonhuman primate tail-withdrawal assay that are normalized across different dosing conditions.

      Table S5. Raw data.

      References (74, 75)

    • The PDF file includes:

      • Materials and Methods
      • Fig. S1. Chemical synthesis scheme for compounds 1 to 6.
      • Fig. S2. AT-121 plasma and brain concentrations after a single subcutaneous administration of 3 mg/kg to male Sprague-Dawley rats.
      • Fig. S3. In vitro plasma stability assessment of AT-121 in nonhuman primate plasma.
      • Fig. S4. Effects of systemic administration of AT-121 on physiologic functions of freely moving nonhuman primates implanted with telemetric probes.
      • Table S1. Brain sample records for AT-121 brain penetration after subcutaneous administration of 3 mg/kg to male Sprague-Dawley rats.
      • Table S2. Pharmacokinetic parameters (plasma and brain) for AT-121 after subcutaneous administration in male Sprague-Dawley rats.
      • Table S3. Plasma stability assessment of AT-121 in nonhuman primate plasma.
      • Table S4. Baseline values for the nonhuman primate tail-withdrawal assay that are normalized across different dosing conditions.
      • References (74, 75)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S5 (Microsoft Excel format). Raw data.

    Navigate This Article