Abuse Liability, Anti-Nociceptive, and Discriminative Stimulus Properties of IBNtxA

Abstract

IBNtxA (3-iodobenzoyl naltrexamine) is a novel μ-opioid receptor (MOR) agonist which is structurally related to the MOR antagonist naltrexone. Recent studies suggest IBNtxA preferentially signals through truncated MOR splice variants, resulting in anti-nociception with reduced side effects, including no conditioned place preference (CPP) when tested at a single dose. IBNtxA represents an intriguing lead compound for preclinical drug development targeting truncated MOR splice variants, but further evaluation of its in vivo pharmacological profile is necessary. The purpose of this study was to independently verify the antinociceptive properties of IBNtxA and to examine more completely the rewarding properties and discriminative stimulus effects of IBNtxA, allowing broader assessment of IBNtxA as a candidate for further medications development. A dose of 3 mg/kg IBNtxA was equipotent to 10 mg/kg morphine in a hot-plate analgesia assay. In drug discrimination testing using mice trained to discriminate between 3 mg/kg IBNtxA and vehicle, the κ-agonist U-50488 fully substituted for IBNtxA. MOR agonist morphine, δ-agonist SNC162, NOP agonist SCH 221510, and MOR/NOP partial agonist buprenorphine each partially substituted for IBNtxA. IBNtxA up to 3 mg/kg did not produce a place preference in CPP. Pretreatment with 3 mg/kg IBNtxA but not 1 mg/kg IBNtxA attenuated acquisition of place preference for 10 mg/kg morphine. A dose of 3 mg/kg IBNtxA attenuated morphine-induced hyperlocomotion but did not alter naloxone-precipitated morphine withdrawal. Overall, IBNtxA has a complicated opioid receptor pharmacology in vivo. These results indicate that IBNtxA produces potent anti-nociception and has low abuse liability, likely driven by substantial κ agonist signaling effects.

Figure 1

Structures of IBNtxA, naltrexone, and morphine.

Figure 2

Time-course of hot-plate analgesia tests. Male C57BL/6 mice (n = 6/group) received IBNtxA or morphine at the indicated doses using a hot-plate apparatus set at 55 °C. (A) The time elapsing to the first nocifensive response (jumping, paw licking or fluttering) was scored. A maximal latency of 20 s was used to minimize any tissue damage. (B) Results analyzed as percent maximal effect (%MPE) for each mouse. All results are presented as means ± SEM; ***, p < 0.01 compared to BL of 10 mg/kg morphine; †, p < 0.05 compared to BL of 3 mg/kg IBNtxA.

Figure 3

Drug discrimination results for animals trained to distinguish 3 mg/kg IBNtxA from vehicle. (A) IBNtxA and KOR agonist U-50488 fully substitute for IBNtxA. MOR agonist morphine, DOR agonist SNC162, NOP agonist SCH 221510, and MOR partial agonist/NOP agonist buprenorphine partially substitute for IBNtxA. The psychostimulant cocaine does not substitute for IBNtxA. Because these data ultimately represent the proportion of mice who chose the IBNtxA-paired nose poke hole for their first response, there are no error bars. (B) Behavioral disruption of drug responding as determined by the time to first drug response. These results are presented as means ± SEM. The highest tested doses of buprenorphine (1 mg/kg), morphine (10 mg/kg), SCH 221510 (10 mg/kg), and cocaine (10 mg/kg) each induced a substantial behavioral disruption. Seven out of seven mice failed to respond to 1 mg/kg buprenorphine, and six out of seven mice failed to respond to 10 mg/kg morphine. Therefore, drug substitution data are not available. n = 7 for each drug dose tested, except for 0.3 and 1.0 mg/kg SCH 221510 and 3 and 10 mg/kg cocaine where n = 6.

Figure 4

Conditioned place preferences of varying doses of morphine or IBNtxA compared to vehicle-only treatment. A dose of 10 mg/kg morphine significantly induced a preference for the drug-paired compartment, while IBNtxA did not produce a significant place preference at doses up to 3 mg/kg. DMSO vehicle (n = 17), 3 mg/kg morphine (n = 14), 10 mg/kg morphine (n = 22), 20 mg/kg morphine (n = 12), 0.3 mg/kg IBNtxA (n = 13), 1 mg/kg IBNtxA (n = 16), and 3 mg/kg IBNtxA (n = 14). All results are presented as means ± SEM; *, p < 0.05 compared to vehicle.

Figure 5

IBNtxA pretreatment attenuated acquisition of morphine CPP. CPP training for 10 mg/kg morphine was modified to include an IBNtxA or vehicle pretreatment prior to standard side training. Pretreatment with 3 mg/kg IBNtxA significantly attenuated acquisition of morphine-induced place preference (n = 10/group). All results are presented as means ± SEM; *, p < 0.05 compared to vehicle pretreatment.

Figure 6

Male mice (n = 8–11/group) received DMSO vehicle or 3 mg/kg IBNtxA pretreatments 15 min prior to DMSO vehicle or 10 mg/kg morphine administration. (A) Time course of locomotor activity immediately following the second injection. (B) Overall distance traveled of each group. IBNtxA pretreatment significantly attenuated morphine-induced hyperlocomotion but did not significantly alter locomotion following vehicle injection. All results are presented as means ± SEM; ****, p < 0.0001 compared to all other groups.

Figure 7

Male mice (n = 8/group) received 50 mg/kg morphine. To test whether IBNtxA might induce or induce withdrawal, or if blunt naloxone induced withdrawal, each group received a DMSO vehicle or 3 mg/kg IBNtxA pretreatment. Fifteen minutes later, both groups received 50 mg/kg naloxone. Jumps were counted over 10 min, defined as all four paws leaving the floor of a glass cylinder. IBNtxA administration did not induce withdrawal or attenuate naloxone-induced withdrawal jumping behaviors. PTX = pretreatment. All results are presented as means ± SEM.