KOR/κ Opioid Receptor

Norbinaltorphimine, with pA2 values of 10.2-10.4, reversibly opposes the effects of kappa agonists. Venatorfimin has pA2 values of 7.4-7.6 and 7.6-7.8, respectively, making it a substantially less effective antagonist of the mu and delta receptors [1].

In adolescent rats, nortrophimine weakly and inconsistently boosts and attenuates taste avoidance based on the dose and trial, which results in inconsistent taste avoidance in response to THC. Nortropenine did not affect the two-bottle preference test and only slightly affected final bottle avoidance. It's interesting to note that nortrophenine had no effect on adult rats' THC-induced taste avoidance [2]. Pretreatment with nobinatofamine had no effect on nicotine-induced recovery, but it dramatically reduced the recovery caused by stress-induced nicotine-CPP [3].

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Objectives: The present study was designed to assess the impact of KOR antagonism on the aversive effects of THC in adolescent and adult rats using the conditioned taste avoidance (CTA) procedure. [2]
Methods: Following a single pretreatment injection of norbinaltorphimine (norBNI; 15 mg/kg), CTAs induced by THC (0, 0.56, 1.0, 1.8, and 3.2 mg/kg) were assessed in adolescent (n = 84) and adult (n = 83) Sprague-Dawley rats. [2]
Results: The KOR antagonist, norBNI, had weak and inconsistent effects on THC-induced taste avoidance in adolescent rats in that norBNI both attenuated and strengthened taste avoidance dependent on dose and trial. norBNI had limited impact on the final one-bottle avoidance and no effects on the two-bottle preference test. Interestingly, norBNI had no effect on THC-induced taste avoidance in adult rats as well. [2]
Conclusions: That norBNI had no significant effect on THC-induced avoidance in adults, and a minor and inconsistent effect in adolescents demonstrates that the aversive effects of THC are not mediated by KOR activity as assessed by the CTA design in Sprague-Dawley rats.

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Objectives: In the current study, we determined whether the selective KOR antagonist, norbinaltorphimine (nor-BNI), would block stress-induced reinstatement of nicotine preference. [3]
Methods: Adult Institute of Cancer Research mice were conditioned with 0.5 mg/kg nicotine, injected subcutaneously (s.c.) for 3 days and tested in the nicotine-conditioned place preference (CPP) model. After 3 days extinction, nor-BNI (10 mg/kg, s.c.) was administered 16 h prior to a priming dose of nicotine (0.1 mg/kg, s.c.), and mice were tested in the CPP model for nicotine-induced reinstatement of CPP. A separate group of mice was subjected to a 2-day modified forced swim test (FST) paradigm to induce stress after 3 days extinction from CPP. Mice were given vehicle or nor-BNI (10 mg/kg, s.c.) 16 h prior to each FST session.[3]
Results: Nor-BNI pretreatment significantly attenuated stress-induced reinstatement of nicotine-CPP, but had no effect on nicotine-primed reinstatement.[3]
Conclusions: Blockade of KORs by selective antagonists attenuates stress-induced reinstatement of nicotine-CPP. Overall, the kappa opioid system may serve as a therapeutic target for suppressing multiple signaling processes which contribute to maintenance of smoking, smoking relapse, and drug abuse in general.[3]

The pharmacological profile of the opioid antagonist norbinaltorphimine has been characterised in vitro and in vivo. In vitro, norbinaltorphimine reversibly antagonised the effects of kappa agonists with pA2 values of 10.2-10.4. Norbinaltorphimine was much less potent as an antagonist at mu and delta receptors, pA2 values were 7.4-7.6 and 7.6-7.8, respectively. In all cases Schild slopes were unity. In vivo, norbinaltorphimine was an effective antagonist only at high dose levels and was not very selective between mu and kappa. The results indicate that in vitro norbinaltorphimine is a potent selective kappa antagonist; however, this antagonist profile is not maintained in vivo[1].

Norbinaltorphimine was dissolved in sterile H2O at a concentration of 15 mg/ml and administered subcutaneously (SC) at a dose of 15 mg/kg. [2]
Norbinaltorphimine dihydrochloride was dissolved in a physiological saline solution (0.9 % sodium chloride) and injected subcutaneously (s.c.) at a volume of 10 ml/kg body weight.[3]

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Experiment 1: Adolescents [2]
Phase I: Habituation Abbreviated CTA procedures designed to maintain proper growth rates in adolescent animals were utilized as published previously (Hurwitz et al. 2012; Wetzell et al. 2014). Beginning on PND 22, subjects (n = 84) were weighed daily to acclimate them to experimenter handling. On PND 28, they were deprived of water for 24 h prior to the start of water habituation. On PND 29, they were transferred to test cages and given 45-min access to water presented in graduated 50 ml Nalgene centrifuge tubes each affixed with a rubber stopper and sipper tube, after which they had ad-libitum access to water in their home bins for 23¼ h. This 2-day cycle (23¼-h water deprivation/45-min test cage access followed by 24-h water access) was repeated two more times (final day on PND 33).

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Phase II: Drug pretreatment [2]
Animals were ranked according to average water consumption on all habituation cycles and assigned to one of two groups [norbinaltorphimine (nor-BNI or norBNI) (n = 42) and Vehicle (n = 42)], such that mean water intake was comparable among groups. On PND 34 (approximately 24 h prior to conditioning, see below), subjects assigned to the norBNI group were injected with norBNI (15 mg/kg) and subjects assigned to the Vehicle group were injected with the norBNI vehicle at an equal volume. The timing of the norBNI injection was based on research by Munro and colleagues (2012) demonstrating that SC administration of norBNI produced long-lasting antagonistic effects that were selective to KOR.

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Phase III. Taste avoidance conditioning [2]
Following the injection of norbinaltorphimine (nor-BNI or norBNI) or vehicle, animals were deprived of water for 23¼ h (PND 34). A novel saccharin solution was then presented instead of water for 45 min on PND 35. Following saccharin access, animals in each pretreatment group (norBNI and Vehicle) were immediately rank-ordered according to saccharin consumption and assigned to one of five groups [0 (n = 18), 0.56 (n = 16), 1.0 (n = 16), 1.8 (n = 16), 3.2 (n = 18)], such that mean saccharin intake was comparable. This procedure yielded a total of 10 groups; N0, N0.56, N1.0, N1.8, N3.2, V0, V0.56, V1.0, V1.8 and V3.2, where N or V refers to the pretreatment group (norBNI or Vehicle) and the number refers to the dose of THC administered during conditioning. THC doses were chosen based on their range from potentially rewarding to aversive (Braida et al. 2004; Wakeford and Riley 2014). Within 20 min of saccharin access, subjects were injected with drug or vehicle and given ad-libitum water access in their home bins for 23¼ h. This 2-day cycle was repeated three more times for a total of four conditioning trials (final day on PND 41).

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Phase IV. Two-bottle test [2]
Following the final two-day cycle during conditioning, animals were deprived of water on Day 42 for 24h prior to being given 45-min access to two Nalgene tubes (one containing tap water and the other containing the saccharin solution) in a two-bottle assessment of the CTA (Day 43). Bottle placement was counterbalanced to control for positioning effects on this test. Following this access, the animals were returned to their home bins and no injections were administered.

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Experiment 2: Adults [2]
The procedures for the adults were identical to those described above with the following exceptions: 83 subjects were brought into the facility on PND 21 and maintained on ad-libitum food and water with no manipulations until PND 76 when daily weighing began. Water bottles were removed on PND 83, and the habituation phase began on PND 84. The norbinaltorphimine (nor-BNI or norBNI) or vehicle injections were given on PND 89, and the four CTA conditioning trials occurred from PND 90 to 96. The final two-bottle test was performed on PND 98.

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Nicotine-primed reinstatement of nicotine-CPP [3]
Two individual cohorts of seven to nine mice were tested in the CPP paradigm and classified into groups A and B. On day 6, 1 day after test day, the test day procedure was repeated in both groups for three additional days to measure extinction of nicotine-CPP. All mice received saline injections (s.c.) prior to entering the chambers on these days. After day 8 test session, group A mice received an injection of norbinaltorphimine (nor-BNI or norBNI) (10 mg/kg, s.c.), and group B mice received an injection of vehicle, 16 h prior to day 9 test session. On day 9, mice received an injection of nicotine (0.1 mg/kg, s.c.), and a subset of animals from group A, the nor-BNI pre-treated group (n=4 for saline and nicotine) received vehicle, and were immediately placed in the chamber for testing. A timeline for the experiment is shown in Fig. 1a.

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Stress-induced reinstatement of nicotine-CPP [3]
As in the nicotine-primed reinstatement study, two individual cohorts of six to eight mice, groups A and B, were tested in the CPP paradigm. After three CPP extinction days (day 8), nicotine-conditioned mice from group B received injections of norbinaltorphimine (nor-BNI or norBNI) (10 mg/kg, s.c.), while nicotine-conditioned mice from group A received vehicle. Saline-conditioned mice served as a control group (unstressed) and were treated with vehicle (group A) or norbinaltorphimine (nor-BNI or norBNI) (group B) in the home cage, according to the same treatment schedule as mice subjected to the FST. On day 9, 16 h after nor-BNI pretreatment, to induce stress, mice were exposed to a 2-day modified forced swim test (Porsolt et al. 1977; McLaughlin et al. 2003). Briefly, mice swam in opaque 5-l beakers filled with 3.5 l of 30°C water. After each trial, the mice were dried with towels and returned to their home cages before further testing. On day 1 of the FST (experiment day 9), mice were placed in water to swim for a single trial of 15 min. At the end of FST day 1, mice were again pretreated with nor-BNI or vehicle 16 h prior to FST day 2. On day 2 of the FST (experiment day 10), mice were placed in water to swim through a series of four trials, each 6 min long. Trials were separated by a 10-min return to the home cage. Difficulties in swimming or staying afloat were the criteria for exclusion in this study; however, no mice met these criteria. On day 11, mice were reexposed to the CPP chambers for testing. A timeline for the experiment is shown in Fig. 1b.

[1]. Norbinaltorphimine: antagonist profile at kappa opioid receptors. Eur J Pharmacol. 1987 Dec 15;144(3):405-8.

[2]. Effect of norbinaltorphimine on ∆⁹-tetrahydrocannabinol (THC)-induced taste avoidance in adolescent and adult Sprague-Dawley rats. Psychopharmacology (Berl). 2015 Sep;232(17):3193-201.

[3]. Effects of the kappa opioid receptor antagonist, norbinaltorphimine, on stress and drug-induced reinstatement of nicotine-conditioned place preference in mice. Psychopharmacology (Berl). 2013 Apr;226(4):763-8.

Similar to previous findings with cocaine and ethanol, nor-BNI did not block nicotine-primed reinstatement of nicotine-CPP, indicating an effect specific to stress-induced responses and implicating differential mechanisms in stress-induced vs. drug-induced reinstatement of drug relapse. The KOR antagonists JDTic and nor-BNI also had no effect on the expression of nicotine-CPP (Jackson et al. 2010), and a follow-up study from our lab revealed that JDTic, when administered to mice conditioned with a dose of nicotine that does not produce significant CPP in mice (nicotine, 0.1 mg/kg, s.c.), did not enhance nicotine-CPP (nicotine (0.1)=52±10 vs. JDTic (16) – (nicotine (0.1)=75±22), suggesting that KORs are not involved in nicotine reward pathways. However, at higher, aversive nicotine doses, nor-BNI pretreatment switched the aversive response to a place preference (Smith et al. 2012), further supporting a role for the kappa opioid receptor system in mediating aversive effects of nicotine (Jackson et al. 2010). In summary, the KOR antagonist nor-BNI blocks stress-induced reinstatement of nicotine-CPP, but not nicotine-primed reinstatement of nicotine-CPP. Taken together with previous studies, the kappa opioid system serves as a potential therapeutic target for suppressing signaling processes associated with multiple aspects of drug abuse maintenance and relapse.[3]

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Although the aversive effects of THC appear to be mediated by KOR activity in adults (Cheng et al. 2004; Ghozland et al. 2002; Zimmer et al. 2001), it is unknown to what extent, if any, kappa receptor activity is involved in such effects in adolescents. Given that adolescent rats display relative insensitivity to the aversive effects of the kappa agonist U62-066 (Anderson et al. 2014), the aversive effects of THC may be mediated differently in this age group. As described, norBNI's effect on taste avoidance in adolescents was inconsistent in that norBNI had no effect on avoidance induced by the lowest (0.56 mg/kg) and highest (3.2 mg/kg) doses of THC and opposite effects at the intermediate doses (strengthening of avoidance at 1.0 mg/kg and attenuation of avoidance at 1.8 mg/kg). For all cases in which norBNI significantly impacted THC-induced avoidance, the effects were modest and trial dependent. On the final one-bottle avoidance test, norBNI pretreatment significantly attenuated taste avoidance only in the group of animals conditioned with 1.8 mg/kg of THC. There was no evidence of any pretreatment effect of norBNI on the two-bottle assessment as vehicle and norBNI-treated subjects displayed comparable dose-dependent avoidance of the THC-associated saccharin. Such findings are consistent with the initial prediction that adolescents are relatively insensitive to activity at the kappa receptor and that taste avoidance induced by THC would be mediated by a non-KOR related mechanism. Similar assessments were made in adults, primarily to replicate previous research and confirm that THC's aversive effects were kappa mediated in this population when assessed using the CTA procedure. Interestingly, norBNI was without effect in the adult subjects on any analysis run (i.e., acquisition, final one-bottle avoidance test or two-bottle test). One immediate issue is whether norBNI at this dose and under the specific parameters tested is behaviorally active. As noted, although norBNI had no consistent effect in adolescent subjects, there were significant changes in THC-induced avoidance between saline- and norBNI-treated animals indicative of a behaviorally active dose. Interestingly, recent work from a number of laboratories report that norBNI as administered in the present study is capable of affecting drug-induced taste avoidance, as well as other behavioral effects. Specifically, subcutaneous norBNI pretreatment at a similar dose range used here and administered 24 h prior to the beginning of experimental procedures significantly affects ethanol-induced taste avoidance in stressed mice (Anderson et al. 2013), ethanol intake in adult mice (Morales et al. 2014), cocaine self-administration in rats given 6-h access to cocaine (Wee et al. 2009) and heroin self-administration in rats given 6-h access to heroin (Schlosburg et al. 2013). Although norBNI has been reported to impact a variety of behavioral endpoints, it is important to note that others have also found null effects when assessing the role of KOR activity with norBNI administration in the behavioral effects of other drugs of abuse (Hutsell et al. 2015; Negus 2004).[2]

C40H45CL2N3O6

734.707809209824

733.268

113158-34-2

105618-26-6

11957626

Light yellow to gray solid powder

7

8

4

51

1340

8

Cl.Cl.O1C2=C(C=CC3C[C@@H]4[C@@]5(CC6=C([C@H]1[C@@]5(C=32)CCN4CC1CC1)NC1=C6C[C@]2([C@H]3CC4C=CC(=C5C=4[C@@]2(CCN3CC2CC2)[C@H]1O5)O)O)O)O

JOJPJLHRMGPDPV-LZQROVCBSA-N

InChI=1S/C40H43N3O6.2ClH/c44-25-7-5-21-13-27-39(46)15-23-24-16-40(47)28-14-22-6-8-26(45)34-30(22)38(40,10-12-43(28)18-20-3-4-20)36(49-34)32(24)41-31(23)35-37(39,29(21)33(25)48-35)9-11-42(27)17-19-1-2-19;;/h5-8,19-20,27-28,35-36,41,44-47H,1-4,9-18H2;2*1H/t27-,28-,35+,36+,37+,38+,39-,40-;;/m1../s1

(1S,2S,7S,8S,12R,20R,24R,32R)-11,33-bis(cyclopropylmethyl)-19,25-dioxa-11,22,33-triazaundecacyclo[24.9.1.18,14.01,24.02,32.04,23.05,21.07,12.08,20.030,36.018,37]heptatriaconta-4(23),5(21),14(37),15,17,26,28,30(36)-octaene-2,7,17,27-tetrol;dihydrochloride

nor-Binaltorphimine dihydrochloride; 113158-34-2; Norbinaltorphimine dihydrochloride; Nor-bni; nor-BNI dihydrochloride; Y8WNP37PIV; NorbinaltorphimineDihydrochlorideSalt; 105618-26-6;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~136.11 mM)
H2O : ~33.33 mg/mL (~45.36 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.40 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.40 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)