NOP Receptor/ORL1

Behavioral studies in pain models and pharmacokinetic evaluations were carried out in Sprague-Dawley rats (weight range 134−423 g); Iffa Credo, Brussels, Belgium provided the tail-flick model; Harlan Laboratories, Indianapolis, IN provided the bone cancer model; Janvier Laboratories, Le Genest Saint Isle, France provided the other pain models and pharmacokinetics); male rats were used in the majority of the experiments, with the exception of the bone cancer and tail-flick models, which used female Sprague-Dawley rats. Male Wistar rats, weighing between 150 and 375 grams, were used in side effect model studies (Depré, Saint Doulchard, France). The rats were kept in standard housing (20–24°C, 12 hours of light and dark, 35–70% relative air humidity, 10–15 air changes per hour, air movement<0.2 m/s), with unlimited access to food and water in their home cage. Except for mononeuropathy models, which required animals to undergo repeated testing with a minimum one-week washout period in between, all in vivo models only employed animals once. With the few exceptions listed below, animal testing was carried out in compliance with German Animal Welfare Law, the International Association for the Study of Pain's guidelines, and recommendations. The local government's animal research committee, which gets advice from a separate ethics committee, approved all study protocols. The treatment groups were assigned to the animals at random. Randomized tests were conducted using various doses and vehicles. The operators conducting the behavioral tests were unaware of the study hypothesis and the nature of drug differences, even though they were not formally "blinded" with regard to the treatment.

Cebranopadol (trans-6'-fluoro-4',9'-dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane-1,1'(3'H)-pyrano[3,4-b]indol]-4-amine) is a novel analgesic nociceptin/orphanin FQ peptide (NOP) and opioid receptor agonist [Ki (nM)/EC50 (nM)/relative efficacy (%): human NOP receptor 0.9/13.0/89; human mu-opioid peptide (MOP) receptor 0.7/1.2/104; human kappa-opioid peptide receptor 2.6/17/67; human delta-opioid peptide receptor 18/110/105].[1]

---

Human MOP, DOP, KOP, and NOP receptor binding assays were run in microtiter plates with wheat germ agglutinin-coated scintillation proximity assay beads. [N-allyl-2,3-3H]naloxone and [tyrosyl-3,5-3H]deltorphin II, [3H]Ci-977, and [leucyl-3H]nociceptin were used as ligands for the MOP, DOP, KOP, and NOP receptor binding studies, respectively. The KD values of the radioligands used for the calculation of Ki values were provided as supplemental information. The assay buffer used for the MOP, DOP, and KOP receptor binding studies was 50 mM Tris-HCl (pH 7.4) supplemented with 0.052 mg/mL bovine serum albumin. For the NOP receptor binding studies, the assay buffer used was 50 mM HEPES, 10 mM MgCl2, 1 mM EDTA (pH 7.4). The final assay volume of 250 μL/well included 1 nM [3H]naloxone, 1 nM [3H]deltorphin II, 1 nM [3H]Ci-977, or 0.5 nM [3H]nociceptin as a ligand and cebranopadol in dilution series. Cebranopadol was diluted with 25% DMSO in water to yield a final 0.5% DMSO concentration, which also served as a respective vehicle control. Assays were started by the addition of beads (1 mg beads/well), which had been preloaded for 15 minutes at room temperature with 23.4 μg of human MOP membranes, 12.5 μg of human DOP membrane, 45 μg of human KOP membranes, or 25.4 µg of human NOP membranes per 250 µL of final assay volume. After short mixing, the assays were run for 90 minutes at room temperature. The microtiter plates were then centrifuged for 20 minutes at 500 rpm, and the signal rate was measured by means of a 1450 MicroBeta Trilux. IC50 values reflecting 50% displacement of [3H]naloxone-, [3H]deltorphin II-, [3H]Ci-977-, or [3H]nociceptin-specific receptor binding were calculated by nonlinear regression analysis. Individual experiments were run in duplicate and were repeated three times in independent experiments[1].

Cebranopadol was tested for its agonistic activity on human recombinant MOP, DOP, or NOP receptor-expressing cell membranes from Chinese hamster ovary K1 cells, or KOP receptor-expressing cell membranes from human embryonic kidney cell line 293 cells. For each assay, 10 µg of membrane proteins was incubated for 45 minutes at 25°C with 0.4 nM [35S]GTPγS (GE Healthcare) and various concentrations of agonists in a buffer containing 20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 1.28 mM NaN3, and 10 µM guanosine diphosphate. The bound radioactivity was calculated using the methods previously mentioned.

Cebranopadol was dissolved in vehicle that consisted of 5% dimethylsulfoxide, 5% Emulphor, and 90% distilled water. The solution was vortexed before filling a 1-ml syringe for oral injection. Cebranopadol was injected orally by gavage at doses of 0.0, 25, and 50 μg/kg. Cocaine HCl was dissolved in 0.9% saline at a dose of 0.5 mg/kg/infusion and self-administered intravenously. Sweetened condensed milk was diluted 2:1 (v/v) in water.[2]

---

Effect of Cebranopadol on the Escalation of Cocaine Self-Administration[2]
Rats (n = 14) were trained to self-administer cocaine under a fixed-ratio 1 (FR1) schedule of reinforcement in daily 6-hour sessions. Each active lever press resulted in the delivery of one cocaine dose (0.5 mg/kg/0.1 ml infusion). A 20-second timeout (TO) period followed each cocaine infusion. During the timeout period, responses on the active lever did not have scheduled consequences. This TO period occurred concurrently with illumination of a cue light that was located above the active lever to signal delivery of the positive reinforcement. The rats were trained to self-administer cocaine in 15 sessions (5 days/week) until a stable baseline of reinforcement was achieved (<10% variation over the last three sessions). A within-subjects Latin-square design was used for the drug treatments. The rats were orally injected with cebranopadol (0, 25, and 50 μg/kg) 30 minutes before beginning the sessions. Oral administration was performed by gavage using a 19-gauge needle and 8 cm of Tygon tubing (0.030 inch inner diameter, 0.090 inch outer diameter). The animals were subjected to cocaine self-administration at 2-day intervals between drug tests.

---

Effect of Cebranopadol on Sweetened Condensed Milk Self-Administration[2]
Rats (n = 12) were trained to self-administer SCM under an FR1 schedule of reinforcement for 6 hours per day to match cocaine self-administration. After each SCM reward delivery, a 20-second TO period occurred, during which responses on the active lever had no scheduled consequences. This TO period occurred concurrently with illumination of a cue light that was located above the active lever to signal delivery of the positive reinforcement. The rats were trained to self-administer SCM for several days until a stable baseline of reinforcement was achieved (<10% variation over the last three sessions). When the stable baseline was reached, the rats orally received cebranopadol (0, 25, and 50 μg/kg) 30 minutes before beginning the next session. The animals were subjected to SCM self-administration at 2-day intervals between drug tests.

---

Cebranopadol-Induced Conditioned Place Preference and Locomotor Activity[2]
Cebranopadol-induced CPP was evaluated using a biased, counterbalanced CPP procedure. Naive rats (n = 9) were handled and habituated to oral administration for 1 week before beginning the study. The experiment consisted of three 30-minute phases: pretest (one session), conditioning (eight sessions), and preference test (one session). The rats were placed in a dim (40 lux) room 30 minutes before starting the tests. A two-chambered (38 cm × 32 cm × 32 cm) place conditioning apparatus was used, with visual cues on the walls (stripes or dots for compartments A and B, respectively) and tactile cues on the floor (smooth or rough for compartments A and B, respectively). On day 1 (pretest), naive rats were placed between the two chambers and allowed to freely explore both chambers for 30 minutes. Individual bias toward either compartment A or B was observed, and the animals were assigned to place conditioning subgroups according to their least-preferred compartment; therefore, biased assignment was used. Conditioning was performed within subjects. Each rat received cebranopadol (25 μg/kg orally) and vehicle on alternating days in a counterbalanced design 30 minutes before being placed in the conditioning chamber. The preference test was performed 24 hours after the last conditioning session. Thirty minutes after vehicle administration, the rats were placed in the nonconditioned side of the apparatus with free access to both chambers. The time spent in the different chambers was recorded. Locomotor activity was recorded in each phase (pretest, conditioning, and preference test) using a video camera that was connected to the ANY-maze Video Tracking System 5.11.

---

Effect of Cebranopadol on Conditioned Reinstatement of Cocaine-seeking Behavior[2]
Cocaine Self-Administration Training.[2]
Rats (n = 10) were surgically prepared with indwelling microurethane catheters that were inserted in the right jugular vein. After 7 days of postsurgical recovery, the rats began self-administration training. The rats were trained to self-administer cocaine (0.5 mg/kg/0.1 ml infusion, i.v.) for 6 hours/day on an FR1 schedule in the presence of a contextual/discriminative stimulus (SD). Each session was initiated by extending two retractable levers into the operant conditioning chamber. Constant 70-dB white noise served as a discriminative stimulus that signaled availability of the reinforcer throughout the session. Responses on the right, active lever were reinforced with a dose of cocaine, followed by a 20-second TO period that was signaled by illumination of a cue light above the active lever. During this TO period, the lever remained inactive to prevent accidental overdosing with cocaine. Responses on the left, inactive lever had no scheduled consequences.

---

Conditioned Reinstatement.[2]
Two days later, the rats were presented with the SD. To evaluate the effect of cebranopadol on the conditioned reinstatement of cocaine seeking, the rats were treated with cebranopadol (0, 25, and 50 μg/kg) in a counterbalanced Latin-square design 30 minutes before the reinstatement test. The reinstatement test lasted 2 hours under SD conditions, except that cocaine was unavailable. Cebranopadol was administered only in the SD conditions, with a 2-day interval between tests.

8.9 and 26.6 mg/kg s.c. for whole-body plethysmography test in conscious rats.

Sprague-Dawley rats

[1]. J Pharmacol Exp Ther . 2014 Jun;349(3):535-48.

[2]. J Pharmacol Exp Ther . 2017 Sep;362(3):378-384.

Cebranopadol is a member of indoles.
Cebranopadol has been used in trials studying the treatment of Pain, Neoplasms, and Chronic Pain.
Cebranopadol is an orally available benzenoid that acts as an opioid peptide receptor agonist for the nociceptin/orphanin FQ peptide receptor (opioid receptor like -1; OPRL1; ORL-1; NOP; kappa-type 3 opioid receptor) and the classical opioid receptors, mu, delta and kappa, with potential anti-nociceptive activity. Upon oral administration, cebranopadol binds to NOP and the mu, delta and kappa opioid receptors, which enhances NOP- and opioid receptor-mediated signaling, interferes with the sensation of pain and results in an analgesic effect. NOP, a member of the opioid receptor family, and its endogenous ligand nociceptin play key roles in the regulation of various brain activities including pain, and some inflammatory and immune responses.

---

Drug Indication
Treatment of chronic pain.

---

Cebranopadol (trans-6'-fluoro-4',9'-dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane-1,1'(3'H)-pyrano[3,4-b]indol]-4-amine) is a novel analgesic nociceptin/orphanin FQ peptide (NOP) and opioid receptor agonist [Ki (nM)/EC50 (nM)/relative efficacy (%): human NOP receptor 0.9/13.0/89; human mu-opioid peptide (MOP) receptor 0.7/1.2/104; human kappa-opioid peptide receptor 2.6/17/67; human delta-opioid peptide receptor 18/110/105]. Cebranopadol exhibits highly potent and efficacious antinociceptive and antihypersensitive effects in several rat models of acute and chronic pain (tail-flick, rheumatoid arthritis, bone cancer, spinal nerve ligation, diabetic neuropathy) with ED50 values of 0.5-5.6 µg/kg after intravenous and 25.1 µg/kg after oral administration. In comparison with selective MOP receptor agonists, cebranopadol was more potent in models of chronic neuropathic than acute nociceptive pain. Cebranopadol's duration of action is long (up to 7 hours after intravenous 12 µg/kg; >9 hours after oral 55 µg/kg in the rat tail-flick test). The antihypersensitive activity of cebranopadol in the spinal nerve ligation model was partially reversed by pretreatment with the selective NOP receptor antagonist J-113397[1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] or the opioid receptor antagonist naloxone, indicating that both NOP and opioid receptor agonism are involved in this activity. Development of analgesic tolerance in the chronic constriction injury model was clearly delayed compared with that from an equianalgesic dose of morphine (complete tolerance on day 26 versus day 11, respectively). Unlike morphine, cebranopadol did not disrupt motor coordination and respiration at doses within and exceeding the analgesic dose range. Cebranopadol, by its combination of agonism at NOP and opioid receptors, affords highly potent and efficacious analgesia in various pain models with a favorable side effect profile.[1]

---

Cebranopadol is a novel agonist of nociceptin/orphanin FQ peptide (NOP) and opioid receptors with analgesic properties that is being evaluated in clinical Phase 2 and Phase 3 trials for the treatment of chronic and acute pain. Recent evidence indicates that the combination of opioid and NOP receptor agonism may be a new treatment strategy for cocaine addiction. We sought to extend these findings by examining the effects of cebranopadol on cocaine self-administration (0.5 mg/kg/infusion) and cocaine conditioned reinstatement in rats with extended access to cocaine. Oral administration of cebranopadol (0, 25, and 50 μg/kg) reversed the escalation of cocaine self-administration in rats that were given extended (6 hour) access to cocaine, whereas it did not affect the self-administration of sweetened condensed milk (SCM). Cebranopadol induced conditioned place preference but did not affect locomotor activity during the conditioning sessions. Finally, cebranopadol blocked the conditioned reinstatement of cocaine seeking. These results show that oral cebranopadol treatment prevented addiction-like behaviors (i.e., the escalation of intake and reinstatement), suggesting that it may be a novel strategy for the treatment of cocaine use disorder. However, the conditioned place preference that was observed after cebranopadol administration suggests that this compound may have some intrinsic rewarding effects.[2]

---

One limitation of the present study was the lack of full characterization of the pharmacokinetics and pharmacodynamics of cebranopadol. We also did not evaluate the effects of cebranopadol on the pharmacokinetics of cocaine. However, we do not believe that the reduction of cocaine escalation was related to possible pharmacokinetic effects on blood cocaine levels because cebranopadol effectively reduced conditioned reinstatement. In this case, cocaine was unavailable, thus excluding possible effects on blood cocaine levels. We also did not identify shifts in the dose-response curve or specific receptors that mediate its preclinical efficacy. Follow-up studies are needed to fully characterize the reinforcing properties and possible abuse potential of cebranopadol, particularly considering that we found that cebranopadol produced conditioned place preference. However, although such characterization studies are important from a theoretical perspective to understand the precise mechanisms of action and facilitate medication development, cebranopadol has already been shown to be well tolerated in humans and is already being tested in several clinical trials for the treatment of pain. In summary, the present study provides preclinical evidence of the efficacy of cebranopadol in reversing compulsive-like responding for cocaine and cue-induced reinstatement of cocaine seeking. Cebranopadol may be a new therapeutic option for the prevention of cocaine abuse and relapse.[2]

C24H27FN2O

378.482389688492

378.21

C, 68.34; H, 6.58; F, 4.00; N, 5.90; O, 15.17

863513-93-3

Cebranopadol; 863513-91-1

11848225

White to gray solid powder

4.3

1

3

2

28

553

0

N([C@@]1(CC[C@]2(OCCC3C4C=C(F)C=CC=4NC2=3)CC1)C1C=CC=CC=1)(C)C

CSMVOZKEWSOFER-UHFFFAOYSA-N

InChI=1S/C24H27FN2O/c1-27(2)23(17-6-4-3-5-7-17)11-13-24(14-12-23)22-19(10-15-28-24)20-16-18(25)8-9-21(20)26-22/h3-9,16,26H,10-15H2,1-2H3

6-fluoro-N,N-dimethyl-1'-phenylspiro[4,9-dihydro-3H-pyrano[3,4-b]indole-1,4'-cyclohexane]-1'-amine

GRT6005; GRT 6005; GRT-6005; Cebranopadol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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