Nalfurafine esters and related formulations for promoting stress-resilience

Abstract

Nalfurafine ester derivatives, formulations that include the nalfurafine ester derivatives, and methods for using the nalfurafine ester derivatives and formulations for promoting stress-resilience.

Description

[0001] NALFURAFINE ESTERS AND RELATED FORMULATIONS

[0002] FOR PROMOTING STRESS-RESILIENCE

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims the benefit of U.S. Patent Application No. 63 / 602952, filed November 27, 2023, expressly incorporated herein by reference in its entirety.

[0005] BACKGROUND

[0006] The role of dynorphins in human pathologies has been suggested by extensive preclinical studies, but safe and effective selective kappa antagonists are not yet available for human studies. Thus, the role of dynorphins in humans has not been demonstrated and the utility of kappa receptor antagonists in humans has not been established.

[0007] Nalfurafine is a functionally selective, G-biased kappa opioid receptor agonist. Sustained activation of kappa receptors by nalfurafine causes receptor inactivation (desensitization) and results in functional antagonism rather than competitive antagonism.

[0008] Nalfurafine has been safely used in Japan for the treatment of pruritis in patients with renal failure; no adverse effects have been reported. Nalfurafine prodrugs are predicted to be safe and effective, but this has not been established. Nalfurafine has not been approved for use in the US or Europe.

[0009] A need exists for safe and effective selective kappa antagonists for inactivating kappa opioid receptors for sustained antagonism of dynorphin effects. The present disclosure seeks to fulfill this need and provides further related advantages.

[0010] BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1 compares the analgesic response to the kappa opioid receptor agonist U50,488 as a function of nalfurafine-induced kappa receptor inactivation. Male mice were intraperitoneally (i.p.) injected with nalfurafine at the doses shown (x-axis) daily for 7 days, allowed to recover for 7 days, and then challenged with the kappa opioid agonist U50,488 at 10 mg / kg (i.p.). The latency of the mice to withdraw their tails from 52.5°C water is a standard measure of antinociception. Daily treatment for 7 days with 1 ug or 10 ug / kg nalfurafine significantly blocked U50,488 antinociception. The results indicate that microdosing mice with nalfurafine caused inactivation of the kappa receptors responsible for the analgesic effects of kappa agonists.

[0012] FIG. 2 compares odorant score as a function of nalfurafine dose. Male mice were stressed by repeated forced swim in 30°C water over two days (15 min day 1 and 6 min four times on day 2). This stress paradigm is known to cause the release of the endogenous dynorphin stress-peptides. The swim episode was paired with exposure to a distinct, but neutral odorant (a dilute almond scent). Mice were later given a choice between being in a compartment containing the scent or one without the almond scent. In the absence of stress pairing, mice spent equal times in the two compartments (left bar). Mice that had the stress-scent pairing avoided the compartment paired with the scent (saline bar). Mice injected for 7 days with nalfurafine (1 or 10 pg / kg) and then allowed to recover for 7 days before the 2-day swim stress showed a significant block of the odorant aversion.

[0013] FIG. 3 compares urine output after nalfurafine dose compared to control as a function of time. Male mice were injected with 100 pg / kg nalfurafine (i.p.) or saline daily for 30 days then placed for 60 min each day in a chamber with absorbant pad to catch the urine. Nalfurafine-treated mice significantly increased their urine excretion compared with saline-treated mice. The diuresis response did not show tolerance. The results show that nalfurafine treatment did not inactivate the kappa receptors responsible for the diuretic response.

[0014] FIG. 4 illustrates pharmacokinetics of nalfurafine decanoate and compares nalfurafine serum levels (ng / mL) in mice over time (0.5-12 hr) after intraperitoneal injection with nalfurafine decanoate or nalfurafine-HCl (100 pg / kg). Mice were injected i.p. with either 100 ug / kg nalfurafine-HCl or orally administered 100 pg / kg nalfurafine decanoate and serum samples were taken at intervals between 30 min - 30 hrs. Each mouse was bled once and 4 mice were used per time point. The results show that the nalfurafine decanoate is absorbed from the gut after oral administration, serum levels peak more slowly than after i.p. administration, and drug is cleared within 12 hrs.

[0015] FIG. 5 illustrates dose-dependent Kappa Receptor inactivation by nalfurafine decanoate and compares tail withdrawal latency for male mice after 7 days for various nalfurafine decanoate doses (5 pg, 500 ng, 50 ng, 5 ng, and 500 pg per mouse with vehicle control). Vehicle is reagent grade sesame oil, and the doses were administered i.m. in 50 pl volume. The results show that the nalfurafine decanoate inactivated the kappa receptors in a dose-dependent manner to block the analgesic effects of U50,488 in male C57BL6 mice.

[0016] FIG. 6 compares tail withdrawal latency for male mice after 7, 14, 21, and 27 days for various nalfurafine decanoate doses (5 pg, 500 ng, 50 ng, 5 ng, and 500 pg per mouse administered in 50 pl volumes i.m. with vehicle (sesame oil) control). The results show that nalfurafine decanoate was effective in male C57BL / 6 mice and inactivated the kappa opioid receptors for over 21 days following a single administration.

[0017] FIG. 7 compares tail withdrawal latency for female mice after 7, 14, and 21 days for various nalfurafine decanoate doses (5 pg, 500 ng, 50 ng, 5 ng, and 500 pg per mouse administered in 50 pl volumes i.m. with vehicle (sesame oil) control). The results show that nalfurafine decanoate was also effective in female C57BL / 6 mice and inactivated the kappa opioid receptors for over 21 days following a single administration.

[0018] FIGS. 8A-8G demonstrate that repeated administration of nalfurafine and nalmefene block stress-paired cue aversion. FIGS. 8A-8C present odorant scores for norBNI, nalfurafine and nalmefene, respectively, in a swim stress assay. Odorant score were determined by the amount of time spent in the null odorant side subtracted from the paired odorant side (a negative score representing aversion). Unstressed animals (A = 10) had neutral scores while stressed animals pretreated with saline (A = 13) showed an aversion. 7 daily injections of norBNI (330 pg / kg (A = 7) or 1 mg / kg (A= 11)), nalfurafine (1 pg / kg (A = 11) or 10 pg / kg (A = 6)), and nalmefene (1 mg / kg (A = 12)) blocked stressodor paired aversion. FIG. 8D is a schematic illustration of almond pairing with either repeated force swim-stress or naloxone precipitated withdrawal, followed by 3 -chamber aversion assay with the scent on one side. FIG. 8E is a timeline illustrating differences between acute and protracted withdrawal groups in the naloxone-precipitated withdrawalodorant pairing and testing. FIGS. 8F and 8G compare odor preferences after norBNI, nalfurafine and nalmefene administration versus saline control. Mice that underwent precipitated withdrawal during the acute withdrawal phase (2 days after pump removal) and were treated daily for 7 days with saline (A = 10) showed a strong aversion to the almond scent which was significantly attenuated in groups receiving daily injections of norBNI (A = 8), nalfurafine (A = 10), and nalmefene (A = 10) (FIG. 8F). Mice that underwent precipitated withdrawal during the protracted withdrawal phase (10 days after pump removal) and were treated daily for 7 days with saline (A = 10) showed a strong aversion to the almond scent which was fully blocked in groups receiving daily injections of norBNI (A = 10), nalfurafine (A = 10), and nalmefene (A = 15) (FIG. 8G). Statistical comparisons are shown * p <0.05, ** p <0.01. DETAILED DESCRIPTION

[0019] The present disclosure provides nalfurafine ester derivatives, formulations that include nalfurafine ester derivatives, and methods for using the nalfurafine ester derivatives and formulations for promoting stress-resilience.

[0020] In one aspect, the present disclosure provides nalfurafine ester derivatives of formula (I): or a stereoisomer or pharmaceutically acceptable salt thereof.

[0021] Nalfurafine is a compound of formula (I) wherein R is H.

[0022] The compounds of the present disclosure are esters (i.e., R is C(=O)R1, where R1is a lipophilic alkyl group that imparts solubility of the nalfurafine derivative in a lipophilic vehicle. The lipophilic alkyl group does not interfere with cleavage of R from the nalfurafine derivative by endogenous esterases to produce nalfurafine.

[0023] In certain embodiments R1is a C5-C11 alkyl group.

[0024] In one embodiment, the present disclosure provides nalfurafine decanoate, a compound of formula (I) where R is C(=O)(CH2)§CH3(R1is C9 alkyl).

[0025] In another embodiment, the present disclosure provides nalfurafine octanoate, a compound of formula (I) where R is C(=O)(CH2)6CH3) (R1is C7 alkyl).

[0026] In a further embodiment, the present disclosure provides nalfurafine dodecanoate, a compound of formula (I) where R is C(=O)(CH2)K)CH3) (R1is Cl l alkyl).

[0027] The nalfurafine ester derivatives of the present disclosure are nalfurafine prodrugs. As used herein, the term “nalfurafine prodrug” refers to a nalfurafine derivative that delivers nalfurafine in vivo, (i.e., upon administration of the prodrug to a subject the prodrug provides nalfurafine).

[0028] The nalfurafine prodrugs described herein (e.g., nalfurafine decanoate) are medications designed to inhibit kappa opioid receptor activation (e.g., aticaprant, navacaprant). The nalfurafine prodrugs described herein have a novel mechanism of action that is distinct from other medications in this class. Whereas aticaprant and navacaprant are competitive antagonists with zero kappa receptor efficacy, the nalfurafine prodrugs described herein are converted in vivo to nalfurafine, which is a functionally selective, G- biased kappa opioid receptor agonist. Sustained activation of kappa receptors by nalfurafine causes receptor inactivation (desensitization). The nalfurafine prodrugs described herein provide functional antagonism rather than competitive antagonism.

[0029] In another aspect, the present disclosure provides formulations of the nalfurafine ester derivatives (nalfurafine prodrug) described herein. The formulations include one or more of the nalfurafine ester derivatives and a hydrophobic vehicle. The nalfurafine prodrugs described herein are highly lipophilic and can be formulated in a hydrophobic vehicle (e.g., sesame oil) as a depot intramuscular injection. Alternatively, the nalfurafine prodrugs described herein in sesame oil or similar vehicle are formulated as a gel-cap for oral administration. This formulation slowly releases the nalfurafine prodrug into the blood where it is hydrolyzed by plasma esterases to release the active compound nalfurafine. The formulation is designed to provide sustained release lasting for weeks after injection or oral administration.

[0030] Nalfurafine released from the nalfurafine prodrugs described herein by esterases acting on the prodrug binds to kappa opioid receptors in brain and peripheral tissues. Nalfurafine is a G-biased agonist meaning that it will produce analgesia and anti-pruritic effects typical of kappa opioid agonists without producing the dysphoria and aversive effects characteristic of un-biased kappa agonists (e.g., pentazocine, salvinorin A). Because nalfurafine is G-biased, it does not efficiently activate G-protein receptor kinase (GRK3) and p-arreslin-mediaied receptor desensitization. Instead, sustained activation of kappa opioid receptors by nalfurafine stimulates the eJun kinase / Peroxiredoxin 6 pathway to inactivate the kappa receptor. Kappa receptor inactivation by this mechanism results in long-lasting inhibition (>4 weeks). The amount of the nalfurafine prodrug present in the depot injection can be optimized during the initial human studies by determining the dose range useful to inactivate kappa receptors after 7 days of exposure. Oral administration is also expected to produce sustained elevation of nalfurafine blood levels but with a shorter duration of effect. The dose chosen will be low (non-saturating) because low levels of receptor activation are sufficient to cause slow accumulation of receptor inactivation). The optimized formulation slowly releases prodrug to achieve sufficient drug concentration useful to slowly inactivate kappa opioid receptors for sustained antagonism (lasting weeks) of dynorphin effects. Dynorphin is an endogenous stress hormone I neuropeptide that encodes the dysphoric, anxiogenic, and cognitive disrupting effects of stress.

[0031] In a further aspect, the present disclosure provides therapeutic uses of the nalfurafine ester derivatives (nalfurafine prodrugs), and their formulations described herein.

[0032] In certain embodiments, the disclosure provides a method for inactivating kappa opioid receptors mediating the dysphoric, anxiogenic, and proaddictive effects of the stress hormone / neuropeptide dynorphin transmitters, comprising administering a therapeutically effective amount of a nalfurafine ester derivative (nalfurafine prodrug), or a stereoisomer or pharmaceutically acceptable salt thereof. In the methods, inactivating kappa opioid receptors mediating the dysphoric, anxiogenic, and proaddictive effects of the stress hormone / neuropeptide dynorphin transmitters is a selective inactivation of kappa opioid receptors. Mu opioid receptors are not inactivated by nalfurafine prodrug treatment. Besides receptor specificity, the nalfurafine prodrugs are predicted to show tissue selectivity. Daily nalfurafine treatment inactivates the kappa receptors mediating the analgesic, dysphoric, cognitive disrupting and proaddictive effects of kappa agonists, but does not inactivate the kappa receptors medicating the anti-pruritic or diuretic effects of kappa agonists.

[0033] In certain embodiments of the method, the nalfurafine ester derivative, or a stereoisomer or pharmaceutically acceptable salt thereof, is administered in a hydrophobic vehicle.

[0034] In certain embodiments of the method, the nalfurafine ester derivative, or a stereoisomer or pharmaceutically acceptable salt thereof, is administered intramuscularly by injection.

[0035] In other embodiments of the method, the nalfurafine ester derivative, or a stereoisomer or pharmaceutically acceptable salt thereof, is administered orally. In certain of these methods, the nalfurafine ester derivative is administered as a capsule (e.g., gel cap).

[0036] The nalfurafine prodrugs described herein is a first-in-class medication able to selectively inactivate the kappa opioid receptors mediating the dysphoric, anxiogenic, and proaddictive effects of the stress hormone I neuropeptide dynorphin transmitters. Dynorphins are a group of structurally related neuropeptides synthesized in neurons and released when the person experiences stressful circumstances. Dynorphins are the endogenous neurotransmitters of the kappa opioid receptors. Kappa opioid receptors are in the same receptor family as mu opioid receptors which mediate the effects of morphine- like opioids, but unlike mu agonists kappa opioid drugs are dysphoric and non-addictive.

[0037] Preclinical studies using rats, mice, and nonhuman primates have shown that stress induces the release of dynorphins in brain to produce depression-like, anxiety-like, cognitive disruption (confusion), and proaddictive behaviors. In humans, the dynorphins are predicted to be responsible for the anhedonic component of depression, the anxiogenic components of stress-disorders, and some of the adverse cognitive effects of psychosis.

[0038] Stress increases drug seeking behaviors in animal models of addiction, and dynorphin has a clear role in the adverse effects evident during drug withdrawal and abstinence. This is evident in persons with a history of opioid use (heroin, fentanyl, and oxycodone) and is also prominent in persons in withdrawal from excessive alcohol use, nicotine dependence, and psychostimulant use (cocaine and methamphetamine). The nalfurafine derivatives (nalfurafine prodrugs) described herein would be used to promote abstinence in persons recovering from opioid, alcohol, nicotine, and psychostimulant use. Individuals in recovery from substance use experience profound dysphoria, anxiety, and drug-craving that trigger further drug-seeking.

[0039] In certain embodiments, the nalfurafine ester derivatives (nalfurafine prodrugs) described herein are administered in a depot formulation rather than by a daily oral route. The depot injection provides stable protection and better compliance as compared to daily oral dosing schedules.

[0040] The nalfurafine ester derivatives (nalfurafine prodrugs) described herein would be given in low, non-receptor-saturating doses (microdosing). This is in contrast to competitive kappa receptor antagonists that theoretically would need to be given at doses useful to saturate the kappa receptor in order to prevent dynorphin binding. High concentrations of a competitive antagonist that would be required for effective treatment increase the probability of off-target adverse effects.

[0041] The nalfurafine ester derivatives (nalfurafine prodrugs) described herein activate eJun kinase / PRDX6 mechanism of kappa receptor inactivation. That mechanism regulates the kappa receptors mediating the dysphoric and proaddictive effects of dynorphin. Other kappa opioid receptors are not regulated by the eJun kinase / PRDX6 mechanism, and the antipruritic and diuretic effects of dynorphins would not be affected by the nalfurafine derivatives (nalfurafine prodrugs) described herein and therefore are more selective than the competitive kappa antagonists because of this tissue selectivity.

[0042] The nalfurafine ester derivatives (nalfurafine prodrugs) described herein can be prepared by, for example, esterification of nalfurafine using the appropriate carboxylic acid or reactive carboxylic acid derivation. Suitable chemistries for achieving this esterification are known to those of skill in the art.

[0043] Pharmacokinetic Analysis

[0044] As described herein, the nalfurafine ester derivatives described herein are nalfurafine prodrugs that release nalfurafine in vivo.

[0045] The pharmacokinetics of a representative nalfurafine ester, nalfurafine decanoate, was evaluated and the results shown in FIG. 4. Referring to FIG. 4, nalfurafine serum levels (mg / mL) in mice over time (0.5-12 hr) after intraperitoneal injection with nalfurafine decanoate compared to nalfurafine-HCl (100 pg / kg). The nalfurafine-decanoate free-base was dissolved in reagent grade sesame oil before animal administration either at 50 pL intramuscularly (i.m.) or 100 pL oral gavage per mouse. Mice dosed orally were fasted for 12 hours.

[0046] Four male mice were injected with 50 pL (i.m.) nalfurafine decanoate (10 mg / mL in reagent grade sesame oil). Their nalfurafine serum levels were 8.4 + 3.7 ng / mL at 1-wk after injection; 1.5 + 1.9 ng / mL at 2-wk after injection; 0.4 +_0.6 ng / mL at 3-wk after injection; and 0.18 + 0.16 ng / mL at 4-wk after injection.

[0047] Dose-dependent Kappa Receptor Inactivation

[0048] The nalfurafine ester derivatives described herein exhibit dose-dependent Kappa Receptor inactivation in vivo.

[0049] The dose-dependent Kappa Receptor inactivation activity of a representative nalfurafine ester, nalfurafine decanoate, was evaluated using a tail withdrawal latency assay. Male and female C57BL / 6 mice were injected once with 50 uL nalfurafine decanoate (i.m.) at various doses. Mice were challenged at weekly intervals with the kappa opioid agonist U50,488h (10 mg / kg, i.p.) and the tail withdrawal latency was measured using the warm water (52.5°C) assay. Inhibition of U50 analgesia was evident at each dose and lasted over 4 weeks for both male and female mice.

[0050] The results are shown in FIGS. 5-7. FIG. 5 illustrates dose-dependent Kappa Receptor inactivation by nalfurafine decanoate and compares tail withdrawal latency for male mice after 7 days for various nalfurafine decanoate doses (5 pg, 500 ng, 50 ng, 5 ng, and 500 pg per mouse with vehicle control). FIG. 6 compares tail withdrawal latency for Group 2 mice after 7, 14, 21, and 27 days for various nalfurafine decanoate doses (5 pg, 500 ng, 50 ng, 5 ng, and 500 pg per mouse with vehicle control). FIG. 7 compares tail withdrawal latency for female mice after 7, 14, and 21 days for various nalfurafine decanoate doses (5 pg, 500 ng, 50 ng, 5 ng, and 500 pg per mouse with vehicle control).

[0051] Nalfurafine Blocks Stress-Paired Cue Aversion

[0052] The nalfurafine ester derivatives described herein are nalfurafine prodrugs that release nalfurafine in vivo. Repeated administration of nalfurafine was found to block stress-paired cue aversion.

[0053] An additional therapeutic utility of KOR antagonists is the potential to promote stress resilience which would be advantageous for the treatment of disorders such as depression or opioid withdrawal syndrome. To determine if administering (microdosing) nalfurafine would block dynorphin-induced stress responses, two different assays of dysphoria were used. In mice, dysphoria is typically measured as aversion conditioned to a cue associated with the stressful experience.

[0054] In a first assay, mice were subjected to a repeated forced swim paradigm previously shown to release dynorphin in the presence of a neutral odorant cue (almond scent). Following conditioning, mice were introduced to a 3-chamber place preference apparatus with the almond scent present in only one compartment (FIG. 8D). Control mice exposed to the almond scent in the absence of the stress-pairing showed no aversion to the almond scent-containing compartment. In contrast, mice experiencing stress-pairing show robust odorant- aversion (FIGS. 8A-8C). Pretreatment of the mice for 7 days with norBNI (either 330 pg / kg or 1 mg / kg) (FIG. 8A), nalfurafine (1 g / kg or 10 pg / kg) (FIG. 8B), or nalmefene (100 pg / kg or 1 mg / kg) (FIG. 8C) to inactivate KOR, blocked odorant aversion. The drug potencies in the swim-odorant assay were comparable to the tail-flick analgesia assay. In previous work, mice that were injected with cocaine in the presence of the almond scent developed robust place preference for the almond-scent paired compartment. NorBNI (10 mg / kg) treatment before cocaine conditioning did not block the development of almond- scent preference.

[0055] In a second stress assay, almond scent was paired with naloxone precipitated withdrawal in opioid-dependent mice (FIGS. 8D and 8E). Acute opioid abstinence causes dynorphin release and includes a profound dysphoric response. Mice were implanted with osmotic mini-pumps containing fentanyl (2 mg / kg / day) to produce opioid dependence. After 7 days, the pumps were removed and 2 days later mice were injected twice with 1 mg / kg naloxone in the presence of the almond scent, once in the morning and once in the evening to precipitate withdrawal. Fentanyl-dependent mice developed a robust aversion to the odorant. In contrast, control mice with saline-filled minipumps did not develop aversion when injected with naloxone in the presence of almond scent. Fentanyl-dependent mice that were pretreated for 7 days with norBNI (1 mg / kg) showed significantly reduced odorant aversion when challenged with naloxone 2-days after pump removal (acute withdrawal). Mice pretreated for 7 days with nalfurafine (5 p g / kg) or nalmefene (5 mg / kg) showed significantly reduced aversion but the decrease was incomplete. Mice that were pretreated for 7 days with norBNI (1 mg / kg), nalfurafine (5 g / kg) , or nalmefene (5 mg / kg) and experienced naloxone-precipitated withdrawal 10 days after pump removal (protracted withdrawal) showed no aversion to the odorant. Naloxone precipitation is not identical to spontaneous withdrawal from opioids - it is more intense and acute; however, the symptoms are qualitatively similar. Although we are ultimately interested in determining the role of dynorphin in humans experiencing opioid abstinence, the conditioning paradigm in mice works better if the cue and the stimulus are discrete and temporally associated. The 1 mg / kg dose of naloxone chosen blocks mu-opioid receptors but is insufficient to block KOR. These results from both stress assays suggest that repeated low-dose treatment with nalfurafine promotes stress resilience and supports their potential therapeutic utilities in preventing relapse during protracted opioid abstinence.

[0056] Materials and methods

[0057] The Preparation and Characterization of a Representative Nalfurafine Ester: Nalfurafine Decanoate

[0058] The preparation of nalfurafine decanoate was adapted from the procedure described in Journal of Controlled Release 232 (2016), 196-202.

[0059] Decanoyl chloride (92.5 mg, 0.485 mmol) was added dropwise at ambient temperature to a stirred mixture of nalfurafine hydrochloride (0.249 g, 0.485 mmol), toluene (7.4 ml) and triethylamine (0.15 ml, 1.07 mmol) under nitrogen atmosphere. Stirring at ambient temperature was continued for 16 hours. The reaction mixture was diluted with ethyl acetate (EtOAc) and washed with water then brine, dried (MgSC ), then concentrated. The resulting yellow oil (240 mg) was analysed by HPLC and LCMS, revealing a late eluting diester impurity (di-decanoyl). The oil was subjected to chromatography on basic alumina, loading and eluting with methylene chloride (DCM) then EtOAc to afford the desired product as a gum which foamed when evaporated from DCM / hexane under vacuum (195 mg, 64% yield).

[0060] :H NMR (400 MHz, methanol-d4): 57.69 - 7.86 (m, 1H), 7.36 - 7.58 (m, 2H), 6.93 (d, J = 8.25 Hz, 1H), 6.71 - 6.86 (m, 2H), 6.67 (s, 1H), 6.49 (d, J = 15.13 Hz, 1H), 4.72 (d, J= 7.64 Hz, 1H), 3.79 (br. s., 1H), 3.08 - 3.25 (m, 3H), 3.00 (s, 3H), 2.62 - 2.82 (m, 2H), 2.38 - 2.54 (m, 2H), 2.21 - 2.37 (m, 3H), 2.07 - 2.20 (m, 1H), 1.60 - 1.77 (m, 2H), 1.41 - 1.59 (m, 4H), 1.14 - 1.40 (m, 12H), 0.78 - 1.00 (m, 4H), 0.55 (d, 7 = 7.49 Hz, 2H), 0.17 (br s, 2H).

[0061] 13C NMR (100 MHz, methoanol-d4): 5 172.9, 169.9, 147.6, 145.9, 145.5, 134.8, 134.0, 133.9, 133.0, 124.8, 123.7, 120.9, 118.6, 109.1, 71.5, 63.5, 60.3, 45.1, 34.8, 33.2, 31.8, 31.6, 30.7, 30.5, 30.4, 30.2, 26.1, 24.0, 23.9, 14.6, 10.4, 4.7, 4.4.

[0062] High Performance Liquid Chromatography (HPLC) / Mass Spectrometry (MS)

[0063] HPLC / MS was used to detect and quantitate free nalfurafine in mouse sera.

[0064] Sample Preparation: 50 pL (45 pL sera + 5 pL naloxone (100 ng / ml in methanol (MeOH) as internal standard. Add 400 pL MeOH; vortex 5 min; centrifuge 5 min; collect supernatant and dry under nitrogen at room temperature; reconstitute in 50 pL H2O / MeOH 1:1; inject 20 pL on Waters Xevo-TQXS HPLC-Mass Spectrometer.

[0065] Analytical conditions: Waters BEH C18 1.7 um 2.1*50mm HPLC; mobile phase A: 0.1% formic acid in H2O; mobile phase B: 0.1% formic acid in MeOH; Needle wash: ACN / MeOH 1:1; flow rate: 0.3 mL / min room temperature. Injection volume: 20 pL. Naloxone: MRM transitions: 328.29 / 212.28, 328.29 / 310.33 Retention time: 2.3 min. Nalfurafine: MRM transitions: 477.25 / 121.10, 477.25 / 459.20; Retention time: 3.2 min.

[0066] Drugs

[0067] Nalfurafine (NIDA Drug Supply Program), nalmefene (Tocris), norBNI (NIDA Drug Supply Program), U50,488 (Tocris), and naloxone (Tocris) were dissolved in saline and administered intraperitoneally (i.p.).

[0068] Animals

[0069] Adult C57BL / 6 wild-type (WT) or KOR-cre male and female were used (The lackson Laboratory). KOR-cre mice were ear punched at least 21 days after birth and genotyped using Transnetyx genotyping services. Surgeries performed on KOR-cre mice occurred between 5 and 7 weeks of age.

[0070] Odorant-paired repeated forced swim stress Mice were treated with saline, norBNI, nalfurafine, or nalmefene daily for 7 days. To allow drug clearance and focus on the long-lasting KOR inactivation, 7 days after their last treatment mice were exposed to one 15 -minute swim on day 1 and four 6-minute swims on day 2, in 30°C water, without opportunity for escape. This repeated swim stress protocol was previously demonstrated to evoke dynorphin release. Odorant-swim stress pairing was conditioned as previously described (B. B. Land, M. R. Bruchas, J. C. Lemos, M. Xu, E. J. Melief, C. Chavkin, The dysphoric component of stress is encoded by activation of the dynorphin kappa-opioid system. J. Neurosci. Off. J. Soc. Neurosci. 28, 407-414 (2008)). N = 6-16 per treatment group.

[0071] Odorant-paired precipitated fentanyl withdrawal

[0072] Mice were treated with saline, norBNI, nalfurafine, or nalmefene daily for 7 days. On day 4 of the 7-day treatment, osmotic pumps (Alzet Model 1007D) filled with either saline or fentanyl (2 mg / kg / day) were implanted under the skin between the scapula to induce opioid dependence. The pumps were removed after 7 days and mice were allowed to recover for 2 days. On either day 3 (acute withdrawal) or day 10 (protracted withdrawal) post-pump removal, they were given injections of 1 mg / kg naloxone in the presence of a Nestlet containing 20 pL of imitation almond extract once in the morning and once in the afternoon. Odorant-conditioned aversion was assessed in the 3 -compartment place preference apparatus 7 days after naloxone-odorant pairing.

[0073] Odorant- aversion test

[0074] Mice underwent two types of odorant-conditioned pairing: stress-odorant pairing in which almond scent was paired with repeated forced swim stress and withdrawal-odorant pairing in which almond scent was paired with two instances of naloxone-precipitated withdrawal (outlined above). Mice were pre-exposed to a 3 -chamber Plexiglas box for 3 min before odorant conditioning. For the odorant-aversion test, mice were placed in a Plexiglas 3 -chamber box with a quarter Nestlet containing 20 pL of almond extract placed on one far side of the chamber, and a quarter Nestlet with no scent placed on the far side of the other chamber. The session in the 3 -chamber box was video recorded for 14 min and analyzed using Ethovision for the time spent in each zone. The odorant aversion was calculated by the time spent in the odor-paired chamber minus the time spent in the opposite chamber. N = 8-15 per treatment group. While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the disclosure.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A compound of formula (I) :or a stereoisomer or pharmaceutically acceptable salt thereof, wherein R is C(=O)R!, where R1is C -C 1 1 alkyl.

2. The compound of Claim 1, wherein R1is C7 alkyl.

3. The compound of Claim 1, wherein R1is C9 alkyl.

4. The compound of Claim 1, wherein R1is Cll alkyl.

5. A formulation comprising the compound of Claim 1, or a stereoisomer or pharmaceutically acceptable salt thereof, and a hydrophobic vehicle.

6. The formulation of Claim 5, wherein the hydrophobic vehicle is a vegetable oil.

7. A method for inactivating kappa opioid receptors mediating the dysphoric, anxiogenic, and proaddictive effects of the stress hormone / neuropeptide dynorphin transmitters, comprising administering a therapeutically effective amount of a compound of any one of Claims 1-4, or a stereoisomer or pharmaceutically acceptable salt thereof.

8. The method of Claim 7, wherein inactivating kappa opioid receptors mediating the dysphoric, anxiogenic, and proaddictive effects of the stress hormone I neuropeptide dynorphin transmitters is a selective inactivation of kappa opioid receptors.

9. The method of Claims 7 or 8, wherein the compound, or a stereoisomer or pharmaceutically acceptable salt thereof, is administered in a hydrophobic vehicle.

10. The method of any one of Claims 7-9, wherein the compound, or a stereoisomer or pharmaceutically acceptable salt thereof, is administered intramuscularly by injection.

11. The method of any one of Claims 7-9, wherein the compound, or a stereoisomer or pharmaceutically acceptable salt thereof, is administered orally.

12. The method of Claim 11, wherein the compound, or a stereoisomer or pharmaceutically acceptable salt thereof, is administered as a capsule.

13. The use of a compound of any one of Claims 1-4, or a stereoisomer or pharmaceutically acceptable salt thereof, for inactivating kappa opioid receptors mediating the dysphoric, anxiogenic, and proaddictive effects of the stress hormone / neuropeptide dynorphin transmitters.

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