Nalfurafine and nalmefene as kappa opioid receptor inactivating therapeutic agents

Abstract

The present disclosure provides a method for using nalfurafine or nalmefene for activating JNK / PRDX6 to stimulate localized peroxide production that causes G-protein depalmitoylation and deactivation of kappa opioid receptors (KORs) in a subject; a method for using nalfurafine or nalmefene for inactivating kappa opioid receptors responsible for dysphoria during mu-opioid withdrawal in a subject; a method for using nalfurafine or nalmefene for promoting stress resilience in a subject recovering from polysubstance withdrawal; a method for using nalfurafine or nalmefene for inactivating kappa opioid receptor responsible for the dysphoria and psychotomimetic effects of kappa opioid agonists administered to a subject for the treatment of pruritus or for the promotion of diuresis; and a method for using nalfurafine or nalmefene in combination with a kappa opioid agonist for the treatment of neuropathic pain in a subject.

Description

NALFURAFINE AND NALMEFENE AS KAPPA OPIOID RECEPTOR INACTIVATING THERAPEUTIC AGENTSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 718385, filed November 8, 2024, expressly incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with Government support under Grant Nos. P30 DA048736 and R56DA030074 awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention.BACKGROUND

[0003] The endogenous dynorphin / kappa opioid receptor (KOR) system in the brain mediates the dysphoric effects of stress, and KOR antagonists may have therapeutic potential for the treatment of drug addiction, depression, and psychosis. One class of KOR antagonists, the long-acting norBNI-like antagonists, have been suggested to act by causing KOR inactivation through a cJun-kinase mechanism rather than by competitive inhibition.

[0004] An essential role for endogenous dynorphin opioids in regulating mood and addiction risk has been suggested by multiple preclinical studies during the last two decades. Pharmacological and genetic disruption of dynorphin actions in the rodent brain block depression-like and anxiety-like behaviors. Stress-induced release of endogenous dynorphins potentiates the rewarding effects of addictive drugs (e.g., cocaine, ethanol, nicotine, and heroin). Stress-induced reinstatement of drug self-administration is blocked by kappa opioid receptor (KOR) antagonists, and KOR antagonists block the dysphoric effects evident during opioid, nicotine, and ethanol abstinence. Kappa agonists have psychotomimetic effects, and the stress-induced release of dynorphins disrupts prefrontal- cortical circuits responsible for cognition. Stress exposure has been shown to increase the risk of mood and substance use disorders in humans, and these preclinical studies strongly suggest that KOR antagonists could be therapeutically useful in promoting stress resilience in vulnerable people. However, selective, safe, and effective KOR antagonists for human studies are still being developed, and the role of dynorphin in human pathophysiology is not yet established.

[0005] The majority of the preclinical studies examining the dyn / KOR system have used the selective KOR antagonists norbinaltorphimine (norBNl) or JDTic. Although these drugs are competitive KOR antagonists in vitro, they both have the unusual properties of delayed onset of action and very long durations of effect when given in vivo. For example, a single injection of norBNl in rodents or nonhuman primates can block KOR activation for weeks. The mechanism for this long duration was initially unclear and attributed to a very slow clearance of the drug from the brain (a pharmacokinetic mechanism). However, a detailed analysis of the molecular pharmacology revealed that both norBNl and JDTic are not conventional antagonists; rather they are functionally selective KOR agonists that activate eJun N-terminal Kinase (JNK), which recruits the phospholipase peroxiredoxin 6 (PRDX6) to the plasma membrane; PRDX6 bound to KOR stimulates NADPH oxidase to locally generate reactive oxygen (ROS) which oxidizes the sulfhydryl in exposed cysteine residues and depalmitoylates the G-protein, Gai. The depalmitoylated Gai binds more tightly to KOR and prevents receptor activation. Pharmacological inhibition of the JNK or PRDX6 components in this signaling pathway or genetic deletion of JNK- 1 blocked the long duration of norBNl action. In this context, the agonist and antagonist labels are misleading, and norBNI-like ligands are better described as KOR-inactivating agents.

[0006] This series of molecular characterization studies helped establish that there are four different types of functionally selective KOR ligands (FIGS. 1A-1D). These include unbiased high-efficacy agonists, competitive antagonists, and long-acting KOR- inactivating agents. A fourth group of functionally selective KOR ligands includes nalfurafine, a highly efficacious but G-biased KOR agonist, and nalmefene, a low-efficacy KOR partial agonist (FIG. ID).

[0007] Despite the advance in the development of KOR ligands noted above, a need exists for new ligands as medications to block KOR and promote stress resilience for the adjunctive treatment of mood disorders, psychosis, and drug addiction. Specifically, although competitive KOR antagonists have been developed, KOR-inactivating antagonists have not yet advanced to human use. KOR-inactivators may have substantial safety and efficacy advantages over competitive KOR antagonists. The present disclosure seeks to fulfill this need and provides further related advantages.SUMMARY

[0008] In one aspect, the disclosure provides a method for selectively inactivating kappa opioid receptors in a subject by activating JNK / PRDX6 to stimulate localized peroxide production that causes G-protein depalmitoylation and deactivation of kappa opioid receptors, the method comprising administering to a subject in need thereof an amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, effective to activate JNK / PRDX6 in the subject.

[0009] In another aspect, the disclosure provides a method for inactivating kappa opioid receptors responsible for dysphoria, anhedonia, and drug craving during mu-opioid withdrawal in a subject, the method comprising administering a therapeutically effective amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, to the subject.

[0010] In a further aspect, the disclosure provides a method for promoting stress resilience in a subject recovering from polysubstance withdrawal, the method comprising administering a therapeutically effective amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, to the subject.

[0011] In another aspect, the disclosure provides a method for selectively treating pruritus in a subject without activating KORs responsible for dysphoric or psychotomimetic effects, comprising administering to a subject in need thereof a therapeutically effective amount of a selective and highly efficacious kappa opioid agonist in combination with a therapeutically masking amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, to effect pruritus treatment.

[0012] In a related aspect, the disclosure provides a method for selectively promoting diuresis in a subject without activating KORs responsible for dysphoric or psychotomimetic effects, comprising administering to a subject in need thereof a therapeutically effective amount of a selective and highly efficacious kappa opioid agonist in combination with a therapeutically masking amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, to promote diuresis.

[0013] In a further related aspect, the disclosure provides a method for the selective treatment of neuropathic pain in a subject without activating KORs responsiblefor dysphoric or psychotomimetic effects, comprising administering to a subject in need thereof a therapeutically effective amount of a selective and highly efficacious kappa opioid agonist and a therapeutically masking amount of nalfurafine or pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof.DESCRIPTION OF THE DRAWINGS

[0014] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.

[0015] FIGS. 1A-1D are schematic illustrations of major pathways in the functionally selective signaling cascade of the kappa opioid receptor. FIG. 1A illustrates highly efficacious agonists including U50,488 and the endogenous dynorphin peptides activate both heterotrimeric G-proteins and [3-arrestin-dependent signaling cascades. G- protein activation releases GPy that activates inwardly rectifying potassium channels and inhibits voltage-gated calcium channels to reduce neuronal excitability and presynaptically inhibit neurotransmitter release. The dysphoric effects of efficacious KOR agonists occur through GRK3 / p-arrestin activation of p38a MAPK in serotonergic neurons in the dorsal raphe nucleus and dopaminergic neurons in the ventral tegmental area. FIG. IB illustrates competitive antagonists, including naloxone binding to the orthosteric site of the receptor and sterically inhibit agonist binding. FIG. 1C illustrates long-lasting, functional antagonists including norBNI proposed to be functionally selective activators of a signaling cascade that leads to the phosphorylation of c-Jun N-temiinal kinase (JNK), recruitment of PRDX6, increased activity of NADPH oxidase, and generation of reactive oxygen species (ROS). Oxidation is proposed to depalmitoylate Gai and shift the orientation of the receptor«G-protein binding interface to irreversibly block guanine nucleotide exchange. Because activated p38a MAPK inhibits JNK, highly efficacious KOR do not cause long- lasting KOR inactivation. FIG. ID illustrates biased and partial agonists causing conformational changes that lead to the G-protein pathway being preferentially activated (nalfurafine) or the GRK3 / P-arrestin pathway being activated weakly (nalmefene) and not causing aversion.

[0016] FIGS. 2A-2I illustrate that nalfurafine and nalmefene cause reactive oxygen species (ROS) generation in a KOR-specific manner. FIG. 2 A illustrates a proposed pathway for nalfurafine and nalmefene leading to the generation of ROS. FIG.2B shows a confocal microscopic image of a horizontal VTA slice showing merged channels of the GFP sensor and the mCherry tag (left image); and a representative 2-photon image of the same live slice at higher magnification (right image). FIG. 2C compares representative images (contrast-enhanced) using 2-photon microscopy of KOR-positive cell bodies in the VTA with oROS expression under buffer-only condition (Baseline), after 100 nM nalfurafine washed on for 20 min (Nalfurafine), followed by H2O2 for positive control (H2O2). FIG. 2D compares AF / F values for 50 min imaging sessions. Wash on of 100 nM nalfurafine (Naif, Naif + NLx, Naif + MJ33, Naif + JNK-IN-8) (V = 10) and 10 mM nalmefene (Naim, Naim + NLx, Naim + MJ33, Naim + JNK-IN-8) (N - 7) both show clear increases in oROS fluorescence that can be blocked by naloxone (Nix) (A = 3), the peroxiredoxin inhibitor MJ33 (N = 4), or the eJun Kinase inhibitor JNK-IN-8 (N = 4). The addition of H2O2 at 40 min confirmed that the sensor was active at the end of the time course. FIG. 2E compares areas under the curve (AUC) quantification for nalfurafine (Naif, Naif + NLx, Naif + MJ33, Naif + JNK-IN-8) (left panel) and nalmefene (Naim, Naim + NLx, Naim + MJ33, Naim + JNK-IN-8) (right panel) of time course data from 0- 40 min. FIG. 2F is a schematic illustration of the fiber photometry setup used in the AUC quantitation in FIG. 2G. FIG. 2G compares AUC quantification of fiber photometry time course for 40 min after injection of various doses of nalfurafine (A = 4-13) (left panel) and nalmefene (N = 3-14) (right panel), which dose-dependently increased oROS fluorescence. FIG. 2H compares AUC quantification of time course data after injection of 100 ug / kg nalfurafine in female mice measured at various points in the estrus cycle (A = 7-16) (left panel). Female mice show no response to nalfurafine at the high estrogen stage (proestrus); and AUC 0-40 min of female mice pretreated with estradiol or progesterone compared to the cycle collapsed into high estrogen stage (proestrus) and low estrogen stages (estrus- diestrus) (A = 9-16) (right panel). Pretreatment with estradiol mimics the proestrus stage, and pretreatment with progesterone keeps the animals in the low estrus state, mimicking the low estrogen stages. FIG. 21 compares representative images of histology from vaginal lavage to determine the estrous cycle stage (proestrus, estrus, metestrus, diestrus). Statistical comparisons are shown * p <0.05, ** p <0.01, *** p <0.001, **** p < 0.0001.

[0017] FIGS. 3A-3H compare tail withdrawal latency (a measure of nociceptive response) shown as a percent of the baseline of the normal increase elicited by U50.488 administration (U50,488 analgesia as % Ctrl). FIGS. 3A-3C compare U50,488 analgesia for 7 days post-last treatment norBNI (100 pg / kg - 1 mg / kg, A = 4-20), nalfurafine (10ng / kg - 100 pg / kg, N = 7-24), and nalmefene (1 pg / kg - 10 mg / kg, N = 8-24), respectively. All 3 treatments dose-dependently block U50,488-induced analgesia. FIGS. 3D-3F compare U50,488 analgesia for the same dosing regimen as in FIGS. 3A-3C. Treatments of norBNI (1 mg / kg, N = 8), nalfurafine (1 mg / kg, N= 11), and nalmefene (10 mg / kg, N = 14) were given once daily for 7 days. Time points are shown as days after the last administration of treatment measured out to 28 days. Repeated treatment with norBNI, nalfurafine, and nalmefene blocks U50,488-induced latency increase. Pretreating with MJ33 1 hour before each daily dose caused KOR function to recover more rapidly. FIGS. 3G and 3H compare 3 and 7 days post-last treatment, respectively, of 7 daily doses of Saline (A = 16), norBNI (1 mg / kg, N = 8), nalfurafine (1 mg / kg, N = 11), nalmefene (10 mg / kg, N = 14), naloxone (10 mg / kg, N = 7), and naltrexone (10 mg / kg, N = 8). Statistical comparisons are shown * p <0.05, ** p <0.01, *** p <0.001, **** p < 0.0001.FIGS. 4A-4G illustrate that repeated administration of nalfurafine and nalmefene block stress-paired cue aversion. FIGS. 4A-4C compare odorant scores 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 (N = 10) had neutral scores while stressed animals pretreated with saline (N = 13) showed an aversion. 7 daily injections of norBNI (330 pg / kg (N = 7) or 1 mg / kg (N = 11)) (FIG. 4A), nalfurafine (1 pg / kg (N = 11) or 10 pg / kg (N = 6)) (FIG. 4B), and nalmefene (1 mg / kg (N = 12)) (FIG. 4C) blocked stress-odor paired aversion. FIG. 4D is a schematic illustration of almond pairing with either repeated force swim-stress or naloxone -induced aversion, followed by 3-chamber aversion assay with the scent on one side. FIG. 4E is a timeline illustrating differences between acute and protracted withdrawal groups in the naloxone-induced aversion odorant pairing and testing. FIG. 4F shows that mice that underwent naloxone- induced aversion during the acute withdrawal phase (2 days after pump removal) and were treated daily for 7 days with saline (N = 10) showed a strong aversion to the almond scent which was significantly attenuated in groups receiving daily injections of norBNI (N = 8), nalfurafine (N = 10), and nalmefene (N = 10). FIG. 4G shows mice that underwent naloxone -induced aversion during the protracted withdrawal phase (10 days after pump removal) and were treated daily for 7 days with saline (N = 10) showed a strong aversion to the almond scent which was fully blocked in groups receiving daily injections of norBNI (N = 10), nalfurafine (N = 10), and nalmefene (N = 15). Statistical comparisons are shown * p <0.05, ** p <0.01.FIGS. 5A-5F illustrates that KOR inactivation demonstrates tissue specificity. FIG. 5A compares serum prolactin dose-response curves 60 min post administration of nalfurafine (N = 4-12), nalmefene (N = 4-8), or U50,488 (N = 4-11). Nalfurafine has greater potency than U50,488, while nalmefene has similar potency but reduced efficacy, highlighting its partial agonism. FIG. 5B is a schematic illustration of KOR activation by nalfurafine or nalmefene showing acutely increasing prolactin, whereas repeated doses cause inactivation of the receptor. FIGS. 5C and 5D compare repeated doses of nalfurafine (N = 4-11) (FIG. 5C) and nalmefene (N = 4-11) (FIG. 5D) blocked the nalfurafine -induced increase in serum prolactin 7 days after last treatment at various doses. FIG. 5E shows that nalfurafine (100 pg / kg) increases diuresis, which can be blocked acutely, but not longterm, by norBNI (10 mg / kg) (N = 10-19). FIG. 5F shows that GNTI (30 ug / kg) induces pruritus, which can be blocked by nalfurafine (50 pg / kg). Daily doses of nalfurafine (7x 50 ug / kg, red bars) do not prevent this effect (N = 7). Statistical comparisons are shown * p <0.05, ** p <0.01, *** p <0.001, **** p < 0.0001.

[0018] FIGS. 6A and 6B illustrate the enhanced sensitivity and kinetics of an oROS sensor. FIG. 6A is a schematic illustration of the crystal structure of reduced and oxidized forms of Regulatory Domain (RD) of Escherichia coli OxyR. C-C pair, fluorescent protein insertion loop, and the newly identified fluorescent protein insertion site for oROS sensors are shown. FIG. 6B shows representative reduction kinetics of oROS-G and HyPer7 after 100 pM H2O2 stimulation followed by media wash (n>100 cells per sensor). HEK293 expressing oROS-G were stimulated with 100 pM H2O2 followed by media wash.

[0019] FIG. 7A-7C illustrate that KOR inactivating agents demonstrate shortacting antagonism in specific tissues. Nalfurafine was given to mice each day for 6 days at various doses with pretreatment of either saline or low-dose norBNI (1 mg / kg) for 7 days and 7 days of recovery (FIG. 7A). No differences were observed between saline and norBNI repeated dose pretreatment. Doses of nalfurafine were given daily, and tolerance to its diuretic action was not observed. Mice treated daily with 100 mg / kg nalfurafine (n=4) showed no tolerance to the diuresis effect of nalfurafine (FIG. 7B). Saline (n=3) had no effect on diuresis. GNTI-induced itch data from FIG 4F displayed in 5 min bins (FIG. 7C).

[0020] FIGS. 8 A and 8B illustrate that KOR-inactivating agents do not increase pain sensitivity. FIG. 8A compares baseline tail withdrawal latencies before administration of U50,488. norBNI increased baseline latency, while nalfurafine and nalmefene did not.FIG. 8B compares jumping behavior during hot plate test 1-2 hours after pump removal. Mice in spontaneous withdrawal from fentanyl show increased jumping behavior. Treatment with KOR-inactivating agents does not significantly change hyperalgesia.DETAILED DESCRIPTIONThe present disclosure provides the use of nalfurafine or nalmefene for activating JNK / PRDX6 to stimulate localized peroxide production necessary for G-protein depalmitoylation and deactivation of kappa opioid receptors (KORs) in a subject; the use of nalfurafine or nalmefene for inactivating kappa opioid receptors responsible for dysphoria during mu-opioid withdrawal in a subject; the use of nalfurafine or nalmefene for promoting stress resilience in a subject recovering from polysubstance withdrawal; the use of nalfurafine or nalmefene for inactivating kappa opioid receptor responsible for the dysphoria and psychotomimetic effects of kappa opioid agonists administered to a subject for the treatment of pruritus or for the promotion of diuresis; and the use of nalfurafine or nalmefene in combination with a kappa opioid agonist for the treatment of neuropathic pain in a subject.

[0021] Nalfurafine is a drug that acts as a highly selective kappa-opioid receptor (KOR) agonist, used to treat pruritus (itching) in patients with renal failure and chronic liver diseases who are undergoing dialysis or have conditions like Primary Biliary Cholangitis (PBC). The chemical structure of nalfurafine is shown below.

[0022]

[0023] Nalmefene is an opioid antagonist medication used to reverse opioid overdose and to treat alcohol dependence by blocking opioid receptors in the brain, whichcounters the effects of both natural and synthetic opioids. The chemical structure of nalmefene is shown below.

[0024] In the methods described herein, nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, are administered to a subject in need thereof. As used herein, the term “pharmaceutically acceptable salt” refers to a salt of nalfurafine or nalmefene acceptable for pharmaceutical administration. Suitable pharmaceutically acceptable salts are known in the art. Nalfurafine and nalmefene salts can be formed by the neutralization reaction of an acid and a base. For nalfurafine’ s or nalmefene’ s phenolic functionality, salts can be derived from a variety of organic and inorganic counter ions well known in the art and include sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium; and for nalfurafine’ s or nalmefene’ s basic functionality (amine), salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate.

[0025] Kappa opioid receptor inactivating agents are a newly described class of antagonists whose novel mechanism of action provides a new approach for medication development. No medication of this class has been developed for therapeutic use.

[0026] Kappa receptors in brain are activated by the dynorphin neuropeptides and mediate many of the pathophysiological effects of stress. Novel therapeutics able to effectively block the effects of dynorphin would promote stress-resilience and have not yet been approved for therapeutic use.

[0027] Kappa receptors can be blocked by both conventional competitive antagonists and by receptor inactivating agents. Receptor inactivation has several potential advantages highlighted in this application.

[0028] A recently completed drug screen identified two medications that are selective Kappa opioid receptor (KOR) inactivating agents: nalfurafine and nalmefene. They have not previously been identified as KOR inactivating agents.

[0029] Functionally selective G-biased KOR agonist nalfurafine and the low- efficacy KOR partial agonist nalmefene activate JNK / PRDX6 to stimulate peroxide production necessary for G-protein depalmitoylation. Because KOR inactivation caused by JNK / PRDX6 recovers slowly, daily treatment with submaximal doses of nalfurafine or nalmefene caused accumulating inactivation of KORs responsible for stress-induced aversion and dysphoria during mu-opioid withdrawal. The dysphoria evident during protracted abstinence from nicotine, ethanol, and psychostimulants is similarly inhibited by KOR inactivation and is predicted to be blocked by nalfurafine or nalmefene treatment in dependent persons.

[0030] Theoretically, KOR-inactivation by nalfurafine or nalmefene confers several potential advantages as medications. First, these two medications have histories of safety and tolerability in human use. Nalfurafine has been approved in Japan as an antipruritic in uremic itch. Nalmefene is used as an alternative to naloxone and naltrexone in the treatment of alcohol use disorder and to reverse respiratory depression during acute opioid overdose. In C57BL6 mice, doses of 50 pg / kg (i.p.) are needed to block scratching caused by the irritant GNTI. Other strains of mice and non-human primates also show dose-dependent antipruritic responses. Inactivation of KOR responsible for stress-induced aversion is evident at 1 pg / kg daily dosing for 7 days, and still lower doses given for longer are likely to be effective. Dosing at 2.5-5 pg per day to treat pruritus in humans produces only mild adverse reactions reducing the dose by 10 to 20-fold is predicted to cause negligible acute adverse effects. Slow accumulation of inactivation would allow the titration of effect, and slow recovery would allow stable antagonism.

[0031] In contrast, competitive antagonists would need to be taken at supersaturating doses to prevent dynorphin activation of KOR. High doses increase the probability of off-target effects and toxicity. The competitive KOR antagonist Aticaprant developed by Eli Lilly (LY-2456302) was recently evaluated by Janssen Pharmaceuticals in a Phase 2 clinical trial in humans with treatment-resistant anhedonia. Anhedonia was significantly reduced by treatment with 10 mg / day of Aticaprant in conjunction with SSRI / SNRI treatment, but the anhedonia measured by the Snaith-Hamilton Pleasure Scale (SHAPS) rating scale was reduced from a score of 41 to a score of 34 which was not significantly different from placebo and may not be a clinically meaningful effect. There are many reasons why the treatment may have only produced a modest effect, but one concern is that the dose used only occupies about 70% of the receptors after 24 hours.Higher drug doses have known off-target effects and because dynorphins have very high efficacies, they may need only occupy 10% of the receptors to produce a maximal effect.

[0032] The results described herein highlight the potential of leveraging functional selectivity. With the identification and characterization of four signaling pathways at KOR, we can expand past the more traditional idea of what a partial agonist is and continue to use these mechanisms to our advantage with drugs that are already available and shown to be safe and well tolerated in humans. Tn the case of nalfurafine and nalmefene in combination with repeated low-dosing regimens, KOR blockade can build slowly to promote stress-resilience.

[0033] In one aspect, the disclosure provides a method for selectively inactivating kappa opioid receptors in a subject by activating JNK / PRDX6 to stimulate localized peroxide production that causes G-protein depalmitoylation and deactivation of kappa opioid receptors. In the method, an amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, effective to activate JNK / PRDX6 in the subject is administered. The method achieves deactivation of kappa opioid receptors via localized peroxide production necessary for G-protein depalmitolylation as a result of JNK / PRDX6 activation.

[0034] As used herein, in the context of this method, the term “subject in need thereof’ refers to persons who are stress-sensitive because of hyperactivity of their dynorphin systems. Examples include persons with anxiety disorders, clinical depression involving anhedonia, persons experiencing anhedonia during opioid abstinence, alcohol use disorder, nicotine use, psychostimulant use, and polysubstance use disorders. In addition, persons experiencing hallucinations during psychosis would benefit from the method.

[0035] In another aspect, the disclosure provides a method for inactivating kappa opioid receptors responsible for dysphoria (and anhedonia and drug craving) during mu- opioid withdrawal in a subject. In the method, a therapeutically effective amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered to the subject. The method achieves reduction of dysphoria (and anhedonia and drug craving) during mu-opioid withdrawal.

[0036] As used herein in the context of this method, the term “subject” refers to a person experiencing mu-opioid withdrawal or withdrawal from the other drags of abuse.

[0037] In a related aspect, the disclosure provides a method for promoting stress resilience in a subject recovering from polysubstance withdrawal. In the method, a therapeutically effective amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered to the subject.

[0038] It will be appreciated that in addition to inactivating kappa opioid receptors responsible for dysphoria during mu-opioid withdrawal, the method is effective for inactivating kappa opioid receptors responsible for dysphoria during withdrawal from polysubstances, for example, a subject withdrawing from use of opiates and psychostimulants, or a subject withdrawing from nicotine and alcohol (e.g., a subject simultaneously using opiates and psychostimulants, or a subject using nicotine and alcohol).

[0039] As used herein in the context of this method, the term “subject” refers to a person recovering from poly substance abuse. It will be further appreciated that a subject recovering from abuse of a single substance would also benefit from the method.

[0040] In another aspect, the present disclosure provides a method for inactivating kappa opioid receptor (KOR) responsible for the dysphoria and psychotomimetic effects of kappa opioid agonists administered for the treatment of pruritus or for the promotion of diuresis. Thus, in this aspect, the disclosure provides a method for the treatment of pruritus or for the promotion of diuresis comprising administering a kappa opioid agonist in combination with nalfurafine or nalmefene.

[0041] In certain embodiments, the disclosure provides a method for selectively treating pruritus in a subject without activating KORs responsible for dysphoric or psychotomimetic effects, comprising administering to a subject in need thereof a therapeutically effective amount of a selective and efficacious kappa opioid agonist in combination with a therapeutically masking amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, to effect pruritus treatment. In other embodiments of this method, the disclosure provides a method for treating pruritus in a subject. In this method, a therapeutically effective amount of a kappa opioid agonist in combination with a therapeutically effective amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof to effect pruritustreatment. In the context of this method, as used herein, the term “effective” refers to clinical reduction of itch.

[0042] In certain embodiments, the disclosure provides a method for selectively promoting diuresis in a subject without activating KORs responsible for dysphoric or psychotomimetic effects, comprising administering to a subject in need thereof a therapeutically effective amount of a selective and efficacious kappa opioid agonist in combination with a therapeutically masking amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, to promote diuresis. In other embodiments of this method, the disclosure provides a method for promoting diuresis in a subject. In this method, a therapeutically effective amount of a kappa opioid agonist in combination with a therapeutically effective amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof to promote diuresis. Diuresis is clinically important in the treatment of hypertension. Thus, the therapeutic use reduces blood pressure in persons with hypertension and the therapeutic amount achieves this result.

[0043] In a further related aspect, the disclosure provides a method for the selective treatment of neuropathic pain in a subject without activating KORs responsible for dysphoric or psychotomimetic effects, comprising administering to a subject in need thereof a therapeutically effective amount of a selective and efficacious kappa opioid agonist and a therapeutically masking amount of nalfurafine or pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof. In other embodiments of this method, the disclosure provides a method for the treatment of neuropathic pain in a subject. In this method, a therapeutically effective amount of a kappa opioid agonist and a therapeutically acceptable amount of nalfurafine or pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof (i.e., a subject suffering from neuropathic pain).

[0044] In the context of the above methods, as used herein, the term “masking amount” refers to an amount of nalfurafine or pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, effective to selectively block of the KORs responsible for the undesired effects (e.g., dysphoric or psychotomimetic effects). Using the masking amount would effectively mask these KORs.

[0045] The method for treating neuropathic pain described herein is an improvement in the treatment of neuropathic pain with a kappa opioid agonist. Administration of a kappa opioid agonist for the treatment of pain by highly efficacious kappa agonists is typically accompanied by psychotomimetic and dysphoric responses. KOR agonists do not have the addiction risk that Mu opioid agonists have. However, the psychotomimetic and dysphoric adverse effects limit their therapeutic utility. As described herein, treatment with nalmefene or nalfurafine would selectively inactivate the Kappa receptors responsible for the psychotomimetic and dysphoric effects of highly efficacious KOR agonists and allow their use in the treatment of itch and hypertension. In the method described, the kappa opioid agonist is the pain reliever and nalfurafine or nalmefene, or their pharmaceutically acceptable salts, treat or prevent pruritus and promote diuresis. In the method, co-administration of nalfurafine or nalmefene, or their pharmaceutically acceptable salts, overcomes or precludes the adverse side effects associated with administration of kappa opioid agonists for the treatment of pain.

[0046] In certain embodiments of the methods described herein, nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered at a microdose.

[0047] As used herein, the microdose for nalfurafine is less than the maximal dose for treating uremic itch (e.g., about 2-5 pg per day) and the microdose for nalmefene is less than the maximal dose for treating alcohol use disorder or for reversing respiratory depression during acute opioid overdose (e.g., about 20 mg / day or about 1-3 mg per infusion).

[0048] In certain embodiments of the methods described herein, nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered at a dose that is about 10 to about 100 times, or about 10 to about 50 times, less than the standard dose. In certain of these embodiments, nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered at a dose that is from about 100 pg to about 500 pg.

[0049] In certain embodiments of the methods described herein, nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered orally.

[0050] In certain embodiments of the methods described herein, nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered daily.

[0051] In certain embodiments of the methods described herein, nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered in a depot formulation, for example, intramuscularly injected monthly.

[0052] In other related aspects, the disclosure provides the use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, for activating JNK / PRDX6 to stimulate localized peroxide production that causes G-protein depalmitoylation and deactivation of kappa opioid receptors in a subject; the use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, for inactivating kappa opioid receptors responsible for dysphoria during mu-opioid withdrawal in a subject; the use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, for promoting stress resilience in a subject recovering from polysubstance withdrawal; the use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, in combination with a kappa opioid agonist for treating pruritus in a subject; the use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, in combination with a kappa opioid agonist for promoting diuresis in a subject; and the use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, in combination with a kappa opioid agonist for the treatment of neuropathic pain in a subject.

[0053] The novel sensor oROS-Gr detects increases in ROS generation in the presence of nalfurafine and nalmefene.

[0054] Because the INK / PRDX6 mechanism for receptor inactivation is dependent on the generation of ROS, monitoring ROS generation was determined to be an effective means to identify compounds having norBNI-like effects. To achieve this, a ROS sensor using a structure-based design was optimized (see FIGS. 6A and 6B). The resulting sensor oROS-Gr was expressed in a cre-dependent manner in the ventral tegmental area (VTA) of KORCremice. Ex vivo horizontal slices were used for live-cell 2-photon fluorescence imaging (FIG. 2B). Consistent with the G-biased KOR agonists nalfurafineand nalmefene producing long-lasting norBNI-like effects, it was observed that nalfurafine (100 nM) and nalmefene (10 p.M) significantly increased oROS fluorescence (FIGS. 2D and 2E). The fluorescence increase was blocked by the competitive opioid receptor antagonist naloxone (10 pM), the PRDX6 inhibitor MJ33 (10 pM), and the JNK inhibitor JNK-IN-8 (1 pM) (FIG. 2D). Activation of oROS-Gr in the VTA was also imaged using in vivo fiber photometry (FIG. 2F). Both nalfurafine and nalmefene caused dose-dependent increases in fluorescence, and the response was blocked by pretreatment with 10 mg / kg naloxone (FIG. 2G). Although naloxone is not a KOR-selective antagonist, response mediation by KOR is suggested because the sensor was expressed in KOR-cre VTA neurons, neither drug has significant affinity for DOR, and nalmefene is a MOR antagonist, not agonist. These results suggest that both nalfurafine and nalmefene stimulate the generation of ROS through the KOR / INK / PRDX6 signaling cascade in KOR-expressing VTA neurons.

[0055] KOR-induced ROS generation increase is estrus cycle dependent in female mice.

[0056] The generation of oROS-Gr allowed for addressing an additional fundamental question relating to the regulation of KOR sensitivity to the long-lasting effects of norBNI. It has been previously reported that female rodents have estrus statedependent insensitivity to KOR activation because estrogen stimulates G protein Receptor Kinase 2 (GRK2) to block Gpy signaling. Through this proposed mechanism, estrogen blocks the long-lasting effects of norBNI. To determine if the activation of oROS was similarly affected, was expressed oROS-Gr was expressed in VTA neurons of adult female KOR-Cre mice and the fluorescence responses to nalfurafine during different estrus stages was measured. Nalfurafine was chosen to stimulate ROS because it has higher potency than nalmefene (FIG. 2G). Nalfurafine significantly increased oROS fluorescence during estrus (lowest estrogen state) but had no effect during proestrus (highest estrogen state) (FIG. 2H). Female mice pretreated with estradiol had no significant oROS response to nalfurafine, whereas female mice pretreated with progesterone showed a robust increase in fluorescence after nalfurafine injection (FIG. 21). These results are consistent with previously suggested mechanisms of inconsistent G protein-mediated KOR agonist effects in females.

[0057] Repeated administration of nalfurafine or nalmefene block kappa agonist- induced analgesia in a JNK-pathway-specific manner.

[0058] A single injection of 10 mg / kg norBNI completely inactivates KOR for weeks in mice. A single injection of a 100-fold lower dose of norBNI was ineffective, but daily injection of 0.1 mg / kg norBNI for 3 weeks produced KOR inactivation in both male and female mice. Presumably, the lower dose inactivates a small percentage of the receptors each day, and because KOR recovery takes weeks, receptor inactivation accumulates. Reducing a drug dose by 100-fold reduces the risk of adverse and off-target effects and would be potentially advantageous in human therapeutics. To determine the abilities of nalfurafine and nalmefene to produce norBNI-like cumulative KOR inactivation, various doses were injected daily for 7 days and then waited for 7 days to allow drug clearance before assessing the degree of KOR inactivation using the standard warm-water (52.5°C) tail withdrawal assay. Treatment for 7 days with low doses of norBNI (330 pg / kg), nalfurafine (100 ng / kg), or nalmefene (100 pg / kg) significantly inhibited the antinociceptive effects of the KOR agonist U50.488 (FIGS. 3A-3C). The inactivation was long-lasting: norBNI-treated mice (1 mg / kg) showed only partial recovery after 28 days (FIG. 3D). Nalfurafine-treated mice recovered only after 21 days (FIG. 3E), and nahuefene-treated mice required more than 14 days to recover (FIG. 3F). In contrast, the effects of naloxone (10 mg / kg) and naltrexone (10 mg / kg) completely reversed in 3 days (FIGS. 3G and 3H). As previously shown, twice daily treatment with escalating doses of U50,488 (10-75 mg / kg), which does not activate JNK / ROS, produced analgesic tolerance that fully reversed within 14 days and was not evident in GRK3 knockout mice. When PRDX6 was inhibited by injection with MJ33 (1.25 mg / kg) before each injection of norBNI, nalfurafine, or nalmefene, the rates of recovery of the U50488 analgesic response were significantly increased. The sensitivity to the selective PRDX6 inhibitor supports the conclusion that long-acting KOR inactivation measured in vivo results from the JNK / PRDX6 / ROS mechanism proposed. The difference between naltrexone and nalmefene in their durations of action is striking since these two drugs have very similar structures (a ketone versus a methylene group on ring carbon 6). These results support the conclusion that microdosing with nalfurafine or nalmefene can inactivate KOR in a norBNI-like manner through the JNK / PRDX6 cascade.

[0059] Repeated administration of nalfurafine and nalmefene block stress-paired cue aversion.

[0060] To determine if microdosing nalfurafine or nalmefene would block dynorphin-mediated 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. In the 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. 4D). 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. 4A-4C). Pretreatment of the mice for 7 days with norBNI (either 330 pg / kg or 1 mg / kg), nalfurafine (1 pg / kg or 10 pg / kg), or nalmefene (100 pg / kg or 1 mg / kg) to inactivate KOR, blocked odorant aversion. The drug potencies in the swim-odorant assay were comparable to the tail-flick analgesia assay. Because the place aversion assay requires conditioning, it is important to note that norBNI does not affect learning. In previous work, mice that were injected with cocaine in the presence of the almond scent developed robust place preferences for the almond-scent paired compartment. NorBNI (10 mg / kg) treatment before cocaine conditioning did not block the development of almond-scent preference.

[0061] In the second stress assay, almond scent was paired with naloxone- induced aversion in fentanyl-dependent mice (FIGS. 4D and 4E). The 1 mg / kg dose of naloxone chosen will block mu-opioid receptors but is insufficient to block KOR. Acute opioid abstinence causes dynorphin release and includes a profound dysphoric response. Mice were implanted with osmotic minipumps containing fentanyl (2 mg / kg / day) to produce opioid dependence. After 7 days, the pumps were removed, and 2 days later, the 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. Fentanyl-dependent mice developed a robust aversion to the odorant (FIG. 4F). 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 pg / kg) ornalmefene (5 mg / kg) showed significantly reduced aversion, but the decrease was incomplete (FIG. 4F). Mice that were pretreated for 7 days with norBNl (1 mg / kg), nalfurafine (5 pg / kg), or nalmefene (5 mg / kg) and experienced naloxone-induced aversion 10 days after pump removal (protracted withdrawal) showed no aversion to the odorant (FIG. 4G). These results from both stress assays suggest that repeated low-dose treatment with nalfurafine or nalmefene may block the dynorphin component of the stress response and supports their potential therapeutic utilities in preventing relapse during protracted opioid abstinence.

[0062] Long-term KOR antagonism is tissue-specific.

[0063] The long-lasting inactivation of KOR that results from ROS generation and subsequent receptor inactivation has been previously shown to have tissue selectivity; norBNl inactivates KOR located on VTA somatic membranes but not on nerve terminals of the same neurons. The regulation of KOR controlling pituitary function, diuresis, and itch behaviors was determined (FIGS. 5A and 5C-5F). Nalfurafine, nalmefene, and U50,488 each dose-dependently increase serum prolactin (FIG. 5A). Acute nalfurafine is a high potency, full agonist (EC50 = 2.9 pg / kg) and nalmefene is a low potency partial agonist in this assay (FIG. 5A). Pretreatment with norBNl (10 mg / kg, 7 days prior) blocked the increase in prolactin seen with U50,488 and nalfurafine (FIG. 5A). Similarly, 7 days of treatment with nalfurafine or nalmefene dose-dependently blocked the increase in serum prolactin stimulated by an acute challenge with nalfurafine (100 pg / kg) (FIGS. 5B and 5C).

[0064] Kappa agonists promote diuresis, and 100 pg / kg nalfurafine increases urine output (FIG. 5E). Acute norBNl (10 mg / kg) blocked nalfurafine-induced diuresis at Ihr and 24hr post-administration, but norBNl antagonism was not evident 7- or 14-days post-administration (FIG. 5E). Nalfurafine stimulation of urine output was dosedependent, but norBNl (1 mg / kg) treatment for 7 days did not shift the dose-response curve (FIGS 7A and 7B). Daily injection of 100 pg / kg nalfurafine for 30 days produced a consistent increase in urine output with no evidence of tolerance (i.e., KOR inactivation) (FIGS. 7A and 7B).

[0065] Repeated dosing of nalmefene or nalfurafine will cause KOR inactivation for some, but not all KOR. With repeated dosing of nalmefene or nalfurafine the analgesic and dysphoric responses to KOR agonists are blocked, but the diuretic and antipruritic effects of KOR agonists are spared. This is likely because the eJun Kinase / PRDX6 mechanism is not expressed in the cells responsible for the regulation of vasopressin andthe itch responses. Selective, high efficacy KOR agonists that have been developed for use in humans for the treatment of pain (including U-50488, spiradoline, enadoline, and niravoline) produce dysphoric and psychotomimetic effects. These adverse effects have stopped their development. Repeated (daily) dosing of nalfurafine or nalmefene is combined with a selective kappa agonist to reduce the adverse effects of the KOR agonist. In this way, KOR agonists in combination with nalfurafine or nalmefene can be used to selectively produce diuretic, antipruritic, and anti -neuropathic effects without causing psychotomimetic or dysphoric effects.

[0066] The selectivity of the combination of a kappa opioid agonist with nalfurafine is illustrated in FIGS. 5E, 5F, 7A and 7B. FIGS. 5E, 7A, and 7B show that KOR inactivation by norBNI, which acts like nalfurafine and nalmefene to inactivate KOR, or daily treatment with nalfurafine does not inactivate the kappa receptors responsible for diuresis. FIG. 5E shows that nalfurafine increases diuresis, which can be blocked acutely, but not long-term, by norBNI.

[0067] FIGS. 5F and 7C similarly show that KOR inactivation by daily nalfurafine does not inactivate kappa receptors responsible for the antipruritic effects of KOR activation. FIG. 5F shows that GNTI induces pruritus, which can be blocked by nalfurafine. Daily doses of nalfurafine do not prevent this effect.

[0068] It has also been shown that nalfurafine can block GNTI-induced pruritis in a KOR-dependent manner. After 7 days of daily nalfurafine (50 ug / kg), nalfurafine is still able to block GNTI-induced itch (FIG. 5F). These findings suggest that the ROS- induced KOR inactivation is restricted to a subset of KOR responses. The basis for this tissue selectivity has not been established but presumably results from differences in INK / PRDX6 expression or receptor regeneration rate.

[0069] Activation of the INK / PRDX6 pathway by nalfurafine and nalmefene results in norBNI-like KOR inactivation. The neuroimaging and behavioral pharmacological results provide substantial validation of the novel JNK signaling and ROS production mechanism proposed to be responsible for the long duration of KOR inactivation, which may be a general regulatory mechanism of Gi / o coupled receptor function. Because KOR inactivation is long-lasting, daily microdosing of either nalfurafine or nalmefene allows slow accumulation of inactivation of KOR necessary to block the pro- depressive effects of elevated dynorphin tone evident during stress exposure and abstinence in opioid-dependent mice. Estrogen regulation of the INK / PRDX6 signaling pathway wasconfirmed in female mice, and progesterone treatment may be useful in enhancing treatment efficacy in premenopausal women. Most importantly, the KOR-inactivating medications nalfurafine and nalmefene, which have demonstrated safety in humans, have potential advantages over conventional competitive antagonists in the treatment of depression and addiction disorders.

[0070] The results described herein provide validation of the concept that KOR activates three different signaling pathways: GPy for membrane-delimited regulation of ion channel conductance; GRK3 / p-arrestin regulation of p38 MAPK mechanisms controlling mood; and JNK / PRDX6 controlling Gai coupling to KOR. KOR ligands differ in their efficacies at each pathway, and functional selectivity needs to be considered in at least these three dimensions. The long duration of norBNI was initially thought to be caused by slow pharmacokinetic clearance; however, an alternative explanation was suggested when norBNI was found to stimulate the phosphorylation of eJun N-terminal kinase (JNK) in KOR-transfected HEK293 cells and mouse brain. The long duration of norBNI action was prevented if JNK was pharmacologically inhibited in vivo or if the JNK1 gene isoform was deleted. Additionally, the pharmacokinetic explanation was excluded by a receptor occlusion experiment, and the duration of action of a broad range of KOR antagonists was tightly correlated with their abilities to stimulate JNK phosphorylation in the spinal cord.

[0071] NorBNI enhances the association of KOR with GPy and PRDX6 which is known to stimulate NADPH generation of ROS through its phospholipase activity. ROS can oxidize sulfhydryl groups on exposed cysteines, and the amino-terminal cysteine residue in Gai is reversibly palmitoylated. This palmitoylation controls the binding orientation of the Gai in the receptor signaling complex, and norBNI generation of ROS depalmitoylates the Gai. This depalmitoylation and the resulting inactivation are irreversible, requiring full resynthesis of the receptor (over 3 weeks) to recover the KOR response. The results using the novel oROS sensor showed that ROS is generated by VTA KOR activation with both nalfurafine and nalmefene in ex vivo slice and in vivo fiber photometry and that KOR inactivation can be perturbed by blocking the JNK / PRDX6 pathway.

[0072] There is now a large body of preclinical literature suggesting that KOR antagonists may prove useful in the treatment of the anhedonic component of many mental disorders. NorBNI and JDTic, long-lasting KOR antagonists, have been shown to block stress-induced drug-seeking and drug self-administration. Dynorphin is elevated in animalmodels of stress behaviors and in the CSF of persons experiencing psychosis. As described herein, nalfurafine and nalmefene have the ability to block stress-cue-paired aversion. In the odor-swim stress pairing, there is a full block of odorant aversion, consistent with what has been shown with a single large dose of norBNI. In the odorant-opioid withdrawal pairing, KOR inactivation caused a partial block during acute withdrawal and a complete block during protracted withdrawal. Acute opioid withdrawal includes a profound contribution of a hyperactive sympathetic nervous system, and the physical symptoms can be ameliorated by a2A adrenergic receptor agonists clonidine or lofexidine. Naloxone precipitation is not identical to spontaneous withdrawal from opioids - it is more intense and acute; however, the symptoms are qualitatively similar. The results described herein indicate that during the protracted withdrawal phase, the dysphoric effects are largely mediated by residual hyperactivity of the dynorphin / KOR system. Therefore, KOR antagonists have therapeutic utility to decrease negative affect during protracted abstinence in humans.

[0073] Theoretically, KOR-inactivation by nalfurafine or nalmefene confers several potential advantages as medications. First, these two medications have histories of safety and tolerability in human use. Nalfurafine has been approved in Japan as an antipruritic in uremic itch. Nalmefene is used as an alternative to naloxone and naltrexone in the treatment of alcohol use disorder and to reverse respiratory depression during acute opioid overdose. Slow accumulation of inactivation would allow the titration of effect, and slow recovery would allow stable antagonism. In contrast, competitive antagonists would need to be taken at supersaturating doses to prevent dynorphin activation of KOR. Dynorphins have very high affinities and receptor efficacies; they may need only occupy a fraction of the receptors to produce a maximal effect. The results described herein highlight the potential of leveraging functional receptor selectivity insights for therapeutic advantage. With the identification and characterization of these three signaling pathways at KOR, the traditional idea of partial agonism can be expanded to generate novel medications to promote stress resilience.

[0074] Neither nalfurafine nor nalmefene are completely selective KOR ligands, but KOR inactivation was observed at doses 10-100 fold lower than necessary for mu opioid receptor actions. KOR inactivation is sex-dependent, and as described herein, nalfurafine causes peroxide production only during estrus (low-estrogen state) or following progesterone treatment of female mice. Because KOR inactivation recovers slowly, dailytreatment with submaximal drug doses causes accumulating inhibition. Daily microdosing with nalfurafine or nalmefene blocked KORs responsible for antinociceptive effects, blocked KORs mediating stress -induced aversion, mitigated KOR-mediated dysphoria during acute and protracted withdrawal in opioid-dependent mice, and blocked KOR- induced prolactin secretion. In contrast, KORs mediating the diuretic and anti-pruritic effects were not regulated by JNK. Both nalfurafine and nalmefene have long histories of safety and use in humans and as described herein, can be repurposed for the treatment of dynorphin-mediated stress disorders.

[0075] Materials and Methods

[0076] Animals

[0077] Adult C57BL / 6 wild-type (WT) or KOR-cre male and female mice were used (Jackson Labs). Animal studies were approved by the University of Washington IACUC. Detailed descriptions of the stereotaxic injection procedures, fiber photometry recording methods, and estrous cycle stage determination are described below.

[0078] Drugs

[0079] Nalfurafine (NIDA Drug Supply Program), nalmefene (Tocris), norBNI (NIDA Drug Supply Program), U50,488 (Tocris), naloxone (Tocris), naltrexone (Tocris), GNTI (Tocris), and MJ33 (Sigma Aldrich) were dissolved in saline and administered intraperitoneally (i.p.). Estradiol (50 ug / kg; Cayman Chemical) and progesterone (5 mg / kg; ThermoFisher) were dissolved in 0.1% ethanol / 0.1% Cremophor EL / 99% saline and administered subcutaneously (s.c.) in a volume of 10 ml / kg.

[0080] In vitro neuron imaging

[0081] Brains were collected 2-4 weeks after viral injection, and 200 pm horizontal slices were obtained using a vibratome as described in Reichard KL, Newton KA, Rivera ZMG, Menezes PMS de, Schattauer SS, Land BB, Chavkin C (2020): Regulation of Kappa Opioid Receptor Inactivation Depends on Sex and Cellular Site of Antagonist Action. Mol Pharmacol 98: 548-558. Image collection was done on a Broker Investigator 2-photon microscope, with software Prairie View 5.5, simultaneously collecting the mCherry (1040 nm fixed) and GFP (920 nm tunable) signals with a Nikon 16X water immersion objective, as well as a z-stack spanning 60 pm across an hour time course. During this collection time, a baseline was established for 7 minutes in ACSF buffer solution (124 mM NaCl, 3 mM KC1, 2 mM MgSO4, 1.25 mM NaH2PO4, 2.5 mM CaCh, 26 mM NaHCCh, 10 mM Glucose) or ACSF with naloxone (10 pM), MJ33 (10pM), or JNK-IN-8 (1 pM) at 32°C followed by treatment with nalfnrafine (100 nM), nalmefene (10 pM), or those in addition to previously mentioned inhibitors for 30 min, followed by H2O2 (590 pM) for 5 minutes. Analyses were completed by creating a hyperstack in ImageJ and then creating a Z-proj ection for both signal channels. Regions of interest (ROIs) were drawn broadly over the entire slice, and values were collected using ImageJ for each channel with the same ROI from raw, unaltered images. The mCherry signal value was subtracted from the GFP signal value, and the resulting data were fit so that the lowest value was adjusted to 1. Then AF / F was calculated using the average value of the baseline (7 minutes of buffer solution, or 7 minutes of only inhibitor treatment) for the rest of the time course. Cells that showed no response to H2O2 wash were excluded from the analysis. Representative 2p images (FIGS. 2A and 2B) have enhanced contrast for visual representation. N = 3-10 per treatment group, with each data point representing the average of 15-30 individual cells in a single slice. Horizontal images were collected using slices that underwent 2Photon imaging and were put into 10% formalin. Confocal images were taken with a Leica SP8x Confocal microscope.

[0082] Prolactin determination

[0083] Blood samples of up to 500 pL were taken from mice 1 hour after drug treatment and allowed to clot for 5 min at room temperature before being centrifuged for 15 min (2000 x g) at 4°C. Serum was immediately frozen on crushed dry ice and stored at -80°C until assayed in Mouse Prolactin (PRL) ELISA Kit PicoKine® from Boster Biological Technology. PRL assay was performed following the manufacturer’s instructions at 1 : 10 dilution of sera. Contrary to the instructions, sera was found to reduce the slope of the PRL standard curve. 10 pL of pooled sera from control, uninjected mice was added to each 100 p L of diluted PRL standard to correct for this. Thus, sera PRL assay values reported are ng / ml over baseline. N = 4-16 per treatment group.

[0084] Statistical analysis

[0085] All data were analyzed using either one- or two-way ANOVA or nonparametric tests appropriate for variables and normality was assessed using GraphPad Prism unless otherwise stated. Post -hoc tests for multiple comparisons were performed to determine differences from controls. Initial analysis was done with experimenters blinded to treatment groups. Exclusion criteria for all experiments were based on statistical outliers or insufficient responses during baseline measures or compared to positive or negative controls. The number of animals used in each treatment group was determined by the priorresults for similar assays. Animals in each study were age -matched and randomized before the start of treatments. Studies were designed so that all animals in a single cage received the same treatment as their cage mates.

[0086] Stereotaxic injection

[0087] Male KOR-cre mice between 5-7 weeks were anesthetized with 2.5% isoflurane and head fixed on a Kopf Model 942 stereotaxic alignment system. A 2.0 uL model 7002 KH Neuros Syringe (Hamilton, NV, USA) was lowered for viral injection of 0.5 uL of AAVl-FLEX-oROS-mCheny. For in vitro brain slice experiments, bilateral injection into the VTA using coordinates: ML: + / - 0.5 mm, AP: -3.28 mm, DN: -4.5mm, and for fiber photometry experiments, unilateral injection using coordinates AP: -3.28 mm, ML: -1.71 mm, DV: -4.67 mm at a 15-degree angle; injections were done 2-4 weeks prior to imaging. An implantable fiber optic cannula (400 / 430 core, 0.57 NA; Doric Lenses, Quebec, CA) was placed at the same coordinates as the viral injection site, and then C&B Metabond was used to secure the cannula to the skull. After injection, the needle was kept at the injection site for 5 minutes before removal. All mice were monitored and given 10 mg / kg carprofen daily for 7 days for post-surgical analgesia.

[0088] Fiber photometry

[0089] A real-time signal processor (RZ5P; Tucker-Davis Technologies) was connected to Synapse Software (Fiber Photometry) to set the frequency of light stimulation and record input from photodetectors as described in Abraham AD, Casello SM, Schattauer SS, Wong BA, Mizuno GO, Mahe K, et al. (2021): Release of endogenous dynorphin opioids in the prefrontal cortex disrupts cognition. Neuropsychophannacol 46: 2330-2339. For in vivo experiments, mice were run with randomized treatments on separate days. Mice received saline, nalfurafine (100 pg / kg), or nalfurafine (100 pg / kg) with naloxone (10 mg / kg). For 100 pg / kg nalfurafine experiments, after baseline, the recording session lasted for 45 minutes. For 10 mg / kg naloxone pretreatment to 100 pg / kg nalfurafine experiments, after baseline, mice were given administration of naloxone, and after 15 minutes 100 pg / kg nalfurafine was administered. Mice were allowed to freely explore the chamber during in vivo recordings. N = 3-13 per treatment group.

[0090] Estrous cycle stage determination

[0091] Vaginal lavage for the estrous cycle stage determination was done following behavioral testing; 50 mL of deionized water was pipetted to collect epithelial cells and placed on glass slides for cytology. The estrous cycle stage was classified usingthe estrous cycle identification tool (Byers SL, Wiles MV, Dunn SL, Taft RA (2012): Mouse Estrous Cycle Identification Tool and Images. PLOS ONE 7: e35538) and then quantified by the relative ratio of nucleated epithelial cells, cornified epithelial cells, and leukocytes. Cytology was determined with the investigator blind to treatment.

[0092] Hormone treatment

[0093] Female mice were injected subcutaneously with 5 mg / kg progesterone (ThermoFisher) or 50 pg / kg estradiol (Cayman Chemical). 3 days later, the mice were tethered for fiber photometry (as described above) and given an intraperitoneal injection of nalfurafine (100 pg / kg), and the oROS response was measured. Estrous states were determined after the recording session. Following lavage, mice were given a second subcutaneous injection of the treatment they received prior and 3 days later, repeated the assessment of their estrous state and fiber photometry experiments. Both treatment groups had a two-week recovery period with no treatment. Following the recovery period, the treatments were switched. Data analyses were done by an investigator blind to treatment. N = 7-12 per treatment group.

[0094] Warm water tail withdrawal

[0095] Mice were tested for U50,488 (10 mg / kg i.p.) antinociception using a 52.5°C warm water tail withdrawal test as described in Bruchas MR, Yang T, Schreiber S, DeFino M, Kwan SC, Li S, Chavkin C (2007): Long-Acting K Opioid Antagonists Disrupt Receptor Signaling And Produce Noncompetitive Effects By Activating C-Jun N-Terminal Kinase. Journal of Biological Chemistry 282: 29803-29811. Scores were calculated as the difference between the before and 30 min after U50,488 injection. N = 6-24 per treatment group.

[0096] Odorant-paired repeated forced swim stress

[0097] 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 (McLaughlin JP, Marton-Popovici M, Chavkin C (2003): K Opioid Receptor Antagonism and Prodynorphin Gene Disruption Block Stress-Induced Behavioral Responses. JNeurosci 23: 5674-5683). Odorant-swim stress pairing was conditioned as described in Land BB, Bruchas MR, Lemos JC, Xu M, Melief EJ, Chavkin C (2008): The dysphoric component of stress isencoded by activation of the dynorphin kappa-opioid system. J Neurosci 28: 407-414. N = 6-16 per treatment group.

[0098] Post fentanyl naloxone induced aversion - odorant pairing

[0099] 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.

[0100] Hot plate Nociception

[0101] 1-2 hours after the removal of the osmotic mini pump, mice were placed on a hot plate held at 55°C for 30 seconds. Jumping behavior was used as a measure of thermal hyperalgesia.

[0102] Odorant-aversion test

[0103] Mice underwent two types of odorant-conditioned pairing: stress-odorant pairing in which almond scent was paired with repeated forced swim stress and postfentanyl naloxone-odorant pairing in which almond scent was paired with two instances of naloxone -precipitated aversion (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 Etho vision 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.

[0104] Diuresis assay

[0105] Mice were habituated for 20 min in 4x4 inch plexiglass boxes with preweighed paper towels below them before being injected with saline or 100 pg / kg nalfurafine. 60 min after injection, mice were removed, and the change in weight of the paper towel was measured. Mice were also tested 1 and 24 hours, 7- and 14-days post 10mg / kg norBNI treatment or 7 days after the last administration of 7 daily treatments of 1 mg / kg norBNI. For the 30-day study, mice were injected with either saline or nalfurafine before urine output measurement every day for 30 days. N = 10-20 per treatment group and N = 3-4 for 30-day study.

[0106] GNTI-induced pruritus

[0107] Mice were injected with saline (s.c.) 20 min prior to 30 pg / kg GNTI (s.c.), and scratching behavior was recorded for 30 min as described in Schattauer SS, Kuhar JR, Song A, Chavkin C (2017): Nalfurafine is a G-protein biased agonist having significantly greater bias at the human than rodent form of the kappa opioid receptor. Cell Signal 32: 59-65. Mice were then given daily treatments of either saline or nalfurafine (50 pg / kg) for 7 days. The following day, mice were injected with nalfurafine (50 pg / kg) 20 min prior to 30 pg / kg GNTI to determine nalfurafine block of scratching behavior.

[0108] 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 invention.

Claims

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

1. A method for selectively inactivating kappa opioid receptors in a subject by activating JNK / PRDX6 to stimulate localized peroxide production that causes G-protein depalmitoylation and deactivation of kappa opioid receptors, the method comprising administering to a subject in need thereof an amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, effective to activate JNK / PRDX6 in the subject.

2. A method for inactivating kappa opioid receptors responsible for dysphoria, anhedonia, and drug craving during mu-opioid withdrawal in a subject, the method comprising administering a therapeutically effective amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, to the subject.

3. A method for promoting stress resilience in a subject recovering from polysubstance withdrawal, the method comprising administering a therapeutically effective amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, to the subject.

4. A method for selectively treating pruritus in a subject without activating KORs responsible for dysphoric or psychotomimetic effects, comprising administering to a subject in need thereof a therapeutically effective amount of a selective and efficacious kappa opioid agonist in combination with a therapeutically masking amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, to effect pruritus treatment.

5. A method for selectively promoting diuresis in a subject without activating KORs responsible for dysphoric or psychotomimetic effects, comprising administering to a subject in need thereof a therapeutically effective amount of a selective and efficacious kappa opioid agonist in combination with a therapeutically masking amount of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, to promote diuresis.

6. A method for the selective treatment of neuropathic pain in a subject without activating KORs responsible for dysphoric or psychotomimetic effects, comprising administering to a subject in need thereof a therapeutically effective amount of a selective and efficacious kappa opioid agonist and a therapeutically masking amount of nalfurafine or pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof.

7. The method of any one of Claims 1-6, wherein nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered at a microdose.

8. The method of any one of Claims 1-6, wherein nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered at a dose that is about 10 to about 100 times less than the standard dose.

9. The method of any one of Claims 1-6, wherein nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered at a dose that is from about 100 pg to about 500 pg.

10. The method of any one of Claims 1-6, wherein nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered orally.

11. The method of any one of Claims 1-6, wherein nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, is administered daily.

12. The use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, for activating JNK / PRDX6 to stimulate localized peroxide production that causes G-protein depalmitoylation and deactivation of kappa opioid receptors in a subject.

13. The use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, for inactivating kappa opioid receptors responsible for dysphoria during mu-opioid withdrawal in a subject.

14. The use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, for promoting stress resilience in a subject recovering from polysubstance withdrawal.

15. The use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, in combination with a kappa opioid agonist for treating pruritus in a subject.

16. The use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, in combination with a kappa opioid agonist for promoting diuresis in a subject.

17. The use of nalfurafine or a pharmaceutically acceptable salt thereof, or nalmefene or a pharmaceutically acceptable salt thereof, in combination with a kappa opioid agonist for the treatment of neuropathic pain in a subject.

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