Reversal agents for ultra-potent synthetic opioid-induced overdose and respiratory depression

Abstract

The present invention provides compositions and methods for the reversal of ultra-potent synthetic opioid-induced overdose and respiratory depression in a subject in need thereof.

Description

[0001] REVERSAL AGENTS FOR ULTRA-POTENT SYNTHETIC OPIOID-INDUCED

[0002] OVERDOSE AND RESPIRATORY DEPRESSION

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 560,032, filed March 1, 2024, the contents of which are incorporated by reference herein in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under DA054921, DA056729, and DA013429 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND OF THE INVENTION

[0008] Ultra-potent synthetic (UPS) opioids (e.g., fentanyl, carfentanil) have been designated as high consequence chemicals of concern by United States Government agencies. UPS opioids have high in vivo potency, rapid onset of action, and high toxicity; they are considered public health risks under the ongoing opioid epidemic and chemical threats by both military and civilian agencies. Acute UPS opioid exposure and overdose have high lethality due to Opioid-Induced Respiratory Depression (OIRD), Opioid- Induced Persistent Apnea (OIPA), decreased brain and nerve function, muscle rigidity and bradycardia. Mu-opioid receptor (MOR) antagonists such as naloxone are used as an emergency treatment; however, they have limited ability to reverse the effects of UPS opioids.

[0009] Thus, there is a need in the art for improved compositions and methods for the reversal of ultra-potent synthetic opioid-induced overdose and respiratory depression. This invention satisfies this unmet need.

[0010] SUMMARY OF THE INVENTION In one embodiment, the invention relates to a composition for treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative agonist, wherein the composition comprises at least one inhibitor of Mu opioid receptor (MOR) signaling.

[0011] In one embodiment, the at least one inhibitor of MOR signaling comprises at least one inhibitor of MOR C-motif PDZ-binding domain signaling. In one embodiment, the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one inhibitor of PDZ-RhoGEF, at least one inhibitor of RhoA, at least one inhibitor of phospholipase D (PLD), at least one inhibitor of Sigma- 1 -receptor, or any combination thereof. In one embodiment, the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one PDZ-RhoGEF inhibitor, at least one RhoA inhibitor, at least one Sigma- 1 receptor (Sig-IR) inhibitor, or any combination thereof. In one embodiment, the at least one PDZ-RhoGEF inhibitor is Y16, the at least one RhoA inhibitor is CCG 1423, and the at least one Sig-IR inhibitor is BD1047.

[0012] In one embodiment, the at least one inhibitor of MOR signaling comprises naloxone and at least one inhibitor of MOR C-motif PDZ-binding domain signaling, wherein the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one inhibitor of PDZ-RhoGEF, at least one inhibitor of RhoA, at least one inhibitor of phospholipase D (PLD), at least one inhibitor of Sigma- 1 -receptor, or any combination thereof. In one embodiment, the at least one inhibitor of MOR signaling comprises naloxone and at least one inhibitor of MOR C-motif PDZ-binding domain signaling, wherein the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one inhibitor of PDZ-RhoGEF, at least one inhibitor of RhoA, at least one inhibitor of Sigma- 1 -receptor, or any combination thereof. In one embodiment, the at least one PDZ-RhoGEF inhibitor is Y16, the at least one RhoA inhibitor is CCG 1423, and the at least one Sig-IR inhibitor is BD1047.

[0013] In one embodiment, the at least one inhibitor of MOR C-motif PDZ- binding domain signaling comprises at least one PDZ-RhoGEF inhibitor. In one embodiment, the at least one PDZ-RhoGEF inhibitor is Y16. In one embodiment, the opioid or opioid-derivative comprises an ultra-potent synthetic (UPS) opioid. In one embodiment, the UPS opioid is fentanyl.

[0014] In one embodiment, the opioid- or opioid-derivative-induced overdose or a symptom there of comprises respiratory depression, central nervous system depression, cardiovascular depression, altered level consciousness, miotic pupils, hypoxemia, acute lung injury, aspiration pneumonia, sedation, hypotension, unresponsiveness to stimulus, unconsciousness, stopped breathing, erratic or stopped pulse, choking or gurgling sounds, blue or purple fingernails or lips, slack or limp muscle tone, contracted pupils, vomiting, or any combination thereof.

[0015] In one embodiment, the treating comprises prevention and / or reversal of respiratory depression. In one embodiment, the composition is formulated for administration by nasal delivery.

[0016] In one embodiment, the invention relates to a method of treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative comprising administering a therapeutically effective amount of the compositions described herein. In one embodiment, the administration comprises nasally administering to the subject, via a device adapted for nasal delivery of a formulation by actuation of the device into at least one nostril of the subject, a nasal delivery formulation comprising a therapeutically effective amount of the compositions described herein. In one embodiment, the administration occurs prior to exposure to the opioid or opioid derivative. In one embodiment, the administration occurs about 10 min to about 2 hours prior to exposure to the opioid or opioid derivative. In one embodiment, the administration occurs after exposure to the opioid or opioid derivative. In one embodiment, the administration occurs about 10 min to about 2 hours after exposure to the opioid or opioid derivative.

[0017] In one embodiment, the subject is free from opioid- or opioid-derivative- induced overdose or a symptom thereof for at least about 4 hours following administration. In one embodiment, the subject is free from opioid- or opioid-derivative- induced overdose or a symptom thereof for at least about 8 hours following administration.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0020] Figure 1, comprising Figure 1A and Figure IB, presents a representative illustration depicting that activation of MOR by fentanyl in addition to Gai / o-mediated cAMP inhibition and P-arrestin signaling, elicits signaling through its C-terminal tail and activates RhoGEF / RhoA / PLD / choline / Sigma-1 receptor (Sig-IR). An enlarged view of the human, rat and mouse MOR receptor C-terminus sequence illustrating the last three amino acids PLP are conserved between species (C-motif, PDZ-binding domain).

[0021] Figure 2 depicts representative data demonstrating that stimulation of MOR-WT or MOR- AAA transfected HEK293 cells with morphine, oxycodone or fentanyl significantly inhibited cAMP Response Element (CRE). See also Figure 23.

[0022] Figure 3 depicts representative data demonstrating that at the lowest concentration tested (0.1 pM), fentanyl increased RhoA activity about 2 times higher than oxycodone and 3.5 times higher than morphine, and that that fentanyl (10’7-10’5M) increased RhoA activity with higher efficacy and potency than oxycodone and morphine in MOR-WT cells. See also Figure 25.

[0023] Figure 4 depicts representative data demonstrating that fentanyl (10’7-l O’5M) increased choline with higher efficacy and potency than oxycodone and morphine.

[0024] Figure 5 depicts representative data demonstrating that treatment with pertussis toxin (PTX, 1 pM), Gai / o inhibitor, did not significantly reduced RhoA activity induced by fentanyl (0.81 ±0.087 in the presence of PTX, versus 0.84 ±0.083 in the absence of PTX). See also Figure 27.

[0025] Figure 6 depicts representative data demonstrating that fentanyl significantly increased choline level to 1298 ± 67 pM in MOR-WT, while did not affect it in MOR-AAA (136 ± 14 pM) (n =6); similarly oxycodone and morphine increased choline in MOR-WT, but not in MOR-AAA. See also Figure 30. Figure 7 depicts representative data demonstrating that RhoGEF inhibitor before treatment with fentanyl, oxycodone or morphine (1 pM). Y16 (10 pM) reduced the RhoA activity produced by MOR agonists in MOR-WT cells to levels similar to control (n = 6), indicating that the increase in RhoA activity is RhoGEF-dependent.

[0026] Figure 8 depicts representative data demonstrating that pretreatment of MOR-WT cells with Y16 (10 pM), RhoGEF inhibitor, abolished the increase in choline production by fentanyl, oxycodone and morphine (1 pM), supporting a RhoGEF- dependent mechanism.

[0027] Figure 9 depicts representative data demonstrating that fentanyl produced a prolonged increase in choline.

[0028] Figure 10 depicts representative data demonstrating that in primary PAG neurons fentanyl (1 pM) increased RhoA activity with higher efficacy than oxycodone and morphine. Gai / o-inhibition with pertussis toxin (PTX, 1 pM) did not significantly affect the effect of any of three MOR agonists; however, their effect was reduced to basal level by Y16 (10 pM), RhoGEF inhibitor. These results indicate that the increase in RhoA activity produced by MOR agonists is mediated by RhoGEF and not produced via Gai / o-dependent mechanism.

[0029] Figure 11 depicts representative data demonstrating that in primary PAG neurons fentanyl, oxycodone and morphine (1 pM) increased choline levels, with fentanyl having the highest efficacy. Gai / o-inhibition with PTX (1 pM) did not significantly affect the effect of any of three MOR agonists; however, their effect was reduced to basal levels by Y16 (10 pM), RhoGEF inhibitor, indicating that it is mediated by RhoGEF and not produced via Gai / o-dependent mechanism.

[0030] Figure 12 depicts representative data demonstrating that similarly to the MOR-WT cells, a dose-response relationship was determined with fentanyl being more potent and more efficacious than morphine (n=6) and that in primary PAG neurons, MOR agonists increased choline level with fentanyl being more potent and more efficacious than morphine and oxycodone.

[0031] Figure 13 depicts representative data demonstrating that fentanyl produced a prolonged and sustained increase in choline level to 1016 ± 72 pM (1 min), 987 ± 96 pM (2 min), 964 ± 93 pM (5 min) and 842 ± 86 pM (10 min) (n=6). Figure 14 depicts representative data demonstrating that stimulation of MOR-WT cells with morphine or fentanyl (10_11-10'5M) significantly inhibited CRE; naloxone reversed the CRE inhibition by morphine and fentanyl (n=6). See also Figure 37.

[0032] Figure 15 depicts representative data demonstrating that in MOR-WT cells naloxone (10 pM) abolished the RhoA activity increased by morphine (1 pM), reduced the effect of oxycodone (1 pM) but produced only a slight reduction in fentanyl (1 pM)- induced increase RhoA activity (by 21%), indicating an additional mechanism responsible for fentanyl -induced RhoA activity (n=6). See also Figure 38.

[0033] Figure 16 depicts representative data demonstrating that morphine (1 pM) increased choline level (334 ±32 pM) to about one third of that increased by fentanyl (1298 ± 67 pM); the increase in choline produced by morphine was abolished by naloxone (31± 14 pM) (n = 6). See also Figure 39.

[0034] Figure 17 depicts representative data demonstrating that naloxone (10 pM) abolished the increase of choline produced by morphine (28 ± 24 pM) similarly to basal level (21 ± 11 pM) (n = 6).

[0035] Figure 18, comprising Figure 18A and Figure 18B, depicts representative data demonstrating that morphine (1 pM) produced a sustained hyperpolarization of neurons by 5.58 ± 0.61 mV (n = 6) which was abolished by naloxone (10 pM).

[0036] Figure 19, comprising Figure 19A and Figure 19B, depicts representative data demonstrating that in neurons, application of naloxone (10 pM) on the plateau of the hyperpolarization produced by fentanyl reduced the amplitude of the hyperpolarization. Application of a combination of RhoGEF inhibitor Y16 (10 pM) and Sigma-IR antagonist, BD1047 (20 pM) on the plateau of hyperpolarization produced by fentanyl abolished the hyperpolarization.

[0037] Figure 20 depicts representative data demonstrating that administration of Y16 (20 mg / kg), RhoGEF inhibitor slowly reversed the respiratory depression produced by fentanyl, while administration of Y16 (20 mg / kg) and BD 1047 (10 mg / kg), Sig-IR antagonist, 6 min after fentanyl reversed the respiratory depression faster (n=4) (Figure 20), indicating that blocking both the beginning of the pathway (RhoGEF inhibition) and the end of the pathway (Sig-IR antagonism) was more efficacious. Figure 21 depicts representative data demonstrating that administration of naloxone (Img / kg) and Y16 (20 mg / kg), administered 6 min after fentanyl reversed the fentanyl-induced respiratory depression within 1 min (Figure 21) (n=4); the bottom graph indicate the area outlined (red rectangle) in the top graph.

[0038] Figure 22 depicts representative data demonstrating that IP injection of saline-vehicle-saline or saline-Y16 (20 mg / kg)-saline alone did not affect the oxygen saturation (n=6).

[0039] Figure 23 depicts representative data demonstrating that the mutation of the C-terminal of MOR does not affect Gai / o signaling assessed with CRE assay. Stimulation of MOR-WT or MOR-AAA HEK293 cells with morphine, or fentanyl significantly inhibited CRE. In M0R-D116 cells, morphine and fentanyl did not produce CRE inhibition indicating that the 116 aspartate of MOR is critical for Gai / o signaling.

[0040] Figure 24 depicts representative data demonstrating that the mutation of MOR aspartate 116 (M0R-D116) but not of C-terminal (MOR-AAA) impairs Gai / o- mediated signaling. In forskolin-stimulated MOR-WT and MOR-AAA cells, but not in MOR-D116 cells, fentanyl (1 pM) and morphine (1 pM) produced a decrease in cAMP accumulation assessed using a cADDis™ assay.

[0041] Figure 25 depicts representative data demonstrating that fentanyl increases RhoA activity via MOR C-terminal with higher potency and efficacy than morphine. Dose-response relationships of RhoA activity produced by fentanyl, and morphine (10‘7- 10-sM) in MOR-WT, MOR-D116 and MOR-AAA cells.

[0042] Figure 26 depicts representative data demonstrating that fentanyl (1 pM) but not morphine (1 pM) produced a long-lasting, sustained increase in RhoA activity in MOR-WT cells; the effect was abolished in MOR-AAA cells indicating that the C- terminal of MOR was critical for the increase in RhoA activity produced by fentanyl.

[0043] Figure 27 depicts representative data demonstrating that fentanyl (1 pM) increased RhoA activity with higher efficacy than oxycodone and morphine (1 pM) in MOR-WT cells; Gai / o-inhibition with pertussis toxin (PTX, 1 pM) did not significantly affect the effect of any of three MOR agonists; however, their effect was reduced to basal level by Y16, (10 pM), RhoGEF inhibitor, indicating that it is mediated by RhoGEF and not produced via Gai / o protein. Figure 28 depicts representative data demonstrating that fentanyl produced a prolonged increase in RhoA activity via a RhoGEF-dependent mechanism in MOR-WT cells. Pretreatment of MOR-WT cells with Y16 (10 pM), RhoGEF inhibitor, or CCG 1423 (10 pM), RhoA inhibitor abolished the increase in RhoA activity induced by fentanyl.

[0044] Figure 29 depicts representative data demonstrating that fentanyl increases choline level via C-terminal MOR. Fentanyl increased choline in MOR-WT and MOR- D116 cells with higher efficacy than morphine. In MOR- AAA cells fentanyl and morphine did not increase choline level indicating that the C-terminal of MOR is critical for this effect.

[0045] Figure 30 depicts representative data demonstrating that in MOR-WT cells fentanyl, oxycodone and morphine (1 pM) increased choline levels, with fentanyl having the highest efficacy. Gai / o-inhibition with PTX (1 pM) did not significantly affect the effect of any of three MOR agonists; however, their effect was reduced to basal levels by Y16 (10 pM), RhoGEF inhibitor, indicating that it is mediated by RhoGEF and not produced via Gai / o-dependent mechanism.

[0046] Figure 31 depicts representative data demonstrating that fentanyl produced a prolonged increase in choline level via a RhoGEF-dependent mechanism in MOR-WT cells. Pretreatment of MOR-WT cells with Y16 (10 pM), RhoGEF inhibitor, or CCG 1423 (10 pM), RhoA inhibitor, abolished the increase in RhoA activity induced by fentanyl.

[0047] Figure 32 depicts representative data demonstrating that in primary neurons of periaqueductal gray (PAG) that express MOR and are involved in central pain regulation, fentanyl (10'7- 10'5M) increased RhoA activity with higher efficacy and potency than oxycodone and morphine.

[0048] Figure 33 depicts representative data demonstrating that in primary PAG neurons fentanyl (1 pM) produced a prolonged increase in RhoA activity as compared to morphine (1 pM).

[0049] Figure 34 depicts representative data demonstrating that fentanyl produced a prolonged increase in RhoA activity via a RhoGEF-dependent mechanism in PAG neurons. Pretreatment of neurons with Y16 (10 pM), RhoGEF inhibitor, or CCG 1423 (10 pM), RhoA inhibitor, abolished the increase in RhoA activity induced by fentanyl.

[0050] Figure 35 depicts representative data demonstrating that fentanyl (1 pM) produced a prolonged and sustained increase in choline level in PAG neurons as compared to morphine (1 pM).

[0051] Figure 36 depicts representative data demonstrating that the long-lasting increase in choline level produced by fentanyl (1 pM) in PAG neurons was abolished by Y16 (10 pM), RhoGEF inhibitor, or CCG 1423 (10 pM), RhoA inhibitor.

[0052] Figure 37 depicts representative data demonstrating that stimulation of MOR-WT cells with morphine or fentanyl (10_11-10'5M) significantly inhibited CRE; naloxone (10 pM) reversed the CRE inhibition by morphine and fentanyl.

[0053] Figure 38 depicts representative data demonstrating that in MOR-WT cells naloxone (10 pM) abolished the increase in RhoA activity produced by morphine (1 pM), reduced the effect of oxycodone (1 pM) but produced only a slight reduction in fentanyl (1 pM)-induced increase RhoA activity, indicating an additional mechanism responsible for fentanyl-induced RhoA activity.

[0054] Figure 39 depicts representative data demonstrating that in MOR-WT cells naloxone (10 pM) abolished the morphine (1 pM)-induced increase in choline level, reduced the effect of oxycodone (1 pM) and produced only a slight reduction in the effect of fentanyl (1 pM).

[0055] Figure 40 comprising of Figures 40A and 40B, depicts representative data demonstrating that morphine (1 pM) and fentanyl produced a sustained hyperpolarization of neurons; the hyperpolarization produced by fentanyl had a higher amplitude. Application of naloxone (10 pM) on the plateau of the hyperpolarization, abolished the hyperpolarization produced by morphine and reduced the amplitude of the hyperpolarization produced by fentanyl.

[0056] Figure 41 depicts representative in vivo data demonstrating that the administration of fentanyl (200 pg / kg, subcutaneous, SC) reduced the oxygen saturation in rats, indicative of respiratory depression. Vehicle (control) did not elicit an effect.

[0057] Figure 42 depicts representative in vivo data demonstrating that in rats the reduction in oxygen saturation produced by fentanyl (200 pg / kg, SC) is restored by RhoGEF inhibitor, Y 16 (20 mg / kg, IP) as compared to vehicle (1 :1 : 18; ETOH: cremophor: saline). Y16 by itself did not affect the oxygen saturation.

[0058] Figure 43 depicts representative in vivo data demonstrating that in rats the reduction in oxygen saturation produced by fentanyl (200 pg / kg, SC) is restored by RhoA inhibitor, CCG1423 (5 mg / kg, IP) as compared to vehicle. CCG1423 by itself did not affect the oxygen saturation.

[0059] Figure 44 depicts representative in vivo data demonstrating that in rats the reduction in oxygen saturation produced by fentanyl (200 pg / kg, SC) is restored by Sigma-IR antagonist BD-1047 (10 mg / kg, IP); as compared to vehicle. BD-1047 (10 mg / kg, IP) by itself did not affect the oxygen saturation.

[0060] Figure 45 depicts representative in vivo data demonstrating in rats that the reduction in oxygen saturation produced by fentanyl (200 pg / kg, SC) is reversed by the combination of RhoGEF inhibitor, Y16 (20 mg / kg, IP) and Sigma- 1R antagonist, BD- 1047 (10 mg / kg, IP).

[0061] Figure 46 depicts representative in vivo data demonstrating that in rats the reduction in oxygen saturation induced by fentanyl (200 pg / kg, SC) is reversed by naloxone (Nix, 1 mg / kg, IP), while naloxone (0.5 mg / kg, IP) only partially ameliorated the decrease in oxygen saturation produced by fentanyl.

[0062] Figure 47 depicts representative in vivo data demonstrating that in rats the reduction in oxygen saturation induced by fentanyl (200 pg / kg, SC) is reversed by a combination of naloxone (Nix, 0.5 mg / kg, IP) and of RhoGEF inhibitor, Y16 (20 mg / kg, IP).

[0063] Figure 48 depicts representative in vivo data demonstrating that in rats the reduction in oxygen saturation induced by fentanyl (200 pg / kg, SC) is reversed by a combination of naloxone (Nix, 0.5 mg / kg, IP) and RhoA inhibitor, CCG1423 (CCG, 5 mg / kg, IP).

[0064] Figure 49 depicts representative in vivo data demonstrating that in rats the reduction in oxygen saturation induced by fentanyl (200 pg / kg, SC) is reversed by a combination of naloxone (0.5 mg / kg, IP) and Sigma-IR antagonist, BD-1047 (10 mg / kg, IP). Figure 50 depicts a representative illustration summarizing MOR signaling pathways: in addition to well-known canonical and beta-arrestin-mediated signaling, the new PDZ-RhoGEF / RhoA / Sigma-lR pathway is illustrated and agents that can block it.

[0065] DETAILED DESCRIPTION

[0066] The present invention is based upon the discovery that opioid and nonopioid inhibitors of a new signaling pathway initiated at the C-terminal PDZ-binding domain of MOR (Mu-opioid receptor) reverse within minutes all ultra-potent synthetic (UPS) opioid induced adverse effects (e.g., respiratory depression, etc.). Accordingly, this invention relates to methods and compositions for the reversal of UPS opioid- induced adverse effects in a subject in need thereof.

[0067] In one embodiment, the present invention provides compositions for treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative, wherein the composition comprises at least one inhibitor of Mu opioid receptor (MOR) signaling, and wherein the at least one inhibitor of MOR signaling comprises at least one inhibitor of MOR C-motif PDZ-binding domain signaling. In one embodiment, the at least one inhibitor of MOR C- motif PDZ-binding domain signaling comprises at least one inhibitor of PDZ-RhoGEF, at least one inhibitor of RhoA, at least one inhibitor of phospholipase D (PLD), at least one inhibitor of Sigma- 1 -receptor, or any combination thereof. In one embodiment, the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one PDZ-RhoGEF inhibitor, at least one RhoA inhibitor, at least one Sigma- 1 receptor (Sig- 1R) inhibitor, or any combination thereof, wherein the at least one PDZ-RhoGEF inhibitor is Y16, the at least one inhibitor of RhoA is CCG 1423, the at least one Sig-IR inhibitor is BD1047.

[0068] In one embodiment, the present invention provides compositions for treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative, wherein the composition comprises at least one inhibitor of Mu opioid receptor (MOR) signaling, wherein the at least one inhibitor of MOR signaling comprises naloxone and at least one inhibitor of MOR C-motif PDZ-binding domain signaling, wherein the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one PDZ-RhoGEF inhibitor, at least one Rho inhibitor, at least one Sig-IR inhibitor, or any combination thereof, wherein the at least one PDZ-RhoGEF inhibitor is Y16, the at least one Rho inhibitor is CCG 1423, the at least one Sig-IR inhibitor is BD1047.

[0069] In one embodiment, the present invention provides methods for treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative comprising administering the compositions of the present invention to the subject wherein the subject is free from opioid- or opioid- derivative-induced overdose or a symptom thereof following administration.

[0070] In one embodiment, the opioid or opioid-derivative comprises an ultrapotent synthetic (UPS) opioid. In one embodiment, the UPS opioid is fentanyl.

[0071] Definitions

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0073] As used herein, each of the following terms has the meaning associated with it in this section.

[0074] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0075] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0076] The terms “cells” and “population of cells” are used interchangeably and generally refer to a plurality of cells, i.e., more than one cell. The population may be a pure population comprising one cell type. Alternatively, the population may comprise more than one cell type. In the present invention, there is no limit on the number of cell types that a cell population may comprise. A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0077] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0078] “Inhibitor” as used herein describes an agent which directly or indirectly prevents the functioning of its target, including, without limitation, by reducing the mRNA or protein levels of its target, by physically preventing the target from performing its function, or by degrading or causing the degradation of the target.

[0079] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living organism is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0080] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0081] By the term “specifically binds,” as used herein with respect to an affinity ligand, in particular, an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0082] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder.

[0083] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0084] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject or to prevent the occurrence of at least one sign or symptom of a disease or disorder experienced by a subject, wherein the at least one sign or symptom of a disease or disorder includes an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative such as but not limited to respiratory depression, central nervous system depression, cardiovascular depression, altered level consciousness, miotic pupils, hypoxemia, acute lung injury, aspiration pneumonia, sedation, hypotension, unresponsiveness to stimulus, unconsciousness, stopped breathing, erratic or stopped pulse, choking or gurgling sounds, blue or purple fingernails or lips, slack or limp muscle tone, contracted pupils, vomiting, or any combination thereof. “Wild type activity” as used herein refers to the activity (e.g., catalytic, structural, etc.) displayed by a biological molecule as it occurs in nature. As used herein, “wild type activity” refers to any documented, or yet undocumented, biological activity of a molecule as determined by techniques or assays commonly used in the field to determine the function of a molecule (e.g., catalytic, structural, etc.).

[0085] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0086] Description

[0087] The present invention provides methods and compositions for treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative. In one embodiment, the opioid or opioid- derivative comprises an ultrapotent synthetic (UPS) opioid.

[0088] Compositions

[0089] In one embodiment, the present invention provides compositions for treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative, wherein the composition comprises at least one inhibitor of Mu opioid receptor (MOR) signaling.

[0090] In one embodiment, the at least one inhibitor of MOR signaling comprises at least one inhibitor of MOR C-motif PDZ-binding domain signaling. In one embodiment, the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one inhibitor of PDZ-RhoGEF, at least one inhibitor of RhoA, at least one inhibitor of phospholipase D (PLD), at least one inhibitor of Sigma- 1 -receptor, or any combination thereof.

[0091] In one embodiment, the at least one inhibitor of MOR signaling comprises naloxone and at least one inhibitor of MOR C-motif PDZ-binding domain signaling, wherein the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one inhibitor of PDZ-RhoGEF, at least one inhibitor of RhoA, at least one inhibitor of phospholipase D (PLD), at least one inhibitor of Sigma- 1 -receptor, or any combination thereof.

[0092] MOR PDZ-domain pathway inhibitors

[0093] In one embodiment, the compositions of the present invention comprise at least one inhibitor of MOR C-motif PDZ-binding domain signaling.

[0094] PDZ domains are structural protein domains that recognize simple linear amino acid motifs (also called receptor’s PDZ-binding domain), often at the protein C- terminal, that mediate protein-protein interactions. PDZ proteins (including PDZ- RhoGEF) bind through a well-defined pocket to linear motifs that are mostly situated at the end of the C-termini of receptors.

[0095] In one embodiment, the at least one inhibitor of MOR C-motif PDZ- binding domain signaling comprises at least one inhibitor of PDZ-RhoGEF, at least one inhibitor of RhoA, at least one inhibitor of phospholipase D (PLD), at least one inhibitor of Sigma- 1 -receptor, or any combination thereof.

[0096] In one embodiment, the compositions comprise multiple inhibitors of MOR C-motif PDZ-binding domain signaling in combination. In one embodiment, the compositions of the present invention comprise at least one inhibitor of MOR C-motif PDZ-binding domain signaling in combination with at least one MOR antagonist (e.g., naloxone, etc.). A skilled artisan will appreciate that the compositions of the present invention comprise any combination of MOR antagonists and inhibitors of MOR C-motif PDZ-binding domain signaling that, at concentration ranges known to be suitable in the field, achieve the effect of treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative. RhoA and PDZ Rho-GEF Inhibitors

[0097] In one embodiment, the at least one inhibitor of MOR C-motif PDZ- binding domain signaling comprises at least one inhibitor of RhoA.

[0098] RhoA is a member of the Ras homology family of small GTPases. These proteins cycle from their active (GTP-bound) to their inactive (GDP -bound) conformation by hydrolyzing GTP to GDP. RhoA's functions in the cell are primarily related to cytoskeletal regulation. Recent studies have shown its indirect involvement in myosin phosphorylation and cellular responses to stress, such as the formation of focal adhesions and actin stress fibers. It has also been shown to be directly related to myosin chain elongation, actin filament rearrangement, gene expression, cell-shape determination and cell proliferation.

[0099] RhoA has three known main effectors, which include the ROCK I, II family, which are kinases that cause actomyosin contraction, transformation, and transcription of the SRF gene. Also, these effectors show scaffolding properties that function to polymerize actin and affect the formation of microtubules. The second effector is the PRK1 / PKN proteins that cause endocytosis. And lastly RhoA binds to the effector Citron causing cytokinesis.

[0100] In one embodiment, the at least one inhibitor of RhoA is selected from the group comprising: AT-13148, BA-210, P-Elemene, belumosudil, chroman 1, DJ4, GSK- 57637, GSK429286A, C21H16F4N4O2, H-l 152 Inhibitor, hydroxyfasudil, Ibuprofen, LX-7101, netarsudil, RKI-1447, ripasudil, TCS-7001, thiazovivin, verosudil (AR-12286), Y-27632, Y-30141, Y-33075, Y-39983, simvastatin, CCG-1423 and any combination thereof.

[0101] In one embodiment, the at least one inhibitor of RhoA is CCG-1423.

[0102] In one embodiment, the at least one inhibitor of MOR C-motif PDZ- binding domain signaling comprises at least one inhibitor of PDZ Rho-GEF.

[0103] PDZ Rho-GEF, also known as Rho guanine nucleotide exchange factor 11 is a primarily brain specific guanine nucleotide exchange factor (GEF) for the RhoA small GTPase protein. PDZ-RhoGEF can also be activated directly by the C-terminal tail of certain receptors. Rho is a small GTPase protein that is inactive when bound to the guanine nucleotide GDP. But when acted on by Rho GEF proteins such as RhoGEFl, this GDP is released and replaced by GTP, leading to the active state of Rho. In this active, GTP- bound conformation, Rho can bind to and activate specific effector proteins and enzymes to regulate cellular functions. In particular, active Rho is a major regulator of the cell actin cytoskeleton.

[0104] PDZ Rho-GEF is a member of a group of four RhoGEF proteins known to be activated by G protein coupled receptors coupled to the G12 and G13 heterotrimeric G proteins. The others are ARHGEF1 (also known as pl 15-RhoGEF), ARHGEF12 (also known as LARG) and AKAP13 (also known as ARHGEF13 and Lbc). GPCR-regulated PDZ Rho-GEF (and these related GEF proteins) acts as an effector for G12 and G13 G proteins. In addition to being activated by G12 or G13 G proteins, three of these four RhoGEF proteins (ARHGEF1 / 11 / 12) also function as RGS family GTPase-activating proteins (GAPs) to increase the rate of GTP hydrolysis of G12 / G13 alpha proteins (which are themselves GTPase proteins). This action increases the rate of G protein deactivation, limiting the time during which these RhoGEFs activate Rho.

[0105] In one embodiment, the at least one inhibitor of PDZ Rho-GEF is selected from the group comprising: Y16, A13, TRIPa (Trio inhibitory peptide a), TRIPE32G, TRIPa, and any combination thereof.

[0106] In one embodiment, the at least one inhibitor of MOR C-motif PDZ- binding domain signaling comprises at least one inhibitor of PDZ Rho-GEF, wherein the at least one inhibitor of PDZ Rho-GEF is Y16.

[0107] Sigma- 1 Receptor Inhibitors

[0108] In one embodiment, the at least one inhibitor of MOR C-motif PDZ- binding domain signaling comprises at least one inhibitor of sigma-1 receptor (Sig-IR). Sig-IR is a chaperone protein at the endoplasmic reticulum (ER) that modulates calcium signaling.

[0109] In one embodiment, the at least one inhibitor of Sigma- 1 Receptor is selected from the group comprising: methamphetamine, selegiline, D-Deprenyl, sertraline, SIRA, FTC-146, l-benzyl-6'-methoxy-6',7'-dihydrospiro[piperidine-4,4'- thieno[3.2-c]pyran], 5-HT1 A, 5-HT6, 5-HT7, alA and a2 adrenergic, and NMDA receptors, NE-100, BD1047, S 38093, WQ 1 and any combination thereof.

[0110] In one embodiment, the at least one inhibitor of MOR C -motif PDZ- binding domain signaling comprises at least one inhibitor of Sig-IR, wherein the at least one inhibitor of Sig-IR is BD1047.

[0111] Phospholipase D (PLD) Inhibitors

[0112] In one embodiment, the at least one inhibitor of MOR C-motif PDZ- binding domain signaling comprises at least one inhibitor of phospholipase D (PLD). PLD is a transphosphatidylase enzyme of the phospholipase superfamily that catalyzes the production of choline.

[0113] In one embodiment, the at least one inhibitor of PLD is selected from the group comprising: fodrin, synaptojanin, amphiphysins, ceramide, honokiol, VU0155056, VU0359595, VU0285655-1, ML-299, FIPI, VU0155069, VU0364739 and any combination thereof.

[0114] Additional MOR inhibitor

[0115] In one embodiment, the present invention provides compositions for treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative, wherein the composition comprises at least one MOR inhibitor. In one embodiment, the at least one MOR inhibitor is selected from the group comprising: naloxone, buprenorphine, dezocine, eptazocine, butorphanol, levorphanol, nalbuphine, pentazocine, phenazocine, cyprodime, and any combination thereof.

[0116] In one embodiment, the at least one MOR inhibitor comprises naloxone.

[0117] Concentration ranges

[0118] In one embodiment, the compositions comprise Y16 at a concentration of 10 pM and BD1047 at a concentration of 20 pM.

[0119] In one embodiment, the compositions comprise Y16 at a concentration of between about 1 pM and about 5 pM and / or BD1047 at a concentration of between about 1 pM and about 5 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 5 pM and about 10 pM and / or BD1047 at a concentration of between about 5 pM and about 10 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 10 pM and about 15 pM and / or BD1047 at a concentration of between about 10 pM and about 15 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 15 pM and about 20 pM and / or BD1047 at a concentration of between about 15 pM and about 20 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 20 pM and about 25 pM and / or BD1047 at a concentration of between about 20 pM and about 25 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 25 pM and about 30 pM and / or BD1047 at a concentration of between about 25 pM and about 30 pM.

[0120] In one embodiment, the compositions comprise Y16 at a concentration of 10 pM.

[0121] In one embodiment, the compositions comprise Y16 at a concentration of between about 1 pM and about 5 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 5 pM and about 10 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 10 pM and about 15 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 15 pM and about 20 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 20 pM and about 25 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 25 pM and about 30 pM.

[0122] In one embodiment, the compositions comprise BD1047 at a concentration of 20 pM.

[0123] In one embodiment, the compositions comprise BD1047 at a concentration of between about 1 pM and about 5 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 5 pM and about 10 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 10 pM and about 15 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 15 pM and about 20 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 20 pM and about 25 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 25 pM and about 30 pM.

[0124] In one embodiment, the compositions comprise CCG 1423 at a concentration of 10 pM.

[0125] In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 1 pM and about 5 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 5 pM and about 10 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 10 pM and about 15 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 15 pM and about 20 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 20 pM and about 25 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 25 pM and about 30 pM.

[0126] In one embodiment, the compositions comprise Y16 at a concentration of 10 pM and naloxone at a concentration of 10 pM.

[0127] In one embodiment, the compositions comprise Y16 at a concentration of between about 1 pM and about 5 pM and / or naloxone at a concentration of between about 1 pM and about 5 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 5 pM and about 10 pM and / or naloxone at a concentration of between about 5 pM and about 10 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 10 pM and about 15 pM and / or naloxone at a concentration of between about 10 pM and about 15 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 15 pM and about 20 pM and / or naloxone at a concentration of between about 15 pM and about 20 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 20 pM and about 25 pM and / or naloxone at a concentration of between about 20 pM and about 25 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 25 pM and about 30 pM and / or naloxone at a concentration of between about 25 pM and about 30 pM. In one embodiment, the compositions comprise Y16 at a concentration of between about 5 pM and about 15 pM and naloxone at a concentration of between about 5 pM and about 15 pM.

[0128] In one embodiment, the compositions comprise BD1047 at a concentration of 20 pM and naloxone at a concentration of 10 pM.

[0129] In one embodiment, the compositions comprise BD1047 at a concentration of between about 1 pM and about 5 pM and / or naloxone at a concentration of between about 1 pM and about 5 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 5 pM and about 10 pM and / or naloxone at a concentration of between 5 pM and about 10 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 10 pM and about 15 pM and / or naloxone at a concentration of between about 10 pM and about 15 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 15 pM and about 20 pM and / or naloxone at a concentration of between about 15 pM and about 20 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 20 pM and about 25 pM and / or naloxone at a concentration of between about 20 pM and about 25 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 25 pM and about 30 pM and / or naloxone at a concentration of between about 25 pM and about 30 pM. In one embodiment, the compositions comprise BD1047 at a concentration of between about 15 pM and about 25 pM and naloxone at a concentration of between about 5 pM and about 15 pM.

[0130] In one embodiment, the compositions comprise CCG 1423 at a concentration of 10 pM and naloxone at a concentration of 10 pM.

[0131] In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 1 pM and about 5 pM and / or naloxone at a concentration of between about 1 pM and about 5 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 5 pM and about 10 pM and / or naloxone at a concentration of between about 5 pM and about 10 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 10 pM and about 15 pM and / or naloxone at a concentration of between about 10 pM and about 15 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 15 pM and about 20 pM and / or naloxone at a concentration of between about 15 pM and about 20 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 20 pM and about 25 pM and / or naloxone at a concentration of between about 20 pM and about 25 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 25 pM and about 30 pM and / or naloxone at a concentration of between about 25 pM and about 30 pM. In one embodiment, the compositions comprise CCG 1423 at a concentration of between about 5 pM and about 15 pM and naloxone at a concentration of between about 5 pM and about 15 pM.

[0132] In one embodiment, a skilled artisan will appreciate that the compositions of the present invention comprise at least one inhibitor of MOR signaling at a concentration which will achieve the intended aim of treating an opioid- or opioid- derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative.

[0133] Methods

[0134] In one embodiment, the present invention provides methods for treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative comprising administering the compositions of the present invention to the subject wherein the subject is free from opioid- or opioid- derivative-induced overdose or a symptom thereof following administration.

[0135] Administration

[0136] In one embodiment, the present invention relates to methods of treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative comprising administering a therapeutically effective amount of the compositions described herein.

[0137] In one embodiment, the compositions comprise Y16 administered to a subject at 20 mg / kg and BD1047 administered to a subject at 10 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 1 mg / kg and about 5 mg / kg and / or BD1047 administered to a subject at between about 1 mg / kg and about 5 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 5 mg / kg and about 10 mg / kg and / or BD1047 administered to a subject at between about 5 mg / kg and about 10 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 10 mg / kg and about 15 mg / kg and / or BD1047 administered to a subject at between about 10 mg / kg and about 15 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 15 mg / kg and about 20 mg / kg and / or BD1047 administered to a subject at between about 15 mg / kg and about 20 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 20 mg / kg and about 25 mg / kg and / or BD1047 administered to a subject at between about 20 mg / kg and about 25 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 25 mg / kg and about 30 mg / kg and / or BD1047 administered to a subject at between about 25 mg / kg and about 30 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 30 mg / kg and about 35 mg / kg and / or BD1047 administered to a subject at between about 30 mg / kg and about 35 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 35 mg / kg and about 40 mg / kg and / or BD1047 administered to a subject at between about 35 mg / kg and about 40 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 40 mg / kg and about 45 mg / kg and / or BD1047 administered to a subject at between about 40 mg / kg and about 45 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 45 mg / kg and about 50 mg / kg and / or BD1047 administered to a subject at between 4 about 5 mg / kg and about 50 mg / kg, about 50 mg / kg and about 55 mg / kg, 5 about 5 mg / kg and about 60 mg / kg, about 60 mg / kg and about 65 mg / kg, about 65 mg / kg and about 70 mg / kg, about 70 mg / kg and about 75 mg / kg, about 75 mg / kg and about 80 mg / kg, about 80 mg / kg and about 85 mg / kg, about 85 mg / kg and about 90 mg / kg, about 90 mg / kg and about 95 mg / kg, or about 95 mg / kg and about 100 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at 20 mg / kg.

[0138] In one embodiment, the compositions comprise Y16 administered to a subject at between about 1 mg / kg and about 5 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 5 mg / kg and about 10 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 10 mg / kg and about 15 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 15 mg / kg and about 20 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 20 mg / kg and about 25 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 25 mg / kg and about 30 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 30 mg / kg and about 35 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 35 mg / kg and about 40 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 40 mg / kg and about 45 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 45 mg / kg and about 50 mg / kg.

[0139] In one embodiment, the compositions comprise BD1047 administered to a subject at 10 mg / kg.

[0140] In one embodiment, the compositions comprise BD1047 administered to a subject at between about 1 mg / kg and about 5 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 5 mg / kg and about 10 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 10 mg / kg and about 15 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 15 mg / kg and about 20 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 20 mg / kg and about 25 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 25 mg / kg and about 30 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 30 mg / kg and about 35 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 35 mg / kg and about 40 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 40 mg / kg and about 45 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 45 mg / kg and about 50 mg / kg, about 50 mg / kg and about 55 mg / kg, about 55 mg / kg and about 60 mg / kg, about 60 mg / kg and about 65 mg / kg, about 65 mg / kg and about 70 mg / kg, about 70 mg / kg and about 75 mg / kg, about 75 mg / kg and about 80 mg / kg, about 80 mg / kg and about 85 mg / kg, about 85 mg / kg and about 90 mg / kg, about 90 mg / kg and about 95 mg / kg, or about 95 mg / kg and about 100 mg / kg.

[0141] In one embodiment, the compositions comprise CCG 1423 administered to a subject at 5 mg / kg.

[0142] In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 1 mg / kg and about 2 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 2 mg / kg and about 3 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 3 mg / kg and about 4 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 4 mg / kg and about 5 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 5 mg / kg and about 6 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 6 mg / kg and about 7 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 7 mg / kg and about 8 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 8 mg / kg and about 9 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 9 mg / kg and about 10 mg / kg.

[0143] In one embodiment, the compositions comprise Y16 administered to a subject at 20 mg / kg and naloxone administered to a subject at 1 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at 20 mg / kg and naloxone administered to a subject at 0.5 mg / kg.

[0144] In one embodiment, the compositions comprise Y16 administered to a subject at between about 1 mg / kg and about 5 mg / kg and / or naloxone administered to a subject at between about 0.1 mg / kg and about 0.5 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 5 mg / kg and about 10 mg / kg and / or naloxone administered to a subject at between about 0.5 mg / kg and about 1 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 10 mg / kg and about 15 mg / kg and / or naloxone administered to a subject at between about 1 mg / kg and about 1.5 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 15 mg / kg and about 20 mg / kg and / or naloxone administered to a subject at between about 1.5 mg / kg and about 2 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 20 mg / kg and about 25 mg / kg and / or naloxone administered to a subject at between about 2 mg / kg and about 2.5 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 25 mg / kg and about 30 mg / kg and / or naloxone administered to a subject at between about 2.5 mg / kg and about 3 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 30 mg / kg and about 35 mg / kg and / or naloxone administered to a subject at between about 3 mg / kg and about 3.5 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 35 mg / kg and about 40 mg / kg and / or naloxone administered to a subject at between about 3.5 mg / kg and about 4 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 40 mg / kg and about 45 mg / kg and / or naloxone administered to a subject at between about 4 mg / kg and about 4.5 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 45 mg / kg and about 50 mg / kg and / or naloxone administered to a subject at between about 4 mg / kg and about 4.5 mg / kg, about 4.5 mg / kg and about 5 mg / kg, about 5 mg / kg and about 5.5 mg / kg, about 5.5 mg / kg and about 6 mg / kg, about 6 mg / kg and about 6.5 mg / kg, about 6.5 mg / kg and about 7 mg / kg, about 7 mg / kg and about 7.5 mg / kg, about 7.5 mg / kg and about 8 mg / kg, about 8 mg / kg and about 8.5 mg / kg, about 8.5 mg / kg and about 9 mg / kg, about 9 mg / kg and about 9.5 mg / kg, or about 9.5 mg / kg and about 10 mg / kg. In one embodiment, the compositions comprise Y16 administered to a subject at between about 15 mg / kg and about 25 mg / kg and / or naloxone administered to a subject at between about 0.1 mg / kg and about 2 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at 10 mg / kg and naloxone administered to a subject at 0.5 mg / kg.

[0145] In one embodiment, the compositions comprise BD1047 administered to a subject at between about 1 mg / kg and about 5 mg / kg and / or naloxone administered to a subject at between about 0.1 mg / kg and about 0.5 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 5 mg / kg and about 10 mg / kg and / or naloxone administered to a subject at between about 0.5 mg / kg and about 1 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 10 mg / kg and about 15 mg / kg and / or naloxone administered to a subject at between about 1 mg / kg and about 1.5 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 15 mg / kg and about 20 mg / kg and / or naloxone administered to a subject at between about 1.5 mg / kg and about 2 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 20 mg / kg and about 25 mg / kg and / or naloxone administered to a subject at between about 2 mg / kg and about 2.5 mg / kg. In one embodiment, the compositions comprise BD1047 administered to a subject at between about 5 mg / kg and about 15 mg / kg and / or naloxone administered to a subject at between about 0.1 mg / kg and about 2 mg / kg.

[0146] In one embodiment, the compositions comprise CCG 1423 administered to a subject at 5 mg / kg and naloxone administered to a subject at 0.5 mg / kg.

[0147] In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 1 mg / kg and about 5 mg / kg and / or naloxone administered to a subject at between about 0.1 mg / kg and about 0.5 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 5 mg / kg and about 10 mg / kg and / or naloxone administered to a subject at between 0.5 mg / kg and 1 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 10 mg / kg and about 15 mg / kg and / or naloxone administered to a subject at between about 1 mg / kg and about 1.5 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 15 mg / kg and about 20 mg / kg and / or naloxone administered to a subject at between about 1.5 mg / kg and about 2 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 20 mg / kg and about 25 mg / kg and / or naloxone administered to a subject at between about 2 mg / kg and about 2.5 mg / kg. In one embodiment, the compositions comprise CCG 1423 administered to a subject at between about 1 mg / kg and about 10 mg / kg and / or naloxone administered to a subject at between about 0.1 mg / kg and about 2 mg / kg.

[0148] In one embodiment, a skilled artisan will appreciate that the compositions of the present invention are administered to a subject at a concentration which will achieve the intended aim of treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative.

[0149] In one embodiment, the administration comprises nasally administering to the subject, via a device adapted for nasal delivery of a formulation by actuation of the device into at least one nostril of the subject, a nasal delivery formulation comprising the compositions of the present invention.

[0150] In one embodiment, the administration of the compositions of the invention to a subject can be by any route, including but not limited to intravenous, intranodal, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via an aerosol, etc.), buccal (e.g., sub-lingual, etc.), topical (i.e., both skin and mucosal surfaces, including airway surfaces, etc.), intrathecal, intraarticular, intraplural, intracerebral, intraarterial, intraperitoneal, oral, intralymphatic, intranasal, rectal or vaginal administration, by perfusion through a regional catheter, or by direct intralesional injection. In one embodiment, the compositions of the invention are administered by intravenous push or intravenous infusion given over defined period (e.g., 0.5 to 2 hours). The compositions of the invention can be delivered by peristaltic means or in the form of a depot, although the most suitable route in any given case will depend, as is well known in the art, on such factors as the species, age, gender and overall condition of the subject, the nature and severity of the condition being treated and / or on the nature of the particular composition (i.e., dosage, formulation) that is being administered. In particular embodiments, the route of administration is via bolus or continuous infusion over a period of time, once or twice a week. In other particular embodiments, the route of administration is by subcutaneous injection given in one or more sites (e.g., thigh, waist, buttocks, arm), optionally once or twice weekly. In one embodiment, the compositions, and / or methods of the invention are administered on an outpatient basis.

[0151] In one embodiment, the administration occurs prior to exposure to the opioid or opioid derivative.

[0152] In one embodiment, the subject is free from opioid- or opioid-derivative- induced overdose or a symptom thereof for at least about 4 hours following administration.

[0153] Respiratory depression

[0154] In one embodiment, the methods and compositions of the present invention are directed toward treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative. In one embodiment, the opioid- or opioid-derivative-induced overdose or a symptom thereof comprises respiratory depression.

[0155] The causes of respiratory depression that can be treated with the methods and compositions disclosed herein are varied, and include drug overdose, pharmaceutical use of central respiratory depressants, and medical conditions, including trauma.

[0156] In some embodiments, the respiratory depression results from a drug overdose. Drug classes that are known to cause respiratory depression when taken in excess include narcotics, benzodiazepines, opiates, opioids, barbiturates and alcohol.

[0157] Non-limiting exemplary opioids that can cause respiratory depression when taken in excess can include codeine, fentanyl (DURAGESIC®, ACTIQ®), heroin, hydrocodone (VICODIN®), hydromorphone (DILAUDID®), meperidine (DEMEROL®), methadone (METHADOSE®), morphine, oxycodone (OXYCONTIN®, ROXICODONE®), oxymorphone (OP ANA®), and dextropropoxyphene (DARVOCET®).

[0158] Non-limiting exemplary barbiturates that can cause respiratory depression when taken in excess include amobarbital (SODIUM AMYTAL®), aprobarbital (ALURATE®), butabarbital (BUTISOL®), butalbital (FIORINAL®), hexobarbital (SOMBULEX®), methylphenobarbital (MEBARAL®), pentobarbital (NEMBUTAL®), phenobarbital (LUMINAL®), secobarbital (SECONAL®), sodium thiopental (SODIUM PENTOTHAL®), and talbutal (LOTUSATE®).

[0159] Non-limiting exemplary benzodiazepines include alprazolam (XANAX®), clonazepam (KLONOPIN®), diazepam (VALIUM®), flunitrazepam (ROHYPNOL®), lorazepam (ATIVAN®), nitrazepam (MOGADON®), and temazepam (RESTORIL®). Non-limiting exemplary non-benzodiazepine GABA-A modulators include zaleplon (SONATA®), zopiclone (LUNESTA®), and zolpidem (AMBIEN®).

[0160] Non-limiting exemplary deliriants that can cause respiratory depression when taken in excess include atropine, diphenhydramine hydrochloride (BENADRYL®), dimenhydrinate (DRAMAMINE®), and scopolamine.

[0161] Non-limiting exemplary dissociative anaesthetics that can cause respiratory depression when taken in excess include: fluorathane and related volatile anaesthetics, dextromethorphan (DXM), ketamine (KETASET®), nitrous oxide, phencyclidine (PCP), salvinorin A, (found in Salvia divinorum), and Opium (Papaver somniferum).

[0162] In some embodiments, respiratory depression occurs in a subject having a medical condition. Non-limiting exemplary medical conditions that can cause respiratory depression include central sleep apnea, stroke-induced central sleep apnea, obstructive sleep apnea, sleep apnea resulting from Parkinson's disease, congenital hypoventilation syndrome, sudden infant death syndrome, Retts syndrome, Cheney-Stokes respiration, Ondines curse, and Prader-Willi's syndrome. In some embodiments, the subject has respiratory depression as a result of a traumatic injury or neurodegenerative disease. Nonlimiting exemplary traumatic injuries that can be associated with respiratory depression include spinal cord injury, traumatic brain injury, and drowning. Non-limiting exemplary neurodegenerative diseases include Parkinson's disease, spinal muscular atrophy, amyotrophic lateral sclerosis, Huntington's disease and stroke.

[0163] Exemplary causes of respiratory depression that can be treated using the methods and compositions as disclosed herein are described above. In some embodiments, a person of ordinary skill in the art will be able to recognize a subject having respiratory depression by direct observation. Symptoms of respiratory depression can include hypopnea, which is characterized by a slow or shallow respiratory rate. Clinically significant hypopnea is characterized by a 50% or greater reduction in air flow and is associated with a 3% or greater desaturation in blood 02 levels for 10 seconds or longer. In some embodiments, a subject having respiratory depression will show signs of cyanosis, which is a bluish coloration of the skin due to the presence of deoxygenated hemoglobin in blood vessels near the skin surface. Cyanosis occurs when the oxygen saturation of arterial blood falls below 85%.

[0164] In other embodiments, respiratory depression can be diagnosed using a polysomnogram. This is typically done with subjects suspected of having some form of sleep apnea or another medical condition where respiratory rhythm is disturbed during sleep. In other embodiments, respiratory acidosis (a PaCO2>6.3 kPa or 47 mm Hg and a pH of 7.35) is indicative of respiratory depression. In yet other embodiments, respiratory depression can be routinely monitored using pulse oximetry. Respiratory airflow can be monitored with a nasal cannula connected to a pressure transducer, and thoracic and abdominal respiratory movements are routinely monitored with piezoelectric strain gauges, particularly in newborn infants.

[0165] Using the characteristic observations and tests as described above, a person of ordinary skill in the art will be able to recognize a subject having respiratory depression, and thereby in need of treatment with the methods and compositions of the present invention. In addition, to the observations and tests set forth above, a person of ordinary skill in the art will be aware of other characteristic signs, symptoms and tests to recognize and diagnose respiratory depression suitable for use with the present invention. The next step in the practice of the invention is administering the compositions of the present invention to a subject having respiratory depression as described above.

[0166] Dosage / formulation

[0167] The present invention contemplates a variety of techniques and routes of administration of the compounds used in the practice of the invention. Non-limiting exemplary routes of administration suitable for use with the present invention include nasal, oral, sublingual, rectal, transdermal, vaginal, transmucosal, or enteric. Parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraarterial, intraperitoneal, intranasal, intraocular, as well as direct intraventricular injections among others are contemplated. Indeed, it is not intended that the present invention be limited to any particular route of administration.

[0168] In one aspect of the invention, the amount of the compositions described herein administered to a subject is that of an effective amount to reduce or alleviate respiratory depression in the subject. The quantity of the at least one inhibitor of MOR signaling of the present invention in a unit dose preparation can be from about 0.001 mg / kg body weight to about 100 mg / kg body weight. Preferably the amount of the at least one inhibitor of MOR signaling of the present invention per unit dose will range from about .01 mg / kg to about 50 mg / kg. More preferably, the unit dose of the at least one inhibitor of MOR signaling of the present invention will be in the range of about 0.1 mg / kg to about 10 mg / kg body weight. The composition can, if desired, also contain other compatible therapeutic agents.

[0169] The exact dose to be administered to a subject having respiratory distress will depend on a number of factors unique to the individual subject to be treated, and the particular inhibitor of MOR signaling being administered, and are ultimately the responsibility of the treating physician (or veterinarian). It is contemplated that as part of the subject evaluation, it is well within the skill of the care provider to know how to and when to terminate, interrupt, or adjust administration due to toxicity, etc. Conversely, the care provider will also know how to adjust treatment to higher levels in circumstances where clinical response is inadequate, while precluding toxicity. The magnitude of an administered dose in the management of respiratory depression will vary with the particular agents administered, route of administration, the severity of the respiratory depression and the individual subject’s physiology, biochemistry, etc. The severity of the respiratory depression can be evaluated in part by standard methodologies, and the dose, and frequency of administration will also depend in part on the age, body weight, sex, and response for each individual subject.

[0170] When the compositions of the invention are prepared for administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations include from 0.1 to 99.9% by weight of the formulation. A “pharmaceutically acceptable” is a carrier, diluent, excipient, and / or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. The active ingredient for administration may be present as a powder or as granules; as a solution, a suspension or an emulsion.

[0171] Pharmaceutical formulations containing the compositions of the invention can be prepared by procedures known in the art using well known and readily available ingredients. The compositions of the invention can also be formulated as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.

[0172] The pharmaceutical formulations of the compositions of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

[0173] Thus, the compositions may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0174] It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.

[0175] The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific nonlimiting examples of the carriers and / or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions pH 7.0-8.0. Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.

[0176] Accordingly, the compositions of the present invention may be delivered via various routes and to various sites in a mammal body to achieve a particular effect as is routinely performed in the art. One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.

[0177] The active ingredients of the present invention can be provided in unit dosage form wherein each dosage unit, e.g., a teaspoonful, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. The term “unit dosage form” as used herein refers to physically discrete units suitable as unitary dosages for human and mammal subjects, each unit containing a predetermined quantity of the compositions of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the compositions in the particular host.

[0178] These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

[0179] Kits

[0180] The invention is also directed to kits which comprise the compositions of the present invention and instructions for the methods of their use. Also provided are kits comprising a device for administration of one or more compositions of the present invention as described elsewhere herein by the methods as described elsewhere herein.

[0181] EXPERIMENTAL EXAMPLES

[0182] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0183] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0184] Example 1 : Reversal agents for ultra-potent synthetic opioid-induced overdose and respiratory depression

[0185] Fentanyl is an ultra-potent synthetic (LIPS) opioid designated as one of the high consequence chemicals of concern by United States Government agencies, including the Department of Homeland Security. Fentanyl has high in vivo potency and rapid onset of action that contribute to its therapeutic use as an analgesic. However, its potential for addiction and high toxicity are factors of concern; it is also considered a serious chemical threat by military and civilian agencies. Fentanyl is a p-opioid receptor (MOR) agonist with complex pharmacology. Similarly with other GPCR agonists, fentanyl conveys intracellular signals via two major pathways involving G proteins-second messenger- mediated signaling: 1) inhibition of adenyl cyclase, and 2) P-arrestin-mediated signaling. Respiratory depression, the most severe adverse effects of fentanyl, has a high lethality rate. There is an incomplete understanding of the mechanisms by which fentanyl produces respiratory depression and an urgent need to identify approaches to treat the respiratory depression induced by fentanyl. In the -arrestin KO mouse model but not in the MOR KO mouse model, fentanyl induces respiratory depression, suggesting an additional mechanism involved in fentanyl-induced respiratory depression. These results demonstrated that activation of MOR by fentanyl in addition to Gai / o-mediated cAMP inhibition and P-arrestin signaling, elicits signaling through its C-terminal tail and activates RhoGEF / RhoA / PLD / choline / Sigma-1 receptor (Sig-IR) (Figure 1A and Figure IB). Herein it is demonstrated that SC administered fentanyl-induced respiratory depression is produced via a third signaling pathway initiated by the C-motif PDZ- binding domain of MOR and that blocking this third MOR signaling pathway postexposure to fentanyl can reverse the respiratory depression induced by fentanyl in rats within minutes. These results support new non-opioid agents as novel therapeutic approach to treat / reverse (within minutes) respiratory depression induced by fentanyl. This can be life-saving for vulnerable populations, conferring protection to military personnel and chemical incident responders entering an area contaminated with UPS opioids (fentanyl). Background Fentanyl, an UPS opioid, is a highly potent narcotic analgesic (Kelly et al., 2021, Br J Pharmacol., 180(7) :797-812), used for the management of severe pain in patients for whom alternative treatment options are ineffective, not tolerated, or could not provide sufficient management of pain. Fentanyl and the structurally-related medicinal and illicit drugs are collectively named ‘fentanyls’; they have been responsible for over half of the opioid overdose deaths in the United States in the last few years (Hill et al., 2020, Br J Pharmacol., 177(2):254-266). Overdose deaths increased during the early pandemic period. In 2022, 12-month drug overdose fatalities reached their highest recorded level in the United States, driven primarily by out-of- hospital deaths that were associated with fentanyl, from 46.6 per million in 2016 to 178.0 per million in 2021, a 282% increase (95% confidence interval [CI], (275 to 289) (Jeffery et al. 2023, N Engl J Med 389(1) 87-88). Fentanyl is also a potential chemical weapon. Respiratory depression is one of the life-threatening immediate adverse effects of fentanyl and is more rapid with fentanyl than equipotent doses of heroin or morphine; fentanyl depresses both respiratory rate and tidal volume (Hill et al., 2020, Br J Pharmacol., 177(2):254-266). Fentanyl is an agonist of MOR, a GPCR mainly coupled to Gai / o signaling (Kelly et al., 2021, Br J Pharmacol., 180(7):797-812; Connor & Christie 1999). MOR, similarly with other GPCRs, when stimulated by agonists, convey intracellular signals via two major pathways involving i) G proteins-second messenger mediated signaling producing cAMP inhibition, and ii) [3-arrestin-mediated signaling (Koehl et al., 2018, Nature, 558(7711):547-552; Huang et al., 2015, Nature, 524(7565):315-21; Bouchet et al., 2021, Mol Pharmacol., 100(3):217-223). Fentanyl has a complex pharmacology: in in vitro radioligand binding studies, fentanyl has been shown to have similar affinity for MOR with morphine (McPherson et al. 2010; Schmid et al., 2017, Cell, 171(5): 1165-1175; Toll et al. 1998, NIDA Res Monogr 178: 440-466). In the well-established [35S]GTPyS binding assay to measure potency and efficacy for GPCR activation (Guo et al. 2022) , fentanyl was reported to be only slightly more potent (2.4 fold average) than morphine (Heusler et al. 2016, J Recept Signal Transduct Res 36(2) 158-66; Schmid et al., 2017, Cell, 171(5): 1165-1175; Hassanien et al., 2020, Drug Test Anal., 2(8): 1212- 1221). However, several in vivo studies indicate that fentanyl is at least 50-100 fold more potent than morphine for inducing MOR related behavioral effects such as antinociception or respiratory depression (Sirohi et al. 2008, Pharmacol Biochem Behav 91(1): 115-120; Schwienteck et al., 2019, Neuropharmacology, 150:200-209; Kelly et al., 2021, Br J Pharmacol., 180(7):797-812). While fentanyl and morphine are both agonists of MOR, in silico studies revealed that fentanyl, unlike morphine, may interact with the orthosteric binding pocket of MOR in two or three binding poses (Dosen-Micovic et al. 2006, Bioorg Med Chem 14(9): 2887-2895; Ellis et al. 2018, PLoS One 13(5) e0197734; Eshleman et al. 2020, Biochem Pharmacol 182: 114293). High (3.5A) resolution cryo-electron microscopy (cryo-EM) studies characterized the structure of the MOR bound to the agonist peptide DAMGO clarifying the structural features that contribute to the Gai / o protein coupling specificity of the MOR (Koehl et al., 2018, Nature, 558(771 l):547-552). The higher potency and efficacy of fentanyl in intact cells and in vivo assays despite its similar affinity with morphine at MOR was correlated with its higher lipid solubility, which also explains the ability of fentanyl to rapidly enter the CNS with consequent fast onset of centrally mediated effects relative to morphine and heroin (Hill et al., 2020, Br J Pharmacol., 177(2):254-266). With regard to fentanyl induced cell signaling and potential bias, earlier studies did not support bias signaling (McPherson et al. 2010), while more recent studies reported that fentanyl is arrestin- biased (Schmid et al., 2017, Cell, 171(5): 1165-1175), or G protein-biased (Crowley et al. 2020, ACS Chem Neurosci 11(12): 1781-1790). Respiratory depression results from activation of the MOR (Matthes et al., 1998, J Neurosci., 18( 18): 7285-95; Shook et al. 1990, Am Rev Respir Dis 142(4): 895-909); it is not observed in MOR KO mouse model (Schmid et al., 2017, Cell, 171(5): 1165-1175; Hill et al., 2020, Br J Pharmacol., 177(2):254-266), while it is not attenuated in the P-arrestin2 KO mice (Kliewer et al. 2020, Br J Pharmacol 177(13): 2923-2931). Naloxone, a short-acting MOR competitive antagonist reverses the respiratory depressant effects of morphine and heroin, while multiple doses of naloxone are needed to reverse the effect of fentanyl in people suffering from a fentanyl overdose (Kelly et al., 2021, Br J Pharmacol., 180(7):797-812; Mahonski et al. 2020, Clin Toxicol (phila) 58: 117-123). There is a critical need to better understand the mechanisms of respiratory depression produced by fentanyl in order to provide effective approaches for treatment of overdose.

[0186] In addition to canonical second messengers (cAMP, cGMP and IP3) and P-arrestin signaling, small GTPase proteins are largely involved in GPCR signal transduction (Hilger et al. 2018, Nat Struct Mol Biol 25(1): 4-12; Komolov & Benovic 2018, Cell Signal 41 : 17-24; Yoo et al. 2020, Nat Commun 11(1): 690). Rho GTPases act as molecular switches; they are converted from an inactive GDP -bound state to an active GTP-bound state by guanine nucleotide exchange factors (GEFs) (Wennerberg et al. 2005, J Cell Sci 118(Pt 5) 843-846). RhoGEFs are the best understood regulators of Rho activation (Buchsbaum 2007, J Cell Sci 120 Kim (Pt 7): 1149-1152); they provide a direct link for activation of RhoA by GPCRs (Hart et al. 1996, J Biol Chem 271(41): 25452- 25458; Reuther et al. 2001, J Biol Chem 276(29): 27145-27151 ; Rum enapp et al. 1999, Febs Lett 459(3) 313-318; Togashi et al. 2000, J Biol Chem 275(38): 29570- 29578). Rho-GEFs can be activated by Gaq, Gal2 / 13 and GPy (Aittaleb et al. 2010, Mol Pharmacol 77(2) 111-125).

[0187] PDZ domains are structural protein domains that recognize simple linear amino acid motifs (also called receptor’s PDZ-binding domain), often at the protein C- terminal, that mediate protein-protein interactions. PDZ proteins (including PDZ- RhoGEF) bind through a well-defined pocket to linear motifs that are mostly situated at the end of the C-termini of receptors. PDZ-RhoGEF, a predominantly brain specific Rho- GEF (Fukuhara et al., 1999, J Biol Chem., 274(9):5868-79) can also be activated directly by GPy and by the C-terminal tail of certain receptors (Slepak & Pronin, 2020, JBC, 295(50): 16929-16930). The last residue (referred to as position pO) in PDZ-binding motifs is usually a hydrophobic amino acid (Luck et al., 2011, PLoS One., 6(1 l):e25376), proline in case of MOR. Very recently it has been shown that Gial-WT did not significantly increase Rho-GEFs activity and only the Q204L (Leu) mutant where glutamine (Gin) 204 is mutated to leucine (Leu) (Berman et al., 1996, Cell, 86(3):445-52) can elicit signaling through PDZ-RhoGEF (Chandan et al., 2022, Sci Signal, 15(717):eabi9869). In addition, studies in mutant mouse models with truncated C termini revealed divergent roles for the C termini in morphine-induced behaviors, supporting the importance of C-terminal variants in the complex morphine actions. (Xu et al. 2017, J Clin Invest 127(4): 1561-1573). No signaling role has been ascribed yet to the PDZ- binding domain of the C terminal tail of MOR. RhoGEFs activate RhoA, a regulator of cytoskeletal dynamics (Chircop et al., 2014, SmallGTPases, e29770. doi: 10.4161 / sgtp.29770). RhoA directly activates phospholipase D (PLD) and enhances its Km (Exton et al., 2002, FEBS Lett., 53 l(l):58-61 ; Bruntz et al. 2014). Activation of PLD metabolizes phosphatidylcholine and generates two second messengers: phosphatidic acid (PA) and choline (Exton et al., 2002, FEBS Lett., 531(1):58-61). Relatively recent, it was demonstrated that choline is a second messenger that activates the Sigma- 1 receptor (Sig-IR) (Brailoiu et al., 2019, Cell Rep., 26(2):330-337). MOR has been shown to activate PLD (Mangoura & Dawson 1993, Proc Natl Acad Sci USA. 90(7): 2915-2919; Yang et al. 2010, Exp Neurol 221(1): 166-174), however the mechanism remained unclear. Also, Sig-IR has been involved in the effect of opioids; for example, inhibition of Sig-IR potentiates antinociceptive effects of morphine and disrupts opioid tolerance (Kim et al. 2010, Mol Pharmacol 77(4): 695-703; Montilla-Garcia et al. 2019, Front Pharmacol 10: 136; Mena-Valdes et al. 2021, Eur J Pharmacol 893: 173825; Zhuang et al. 2021, Eur J Med Chem 223: 113658). However, the role of Sig-IR in MOR signaling has not been clearly established.

[0188] The results described herein indicated that i) activation of MOR by fentanyl, oxycodone and morphine activates RhoA, increases choline level in MOR-WT transfected cells but not in cells transfected with MORAAA (C-terminal mutant) and the effect is abolished by inhibition of RhoGEF; ii) fentanyl, oxycodone and morphine activate RhoA and increase choline level in MOR-expressing neurons; iii) naloxone abolishes the effect of morphine, reduces the effect of oxycodone and partially reduces the effect of fentanyl on RhoA activation and choline production indicating an additional mechanism employed by fentanyl; iv) in vivo studies indicate that fentanyl -induced respiratory depression can be reversed by blocking the third MOR signaling pathway using nonopioid agents that inhibit RhoGEF and Sig-IR; combination of naloxone and RhoGEF inhibitor produced a much faster recovery of respiratory function (1 min).

[0189] Results

[0190] Determination of the role of MOR C-terminal PDZ-binding domain in MOR signaling by fentanyl.

[0191] MOR is a GPCR that mainly couples to Gai / o signaling (Kelly et al., 2021, Br J Pharmacol., 180(7):797-812). MOR, similarly with other GPCRs, when stimulated by agonists, convey intracellular signals via two major pathways involving: 1) G proteins-second messenger-mediated signaling producing cAMP inhibition, and 2) P- arrestin-mediated signaling. Inspection of the sequence alignment of the C-terminal tail of MOR indicated the presence of a type II PDZ binding domain conforming to the X— -X-<I» consensus where 0 is a hydrophobic amino acid (Proline [P] in the case of MOR, Figure IB) (Fuh et al. 2000). This observation combined with results of previous studies that PDZ-RhoGEF can be activated by the C-terminal tail of certain receptors (Slepak & Pronin, 2020, JBC, 295(50): 16929-16930) and RhoGEFs activate RhoA, a regulator of cytoskeletal dynamics (Chircop et al., 2014, SmallGTPases, e29770. doi:

[0192] 10.4161 / sgtp.29770) prompted determination of the role of MOR C-terminal PDZ- binding domain in MOR signaling by fentanyl. Moreover, RhoA activates PLD (Exton et al., 2002, FEBS Lett., 531(1):58-61 ; Bruntz et al., 2014, Pharmacol Rev 66(4): 1033- 1079) that can generate choline from phosphatidylcholine (Exton et al., 2002, FEBS Lett., 531(1):58-61). Relatively recent, it was demonstrated that choline is a second messenger that activates the Sig-IR (Brailoiu et al., 2019, Cell Rep., 26(2):330-337). The in vitro experiment results indicated that the MOR through its C-terminal tail activates RhoGEFs / RhoA / PLD / choline / Sig-lR (Figure 1 A and Figure IB) To investigate the role of the C-terminal tail in MOR-induced signaling, the C-terminal motif -PLP (Figure 1A) was mutated to -AAA. HEK293 cells were transiently transfected with MOR plasmids (MOR-WT) or AAA mutant (MOR- AAA) using the Lipofectamine 2000, as reported (Brailoiu et al., 2019, Cell Rep., 26(2):330-337). RhoA activity and choline level in response to morphine and fentanyl treatment in MOR-WT and MOR-AAA cells were determined and compared.

[0193] Mutation of the MOR receptor C-terminal PDZ -binding domain does not affect cAMP inhibition by MOR agonists.

[0194] Since MOR is mainly coupled to Gai / o signaling, the effect of morphine, oxycodone and fentanyl on forskolin-induced cAMP level was investigated using cAMP Response Element (CRE) reporter assays. HEK293 cells were transiently transfected with both CRE-RE reporter and MOR plasmids (WT or AAA mutant) by using Lipofectamine 2000. Cells were cultured in a 96-well plate and grown overnight. After forskolin (1 pM) addition, cells were treated with morphine, oxycodone or fentanyl (10"11-10"5M) for 10 min and incubated for 5 h at 37°C. Cells were lysed and luciferin was added. Luminescence was measured by a plate reader. Luminescence values are given as relative light units (RLU) as % control.

[0195] Stimulation of MORWT or MOR-AAA transfected HEK293 cells with morphine, oxycodone or fentanyl significantly inhibited cAMP Response Element (CRE) (n=6) (Figure 2 and Figure 23). Similar to previous studies (Kelly et al., 2021, Br J Pharmacol., 180(7):797-812), fentanyl was slightly more potent than morphine or oxycodone. The responses were similar in MOR-WT and MOR-AAA cells indicating that the mutation of C-terminal does not affect the ability of MOR to inhibit cAMP; however mutation of aspartate 116 of MOR (M0R-D116) abolished the response (Figure 3.

[0196] MOR activation increases RhoA activity.

[0197] In other experiments the RhoA activity was determined using the RhoA G-LISA Activation assay kit (Cytoskeleton Inc, Cat # BK124-S). MOR-WT and MOR- AAA cells were cultured in a 96-well plate and grown overnight. Fentanyl oxycodone or morphine (10'7-10'5M) were added to the cells for 10 min. RhoA activity was measured by assessing the absorbance at 490 nm. Fentanyl, oxycodone and morphine produced dose-dependent increases in RhoA activity in MOR-WT cells. At the lowest concentration tested (0.1 pM), fentanyl increased RhoA activity about 2 times higher than oxycodone and 3.5 times higher than morphine (n=6) (Figure 3) indicating that in agreement with previous studies (Kelly et al., 2021, Br J Pharmacol., 180(7):797-812) fentanyl had higher efficacy and potency than morphine and oxycodone.

[0198] MOR activation increases choline level.

[0199] As RhoA can activate PLD that produces choline from phosphatidylcholine (Exton et al., 2002, FEBS Lett., 531(1):58-61), other experiments to measure the choline levels as previously reported (Brailoiu et al., 2019, Cell Rep., 26(2):330-337) in response to several concentrations of fentanyl, oxycodone and morphine (10-7-10-5M) was carried out. Fentanyl (0.1 pM, 1 pM, 10 pM) increased choline level to 972 ± 54 pM, 1298 ± 67 pM and 1481 ± 74 pM, respectively; oxycodone in the same concentration, increased choline to 378 ± 46 pM, 834 ± 61 pM and 1138 ± 72 pM while morphine in the same concentrations increased choline just to 27 ± 14 pM, 384 ± 52 pM and 859 ± 63 pM) (n=6) in MOR-WT cells (Figure 4), indicating that fentanyl increased choline with higher efficacy and potency than oxycodone and morphine.

[0200] Mutation of MOR C-terminal abolished the activation of RhoA by fentanyl.

[0201] In these experiments, fentanyl (1 pM) was added to MOR-WT and MOR- AAA cells for 10 min and RhoA activity was determined using the G-LISA RhoA Activation assay kit, as described earlier. In MOR-WT, fentanyl (1 pM) increased the RhoA activity 4.7 times higher than the basal RhoA level (0.84 ± 0.083 versus 0.18 ± 0.011). Treatment with pertussis toxin (PTX, 1 pM), Gai / o inhibitor, did not significantly reduced RhoA activity induced by fentanyl (0.81 ±0.087 in the presence of PTX, versus 0.84 ±0.083 in the absence of PTX) (Figure 5 and Figure 27). In MOR-AAA mutant, in which basal RhoA activity (0.2 ± 0.016) was similar to MOR-WT cells (0.18 ± 0.011), fentanyl did not produce an increase in RhoA significantly different from basal level (0.29 ± 0.043) (n=6) (Figure 5). These results indicate that the C-terminal of MOR is critical for fentanyl-induced RhoA activation that occurs via a Gai / o-independent mechanism.

[0202] Mutation of C-terminal of MOR abolished the increase in choline level by fentanyl, oxycodone and morphine.

[0203] RhoA activation produces an activation of PLD followed by production of choline (Exton et al., 2002, FEBS Lett., 531(1):58-61). The effect of MOR agonists on choline level was then determined. Fentanyl, oxycodone or morphine (1 pM) were added to the MOR-WT and MOR-AAA cells for 10 min; choline was measured as reported (Brailoiu et al., 2019, Cell Rep., 26(2):330-337). Basal choline level in MOR-WT and MOR-AAA cells was 23 ± 6 pM similar to that reported before (Brailoiu et al., 2019, Cell Rep., 26(2):330-337). Fentanyl significantly increased choline level to 1298 ± 67 pM in MOR-WT, while did not affect it in MOR-AAA (136 ± 14 pM) (n =6); similarly, oxycodone and morphine increased choline in MOR-WT, but not in MOR-AAA (Figure 6). Pretreatment with PTX (1 pM) did not reduce the effect of any of the three MOR agonists on choline indicating that was not Gai / o-mediated. MOR activation by MOR agonists increases RhoA activity via RhoGEF. RhoGEFs are the main regulators of RhoA activation (Chircop et al., 2014, SmallGTPases, e29770. doi: 10.4161 / sgtp.29770). The role of RhoGEF in MOR agonists-induced RhoA activity was examined by pretreating MOR-WT cells with Y16 (10 pM), RhoGEF inhibitor (Diviani et al., 2016, Cell Chem Biol., 23(9): 1135-1146) before treatment with fentanyl, oxycodone or morphine (1 pM). Y16 (10 pM) reduced the RhoA activity produced by MOR agonists in MOR-WT cells to levels similar to control (n = 6) (Figure 7), indicating that the increase in RhoA activity is RhoGEF -dependent. MOR activation increased choline production via RhoGEF-dependent mechanism.

[0204] The effect of RhoGEF inhibition on the ability of MOR agonists to increase the choline level was then tested. Pretreatment of MORWT cells with Y16 (10 pM), RhoGEF inhibitor, abolished the increase in choline production by fentanyl, oxycodone and morphine (1 pM), supporting a RhoGEF-dependent mechanism (n = 6) (Figure 8). Fentanyl produced a prolonged increase in choline. Choline level at 1 min, 2 min, 5 min and 10 min after application of fentanyl (1 pM) in MOR-WT cells was examined. Fentanyl increased choline to 1298 ± 67 pM (1 min), 1257 ± 94 pM (2 min), 1139 ± 87 pM (5 min) and 972 ± 79 pM (10 min) (n=6), indicating a prolonged and sustained response (Figure 9).

[0205] Validation of the third MOR signaling pathway in neurons: Fentanyl increased RhoA activity via RhoGEF with higher efficacy than morphine and oxycodone in neurons.

[0206] Experiments were performed in cultured neurons from periaqueductal gray (PAG) an area rich in MOR (Bodnar et al., 1988, Brain Res., 447(l):25-34; Jensen & Yaksh, 1986, Brain Res., 372(2):301-12; Satoh et al., 1983, Life Sci., 1983:33 Suppl 1 : 689-92) and the effect of fentanyl, oxycodone and morphine on RhoA activity was tested and compared. Fentanyl (1 pM) increased RhoA activity with higher efficacy than oxycodone and morphine (1 pM); Gai / o-inhibition with PTX (1 pM) did not significantly affect the effect of any of three MOR agonists; however, their effect was reduced by Y16, indicating that it is mediated by RhoGEF and not produced via Gai / o protein (Figure 10). Basal RhoA activity in neurons was 0.19 ± 0.068; fentanyl (IpM) increased it 5.2 folds (0.61 ± 0.072), PTX did not significantly affect it (0.58 ± 0.081), while Y16 reduced it (0.23 ± 0.054) to levels similar to basal. Morphine in the same concentration (1 pM) increased RhoA activity 2 times over basal level (0.38 ± 0.076) and Y16 reduced it to basal level (0.18 ± 0.036) (n = 6) (Figure 10).

[0207] MOR agonists increased choline level via RhoGEF in neurons. Choline levels were measured in PAG neurons as reported (Brailoiu et al.,

[0208] 2019, Cell Rep., 26(2):330-337). Fentanyl, oxycodone and morphine (1 pM) increased choline levels, with fentanyl having the highest efficacy (Figure 11). The increase in choline produced by fentanyl, oxycodone and morphine was not affected by PTX (1 pM) but significantly reduced by Y16 (10 pM) indicating the participation of RhoGEF and not of Gai / o proteins in this process (n=6) (Figure 11).

[0209] Fentanyl increased choline level with higher potency and efficacy than oxycodone and morphine in neurons.

[0210] Choline level was determined in response to several concentrations of fentanyl, oxycodone and morphine (10’7-10’5M). Similarly to the MOR-WT cells, a doseresponse relationship was determined with fentanyl being more potent and more efficacious than morphine (n=6) (Figurel2). Fentanyl produced a prolonged increase in choline in neurons. Choline level at 1 min, 2 min, 5 min and 10 min after treatment of PAG neurons with fentanyl (1 pM) was examined. Fentanyl produced a prolonged and sustained increase in choline level to 1016 ± 72 pM (1 min), 987 ± 96 pM (2 min), 964 ± 93 pM (5 min) and 842 ± 86 pM (10 min) (n=6) (Figure 13).

[0211] The effect of naloxone on canonical MOR signaling versus C-terminal signaling by fentanyl was determined. Naloxone is a MOR competitive antagonist with higher affinity for the receptor than morphine (Toll et al. 1998, NIDA Res Monogr 178: 440-466). Naloxone is widely used as reversal treatment for opioid overdose (Hill et al.,

[0212] 2020, Br J Pharmacol., 177(2):254-266). A single dose of naloxone can reverse the respiratory depression induced by morphine but multiple doses are needed to reverse the effect of fentanyl (Hill et al., 2020, Br J Pharmacol., 177(2):254-266), indicating that additional mechanisms contribute to the effect of fentanyl. Results demonstrated that naloxone abolishes the effect of morphine and slightly reduces (by about 20%) the effect of fentanyl on RhoA activation and choline production indicating an additional mechanism employed by fentanyl.

[0213] Naloxone abolished the inhibition of cAMP accumulation produced by fentanyl and morphine. The effect of naloxone, MOR competitive antagonist, on the cAMP inhibition by morphine and fentanyl was determined using CRE reporter assays. HEK293 cells were transiently transfected with both CRE-RE reporter and MOR-WT plasmids using Lipofectamine 2000. Cells were cultured in a 96-well plate overnight. After forskolin (1 pM) addition, cells were treated for 10 min with morphine, fentanyl in the absence and presence of naloxone (10 pM). Luminescence values are given as relative light units (RLU) as % control. Stimulation of MOR-WT cells with morphine or fentanyl (10-11-10-5M) significantly inhibited CRE; naloxone reversed the CRE inhibition by morphine and fentanyl (Figure 14 and Figure 37) (n=6). Naloxone abolishes RhoA activity induced by morphine and oxydocone but only slightly reduces RhoA activity induced by fentanyl. In MOR-WT cells naloxone (10 pM) abolished the RhoA activity increased by morphine (1 pM), and oxycodone (1 pM) but produced only a slight reduction in fentanyl (1 pM)-induced increase RhoA activity (by 21%), indicating an additional mechanism responsible for fentanyl-induced RhoA activity (n=6) (Figure 15 and Figure 38). Of note, fentanyl (1 pM) produced an increase in RhoA activity higher than morphine (1 pM).

[0214] Naloxone abolished the increase in choline produced by morphine and oxycodone, but only slightly reduced the increase in choline produced by fentanyl.

[0215] In MOR-WT cells fentanyl (1 pM)-induced increase in choline level was slightly reduced by naloxone (eg, from 1298 ± 67 pM to 1037 ± 82 pM, by 20%). Morphine (1 pM) increased choline level (334 ±32 pM) to about one third of that increased by fentanyl (1298 ± 67 pM); the increase in choline produced by morphine was abolished by naloxone (31± 14 pM) (n = 6) (Figure 16 and Figure 39). Naloxone abolishes the increase in choline produced by morphine and but only slightly the increase in choline by fentanyl in neurons. In cultured PAG neurons that express high level of MOR (Bodnar et al., 1988, Brain Res., 447(l):25-34; Jensen & Yaksh, 1986, Brain Res., 372(2):301-12; Satoh et aL, 1983, Life Sci., 1983:33 Suppl 1 :689-92) fentanyl-induced increase in choline level (1016 ± 72 pM) was slightly reduced by naloxone (784 ± 91 pM, by 22 %). Morphine increased choline with lower efficacy than fentanyl (268 ± 47 pM; 25% of the increase by fentanyl, indicating that fentanyl was 4 times more efficacious than morphine). Naloxone (10 pM) abolished the increase of choline produced by morphine (28 ± 24 pM) similarly to basal level (21 ± 11 pM) (n = 6) (Figure 17).

[0216] The in vivo the role of MOR C-terminal PDZ domain- RhoGEF / RhoA / PLD / choline / Sig-lR pathway in fentanyl-induced respiratory depression.

[0217] Fentanyl overdose induces respiratory depression by acting on respiratory- related areas of brainstem in the dorsolateral pons (Saunders et al., 2022, J Neurophysiol., 128(5): 1117-1132) however the specific mechanism is incompletely understood. MOR binding inhibits adenyl cyclase, thereby reducing cAMP level and facilitating neuronal hyperpolarization via activation of K+ channels (Santhappan et al., 2015, JNC., 135( 1): 76-87). In vitro results indicated that fentanyl signals also through C-terminal PDZ-binding domain via RhoGEF / RhoA / PLD / choline / Sig-lR (Figures 2-16, Figures 23- 39). The results indicated that morphine and fentanyl hyperpolarize cultured brainstem respiratory neurons, while morphine-induced hyperpolarization is abolished by naloxone, the hyperpolarization produced by fentanyl is only reduced by naloxone but abolished by inhibition of RhoGEF and Sig-IR (Figures 18, 19, 40). Moreover, in vivo experiments indicate that non-opioid agents that inhibit the third signaling pathway, for example RhoGEF inhibitor, Y16 (Figure 42), RhoA inhibitor, CCG-1432 (Figure 43), and Sig-IR antagonist, BD-1047 (Figure 44) administered after fentanyl can rescue the respiratory depression induced by fentanyl overdose within minutes. Moreover the combination of low dose naloxone (0.5 mg / kg) with RhoGEF inhibitor, Y16 (Figure 47), with RhoA inhibitor, CCG-1423 (Figure 48) or with Sig-IR antagonist, BD-1047 (Figure 49), produced a faster reversal than naloxone 0.5 mg / kg. Morphine-induced neuronal hyperpolarization is abolished by naloxone. Membrane potential was measured in cultured brainstem respiratory neurons by voltage imaging as previously reported (Brauner et al. 1984, Biochim Biophys Acta 771 : 208-216; Brailoiu et al. 2008, Neuroscience 151(3) 701-710; Brailoiu et al. 2014, Am J Physiol Regul Integr Comp Physiol 306(11): R814-R822). Morphine (1 pM) produced a sustained hyperpolarization of neurons by 5.58 ± 0.61 mV (n = 6) which was abolished by naloxone (10 pM) (Figure 18A and Figure 18B). Fentanyl induced neuronal hyperpolarization was reduced by naloxone and reversed by inhibition of RhoGEF and Sig-IR.

[0218] Fentanyl (1 pM) produced a sustained hyperpolarization of neurons (11 ± 0.82 mV (n =6). Treatment with naloxone (10 pM) on the plateau of fentanyl induced hyperpolarization reduced the amplitude of hyperpolarization (4,2 ± 0.57 mV) (n = 6). Application of RhoGEF inhibitor Y16 (10 pM) and Sig-IR antagonist, BD1047 (20 pM) on the plateau of hyperpolarization produced by fentanyl abolished the hyperpolarization (Figure 19A and Figure 19B).

[0219] RhoGEF inhibitor and Sig-IR inhibitor reversed fentanyl-induced in vivo respiratory depression.

[0220] Experiments were carried out using in vivo measurement of respiratory function (oxygen saturation) in rats using a MouseOx Plus Small Animal Vital Signs Monitor via neck clip as previously described (Wang et al., 2020, Molecules, 25(l l):2640; Inan et al., 2021, Life Sci., 15:285: 120014; Bongiovanni et al., 2022, Drug Alcohol Depend., 1 :238: 109556). Rats were injected with either saline (vehicle for fentanyl) or fentanyl (100 pg / kg) subcutaneously (SC). Six min later, rats were given either Y16 (20 mg / kg) and saline, Y16 (20 mg / kg) and BD 1047 (10 mg / kg), or vehicle (1 : 1 : 18; ETOH: cremophor: saline, as vehicle for Y16) and saline via intraperitoneal (IP) injections (volume of injection, 1 ml / kg). The respiratory function (oxygen saturation) was recorded for 40 min. Fentanyl (100 pg / kg), dose similar to previous studies (Haouzi et al. 2020), induced decrease in oxygen saturation-respiratory depression, and thoracic rigidity, as reported (Saunders et al., 2022, J Neurophysiol., 128(5): 1117-1132). Administration of Y16 (20 mg / kg), RhoGEF inhibitor slowly reversed the respiratory depression produced by fentanyl, while administration of Y16 (20 mg / kg) and BD 1047 (10 mg / kg), Sig-IR antagonist, 6 min after fentanyl reversed the respiratory depression faster (n=4) (Figure 20), indicating that blocking both the beginning of the pathway (RhoGEF inhibition) and the end of the pathway (Sig-IR antagonism) was more efficacious. RhoGEF inhibitor and naloxone reversed fentanyl-induced in vivo respiratory depression.

[0221] Administration of naloxone (Img / kg) and Y16 (20 mg / kg), administered 6 min after fentanyl (200 pg / kg) reversed the fentanyl-induced respiratory depression within 1 min (Figure 21) (n=4); the bottom graph indicates the area outlined (red rectangle) in the top graph. Control experiments, IP injection of saline-vehicle-saline or saline-Y16 (20 mg / kg)-saline alone did not affect the oxygen saturation (n=6) (Figure 22).

[0222] Inhibition of MOR C-terminal PDZ domain-RhoGEF / RhoA / / Sig-lR pathway rescue the fentanyl-induced respiratory depression

[0223] In vivo experiments demonstrate that fentanyl overdose-induced respiratory depression (reduction in oxygen saturation) is reversed by the administration of RhoGEF inhibitor, Y16 (20 mg. kg, IP) (Figure 42), RhoA inhibitor, CCG-1432 (5mg / kg, IP) (Figure 43), and Sig-IR antagonist, BD-1047 (10 mg / kg, IP) (Figure 44) administered after fentanyl (200 pg / kg, SC). In addition, the administration of the combination of RhoGEF inhibitor, Y16 (20 mg. kg, IP) and Sig-IR antagonist, BD-1047 (10 mg / kg, IP) after fentanyl (200 pg / kg, SC) produces a faster recovery of oxygen saturation, as measure of respiratory depression, than each agent alone (Figure 45 compared to Figure 42 and 44). Naloxone (Img / kg, IP) administered after fentanyl (200 pg / kg, SC) restored the oxygen saturation, while lower dose of Naloxone (0.5 mg / kg, IP) improves but does not restore the oxygen saturation within the same time interval with the high dose of Naloxone (Img / kg, IP) (Figure 46). Importantly, the combination of low dose naloxone (0.5 mg / kg) with RhoGEF inhibitor, Y16 (20 mg.kg, IP) (Figure 47), or with RhoA inhibitor, CCG-1423 (5mg / kg, IP) (Figure 48) or with Sig-IR antagonist, BD-1047 (10 mg / kg, IP) (Figure 49), produced a faster reversal than Naloxone 0.5 mg / kg. Reducing the effective dose of Naloxone from Img / kg to 0.5 mg / kg is important to reduce the withdrawal syndrome produced by high dose of naloxone.

[0224] In summary, the results of the experiments demonstrated that blocking this third MOR signaling pathway (Figure 50) post-exposure to fentanyl or combining naloxone with blockers of this new pathway reversed the respiratory depression induced by fentanyl overdose in rats within minutes. This can be life-saving for vulnerable populations, conferring protection to military personnel and chemical incident responders entering an area contaminated with UPS opioids such as fentanyl. The extended significance of this invention is that any compound (opioid, opioid-derived or non-opioid, prodrug administered via intraperitoneal, intravenous, intramuscular, intranasal, sublingual and rectal routes regardless of the formulation) that: i) inhibits the C-terminal- induced signaling and / or ii) inhibitor of RhoGEF / RhoA / PLD / Sigma-1 receptor commercially available (eg, Y16, A13 for RhoGEF’s, all RhoA and PLD inhibitors as well as sigma- 1 receptor antagonists or combinations of these inhibitors) or under development can reverse the adverse effects of UPS opioids such as respiratory depression.

[0225] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention.

[0226] The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A composition for treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid- derivative agonist, wherein the composition comprises at least one inhibitor of Mu opioid receptor (MOR) signaling.

2. The composition of claim 1, wherein the at least one inhibitor of MOR signaling comprises at least one inhibitor of MOR C-motif PDZ-binding domain signaling.

3. The composition of claim 2, wherein the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one inhibitor of PDZ- RhoGEF, at least one inhibitor of RhoA, at least one inhibitor of phospholipase D (PLD), at least one inhibitor of Sigma- 1 -receptor, or any combination thereof.

4. The composition of claim 3, wherein the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one PDZ-RhoGEF inhibitor and at least one Sigma- 1 receptor (Sig-IR) inhibitor.

5. The composition of claim 4, wherein the at least one PDZ- RhoGEF inhibitor is Y16 and the at least one Sig- 1R inhibitor is BD1047.

6. The composition of claim 5, wherein the concentration of Y16 is between aboutl mg / kg and about 50 mg / kg and wherein the concentration of BD1047 is between about 1 mg / kg and about 100 mg / kg.

7. The composition of claim 1, wherein the at least one inhibitor of MOR signaling comprises naloxone and at least one inhibitor of MOR C-motif PDZ- binding domain signaling.

8. The composition of claim 7, wherein the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one PDZ-RhoGEF inhibitor.

9. The composition of claim 8, wherein the at least one PDZ- RhoGEF inhibitor is Y16.

10. The composition of claim 9, wherein the concentration of naloxone is between about 0.3 mg / kg and about 10 mg / kg and the concentration of Y16 is between about 1 mg / kg and about 50 mg / kg.

11. The composition of claim 1, wherein the opioid or opioid- derivative comprises an ultra-potent synthetic (UPS) opioid.

12. The composition of claim 11, wherein the UPS opioid is fentanyl.

13. The composition of claim 1, wherein the opioid- or opioid- derivative-induced overdose or a symptom thereof comprises respiratory depression, central nervous system depression, cardiovascular depression, altered level consciousness, miotic pupils, hypoxemia, acute lung injury, aspiration pneumonia, sedation, hypotension, unresponsiveness to stimulus, unconsciousness, stopped breathing, erratic or stopped pulse, choking or gurgling sounds, blue or purple fingernails or lips, slack or limp muscle tone, contracted pupils, vomiting, or any combination thereof.

14. The composition of claim 13, wherein the opioid- or opioid- derivative-induced overdose or a symptom thereof is respiratory depression.

15. The composition of claim 14, wherein the treating comprises prevention and / or reversal of respiratory depression.

16. The composition of claim 15, wherein the composition is formulated for administration by nasal delivery.

17. A method of treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid-derivative comprising administering a therapeutically effective amount of the composition of any one of claims 1-16 to the subject.

18. The method of claim 17, wherein the administration comprises nasally administering to the subject, via a device adapted for nasal delivery of a formulation by actuation of the device into at least one nostril of the subject, a nasal delivery formulation comprising a therapeutically effective amount of the composition of claim 16.

19. The method of claim 17, wherein the administration occurs prior to exposure to the opioid or opioid derivative.

20. The method of claim 17, wherein the administration occurs about 10 min to about 2 hours prior to exposure to the opioid or opioid derivative.

21. The method of claim 17, wherein the subject is free from opioid- or opioid-derivative-induced overdose or a symptom thereof for at least about 4 hours following administration.

22. The method of claim 17, wherein the subject is free from opioid- or opioid-derivative-induced overdose or a symptom thereof for at least about 8 hours following administration.

23. The method of claim 17, wherein the administration occurs after exposure to the opioid or opioid derivative.

24. The method of claim 17, wherein the administration occurs about 10 min to about 2 hours after exposure to the opioid or opioid derivative.

25. The composition of claim 3, wherein the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one PDZ-RhoGEF inhibitor, at least one RhoA inhibitor, at least one Sigma- 1 receptor (Sig-IR) inhibitor, or any combination thereof.

26. The composition of claim 25, wherein the at least one PDZ- RhoGEF inhibitor is Y16, the at least one RhoA inhibitor is CGG 1423, and the at least one Sig-IR inhibitor is BD1047.

27. The composition of claim 26, wherein the at least one PDZ- RhoGEF inhibitor is Y16, the at least one RhoA inhibitor is CGG 1423, and the at least one Sig-IR inhibitor is BD1047.

28. The composition of claim 27, wherein the concentration of Y16 is between about 1 mg / kg and about 50 mg / kg, the concentration of CGG 1423 is between about 1 mg / kg and about 20 mg / kg, and wherein the concentration of BD 1047 is between about 1 mg / kg and about 100 mg / kg.

29. The composition of claim 7, wherein the at least one inhibitor of MOR C-motif PDZ-binding domain signaling comprises at least one PDZ-RhoGEF inhibitor, at least one RhoA inhibitor, at least one Sig-IR inhibitor, or any combination thereof.

30. The composition of claim 29, wherein the at least one PDZ- RhoGEF inhibitor is Y16, the at least one RhoA inhibitor is CGG 1423, and the at least one Sig-IR inhibitor is BD1047.

31. The composition of claim 30, wherein the concentration of naloxone is between about 0.3 mg / kg and about 10 mg / kg, the concentration of Y16 is between about 1 mg / kg and about 50 mg / kg, the concentration of CGG 1423 is between about Img / kg and about lOmg / kg, and the concentration of BD1047 is between about 5 mg / kg and about 20 mg / kg.

32. A method of treating an opioid- or opioid-derivative-induced overdose or a symptom thereof caused by exposure of a subject to an opioid or opioid- derivative comprising administering a therapeutically effective amount of the composition of any one of claims 25-31 to the subject.

33. The method of claim 30, wherein the administration comprises nasally administering to the subject, via a device adapted for nasal delivery of a formulation by actuation of the device into at least one nostril of the subject, a nasal delivery formulation comprising a therapeutically effective amount of the composition of any one of claims 25-31, wherein the composition is formulated for administration by nasal delivery.

34. The method of claim 32, wherein the administration occurs after exposure to the opioid or opioid derivative.

35. The method of claim 32, wherein the administration occurs about 10 min to about 2 hours after exposure to the opioid or opioid derivative.

36. The method of claim 32, wherein the subject is free from opioid- or opioid-derivative-induced overdose or a symptom thereof for at least about 4 hours following administration.

37. The method of claim 32, wherein the subject is free from opioid- or opioid-derivative-induced overdose or a symptom thereof for at least about 8 hours following administration.

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