WO2021016190A1 - Compositions and methods for modulating drug-use disorders - Google Patents

Abstract

This invention is directed towards compositions and methods for reducing or preventing development of drugs-use disorders that include but not limited by dependence, addiction, desensitization, tolerance, analgesic effect, hyperalgesia, and/or relapse.

Description

COMPOSITIONS AND METHODS FOR MODULATING DRUG-USE DISORDERS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application No.62/876,267, filed on July 19, 2019, the contents of which are hereby incorporated by reference in its entirety.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

FIELD OF THE INVENTION [0004] This invention is directed towards compositions and methods for reducing or preventing development of drugs use disorders that include but not limited by dependence, addiction, desensitization, tolerance, analgesic effect, hyperalgesia, and/or relapse.

BACKGROUND OF THE INVENTION

[0005] Opioids are among the most effective drugs commonly prescribed to treat pain. However, their therapeutic use is limited due to the development of addiction (compulsive engagement in rewarding stimuli, despite adverse consequences), tolerance (reduced responsiveness to the same dose of drug), and withdrawal (an adverse physiological reaction following removal of drug). Adverse effect of opioids and their derivatives burden considerable economic and personal costs to society. SUMMARY OF THE INVENTION

[0006] Aspects of the invention are directed towards a composition comprising a therapeutically effective amount of an analgesic and a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, or any combination thereof.

[0007] Embodiments can further comprise a pharmaceutically acceptable carrier.

[0008] In embodiments, the eEF2 phosphorylation reducer comprises an eEF2 kinase (eEF2K) inhibitor. In embodiments, the eEF2 phosphorylation reducer deceases phosphorylation of eEF2 at Thr56.

[0009] In embodiments, the eEF2K phosphorylation reducer comprises an eEF2K inhibitor.

[0010] In embodiments, the eEF2K inhibitor comprises a compound of Formula I, Formula II, or derivatives thereof:

[0011] R groups for Formula I are described in PCT Publication No. WO2015102752, each of the R groups of which are incorporated by reference in their entireties.

[0012] In embodiments, the analgesic comprises an opioid, opioid peptides, cocaine, ethanol, amphetamine, cannabinoid, a benzodiazepine, derivatives thereof, or any combination thereof.

[0013] Non-limiting examples of an opioid/opiate comprises morphine, fentanyl, methadone, buprenorphine, hydrocodone, oxycodone, heroin, or derivatives thereof.

[0014] Non-limiting examples of a benzodiazepine comprises midazolam, alprazolam, clobazam, clonazepam, clorazepate, chlordiazepoxide, diazepam, estazolam, lorazepam, oxazepam, temazepam, triazolam, or derivatives thereof.

[0015] Aspects of the invention are further directed towards a method of treating a subject afflicted with pain comprising administering to the subject a composition comprising an analgesic and a therapeutically effective amount of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, or any combination thereof.

[0016] Still further, aspects of the invention are directed towards a method of reducing pain in a subject afflicted with a disease comprising administering to the subject a composition comprising an analgesic and a therapeutically effective amount of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, or any combination thereof.

[0017] Yet further, aspects of the invention are directed towards a method of increasing the sensitivity of a subject to an analgesic comprising administering to a subject a composition comprising an analgesic and a therapeutically effective amount of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, or any combination thereof.

[0018] Further aspects of the invention are directed towards a method of reducing or preventing the development of hyperalgesia, reducing or preventing the abuse potential of an analgesic, and/or reducing or preventing a withdrawal symptom after analgesic cessation.

[0019] In embodiments, the analgesic is administered prior to, in conjunction with, or subsequent to administering the eEF2 phosphorylation reducer.

[0020] In embodiments, the subject is administered the composition of claim 1.

[0021] Other objects and advantages of this invention will become readily apparent from the ensuing description. BRIEF DESCRIPTION OF THE FIGURES

[0022] FIG.1 shows chronic Oxycodone administration induces phosphorylation of eEF2 in rat nucleus accumbens. (A) Western blot analysis of P-AMPK, P-PKA, and P-CaMKIIa in lysates containing nucleus accumbens of rats treated with water or oxycodone. (B) Western blot analysis of phospho-eEF2 (also referred to as p-eEF2 or P-eEF2) in lysates containing nucleus accumbens of rats treated with water or oxycodone. Left panel - representative images of western blots of P-eEF2 and total eEF2 nucleus accumbens of animals treated with water or oxycodone. Right panel - graph of the densitometric analysis of phosphor-eEF2 on western blots. The graphs represent the mean ratio of signal of P-eEF2 to total eEF2.

Oxycodone data were normalized to water samples set as 1 (±SEM, n=7, p<0.05).

[0023] FIG.2 shows chronic Oxycodone increases the BDNF protein but not the mRNA level in nucleus accumbens. Rats were treated with water or oxycodone as it is described in FIG.1. (A) Western blot analysis of BDNF in lysates containing nucleus accumbens of rats treated with water or oxycodone. Left panel - representative images of western blots of BDNF and GAPDH in nucleus accumbens of animals treated with water or oxycodone. Right panel - graph of the densitometric analysis of BDNF on western blots. The graphs represent the mean ratio of signal of BDNF to total GAPDH. Oxycodone data were normalized to water samples set as 1 (±SEM, n=3). (B) Real-time PCR analysis of total BDNF mRNA in brain lysates containing nucleus accumbens from rats treated with water or oxycodone. The graphs represent the mean ratio of BDNF mRNA to internal control in each sample.

Oxycodone data were normalized to water samples set as 100% (±SEM, water n=4; oxy n=8).

[0024] FIG.3 shows eEF2K KO mice demonstrate increased analgesic reaction to morphine implant. (A) Measurements of the basal nociceptive reaction using hot plate test in wild type and eEF2K KO mice prior to implantation of morphine or placebo. Hot plate (T=53°C) measurements were taken four days prior to implantation of pellets. Graph represents the mean time it took for the mouse in each group to jump off of the hot plate or to shake or lick its hind paw. The cutoff time for the hot-plate test was 30 seconds. (B)

Comparison of maximum analgesic responses to morphine in wild type and eEF2K KO mice after 4 days of morphine implantation. Graph represents the mean time it took for the mouse in each group to jump off of the hot plate (T=53°C) or to shake or lick its hind paw. The cutoff time for the hot-plate test was 30 seconds. WT+ morphine, n=3; WT + placebo, n=3; KO + morphine, n=1; KO + placebo, n=3. (C) Immuno-fluorescent analysis of µ-opioid receptor-1 (red) in brain sections containing dentate gyrus.

[0025] FIG.4 shows eEF2K KO mice do not develop hyperalgesia or tolerance to chronic morphine injections measured by hot plate test. (A) Development of hyperalgesia following repeated morphine administration. Measurements of the basal nociceptive reaction using hot plate test (T=53°C) in eEF2K KO mice once daily before morphine (KO-M) or vehicle (KO- V) administration. Data for days“-2” and“-1” represent hot plate measurements on days two and one prior to injection experiments. Graph represents the mean time it took for the mouse in each group to jump off of the hot plate or to shake or lick its hind paw. The cutoff time for the hot-plate test was 30 seconds. ; KO + morphine, n=4; KO + vehicle, n=3. (B)

Development of tolerance following repeated morphine administration. Measurements of analgesic reaction using hot plate test (T=53°C) in eEF2K KO mice once daily 30 minutes after morphine (KO-M-30) or vehicle (KO-V-30) administration. Data for days“-2” and“-1” represent hot plate measurements on days two and one prior to injection experiments. Graph represents the mean time it took for the mouse in each group to jump off of the hot plate or to shake or lick its hind paw. The cutoff time for the hot-plate test was 30 seconds. KO-M-30, n=3-4; KO-V-30, n=3.

[0026] FIG.5 shows eEF2K KO mice have similar response in the warm water tail immersion test (TIT) as wild type mice (no treatment). (A) Measurements of the basal nociceptive reaction using TIT in wild type and eEF2K KO mice without any treatment. Time of tail withdrawal from three measurements were averaged for each mouse. Graph represents the mean TIT value for all mice in a corresponding group. (B) Measurements of the basal nociceptive reaction using TIT in wild type and eEF2K KO mice without any treatment. Graph represents the mean value of each of three consecutive measurements for each mouse in a corresponding group.

[0027] FIG.6 shows eEF2K KO mice do not develop hyperalgesia or tolerance after chronic morphine injection measured by TIT. Measurements of the basal nociceptive reaction using TIT (T=49°C) in eEF2K KO mice before morphine (KO-M, n=3-4) or vehicle (KO-V, n=3) administration.

[0028] FIG.7 shows eEF2K KO mice do not develop tolerance after chronic morphine injections measured by TIT. Measurements of analgesic reaction using TIT (T=49°C) in eEF2K KO mice 30 minutes after morphine (KO-M-30, n=3-4) or vehicle (KO-V-30, n=3) administration. TIT measurements were performed as it is described in FIG.6. (A)

Development of tolerance following repeated morphine administration. On days 2, 4, 6, and 8, the TIT measurements were taken three consecutive times with 20 seconds intervals. Time from three measurements were averaged for each mouse. Graph represents the mean TIT value for all mice in a corresponding group. (B) Development of tolerance following repeated morphine administration. On days 10, 11, and 12, the TIT measurements were taken once daily. To avoid tissue damage the cutoff time for the TIT was 30 seconds. Graph represents the mean time it took for the mouse in each group to withdraw a tail.

[0029] FIG.8 shows immuno-staining of hippocampal areas of brain from wild type and eEF2K KO mice. Immunohistochemical analysis of hippocampal areas of brain slices from wild type and eEF2K KO female mice with implanted placebo (described in FIG.3). (A) Brain sections containing hippocampal areas were incubated with primary antibodies against eEF2K (cell signaling, #3692, dilution 1:100). Sections were processed with VectaStain ABC (to detect rabbit primary antibodies) and the signal was visualized by the peroxidase substrate kit DAB (Vector Laboratories, Inc.). The nuclei were stained using the hematoxylin Gill’s 3 formulation. Images were taken using light microscope BX43F (Olympus). Top panel - C3 hippocampal areas. Bottom panel– C1 hippocampal areas. (B) Brain sections containing hippocampal areas were incubated with primary antibodies against eEF2K and phosphor- eEF2 (#PA5-38085, dilution 1:100) and processed as it is described in (A). Images represent C3 hippocampal areas. Scale bar is 20 µm for all images.

[0030] FIG.9 shows immuno-histochemical staining of hippocampal areas of brains from wild type and eEF2K KO mice. Immunohistochemical analysis of hippocampal areas of brain slices from wild type and eEF2K KO male mice after chronic administration of morphine or vehicle (described in FIG.4). Brain slices were prepared as it is described in FIG.8. (A) Brain sections containing hippocampal areas were incubated with primary antibodies against phosphor-eEF2. Sections were processed as it is described in FIG.8(A). Scale bar is 500 µm for all images. (B) Brain sections containing hippocampal areas were incubated with primary antibodies against BDNF (Santa Cruz, # sc-546), dilution 1:100) and processed as it is described in FIG.8(A). Scale bar is 500 µm for all images.

[0031] FIG.10 shows summary of identified eEF2K inhibitors (adapted from Liu, Rui, and Christopher G. Proud. "Eukaryotic elongation factor 2 kinase as a drug target in cancer, and in cardiovascular and neurodegenerative diseases." Acta Pharmacologica Sinica 37.3 (2016): 285.)

[0032] FIG.11 shows confirmed inhibitors of eEF2K activity (adapted from Xiao, Ting, et al. "A high-throughput screening assay for eukaryotic elongation factor 2 kinase inhibitors." Acta Pharmaceutica Sinica B 6.6 (2016): 557-563).

[0033] FIG.12 shows number of wild type or eEF2K KO mice that will be use in each type of analysis. VH– vehicle, and Mph– morphine treated mice; WB lysates - western blot of brain lysates; IHC/IF BS– immunohistochemical or fluorescence analyses of paraffin imbedded brain slices; Frozen BS– frozen brain slices.

[0034] FIG.13 shows eEF2K role in opioid reinforcing effect, dependence and tolerance.

[0035] FIG.14 shows chronic oxycodone administration induces phosphorylation of eEF2 in rat nucleus accumbens. Western blot analyses of P-eEF2 (A) and BDNF (B) in nucleus accumbens lysates of rats treated with water (W) or oxycodone (O). Oxycodone data are normalized to water controls.

[0036] FIG.15 shows A. Hot plate test of the WT and eEF2K KO mice four days after implantation of 25 mg morphine or placebo. B. Immunofluorescence analysis of MOR1 (red) in hippocampal brain sections from WT and KO mice. [0037] FIG.16 shows immunohistochemical analysis of P-eEF2 (A) and BDNF (B) in hippocampal areas of wild type and eEF2K KO mice on 4th day after morphine (M) or placebo (Pl) implants.

[0038] FIG.17 shows warm water tail immersion tests (TIT) in wild type and eEF2K KO mice after morphine administration. A. On days 2, 4, 6, and 8, the TIT were taken three consecutive times with 20 seconds intervals. B. On days 10, 11, and 12, the TIT

measurements were taken once.

[0039] FIG.18 shows IHC staining of brain stem tissues of female WT mice treated with saline or morphine for 30 days. Scale is 100 µm for all images.

[0040] FIG.19 shows (A) hot plate test of WT and eEF2K KO mice prior the morphine administration every 24 hours. (B) Hot plate test of WT and eEF2K KO mice prior the morphine administration on days 3 and 9 after daily morphine. (C) Warm water tail immersion test of WT mice treated with the eEF2K inhibitor A-484954 or its vehicle on days 2 and 10 after daily morphine administration.

[0041] FIG.20 shows western blot analysis of P-eEF2 in MCF7 cells treated with buffer (N) or Paclitaxel (Px).

[0042] FIG.21 shows opioid-induced conditioned place preference (CPP) and relapse. BL – preconditioning baseline session; CPP– conditioned place preference; ET– extinction test; Morph. - morphine. In red-days of the CPP tests.

[0043] FIG. 22 shows opioid dependence. BL– preconditioning baseline session; Nx– naloxone; Sal.– saline; CPA– conditioned place aversion. In red-day when animals will be sacrificed. [0044] FIG.23. shows eEF2K KO mice do not develop anxiety-triggered pain perception. A. 9-20 WT males and 13-15 eEF2K KO males were tested by the TIT daily prior to saline i.p. injection. MPE, maximum possible effect. B.18 WT males and 12-14 KO males were tested by the TIT daily 30 minutes after saline i.p. injection.

[0045] Fig.24. shows suppression of eEF2K delays or prevents development of opioid- induced hyperalgesia. WT and KO male mice were tested by the HP test (A) or by the TIT (B) daily prior to morphine i.p. injection. WT males or females were treated with either vehicle or eEF2K inhibitor and tested by the HP (C) or by TIT (D) prior to oxycodone gavaging.

[0046] Fig.25. shows suppression of eEF2K activity delays or prevents development of morphine-induced tolerance. A. Ten to 20 WT and 13-17 eEF2K KO male mice were tested by the HP (A) or by TIT (B) tests 30 minutes after morphine i.p. injection.

[0047] Fig.26. shows effects of morphine administration on OF locomotor and rearing behavior of mice. Four to 5 WT and 5-6 KO female mice were daily treated with morphine and tested in the open field instrument 15 to 30 minutes after the morphine administration. A. Distance ran by mice in the plastic chamber, cm. B. Resting time - time mice spent without moving, seconds. C. Rearing breaks measured the frequency of mice to stood on hind legs. DETAILED DESCRIPTION OF THE INVENTION

[0048] Abbreviations and Definitions [0049] Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0050] The singular forms“a”,“an” and“the” include plural reference unless the context clearly dictates otherwise. The use of the word“a” or“an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.”

[0051] Wherever any of the phrases“for example,”“such as,”“including” and the like are used herein, the phrase“and without limitation” is understood to follow unless explicitly stated otherwise. Similarly“an example,”“exemplary” and the like are understood to be nonlimiting.

[0052] The term“substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term“substantially” even if the word“substantially” is not explicitly recited.

[0053] The terms“comprising” and“including” and“having” and“involving” (and similarly“comprises”,“includes,”“has,” and“involves”) and the like are used

interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of“comprising” and is therefore interpreted to be an open term meaning“at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example,“a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms“a” or“an” are used,“one or more” is understood, unless such interpretation is nonsensical in context.

[0054] As used herein the term“about” is used herein to mean approximately, roughly, around, or in the region of. When the term“about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0055]

[0056] Compositions of the Invention [0057] Aspects of the invention are directed towards a pharmaceutical composition comprising a therapeutically effective amount of an analgesic and at least one of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, or a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor. In embodiments, the pharmaceutical composition prevents, treats or ameliorates pain in a subject, while preventing or reducing the incidence of abuse of and/or addition the analgesic.

[0058] An“effective amount”,“sufficient amount” or“therapeutically effective amount” can refer to an amount sufficient to effect beneficial or desired clinical results, for example alleviation or reduction in the pain sensation and/or preventing or reducing the incidence of drug-use abuse. An effective amount of a composition as described herein can comprise an amount sufficient to treat, ameliorate, reduce the intensity of or prevent pain of any sort, including acute pain and/or chronic pain. In some embodiments, an effective amount of compositions as described herein can modulate the sensitivity threshold to external stimuli to a level comparable to that observed in healthy subjects. In other embodiments, this level is not be comparable to that observed in healthy subjects, but is reduced compared to not receiving the combination therapy.

[0059] Eukaryotic translation elongation factor 2 (eEF2 or EEF2; PubMed Gene ID 1938; NCBI Reference Sequence NG_042274.1) is a member of the GTP-binding translation elongation factor family, and is an essential factor for protein synthesis. It promotes GTP- dependent translocation of the nascent protein chain from the A-site to the P-site of the ribosome. eEF2 crystal structure and description thereof can be found in Jørgensen R, Ortiz PA, Carr-Schmid A, Nissen P, Kinzy TG, Andersen GR. Nat Struct Biol.2003

May;10(5):379-85 and Gautam Kaul, Gurulingappa Pattan and Towseef Rafeequi. Cell Biochem Funct 2011; 29: 227–234. [0060] SEQ ID NO: 1 - nucleotide sequence of gene encoding Homo sapiens eukaryotic translation elongation factor 2 (EEF2) (NCBI Reference sequence NG_042274.1): >NG_042274.1:5001-14408 Homo sapiens eukaryotic translation elongation factor 2 (EEF2), RefSeqGene on chromosome 19

GG G GG GG G

[0061] SEQ ID NO: 2 - nucleotide sequence of mRNA encoding Homo sapiens eukaryotic translation elongation factor 2 (EEF2) (NCBI Reference Sequence NM_001961.3): >NM_001961.3 Homo sapiens eukaryotic translation elongation factor 2 (EEF2), mRNA

[0062] SEQ ID NO: 3 - amino acid sequence of Homo sapiens eukaryotic translation elongation factor 2 (EEF2) protein. In mature protein, the initiator methionine residue is absent:

[0063] eEF2 is inactivated by EF-2 kinase (eEF2K) phosphorylation. eEF-2 kinase is a highly conserved protein kinase in the calmodulin-mediated signaling pathway that links multiple up-stream signals to the regulation of protein synthesis. Although no crystal structure for the catalytic domain of eEF2K is available, such data are available for two other a-kinases. The a-kinase domains of Dictyostelium myosin-II heavy chain kinase A (MHCK A) and the mouse transient receptor potential ion channels, TRPM7, share substantial similarities, in particular conservation of the spatial positions of their key catalytic residues (Eukaryotic Elongation Factor 2 Kinase (eEF2K) in Cancer. Xuemin Wang, Jianling Xie ID, and Christopher G. Proud. Cancers 2017, 9, 162; doi:10.3390/cancers9120162).

[0064] SEQ ID NO: 4 - Homo sapiens eukaryotic translation elongation factor 2 kinase (eEF2K or EEF2K) mRNA sequence (NCBI Reference Sequence NM_013302.3) >NM_013302.3 Homo sapiens eukaryotic elongation factor 2 kinase (EEF2K), mRNA

[0065]

[0066] SEQ ID NO: 5 - Homo sapiens eukaryotic translation elongation factor 2 kinase (eEF2K or EEF2K) protein sequence (NCBI Reference Sequence NP_037434) >NP_037434.1 eukaryotic elongation factor 2 kinase [Homo sapiens]

[0067] In embodiments, the pharmaceutical composition can comprise an eEF2 phosphorylation reducer which deceases phosphorylation of eEF2. For example, the phosphorylation reducer can decrease phosphorylation at Thr56 of eEF2 or Ser595. Cyclin A- cyclin-dependent kinase 2 (CDK2) phosphorylates eEF2 on Ser595 and directly regulates phosphorylation of Thr56 by eEF2K (see, for example, Hizli, Asli A., et al. "Phosphorylation of Elongation Factor 2 by Cyclin A-CDK2 Regulates Its Inhibition by eEF2

Kinase." Molecular and cellular biology (2012): MCB-01270).

[0068] In embodiments, the eEF2 phosphorylation reducer can comprise a compound or selective inhibitors such as a microRNA, shRNA, siRNA, antisense, or ribozyme molecule specifically targeted to a nucleic acid, such as a nucleic acid that encodes the protein of SEQ ID NO: 3 or SEQ ID NO: 5, or a fragment thereof.

[0069] In embodiments, the eEF2 phosphorylation reducer comprises an eEF2 kinase (eEF2K) inhibitor. For example, the pharmaceutical composition can comprise an eEF2K inhibitor such as a compound of Formula I, Formula II, FIG.10, FIG.11, or derivatives thereof:

[0070] Non-limiting examples of eEF2K inhibitors comprise those described in

WO2015102752; Liu, Rui, and Christopher G. Proud. "Eukaryotic elongation factor 2 kinase as a drug target in cancer, and in cardiovascular and neurodegenerative diseases." Acta Pharmacologica Sinica 37.3 (2016): 285; and Xiao, Ting, et al. "A high-throughput screening assay for eukaryotic elongation factor 2 kinase inhibitors." Acta Pharmaceutica Sinica B 6.6 (2016): 557-563, each of which are incorporated by reference herein in their entireties.

[0071] R groups for Formula I are described in PCT Publication No. WO2015102752, each of the R groups of which are incorporated by reference in their entireties.

[0072] The activity of eEF-2K is dependent on calcium and calmodulin. Activation of eEF-2K proceeds by a sequential multi-step mechanism. First, calcium-calmodulin binds with high affinity to activate the kinase domain, triggering rapid autophosphorylation of Thr-348. Next, autophosphorylation of Thr-348 leads to a conformational change in the kinase likely supported by the binding of phospho-Thr-348 to an allosteric phosphate binding pocket in the kinase domain. This increases the activity of eEF-2K against eEF2. [0073] The pharmaceutical composition described herein can comprise an eEF2K inhibitor, which can refer to any compound or agent that can lead to the suppression of eEF2K activity, such as towards phosphorylation of eEF2. In embodiments, the eEF2K inhibitor can comprise a compound or selective inhibitors such as a microRNA, shRNA, siRNA, antisense, or ribozyme molecule. There are multiple phosphorylation sites on eEF2K, some of which activate eEF2K activity and some of which inhibit eEF2K activity. Thus, any modification that leads to suppression of eEF2K activity toward phosphorylation of eEF2 (including stimulation of phosphorylation of eEF2K that inhibits eEF2K's activity) can be used herein. In embodiments, the eEF2K inhibitor comprises an eEF2K phosphorylation reducer which reduces the phosphorylation, such as at Thr-348 of eEF2K, and subsequence activation of eEF2K. In embodiments, the eEF2K phosphorylation reducer can comprise a compound or selective inhibitors such as a microRNA, shRNA, siRNA, antisense, or ribozyme molecule.

[0074] The pharmaceutical combinations of the invention comprise an analgesic in an admixture with at least one of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, or any combination thereof. The analgesic (commonly referred to as a pain killer) can prevent, reduce, eliminate, ameliorate, or delay the onset of pain. The effect can be temporary or permanent. Non-limiting examples of such analgesics comprise an opioid, opioid peptides, cocaine, ethanol, amphetamine, cannabinoid, a benzodiazepine, derivatives thereof, or any combination thereof. For example, the pharmaceutical composition comprises an eEF2K inhibitor of Formula I or Formula II admixed with at least one opioid.

[0075] Non-limiting examples of an opioid/opiate comprises morphine, fentanyl, methadone, buprenorphine, hydrocodone, oxycodone, heroin, or derivatives thereof. [0076] Non-limiting examples of a benzodiazepine comprises midazolam, alprazolam, clobazam, clonazepam, clorazepate, chlordiazepoxide, diazepam, estazolam, lorazepam, oxazepam, temazepam, triazolam, or derivatives thereof.

[0077] The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier prepared according to conventional pharmaceutical techniques. According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Non- limiting examples of pharmaceutically acceptable carriers comprise solid or liquid fillers, diluents, and encapsulating substances, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl benzoate, propyl benzoate, talc, magnesium stearate, and mineral oil. The amount of the carrier employed in conjunction with the combination is sufficient to provide a practical quantity of material per unit dose of analgesic.

[0078] The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with embodiments herein can be used. Supplementary active compounds can also be incorporated into the pharmaceutical compositions.

[0079] Pharmaceutically acceptable carriers for oral administration comprise sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic saline, and pyrogen-free water. Pharmaceutically acceptable carriers for parenteral administration comprise isotonic saline, propylene glycol, ethyl oleate, pyrrolidone, aqueous ethanol, sesame oil, corn oil, and combinations thereof.

[0080] Various oral dosages forms can be employed, non-limiting examples of which comprise solid forms such as tablets, capsules, granules, suppositories and/or powders.

Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated or multiple compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Liquid oral dosage forms comprise aqueous solutions, emulsions, suspensions, syrups, aerosols and/or reconstituted solutions and/or suspensions. The pharmaceutical composition can alternatively be formulated for external topical application, or in the form of a sterile injectable solution.

[0081] Pharmaceutically effective composition can be provided as a composition comprising between 0.1 and 2000 mg/kg of at least one of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, or a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor. For example, pharmaceutically effective composition can be provided as a composition comprising about 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, 325 mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, 425 mg/kg, 450 mg/kg, 475 mg/kg, 500 mg/kg, 525 mg/kg, 550 mg/kg, 575 mg/kg, 600 mg/kg, 625 mg/kg, 650 mg/kg, 675 mg/kg, 700 mg/kg, 725 mg/kg, 750 mg/kg, 775 mg/kg, 800 mg/kg, 825 mg/kg, 850 mg/kg, 875 mg/kg, 900 mg/kg, 925 mg/kg, 950 mg/kg, 975 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg of an analgesic, a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, or any combination thereof.

[0082] Pharmaceutically effective compositions, such as a pill or tablet, can be comprise between 0.1 and 2000 mg of an analgesic, a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, or any combination thereof. For example, pharmaceutically effective compositions can comprise about 0.1 mg, 1 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg of an analgesic, a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, or any combination thereof.

[0083] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0084] As an exemplary embodiment, a pharmaceutical combination(s) can be administered orally, either in the form of tablets containing excipients such as starch or lactose, or in capsules, either alone or mixed with excipients, or in the form of syrups or suspensions containing coloring or flavoring agents. They can also be injected parenterally, for example intramuscularly, intravenously or subcutaneously. In parenteral administration, they can be used in the form of a sterile aqueous solution which can contain other solutes, such as, for example, any salt or glucose in order to make the solution isotonic.

[0085] The pharmaceutical composition can be administered to a subject for the treatment of pain, for example orally, either covered in gelatin capsules or compressed in lozenges. For oral therapeutic administration, embodiments can be mixed with excipients and used in the form of lozenges, tablets, capsules, elixirs, suspensions, syrups, wafers, chewing gum, and the like. These preparations could contain at least 0.5% of active compound, but can vary depending on each form, in particular between 4% and 75% approximately of the weight of each unit. The amount of active agents in such compositions should be that which is necessary for obtaining the corresponding dosage.

[0086] In parenteral therapeutic administration, the active agent can be incorporated in a solution or suspension. Such preparations, for example, can contain at least 0.1% of the active compound, but can vary between 0.5% and 50% approximately of the weight of the preparation. For example, such preparations comprise about 0.1%, 0.5%, 1%, 5%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, of the weight of the preparation. The amount of the active agent in such compositions should be that which is necessary for obtaining the corresponding dosage. The compositions and preparations as described herein can be prepared in such a way that each parenteral dosage unit can contain between .01 mg and 1000 mg, for example between about 0.5mg and 100 mg of the active compound, for example. While intramuscular administration can be given in a single dose or divided into up to multiple doses, such as three doses, intravenous administration can include a drip device for giving the dose by venoclysis. Parenteral administration can be performed by means of ampoules, disposable syringes or multiple-dose vials made of glass or plastic.

[0087] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers can include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In embodiments, the composition can be sterile and should be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can occur by including an agent in the composition which delays absorption, for example, aluminum monostearate and gelatin. [0088] Sterile injectable solutions can be prepared by incorporating active ingredients in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0089] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active ingredient can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

[0090] Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the pharmaceutical composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0091] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0092] The pharmaceutical composition described herein can be administered to a subject for the treatment of pain in a single dose, or as multiple doses over a period of time. Further, the pharmaceutical composition can be administered at intervals of about 4 hours, 8 hours, 12 hours, 24 hours, or longer. For example, a pill can be administered to a subject prior to the onset of pain to prevent pain, or a multiple pills can be administered over a period of time to ameliorate pain over said period.

[0093] Of necessity, there will be variations which will depend on the weight and conditions of the subject to be treated and on the particular administration route selected.

[0094]

[0095] Method of the Invention

[0096] Pain is associated with a wide range of injury and disease, and is sometimes the disease itself. Some conditions may have pain and associated symptoms arising from a discrete cause, such as postoperative pain or pain associated with a malignancy, or may be conditions in which pain constitutes the primary problem, such as neuropathic pains or headaches.

[0097] Millions suffer from acute or chronic pain every year and the effects of pain exact a tremendous cost on our country in health care costs, rehabilitation and lost worker productivity, as well as the emotional and financial burden it places on patients and their families. The costs of unrelieved pain can result in longer hospital stays, increased rates of rehospitalization, increased outpatient visits, and decreased ability to function fully leading to lost income and insurance coverage. As such, patient's unrelieved chronic pain problems often result in an inability to work and maintain health insurance.

[0098] While acute pain is a normal sensation triggered in the nervous system to alert a subject to possible injury and the need to take care of himself or herself, chronic pain is different. Chronic pain persists. Pain signals keep firing in the nervous system for weeks, months, even years. There may have been an initial mishap (for example, sprained back, serious infection) or there may be an ongoing cause of pain (for example arthritis, cancer, ear infection), but some people suffer chronic pain in the absence of any past injury or evidence of body damage. Many chronic pain conditions affect older adults. Common chronic pain complaints include headache, low back pain, cancer pain, arthritis pain, neurogenic pain (pain resulting from damage to the peripheral nerves or to the central nervous system itself).

[0099] Many opioid-only and opioid combination drugs are used to treat acute and chronic pain when other medications, such as ibuprofen or acetaminophen, aren’t strong

enough. Opioid products come in many different forms. For example, immediate-release products start to work quickly after a subject takes them, but they are effective for shorter periods. Extended-release products release the drugs over longer periods, such as for the treatment of chronic pain.

[00100] Immediate-release opioids are used to treat acute and chronic pain. Extended- release opioids are typically only used to treat chronic pain when immediate-release opioids are no longer enough.

[00101] With prolonged use of an analgesic such as an opioid, pain-relieving effects can lessen and pain can become worse. In addition, the body can develop dependence. Opioid dependence causes withdrawal symptoms, which makes it difficult to stop taking them. Addiction occurs when dependence interferes with daily life. Taking more than the prescribed amount or using illegal opioids like heroin can result in death. Symptoms of addiction include uncontrollable cravings and inability to control opioid use even though it's having negative effects on personal relationships or finances.

[00102] Currently there are no therapies that provide protection against development of opioid-induced hyperalgesia, tolerance, addiction, withdrawal and relapse. Many clinical efforts to prevent development of drug-associated disorders have failed. An alternative approach is to target eEF2K and/or phosphorylation of eEF2 during and after drug administration to alleviate unwanted consequences of drug use.

[00103] Aspects of the invention are directed towards compositions and methods for treating a subject afflicted with pain, wherein the compositions and methods also prevent, reduce, or eliminate addiction and/or dependence to the analgesic.

[00104] In embodiments, the method comprised administering to a subject an analgesic and at least one of a therapeutically effective amount of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor. In embodiments, the components are admixed to provide a pharmaceutical composition, such as those described herein. In embodiments, the analgesic is administered to a subject prior to, in conjunction with, or subsequent to administering the eEF2 phosphorylation reducer.

[00105] “Treatment” can refer to an approach for obtaining beneficial or desired clinical results, for example improvement or alleviation of any aspect of pain, such as acute, chronic, inflammatory, neuropathic, or post-surgical pain. Beneficial or desired clinical results comprise, but are not limited to, one or more of the following: including lessening severity, alleviation of one or more symptoms associated with pain including any aspect of pain (such as shortening duration of pain, and/or reduction of pain sensitivity or sensation).

[00106] Still further, aspects of the invention are directed towards a method of reducing or ameliorating pain in a subject.“Ameliorating” pain or one or more symptoms of pain can refer to a lessening or improvement of one or more symptoms of a pain as compared to not administering a composition as described here.“Ameliorating” can also comprise shortening or reduction in duration of a symptom.

[00107] Aspects of the invention are further directed towards alleviating pain in a subject. The term“alleviate” or“alleviating” can refer to lightening or lessening the severity of a symptom, condition, or disorder. For example, a treatment that reduces the severity of pain in a subject can be said to alleviate pain. For example, a therapeutically effective amount of a pharmaceutical composition described herein can be administered to a subject afflicted with pain, wherein the severity of the pain is lessened. It is understood that, in certain circumstances, a treatment can alleviate a symptom or condition without treating the underlying disorder. In certain aspects, this term can be synonymous with the language “palliative treatment.”

[00108] “Palliating” pain or one or more symptoms of pain can refer to lessening the extent of one or more undesirable clinical manifestations of pain in an individual or population of individuals treated with a composition as described herein.

[00109] Yet further, aspects of the invention are directed towards a method of increasing the sensitivity of a subject to an analgesic, wherein a lower dose of the analgesic is sufficient to treat pain, and/or a method of reducing or preventing the development of hyperalgesia. Hyperalgesia refers to an increased sensitivity to pain, and opioid induced hyperalgesia is a phenomenon in which a subject becomes hypersensitive to painful stimuli due to the use of opioids.

[00110] Still further, aspects of the invention are directed towards reducing or preventing a withdrawal symptom after analgesic cessation. For example, opioid withdrawal refers to the wide range of symptoms that occur after a subject stops the use of opioid drugs. Withdrawal can last up to 10 days, but is most often between 3-5 days. Although it can cause very troubling symptoms (such as vomiting, cramps, and sweating), withdrawal is rarely life- threatening. Non-limiting examples of symptoms of opioid withdrawl comprise anxiety, nausea, vomiting, abdominal pain, diarrhea, restlessness, seating, and tremors.

[00111] Aspects of the invention are also directed towards reducing the incidence of pain or delaying.“Reducing incidence” of pain can refer to any of reducing severity (which can include reducing need for and/or amount of (e.g., exposure to) other drugs and/or therapies generally used for this conditions), duration, and/or frequency (including, for example, delaying or increasing time to pain in an individual). As is understood by those skilled in the art, individuals can vary in terms of their response to treatment, and, as such, for example, a“method of reducing incidence of pain in an individual” reflects administering pharmaceutical compositions as described herein based on a reasonable expectation that such administration can cause such a reduction in incidence in that particular individual.

[00112] "Delaying” the development of pain can refer to deferring, hindering, slowing, retarding, stabilizing, and/or postponing progression of pain. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop pain. A method that“delays” development of the symptom is a method that reduces probability of developing the symptom in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method.

[00113] “Development” or“progression” of pain can refer to initial manifestations and/or ensuing progression of the disorder. Development of pain can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that can be undetectable. For purpose of this invention,

development or progression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or“occurrence” of pain includes initial onset and/or recurrence. For example, pharmaceutical compositions and methods as described herein can be used to prevent the development of pain, or prevent the progression of pain.

[00114] In embodiments, a subject can be administered a therapeutically effective amount of a composition as described herein for the treatment of pain. Compositions as described herein can be administered to a subject by any suitable means, such as oral, intravenous, parenteral, subcutaneous, intrapulmonary, topical, intravitreal, dermal, transmucosal, rectal, and intranasal administration. Parenteral infusions include

intramuscular, intravenous, intraarterial, or intraperitoneal administration.

[00115] Compositions described herein can also be administered transdermally, for example in the form of a slow-release subcutaneous implant or as a transdermal patch. They can also be administered by inhalation. Although direct oral administration can cause some loss of desired activity, for example pain relieving activity, the analgesics can be packaged in such a way to protect the active ingredient(s) from digestion by use of enteric coatings, capsules or other methods known in the art.

[00116] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. The use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects. [00117] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

[00118] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include, for example, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; anti-inflammatory agents; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

[00119] Compositions as described herein can be administered to the subject one time (e.g., as a single injection or deposition). While the term for administering of at least one compound to prevent pain varies depending on species, and the nature and severity of the condition to be prevented or treated, the compound can be administered to humans for a short term or a long term, i.e. for 1 week to 1 year. For example, administration can be once or twice daily to a subject in need thereof for a period of time, such as one week or one month.

[00120] The dosage can vary depending upon known factors such as the

pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion.

[00121] A therapeutically effective dose can depend upon a number of factors known to those of ordinary skill in the art. The dose(s) can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires. These amounts can be readily determined by the skilled artisan.

[00122] In some embodiments, the therapeutically effective amount is at least about 0.1 mg/kg body weight, at least about 0.25 mg/kg body weight, at least about 0.5 mg/kg body weight, at least about 0.75 mg/kg body weight, at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, at least about 100 mg/kg body weight, at least about 200 mg/kg body weight, at least about 250 mg/kg body weight, at least about 300 mg/kg body weight, at least about 3500 mg/kg body weight, at least about 400 mg/kg body weight, at least about 450 mg/kg body weight, at least about 500 mg/kg body weight, at least about 550 mg/kg body weight, at least about 600 mg/kg body weight, at least about 650 mg/kg body weight, at least about 700 mg/kg body weight, at least about 750 mg/kg body weight, at least about 800 mg/kg body weight, at least about 900 mg/kg body weight, or at least about 1000 mg/kg body weight. [00123] In an embodiment, the daily dose range of a composition as described herein lies within the range of from about, a daily dose of about 1 mg/body to about10 g/body, for example about 5 mg/body to about 5 g/body, or for example about 10 mg/body to about 2 g/body of the active ingredient is generally given for treating this disease, and an average single dose of about 0.5 mg to about 1 mg, about 5 mg, about 10 mg, about 50 mg, about 100 mg, about 250 mg, about 500 mg, about 1 g, about 2 g and about 3 g is generally

administered.

[00124] In embodiments, the composition herein is administered to the subject one time (e.g., as a single injection or deposition). Alternatively, administration can be once or twice daily to a subject in need thereof for a period of from about 2 to about 28 days, or from about 7 to about 10 days, or from about 7 to about 15 days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.

[00125] Single unit dosage forms of the disclosure are suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intraarterial), topical (e.g., eye drops or other ophthalmic preparations), transdermal (e.g., cream, lotion, or dermal spray) or transcutaneous administration to a patient. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; powders; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions or solutions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; eye drops or other ophthalmic preparations suitable for topical administration; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms for parenteral administration to a subject.

[00126] The composition, shape, and type of dosage forms of the disclosure will typically vary depending on their use. Further, the dosage can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion.

[00127] For example, a dosage form used in the acute treatment of a disease can contain larger amounts of one or more of the active agents it comprises than a dosage form used in the chronic treatment of the same disease. Similarly, a parenteral dosage form can contain smaller amounts of one or more of the active agents it comprises than an oral dosage form used to treat the same disease. These and other ways in which specific dosage forms encompassed by this disclosure will vary from one another will be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).

[00128] Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a mouse, a rat, a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human. In some embodiments, the subject is a mouse, rat, pig, or human. In some embodiments, the subject is a mouse. In some embodiments, the subject is a rat. In some embodiments, the subject is a pig. In some embodiments, the subject is a human.

[00129]

[00130] Kits of the Invention [00131] A "kit" or "medical kit" of the disclosure comprises a dosage form of a compound as described herein, such as a compound of Formula I or Formula II, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, prodrug, or clathrate thereof. A kit can also include an analgesic, either in combination, such as in a single tablet, or provided separately, such as in two tablets.

[00132] Kits can further comprise additional active agents, for example opioids or non- steroidal anti-inflammatories, examples of which are described herein. For example, an opioid can be provided in a kit described herein at dose lower than that currently used by a subject so as to decrease total body opioid consumption and the deleterious effects associated with prolonged opioid use. Kits of the disclosure can further comprise devices that are used to administer the active ingredients. Examples of such devices include, but are not limited to, syringes, drip bags, patches, and inhalers. Kits can also comprise printed instructions for administering the compound to a subject.

[00133] Kits of the invention can further comprise pharmaceutically acceptable vehicles that can be used to administer one or more active ingredients. For example, if an active ingredient is provided in a solid form that must be reconstituted for parenteral administration, the kit can comprise a sealed container of a suitable vehicle in which the active ingredient can be dissolved to form a particulate-free sterile solution that is suitable for parenteral administration. Examples of pharmaceutically acceptable vehicles include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. EXAMPLES

[00134] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

EXAMPLE 1 [00135] Targeting eEF2K and eEF2 to modulate drug-use disorders

[00136] Use of drugs or processes that inhibit activation of eEF2K and

phosphorylation of eEF2 alone or in combination with drugs or processes that could benefit patients in (1) reducing a dose and duration of opioid/opiate administration to treat pain; (2) prevent development of hyperalgesia, addiction and tolerance to opioid/opiates; and decrease signs of withdrawal after opioid/opiates cessation. Without wishing to be bound by theory, embodiments as described herein will prevent deterioration of quality of life of opioid- dependent patients, and also prevent drug abuse and death from opioid/opiates overdose. This approach also can be applied to other drugs such as but not limited to cocaine, ethanol, amphetamine, cannabinoids and their derivatives and also to other drugs that induce dopamine release (such as but limited by benzodiazepines, midazolam, Xanax, and their derivative).

[00137] Drugs/compounds can be administered in combination with opioid/opiates in order to (1) achieve more potent effect of opioid/opiates that will allow to reduce a dose of opioid/opiates to treat pain; (2) block development of opioid-induce hyperalgesia, tolerance and addiction. Administration of these drugs/compounds can also reduce signs of withdrawal after opioid/opiates cessation. Administration of these drugs/compounds can be combined with administration of other drugs of abuse that include but not limited by opioids/opiates, cocaine, ethanol, amphetamine, cannabinoids and their derivatives and also any other drugs that induce dopamine release (such as but limited by benzodiazepines, midazolam, Xanax, and their derivative).

EXAMPLE 2 [00138] INTRODUCTION

[00139] Embodiments of the invention comprise methods for reducing or preventing development of drugs use disorders that include but not limited by dependence, addiction, desensitization, tolerance, analgesic effect, hyperalgesia, and/or relapse. Drugs of abuse include but not limited by opioids/opiates, cocaine, ethanol, amphetamine, cannabinoids and their derivatives and also any other drugs that induce dopamine release (such as but limited by benzodiazepines, midazolam, Xanax, and their derivative).

[00140] Opioids are among the most effective drugs commonly prescribed to treat pain. However, their therapeutic use is limited due to the development of addiction

(compulsive engagement in rewarding stimuli, despite adverse consequences), tolerance (reduced responsiveness to the same dose of drug), and withdrawal (an adverse physiological reaction following removal of drug). Adverse effect of opioids and their derivatives burden considerable economic and personal costs to society. Thus, it is important to identify genes that contribute to development of drug use disorders.

[00141]

[00142] eEF2K and eEF2

[00143] eEF2K a non-conventional calmodulin-dependent protein kinase CaMK III that belongs to the alpha kinase family [2]. eEF2K is phosphorylated in response to various stimuli such as low cellular energy level, hypoxia, electrical stimulation [3] -[5], genotoxic stress [6], under low pH [7] or low Mg²⁺ [8] conditions, stimulation by hormones (e.g., serotonin [9], phenylephrine [10]), and growth factors (insulin, [11]). eEF2K auto- phosphorylation at Thr-348 (human sequence) [12], and phosphorylation at Ser-398 by AMPK [13], and at Ser499 PKA activates [14] eEF2K and promote phosphorylation of eEF2.

[00144] eEF2 is a general eukaryotic elongation factor-2 that regulates polypeptide chain elongation step during translation [15]. The eEF2 is phosphorylated on Thr56 by eEF2K [16] - [19]. Phosphorylation of eEF2 by eEF2K results in reduction of general protein synthesis [15], [17], [19] - [21]. However, translation of several specific mRNAs is upregulated by phosphor-eEF2. Among them, mRNAs involved in the control of synaptic plasticity: alpha Ca2+/calmodulin dependent kinase II (aCamK II) [21], Arc/Arg3.1 [22], [23], BDNF [24], and microtubule-associated proteins (MAP1B, [25]) [26].

[00145] Both eEF2K and eEF2 are ubiquitous in all eukaryotes and are present in various tissues [27]. Also, both eEF2K and eEF2 can be detected in the postsynaptic neuronal structures [21], [28], [29]. Modulation of eEF2K activity and phosphorylation of eEF2 were linked to changes in synaptic plasticity associated with development of long term depression (LTD), learning and memory, epilepsy and depression [22], [23], [30] - [32]. It was suggested that neuronal activity induces expression of specific proteins at the post- synapses via local activation of the eEF2K/eEF2-P signaling [33] (reviewed in [34]). Indeed, synaptic activity induced changes in dendritic spines morphology were shown to be dependent on eEF2K-driven BDNF expression [24].

[00146]

[00147] Post-Synaptic Activity

[00148] In neurons, synaptic activity triggers the eEF2K/eEF2-P signaling via AMPA and NMDA-type glutamate receptors [21], [33], [35] - [41] and also by activation of the metabolic glutamate receptors (mGluR) 1/5 [23] - [25]. N-Methyl-D-aspartate receptor, NMDAR, activation leads to increased phosphorylation of eEF2 [35] in synaptosomes [21]. Blockade of the NMDAR by ketamine and MK-801 have been found to alter the postsynaptic AMPA receptor compensation under the involvement of eEF2K and BDNF. It was shown that ketamine led to an inhibition of eEF2K followed by enhanced protein synthesis and potentiation of hippocampal synapses that might mediate the antidepressant response of this compound [39], [40]. Activation of metabotropic glutamate receptors, mGluR, increased the phosphorylation of eEF2 in the stratum pyramidal, and stratum radiatum of the hippocampal CA1 region [23]. It was shown that mGluR induces LTD that requires protein synthesis. Arc/Arg3.1 (essential for LTD) is translationally activated by mGluR via eEF2K [23].

Arc/Arg3.1, an activity-regulated cytoskeleton-associated protein, is a localized in dendrites and activated synapses and was shown to be involved in neurotransmitter receptor internalization and trafficking in long term depression [22], [23]. Similarly, MAP1B was suggested to play a role in glutamate receptor trafficking in cultured neurons [25]. Proteomic analysis revealed that b-arrestin interacts with eEF2, Arc family members and MAP1B in HEK293 cells [42]. It was suggested that during synapse specific LTD, neuronal activity induced eEF2K/eEF2-P signaling leads to translational activation of Arc/Arg3.1 that in turn promotes receptor internalization [23].

[00149]

[00150] Pre-Synaptic Activity

[00151] Without wishing to be bound by theory, eEF2K activity in the brain regulates the excitation/inhibition balance by downregulating vesicle release at inhibitory synapses and tonic inhibition. The eEF2K was shown to negatively regulate GABAergic synaptic transmission affecting the excitation/inhibition balance [43]. Loss of eEF2K increased GABAergic synaptic transmission by upregulating expression of presynaptic protein Synapsin 2b and a5-containing GABAA receptors [43]. Inhibition of eEF2K by a potent eEF2K inhibitor A-484954 enhances synaptic transmission by upregulation of the vesicle release probability in hippocampal CA1 neurons [44]. Without wishing to be bound by theory, pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy [43].

[00152]

[00153] eEF2K inhibitors and eEF2K knockout mice

[00154] There are several commercially available eEF2K inhibitors that were tested on the eEF2K activity in neuronal and cancer cells in vitro (reviewed in [45] and [26]).

[00155] There are three mouse models that target the eEF2K activity: (1) eEF2 ki (knock-in) mice bearing inactive eEF2K (kinase-dead mutation of EF2K) show impaired conditioned taste aversion learning [31]. (2) hip-eEF-2K-tg transgenic mice overexpressing EF2K in the hippocampus have impaired consolidation of contextual fear conditioning and LTP [30]; and (3) homozygous eEF2K knockout mice, eEF2K KO, have disrupted eEF2K in all cells [46]. The eEF2K KO mice do not significantly affect normal biological processes. Comparison of eEF2K KO and corresponding wild type littermates showed that gross brain architecture, body weight, food intake, body tone, grip strength, clasping, hanging wire, reflexes, and basic sociability were all normal in the eEF2K KO mice. Behavior tests demonstrated that object recognition ability, anxiety-like behavior, locomotor activity, and novel taste memory as well as spatial memory and reversal learning were normal in eEF2K- KO compared with WT mice. However, eEF2K KO mice exhibited impaired trace fear conditioning and impaired contextual fear conditioning compared with WT mice but normal amygdala-dependent auditory fear conditioning. Baseline synaptic transmission and LTP formation were also normal in eEF2K-KO mice. In addition, eEF2K KO mice demonstrated a strong increase in the miniature inhibitory postsynaptic current (mIPSC) frequency and amplitude in hippocampal granule cells [43]. [00156] Based on the forgoing, there is a strong need in the art to establish the eEF2K and eEF2 relevance with the respect to the drug-use disorders and as a potential target for treatment of drug-use disorders (that include but not limited by addiction and drug dependence, relapse, analgesia and hyperalgesia, desensitization and tolerance, and drug sensitivity and toxicity). Embodiments herein address these needs.

[00157] Although eEF2K loss impairs cocaine-induced locomotor activity in mice [47], the direct link between drug-induced behavior and eEF2K activity/expression has never been established.

[00158] Embodiments herein comprise methods for preventing development of drugs use disorders that include but not limited by drug-induced dependence, addiction, desensitization, tolerance, analgesic effect, and hyperalgesia. Drugs of abuse include but not limited by opioids/opiates, cocaine, ethanol, amphetamine, and their derivatives.

[00159] Described herein, eEF2K KO mice are more sensitive to morphine administration. Also, described herein eEF2K KO mice do not develop (measuring spinal reaction) or exhibit delayed (measuring supra-spinal reaction) signs of tolerance to chronic morphine administration. In addition, eEF2K KO mice do not develop hyperalgesia after prolong morphine administration. Therefore, embodiments herein comprise methods for (1) reduction of opioid/opiate dose necessary for analgesic effect; (2) preventing or delaying development of signs of addictive drugs use disorders, by administering to the patients one or more compounds or using other methods of treatments that inhibit eEF2K activity and/or phosphorylation of eEF2.The eEF2K and/or phosphor-eEF2 inhibitor(s) can be administered orally, intravenously, as nasal spray, and as other ways of treatment.

[00160] The present invention relates to methods for preventing development of drugs use disorders that include but not limited by drug-induced dependence, addiction, desensitization, tolerance, analgesic effect, and hyperalgesia. Specifically, the present invention provides methods for (1) reduction of opioid/opiate dose necessary for analgesic effect; (2) preventing or delaying development of signs of addictive drugs use disorders, by administering to the patients one or more compounds or using other methods of treatments that inhibit eEF2K activity and/or phosphorylation of eEF2.

[00161] As defined herein, eEF2K is a non-conventional calmodulin-dependent protein kinase CaMK III that belongs to the alpha kinase family. eEF2K phosphorylates elongation factor 2, eEF2, a general eukaryotic elongation factor-2 that regulates polypeptide chain elongation step during translation. Phosphorylation of eEF2 by eEF2K results in reduction of general protein synthesis but translational upregulation of several specific mRNAs that are involved in the control of synaptic plasticity: aCamK II, Arc/Arg3.1, BDNF, and

microtubule-associated proteins. In addition, modulation of eEF2K activity was shown to be linked to changes in synaptic plasticity.

[00162] In neurons, the eEF2K/eEF2-P signaling is triggered via AMPA and NMDA- type glutamate receptors and also by activation of the mGluR1/5 that may lead to development of long-term depression. Activation of eEF2K negatively regulates GABAergic synaptic transmission that promotes disinhibition of neuronal excitation leading to long term potentiation (LTP). It was shown that activation of the AMPAR, NMDAR, mGluR1/5, and GABARs by drugs of abuse contribute to development of drug-use disorders, specifically, to development of drug dependence, addiction, hyperalgesia, and tolerance.

[00163]

[00164] Drug Dependence and Addiction

[00165] Drug exposure triggers long term potentiation (LTP) of dopaminergic (DA) neurons in the VTA area. Activation of DA neurons resulting in dopamine release is a key action of drugs of abuse to induce drug-associated positive reinforcement leading to drug dependence and addiction. Brain structures involved in mediating rewarding effect of drugs include nucleus accumbens, ventral tegmental area (VTA), prefrontal cortex and limbic structures (extended amygdala). Increase in dopamine secretion can result from both direct stimulation of dopamine release by DA neurons (cocaine and amphetamine), and an indirect effect (ethanol, opioids). Indirect effect involves modulation of GABAergic interneurons that interact with DA neurons in VTA are (opioids, ethanol). As described herein, increased level of phosphorylated eEF2 in brain lysates nucleus accumbens of rats after chronic oxycodone exposure. In addition increased level of BDNF expression in nucleus accumbens of rats chronically treated with oxycodone and also in hippocampal areas of mice treated with morphine. In VTA, BDNF was linked to development of opioid dependence by switching from a dopamine-independent to a dopamine-dependent response in opiate reward system and thus promoting addiction and signs of withdrawal. Loss or inhibition of eEF2K increased GABAergic synaptic transmission. Thus, loss or inhibition of eEF2K may suppress drug- induced dopamine release and development of rewarding effect and, thus, prevent development of drug addiction and dependence.

[00166]

[00167] Withdrawal and relapse

[00168] Withdrawal from morphine increases glutamate transmission in in the nucleus accumbens and amygdala that may underlie neuroadaptations leading to opiate dependence. Without wishing to be bound by theory, drug-induced LTP of excitatory synapses on VTA DA neurons might be necessary for attributing motivational significance to the drug experience or the learned association between context and drug experience that may lead to relapse. Antagonism of NMDAR or mGluR1/5 inhibited morphine withdrawal signs in rats and in mice. eEF2K is activated by either NMDAR or mGluR1/5 that may contribute to the DA neuron excitation and dopamine release. Thus, without wishing to be bound by theory, inhibition of eEF2K/eEF2-P after drug cessation can suppress drug-induced signs of withdrawal and also prevent relapse.

[00169]

[00170] Tolerance (drug resistance)

[00171] Drug tolerance (or resistance) is characterized by a diminished response of the patient after repeated drug use. As described herein, mice lacking eEF2K exhibit delayed or no signs of tolerance after morphine administration. One of the mechanisms of

desensitization and tolerance development is reduction in receptor density due to receptor internalization. Activation eEF2K/eEF2-P triggers post-synaptic expression of Arc/Arg3.1 and microtubule-associated proteins that were shown to promote internalization of receptors. Increase in Arc expression was shown after chronic morphine treatment and during abstinence. Thus, inhibition of eEF2K/eEF2-P activity during drug administration will prevent drug-induced receptor internalization and, thus, development of tolerance.

[00172]

[00173] Drug potentiation

[00174] As described herein, mice lacking eEF2K were more sensitive to morphine. Further, eEF2K KO mice have higher density of µ–opioid receptor (MOR) expressed in the DG area of the brain. Combination of an opioid and a compound that inhibits eEF2K/eEF2-P activity will increases analgesic effect of opioid. This will allow to reduce a dose of drug to achieve necessary analgesic effect. In addition, combining methadone and a compound that inhibits eEF2K/eEF2-P activity will allow to reduce methadone dose to treat opioid addiction.

[00175]

[00176] Hyperalgesia [00177] Hyperalgesia is characterized increased sensitivity to pain, and is accompanied repeated opioid administration. Hyperalgesia is induced by strong or long-lasting opioid stimuli that induce LTP at C-fiber synapses. Also, opioid-induced hyperalgesia depends on activation of microglia. Microglia-released BDNF promotes development of morphine- induced hyperalgesia in mice. eEF2K/eEF2-P is activated during LTP. Also, eEF2-P promotes BDNF expression. As described herein, mice lacking eEF2K did not develop hyperalgesia after chronic morphine treatment. Without wishing to be bound by theory, inhibition of eEF2K/eEF2-P during repeated drug administration will prevent development of drug-induced hyperalgesia.

[00178]

[00179] MATERIALS & METHODS

[00180] Animal Experiments

[00181] Rats - Female 60 day-old Sprague-Dawley rats (180–240 g) were purchased from Harlan Indianapolis, IN. They were fed chow and water ad libitum and maintained on a 12-h light/dark cycle. The animals were housed three to a cage and allowed to acclimate for at least 1 week before experiments were conducted. The protocol for animal studies was approved by the Louisiana State University Health Science Center, Institutional Animal Care and Use Committee. Rats were assigned to one of two groups (n=3/group) administered either oxycodone (Mallinckrodt Inc., St. Louis MO) or its vehicle water. Oxycodone (15 mg/kg) or water was administered by oral gavage (volume of 1.0 ml/kg) every 24 hours for 30 days. The oxycodone-treated group was compared directly to the water-treated control group, which was handled, treated, and sacrificed at the same time and under the same conditions. For statistical analysis this treatment [three water- (W) and six oxycodone-treated (O1 and O2) animals] was repeated three times. [00182] Mice. Morphine implants - Female wild type (wt) and eEF2K KO (KO) mice (33-54 g) were obtained from the Dr. Ryazanov’s lab (Rutgers, the State University of New Jersey). They were fed chow and water ad libitum and maintained on a 12-h light/dark cycle. The animals were housed one to three animals per cage and allowed to acclimate for at least 6 week before experiments were conducted. Mice were assigned to one of four groups: WT with morphine implant (n=3), WT with placebo implant (n=3), KO with morphine implant (n=4), KO with placebo implant (n=3). Morphine implant: On day 0, mice were briefly anesthetized with isoflurane. For the morphine implant, a small incision, approximately 1 cm, was made at the base of the neck with a surgical blade using aseptic techniques. The subcutaneous space was separated using a forceps to form a small, subcutaneous pocket. Morphine pellets (25 mg) (National Institute of Drug Abuse) was inserted in the dorsal subcutaneous space. The site was closed using surgical glue to seal the wound. Pellets were left in place for 4 days until the end of the experiment. Control mice got an inert placebo pellet (National Institute of Drug Abuse) in the same area on day 1. The protocol for animal studies was approved by the Louisiana State University Health Science Center, Institutional Animal Care and Use Committee. All animals were handled, treated, and sacrificed at the same time and under the same conditions.

[00183] Mice. Morphine injections - Male eEF2K KO (KO) mice (40-45 g) were obtained from Rutgers, the State University of New Jersey. They were fed chow and water ad libitum and maintained on a 12-h light/dark cycle. The animals were housed one to two animals per cage and allowed to acclimate for at least 6 week before experiments were conducted. Mice were assigned to one of two groups: KO mice injected with morphine (n=4) and KO mice injected with vehicle with (n=3). Morphine injections: Morphine sulfate was dissolved in saline solution and injected intraperitoneally (i.p.) in mouse in a volume of 0.03 ml/10 g body weight. Control mice received saline solution (vehicle). Mice received i.p. injection once daily with 15 mg/kg morphine or vehicle for 12 days. The hot plate test was performed every other day starting on day 1 of injections. All animals were handled, treated, and sacrificed at the same time and under the same conditions.

[00184] Hot Plate test - Mice were placed on a heated at 49 or 53°C plate as it is described for each experiment. The time it takes for the mouse to jump off of the hot plate or to shake or lick its hind paw was measured. The cutoff time for the hot-plate test was30 seconds.

[00185] Warm Water Tail Immersion Test (TIT) - See experiments described in FIG 5. Male wild type (WT, n=8) and eEF2K KO (KO, n=10) mice (30-43 g) were obtained from the Dr. Ryazanov’s lab (Rutgers, the State University of New Jersey). All animals were handled, treated, and sacrificed at the same time and under the same conditions. Warm water tail immersion test (TIT): Each mouse was placed in a mouse holder with its tail protruding. Five to two cm of the protruding tail was immersed in a conical tube with warm water (54°C). Time until withdrawal of tail was measured. To avoid tissue damage, the cutoff time of one immersion was 10 seconds. Warm water measurements were taken three times with 15 seconds intervals.

[00186] See experiments described in FIG.6. Measurements of the basal nociceptive reaction using TIT (T=49°C) in eEF2K KO mice before morphine (KO-M, n=3-4) or vehicle (KO-V, n=3) administration. Male eEF2K KO mice (40-45 g) were i.p. injected with 15 mg/kg morphine or vehicle daily for 12 days as it is described in Fig.4. On days 2, 4, 6, and 8, the TIT measurements were taken three consecutive times with 20 seconds intervals. To avoid tissue damage the cutoff time for one TIT was 15 seconds. Time measurements from three measurements were averaged for each mouse. Graph represents the mean TIT value for all mice in a corresponding group. On days 10, 11, and 12, the TIT measurements were taken once daily. To avoid tissue damage the cutoff time for the TIT was 30 seconds. Graph represents the mean time it took for the mouse in each group to withdraw a tail.

Measurements of analgesic reaction using TIT (T=49°C) in eEF2K KO mice 30 minutes after morphine (KO-M-30, n=3-4) or vehicle (KO-V-30, n=3) administration. TIT measurements were performed as it is described in FIG.6.

[00187] Western Blot Analysis - To analyze the expression of proteins in brain tissues, equal amounts of total protein were loaded on a 12% or 4-12% NuPAGE® Novex® Bis-Tris Gel (Invitrogen). The Full-Range Rainbow protein molecular weight marker (GE Healthcare Life Science) was loaded on the same gel to identify the position of specific proteins. Proteins were separated by SDS-PAGE gel and then transferred to a Nitrocellulose membrane (Bio- Rad) using a Mini Trans-Blot cell (Bio-Rad). Expression of specific proteins were determined by probing the membrane with primary antibodies against P-AMPK (Cell Signaling, #2535), AMPK (Cell Signaling, #2603), P-PKA (Cell Signaling, #4781) PKA (Cell Signaling, #4781), and P-CaMKIIa (Cell Signaling, #3361), CaMKIIa (Cell Signaling, #3362), BDNF (Santa Cruz, sc-546), and GAPDH (Fitzgerald, #10R-G109A); and then with secondary anti-bodies: anti-mouse (Vector lab, #PI-2000) and anti-rabbit (Vector lab, #PI- 1000). Blots were developed with the Western Lightning ECL Pro development kit

(PerkinElmer) and exposed to HyBlot CL autoradiography film (Denville Scientific).

Quantitative analysis of Western blot images was performed using the ImageQuant TL software (GE Healthcare Life Science). In experiments involving brain lysates, each sample contained the corresponding brain tissue from three rats.

[00188] Immuno-histochemistry - Immunohistochemical analysis of hippocampal areas of brain slices from wild type and eEF2K KO female mice with implanted placebo (described in FIG.3). Mice were anesthetized with isoflurane and then perfused through the aortic arch with ice-cold saline followed by ice cold 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 (PB). The whole brain was removed and placed in 4% paraformaldehyde in PB overnight at 4°C, transferred to a 15% sucrose in 0.1M PB for 24 hours, then to 30% sucrose in PB for 24 hours and then stored in 50% ethanol at 4°C until further processing. Mouse brain sections were embedded in paraffin according to a standard protocol, cut into 5 µm thick slices and then mounted on glass slides, two consecutive slices on one slide (Millennia 1000). Tissue sections were deparaffinized, rehydrated and stained according to a standard protocol. Brain sections containing hippocampal areas were incubated with primary antibodies against eEF2K (Cell Signaling, #3692, dilution 1:100). Sections were processed with VectaStain ABC (to detect rabbit primary antibodies) and the signal was visualized by the peroxidase substrate kit DAB (Vector Laboratories, Inc.). The nuclei were stained using the hematoxylin Gill’s 3 formulation. Images were taken using light microscope BX43F (Olympus).

[00189] Immuno-fluorescence - Brain sections were incubated with primary antibodies against µ-opioid receptor-1 (Santa Cruz, # sc-7488R, dilution 1:500) The sections were processed with secondary antibody conjugated to biotin (dilution 1:200) followed by incubation with Streptavidin-Alexa Fluor® 594 conjugates. The sections were mounted with VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories Inc., H-1500). Images were taken using AxioObserver with ApoTome microscope (Zeiss). Scale bar is 220 µm for all images.

[00190]

[00191] RESULTS

[00192] Chronic oxycodone treatment activates eEF2K/eEF2-P signaling in rat nucleus accumbens.

[00193] Signaling pathways that activate eEF2K (AMPKa, PKA, CaM and Ca²⁺) and also phosphorylation status of the eEF2K substrate eEF2 were analyzed in rats treated with oxycodone or water to validate whether opioid exposure triggers the eEF2K activation . Referring to FIG.1(A), Western blot analysis of brain lysates containing nucleus accumbens revealed increased phosphorylation of AMPKa, PKA, and CaMKIIa in rats treated with 15 mg/kg oxycodone for 30 days compare to that in rats treated with water. Referring to FIG. 1(B), more than 7-fold increase in phosphorylated eEF2 in nucleus accumbens of rats was observed after chronic oxycodone administration. These data indicates that chronic opioid treatment activates eEF2K/eEF2-P in nucleus accumbens.

[00194] Phosphorylation of eEF2 stimulates translational activation of BDNF mRNA. BDNF mRNA and protein levels in nucleus accumbens of rats treated with oxycodone or water were analyzed. Referring to FIG.2(A), western blot analysis revealed almost 2-fold increase in BDNF protein level in brain lysates containing nucleus accumbens from rats chronically treated with oxycodone compare to that in water treated animals. However, only 1.2-fold increase in BDNF mRNA level in oxycodone exposed nucleus accumbens compare to that in water samples (FIG.2(B)). These data indicates that BDNF mRNA is translationally activated in nucleus accumbens by opioid treatment presumably via activated eEF2K/eEF2-P pathway.

[00195]

[00196] Loss of eEF2K potentiates analgesic effect of morphine implant

[00197] To validate eEF2K's role in opioid-induced analgesic effect, 25 mg morphine or placebo pellet was implanted in wild type mice and mice lacking eEF2K (eEF2K KO mice), and hot plate test was performed. In this test, the time it took for the mouse to jump off of the hot plate (53°C) or to shake or lick its hind paw was measured. Referring to FIG.3A, the basal response measured four days prior to morphine/placebo implantation was similar between wild type and eEF2K KO mice. However, referring to FIG.3B, 25 mg morphine implant drastically affected eEF2K KO mice. First, three out of four eEF2K KO mice died on 2-3 days after surgery. Considering that any other mice (wild type or eEF2K KO) did not die after morphine or placebo pellets implantation, this data indicate higher toxic effect of morphine on mice lacking eEF2K. The surviving eEF2 KO mouse with morphine implant demonstrated almost 2-fold increase in analgesic reaction compare to wild type mice with morphine implants (FIG.3(B). These data invalidate that loss of eEF2K contributes to higher sensitivity to opioids analgesic (and toxic) effects. One of the mechanisms that regulate sensitivity to opioids is the level of µ-opioid receptor (MOR) in brain. Indeed, referring to FIG.3C, immuno-fluorescent analysis of hippocampal brain areas revealed higher MOR expression in eEF2K KO mice compare to that in wild type mice.

[00198]

[00199] eEF2K KO mice do not develop supra-spinal hyperalgesia and tolerance after prolong morphine injections measured by a hot plate test

[00200] A hot plate test (53°C) was performed using eEF2K KO mice prior to (hyperalgesia) and 30 minutes after (tolerance) i.p. injections of 15 mg/kg morphine or vehicle for 12 days to assess the contribution of eEF2K signaling to the development of supraspinal hyperalgesia or tolerance. In general, mice demonstrate shorter time spent on a hot plate starting on a second day of the experiment tested before next morphine injection, which indicates development of hyperalgesia. During all 12 days, eEF2K KO mice that received morphine treatment has similar results in the hot plate test as eEF2K KO mice treated with vehicle (FIG.4(A)), validating that loss of eEF2K prevents development of opioid-induced supra-spinal hyperalgesia. Tolerance is manifested by reduced analgesic reaction after 5-7 days of opioid treatment. As described herein, eEF2K KO mice treated with morphine had higher analgesic response to hot plate for 10 days compare to eEF2K KO mice with vehicle treatment as measured 30 minutes after daily injections (FIG.4(B)). These data indicate that loss of eEF2K prevent or delays development of opioid-induced supra-spinal tolerance. [00201]

[00202] eEF2K KO mice do not develop spinal hyperalgesia and tolerance after prolong morphine injections measures by warm water tail immersion test (TIT)

[00203] To demonstrate whether eEF2K KO and wild type mice have different basal response to pain measured by TIT (54°C), measurements were taken three consecutive times with 15 seconds intervals and the times of tail withdrawal from three measurements were averaged for each mouse. Overall, basal nociceptive values were similar between eEF2K KO and wild type mice (FIG.5(A)). Similarly, there were no difference in nociceptive values of each of three measurements between eEF2K KO and wild type mice (FIG.5(B)).

[00204] To demonstrate whether eEF2K signaling contributes to development of spinal hyperalgesia, TIT (49°C) was performed using eEF2K KO mice prior to daily i.p. injections of 15 mg/kg morphine or vehicle for 12 days. During all 12 days, eEF2K KO mice that received morphine treatment has similar results in the TIT test as eEF2K KO mice treated with vehicle (FIG.6), indicating that loss of eEF2K prevents development of opioid-induced spinal hyperalgesia.

[00205] To demonstrate whether eEF2K signaling contributes to development of spinal tolerance, TIT (49°C) was performed using eEF2K KO mice 30 minutes after daily i.p.

injections of 15 mg/kg morphine or vehicle for 12 days. For the first 8 days, TIT test was performed using three consecutive measurements with cut off time of 15 seconds (FIG.

7(A)). During all period tested, eEF2K KO mice treated with morphine kept their tails in hot water for longer than 15 seconds compare to 4-6 seconds of eEF2K KO mice treated with vehicle (FIG.7(A)). During days 10-12 of morphine injections, one measurement of tail withdrawal with 30 seconds cut off was performed. eEF2K KO mice treated with morphine kept their tails in warm water 3-times longer compare to that of eEF2K KO mice treated with vehicle (FIG.7(B)). These data indicate that loss of eEF2K prevents development of opioid- induced spinal tolerance.

[00206]

[00207] Morphine increases eEF2-P and BDNF expression in hippocampal areas of wild type but not eEF2K KO mice

[00208] Levels of eEF2K and eEF2-P in wild type and eEF2K KO mice were measured. Referring to FIG.8(A), there was a significant reduction in eEF2K signal in C3 and C1 hippocampal areas of eEF2K KO mouse compare to that in wild type mouse.

Similarly, lower phopsho-eEF2 level in eEF2K KO C3 and C1 hippocampal areas compare to wild type mice was measured (FIG.8(B)). These data confirm that eEF2K KO mice have lower eEF2K/eEF2-P signaling.

[00209] To determine whether 15 mg/kg daily morphine i.p. injections activate eEF2K pathway in wild type and eEF2K KO mice, immune-histochemical analysis was performed. Immuno-histochemical analysis of hippocampal areas demonstrated significant increase in phosphorylated eEF2 (FIG.9(A)) and BDNF expression (FIG.9(B)) in wild type mice treated with morphine for 12 days compare to that in eEF2K KO mice treated with morphine or in wild type mice treated with vehicle. These data indicates that (1) prolong morphine administration activates eEF2K/eEF2-P signaling in brain; and that (2) loss of eEF2K attenuates expression of eEF2-P-dependent genes induced by opioid administration.

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47. Shari L. Wiseman, Alexey G. Ryazanov, Jane R. Taylor, Nairn AC: Locomotor senzitization to cocaine using two-injection protocol requires eukaryotic elongation factor-2 kinase (eEF2K)-mediated translational control. In: Poster.2012? EXAMPLE 3 [00211] Specific Aims [00212] Without wishing to be bound by theory, eukaryotic elongation factor-2 kinase (eEF2K) signaling plays a significant role in the development of drug use disorders.

Experiments described herein will validate the role that eEF2K plays in the development of opioid-induced addiction, dependence, tolerance, withdrawal and relapse. eEF2K is a non- conventional calmodulin-dependent protein kinase in the alpha kinase family. It

phosphorylates eEF2, a general translation factor that regulates polypeptide chain elongation. Phosphorylation of eEF2 by eEF2K results in both a reduction in general protein synthesis and upregulation of the specific translation of mRNAs that are involved in the control of synaptic plasticity. In neurons, the eEF2K/eEF2-P signaling pathway is triggered via the activation of glutamate receptors. Activation of eEF2K negatively regulates GABAergic synaptic transmission, promoting disinhibition of neuronal excitation.

[00213] Preliminary evidence indicates that administration of either oxycodone or morphine induces phosphorylation of eEF2 and upregulates translation of P-eEF2-dependent mRNAs in brain. Furthermore, in eEF2K knockout mice, we observed increased analgesic response and delayed development of tolerance after chronic morphine treatment. Based on these observations, inhibition of eEF2K signaling may prevent the development of opioid use disorders. In this proposal, the above will be validated by evaluating behavioral responses to morphine administration in both homozygous eef2k knockout mice and mice treated with an eEF2K inhibitor. In addition, both the area of the brain and cell types in which morphine activates eEF2K signaling will be validated. Lastly, we will validate whether deletion of eEF2K affects the µ-opioid receptor, cAMP and dopamine signaling pathways. Identification of the role that eEF2K plays in opioid-use disorders will promote the development of compounds that can be co-administered with opioids to prevent adverse effects in humans.

[00214] There are three specific aims in this proposal: [00215] Specific Aim 1. Validate the effect that eEF2K deletion has on the

development of opioid analgesic and drug reinforcement behaviors. In this study, wild type and eEF2K KO mice will be i.p. injected with morphine or vehicle. We will measure morphine analgesic efficacy and the development of tolerance by hot plate and warm water tail immersion tests, as well as the development of signs of naloxone-precipitated withdrawal. In addition, we will compare the rewarding effect of morphine in wild type and eEF2K KO mice by monitoring conditioned place preference, conditioned place aversion, and relapse. This study will be performed in collaboration with Dr. Nicholas Goeders (LSUHSC, Shreveport, LA).

[00216] Specific Aim 2. Validate the effect that an eEF2K inhibitor has on the analgesic and rewarding responses of morphine. In this study, we will inject a novel eEF2K inhibitor, LV1053, into the nucleus accumbens of wild type mice. We will monitor the effect that LV1053 has on the analgesic and rewarding effects of morphine using experiments similar to those described in Specific Aim 1.

[00217] Specific Aim 3. Validate the effect that eEF2K deletion or inhibition has on pathways associated with opioid use disorders. In this study we will perform analysis of brain tissues obtained from animals tested in Specific Aims 1 and 2 using animal models for four conditions: acute morphine administration, chronic morphine administration, naloxone- precipitated withdrawal, and relapse. We will perform western blot and

immunohistochemical analyses to map brain areas in which morphine administration induces activation of eEF2K signaling. In addition, we will compare the levels of µ-opioid receptor and dopamine transmission in frozen brain sections and the level of cAMP in brain lysates from wild type, eEF2K KO mice and mice with injected eEF2K inhibitor before and after chronic morphine administration.

[00218] Research Strategy [00219] SIGNIFICANCE.

[00220] Opioids are the most effective prescription drugs used to treat pain. Their therapeutic use is limited, however, due to the development of addiction, tolerance, withdrawal and relapse. These adverse effects of opioids and their derivatives produce considerable economic and personal costs to society. To reduce the adverse effects of opioids and their associated costs, it is important to find the genes and signaling pathways that contribute to the development of the adverse effects.

[00221] Signaling pathways that contribute to the opioid reinforcing effect. Opioids exert their pharmacological effect via binding to the µ-opioid receptor (MOR) [1], which triggers a net of neurotransmitter systems. MOR activation leads to inhibition of GABA release by GABAergic neurons and thus dis-inhibition of excitatory glutamatergic transmission [2]. Without wishing to be bound by theory, glutamate signaling in mesolimbic dopamine structures is associated with the formation of drug-abuse memory and tolerance. Activation of glutamate receptors (NMDA, AMPA, and mGlu) has been shown to be involved in the development of drug reinforcing behavior (reviewed in [3]). Blocking opioid- induced glutamate receptor activation attenuates morphine-related positive memory formation [4] - [6], relapse to heroin [7] and morphine [8], and also attenuates morphine conditioned place aversion [9]. Protein synthesis has been shown to be involved in drug abuse neuroplasticity (reviewed in [10]). In particular, inhibition of the elongation step in protein synthesis by anisomycin or cycloheximide blocks morphine-induced conditioned place preference [11] - [13], suggesting that protein synthesis is a potential target for modulating the underlying neuroplasticity associated with drug abuse.

[00222] eEF2 kinase and neuronal plasticity. Eukaryotic elongation translation factor 2 kinase, eEF2K, is a non-conventional calmodulin-dependent protein kinase (CaMK III) belonging to the alpha kinase family [14]. The eEF2K phosphorylates eEF2 on Thr56 [15] - [18], leading to a reduction in general protein synthesis [16], [18] - [21]. However, translation of several specific mRNAs is upregulated by phosphorylated eEF2. Among them, mRNAs involved in the control of synaptic plasticity: aCamK II [21], activity-regulated cytoskeleton associated protein (Arc/Arg3.1) [22], [23], brain-derived neurotrophic factor (BDNF) [24], and microtubule-associated proteins [25] including MAP1B [26]. Both eEF2K and eEF2 are ubiquitous in all eukaryotes and are present in various tissues including brain [27]. In neurons, synaptic activity has been shown to trigger the eEF2K/eEF2-P signaling pathway via AMPA and NMDA-type glutamate receptors [21], [28] - [35], and also by activation of the class I metabotropic glutamate receptors (i.e., mGluR 1 and 5) [23] - [26]. Activation of eEF2K has been shown to negatively regulate GABAergic synaptic

transmission [36]. It is postulated that neuronal activation of eEF2K/eEF2-P signaling affects synaptic protein synthesis [28] (reviewed in [37]) that is linked to development of long-term depression (LTD) and long term potentiation (LTP), learning and memory, epilepsy and depression [22], [23], [36], [38] - [40].

[00223] Possible link between eEF2K pathway and opioid use disorders. Dis- inhibition of the ventral tegmental area (VTA) dopaminergic neurons has been shown to underlie opioid reinforcement behavior. MOR activation in GABAergic neurons leads to inhibition of their spontaneous inhibitory postsynaptic current (sIPSC) frequency [41].

Similarly, activation of eEF2K has been shown to negatively regulate GABAergic synaptic transmission [36]. Thus, without wishing to be bound by theory, eEF2K/P-eEF2 signaling is underlying the opioid inhibitory effect on GABAergic neurons that is responsible for the dis- inhibition of dopaminergic neurons and dopamine (DA) release (FIG.13A). Opioid activated MOR inhibits GABA release via a b-arrestin2 [41]. Phospho-eEF2 upregulated Arc/Arg3.1 protein binds to the clathrin/b-arrestin complex. It has been reported that Arc/Arg3.1 expression is induced by morphine exposure and also after abstinence [42]. Another phospho- eEF2 upregulated protein, BDNF, is released in an activity-dependent manner and modulates synaptic plasticity associated with addiction and dependence. Chronic exposure to drugs of abuse increases BDNF levels in the VTA neurons (reviewed in [43]). BDNF is linked to the development of opioid dependence by switching from a dopamine-independent to a dopamine-dependent response [44]. Thus, without wishing to be bound by theory, suppression of the eEF2K/phospho-eEF2/Arc/Arg3.1 and eEF2K/phospho-eEF2/BDNF pathways will prevent inhibition of GABAergic sIPSC by opioids and thus will prevent development of the drug rewarding effect (FIG.13A). Another mechanism connecting eEF2K signaling to opioid reinforcing behavior is via glutamate receptor signaling (FIG. 13B), since glutamatergic transmission is involved in the development of drug reinforcing behavior. In addition, upregulation of Arc/Arg3.1 by phospho-eEF2 may play an active role in b-arrestin-dependent MOR and G-proteins uncoupling and receptor internalization [45], pathways involved in the analgesic effect of opioids [46] and the development of drug desensitization and tolerance (FIG.13B).

[00224] The aim of the validation studies of this example is to target a new gene, eEF2K that may contribute to opioid use disorders, including addiction, tolerance, dependence and relapse. Identification of the role that eEF2K plays in opioid-use disorders will promote the development of compounds that can be co-administered with opioids to prevent adverse effects in humans.

[00225] INNOVATION.

[00226] Several studies have shown that chronic opioid exposure alters the gene expression profiles that underlie acute and chronic neuronal plasticity (reviewed in [47]). These changes in gene expression contribute to the opioid-induced analgesic effect, as well as the development of addiction, tolerance, and dependence [48] - [55]. However, the role played by the eEF2K pathway in opioid use disorders has never been reported. The experiments of this example will validate whether loss or inhibition of eEF2K prevents the development of morphine associated adverse effects in mice: addiction, tolerance, signs of withdrawal, and relapse. We will validate the role that eEF2K signaling plays in morphine use disorders using homozygous eef2k knockout mice (eEF2K KO) [56] and a new compound that inhibits eEF2K kinase activity. It has been reported that the eEF2K loss does not significantly affect normal biological processes in mice [36].

[00227] APPROACH

[00228] Preliminary Studies

[00229] Chronic oxycodone treatment activates eEF2K/eEF2-P signaling in rat nucleus accumbens. To validate whether opioid exposure triggers eEF2K activation, we analyzed nucleus accumbens lysates from rats gavaged with either 15 mg/kg oxycodone or water for 30 days. Western blot analysis revealed activation of signaling pathways upstream of eEF2K (i.e., phosphorylation of AMPKa and PKA) in rats treated with oxycodone (data not shown). We also observed a greater than 7-fold increase in phospho-eEF2 in the nucleus accumbens of rats after chronic oxycodone administration (FIG.14A). Phosphorylation of eEF2 is known to stimulate the translation of BDNF mRNA. In agreement with this, oxycodone resulted in a nearly 2-fold increase in BDNF protein levels (FIG.14B) and only a 1.2-fold increase in the BDNF mRNA level suggesting that translation of BDNF mRNA is upregulated. These data indicate that chronic oxycodone treatment activates eEF2K/eEF2-P signaling in the rat nucleus accumbens.

[00230] Loss of eEF2K potentiates the analgesic effect of morphine. To validate whether eEF2K is involved in the opioid-induced analgesic effect, we implanted pellets containing 25 mg of either morphine or placebo in wild type (WT) and eEF2K KO (KO) mice and performed a hot plate (53°C) test. The basal response measured a few days prior to morphine/placebo implantation is similar between WT and KO mice. In contrast, the 25 mg morphine implant drastically affects KO mice, promoting a nearly 2-fold higher analgesic response compared to that of the wild type mice two and four days after surgery (FIG.15A). These data indicate that the loss of eEF2K contributes to a higher sensitivity to the analgesic effects of opioids. One of the mechanisms that regulate sensitivity to opioids is the level of the MOR in the brain. Indeed, immunofluorescence analysis of hippocampal brain areas reveals higher MOR expression in eEF2K KO mice compared to that in wild type mice (FIG. 15B).

[00231] Morphine increases eEF2-P and BDNF levels in the hippocampus of wild type but not eEF2K KO mice. Four days after implantation of the 25 mg morphine pellet, WT mice demonstrated a significant increase in phospho-eEF2 (FIG.16A) and BDNF expression (FIG.16B) compared to that of KO mice with morphine or of WT mice with vehicle implants. These data indicate that: (1) morphine administration activates eEF2K/eEF2-P signaling in the brain; and (2) the loss of eEF2K attenuates opioid-induced expression of eEF2-P-dependent proteins.

[00232] eEF2K KO mice do not develop morphine antinociceptive tolerance. To investigate whether eEF2K signaling contributes to the development of tolerance, we performed warm water tail immersion tests (TIT, 49°C) for WT and KO mice 30 minutes after daily i.p. injections of either 15 mg/kg morphine or vehicle for twelve days. Basal nociceptive values are similar between WT and KO and mice. Wild type mice demonstrate maximal response during the first two days of morphine administration and then their response latencies drop to less than 9 seconds. In contrast, KO mice had average response latencies longer than 15 seconds during all 12 days of morphine administration (FIG.17A and B). These data indicate that the loss of eEF2K prevents the development of opioid antinociceptive tolerance. [00233] A novel eEF2K inhibitor, LV1053, suppresses phosphorylation of eEF2 in tissue culture cells. A compound (LV1053) that inhibits the phosphorylation of eEF2 in tissue culture cells with IC50 = 5 µM was identified. The addition of LV1053 to mouse embryonic fibroblasts efficiently suppresses the doxorubicin-induced phosphorylation of eEF2. Further, this compound is specific for eEF2K and does not inhibit other protein kinases (over 200 protein kinases were tested).

[00234] Without wishing to be bound by theory, the deletion or inhibition of eEF2K will prevent the development of morphine addiction, tolerance, signs of withdrawal and relapse in mice.

[00235] SPECIFIC AIM 1. Validate the effect that eEF2K deletion has on the development of opioid analgesic and drug reinforcement behaviors.

[00236] Objective 1.1. Determine an effect that deletion of eEF2K has on the analgesic efficacy of morphine and the development of tolerance and signs of withdrawal. The analgesic effect of morphine will be measured using hot plate and warm-water tail immersion tests for both WT and eEF2K KO mice. The appropriate temperature for these tests was established in our preliminary study. Base-line measurements for each mouse in the corresponding tests will be determined one or two days prior to the experiment.

[00237] Hot-plate test: the mouse will be placed on a 53°C plate and the time that it takes for the mouse to jump or shake or lick its paws will be measured. The cutoff time for the hot-plate test is 30 seconds.

[00238] Tail-Immersion test (TIT): the mouse will be placed in a mouse holder with its tail protruding. The distal half of the protruding tail will be immersed in a conical tube containing warm water (49°C) and the time until withdrawal of the tail will be measured. The cutoff time of immersion is 30 seconds. Data will be expressed as a percentage of the maximal possible effect (%MPE) calculated as: %MPE=(post-morphine time - base-line time)/(cut-off time - base-line time).

[00239] Acute morphine. First, we will measure the time course for morphine analgesia in WT and KO mice after a single 15 mg/kg morphine i.p. injection. The antinociceptive response values in Animal group I will be determined by a hot plate test every 30 minutes after morphine or vehicle i.p. injections. In this experiment, we will estimate the optimal time point (OTP) with the highest antinociceptive reaction in WT and KO mice, which we will use in our subsequent experiments to measure the analgesic effect of morphine. Eight animals in each group/treatment will be sacrificed immediately after the tests for brain tissue analyses (Acute morphine, FIG.12).

[00240] Chronic morphine. The remaining animals in Animal group I will be used to measure the development of morphine tolerance. WT and KO mice will be i.p. injected with 15 mg/kg morphine or vehicle once a day for another 8 days. Antinociceptive response will be measured by hot plate or by TIT tests on alternate days at the optimal time point described above. Nine-day morphine treatments were chosen based on the preliminary study, which showed a statistically significant difference between the WT and KO mouse antinociceptive responses to 15 mg/kg morphine i.p. injections during days 4-12 (as measured by a TIT). On the last day of morphine administration (day 9), twelve animals in each group/treatment will be sacrificed immediately after the tests for brain tissue analyses (Chronic morphine; FIG. 12).

[00241] Naloxone-precipitated withdrawal. On day 10, the remaining animals in Animal group I will receive one more vehicle or morphine i.p. injection. Two hours after injection, the animals will receive a 5 mg/kg naloxone i.p. injection. Animals will be placed singularly into glass beakers (21 cm diameter and 41 cm height). Withdrawal symptoms (number of jumps higher than 7 cm, wet-dog shakes and paw-tremor) will be monitored over 30-minute periods. The occurrence of diarrhea and mastication will be monitored in six 5- minute intervals. The animals will be sacrificed within 1-2 hours of naloxone administration for brain tissue analyses (Withdrawal, FIG.12).

[00242] In the preliminary studies, both WT and KO mice demonstrated an increase in hot plate response latency after 30 minutes, but not after 24 hours in response to a single 15 mg/kg morphine i.p. injection. Thus, we expect WT and KO mice to demonstrate maximum analgesic response during an overlapping time frame, around 30 minutes. Also, based on our preliminary study, we do not expect KO mice to develop morphine tolerance after 9 days. After naloxone precipitated withdrawal, we expect KO mice to have fewer signs of withdrawal.

[00243] Analgesic efficacy and tolerance. We will determine the dose-response curves measuring antinociceptive response in WT and KO mice on the first and ninth days of daily morphine injections in Animal Group II. These experiments will allow us evaluate whether the deletion of eEF2K affects (1) the animal’s sensitivity to morphine; and (2) the development of morphine tolerance. On day one, WT and KO mice will be treated with morphine using a cumulative dosing scheme (0.1, 0.25, 0.5, 1, 2, 5, 10, 20 mg.kg i.p.

injections) as described in [45] and [57]. Antinociception will be measured by a hot plate test at the optimal time point (OTP) described above. Mice will be injected with the next dose of morphine within 5 minutes of testing and then retested at the OTP again. This procedure will continue until either the mouse does not react to the hot plate within the cutoff time or until there is a plateauing of the dose–response curve, indicating that the latency does not increase from one dose to the next. Each mouse will be injected with a subsequent dose regardless of its response to the hot plate. In this case, all animals will receive the same total amount of morphine during the first day. On day 2 through day 8, mice will be i.p. injected with 15 mg/kg of morphine once a day. On day 9, mice will be treated with morphine using a cumulative dosing scheme similar to the one performed on day 1. The half-maximal antinociceptive dose (AD50) will be determined for day 1 and day 9 by using non-linear regression analysis to fit the sigmoidal curve (GraphPad Prism).

[00244] We expect eEF2K KO mice to have a higher sensitivity to morphine (lower AD50) and to not develop morphine tolerance. We also expect to determine the lowest morphine dose that produces maximal antinociceptive response in KO but not in WT mice. We will use this dose to evaluate the effect of the eEF2K inhibitor as described in Specific Aim 2.

[00245] Objective 1.2 Investigate the effect that deletion of eEF2K has on the development of morphine conditioned place preference, relapse and conditioned place aversion.

[00246] Morphine CPP. The effect that eEF2K deletion has on development of the morphine rewarding effect will be validated in WT and eEF2K KO mice by monitoring the conditioned place preference (CPP) as described in [58]. There is a good correlation between self-administration and CPP tests that measure the rewarding effect of opioids in animal models (reviewed in [59]). CPP will be conducted in Animal group III with a three- compartment place-conditioning apparatus. Before the start of morphine CPP training, initial preferences for the chambers will be determined using a 15-min preconditioning baseline session, in which the mice are placed in the central compartment and allowed to explore all three compartments. The time spent in each compartment and number of compartment entrances will be recorded. The morphine-paired compartment will be randomly assigned for each mouse in both the wild type and KO groups, in a counterbalanced unbiased manner. After the baseline assessment, mice will receive eight conditioning sessions (four morning and four evening sessions). During conditioning, mice in all groups will receive an injection of saline (i.p.) in the morning and will immediately be confined to one compartment (the saline-paired compartment) for 30 min. Four hours later, in the afternoon, mice in the “morphine” groups will receive an injection of morphine (10 mg/kg, i.p.) and immediately be confined to the other compartment (the morphine-paired compartment) for 30 min. Saline controls will be injected with saline only for both the morning and afternoon pairings. On the test day (24 h after the final morphine injection), mice will be placed in the central compartment and allowed to explore all three compartments for 15 min. Place preference scores will be calculated by subtracting the time spent in the drug-paired compartment during baseline testing from the time spent in the drug-paired compartment during the post- conditioning test.

[00247] Relapse; extinction and reinstatement of morphine CPP. The effect that eEF2K deletion has on relapse after morphine administration will be validated by monitoring CPP in the same Animal group III as described in [58]. Extinction of morphine CPP will begin 24 h after morphine CPP is completed. The mice in both groups will receive 10 extinction training sessions (five morning and five evening sessions), during which an injection of saline (i.p.) will be administered and the mice will be confined to the saline- paired compartment for 30 min. During the afternoon extinction training, mice will receive a second injection of saline and will be confined to the morphine-paired compartment for 30 min. Extinction testing occur 24 h after the final conditioning session (7d after morphine treatment); mice will be placed in the central compartment and allowed to explore all three chambers for 15 min. Preference scores will be calculated in the same manner as described above, with extinction of CPP being defined as a preference score that is 15% of the initial preference score. Reinstatement of morphine CPP (relapse) will be tested 24 h after the CPP extinction test. Mice that show extinction of morphine CPP and saline controls will be injected with 10 mg/kg morphine, placed in the central compartment, and allowed to explore all three compartments for 15 min. Morphine reinstatement will be calculated in the same manner as in the morphine CPP experiment. At the end of the experiment, the mice will be sacrificed for brain analysis (Relapse, FIG.12).

[00248] Conditioned place aversion. The effect that eEF2K deletion has on the development of morphine dependence will be validated by monitoring conditioned place aversion (CPA) in Animal group IV as described in [60]. The CPA test will also be conducted with a three-compartment place-conditioning apparatus. A pre-conditioning baseline test will be given on day 0. The mice will be placed in the central compartment and allowed to freely explore all three compartments for 15 min. The naloxone-paired compartment will be randomly assigned for each mouse in both the wild type and KO groups, in a counterbalanced unbiased manner. Prior to conditioning, morphine dependence will be induced by twice daily injections of morphine (i.p.) at 9:00 A.M. and 5:00 P.M. The morphine dose will be progressively increased by 5 mg/kg increments from 5 mg/kg on day 1 to 20 mg/kg on day 4, and this dose will be maintained on days 5 and 6. On day 5, 2 hours after the morning 20 mg/kg morphine injection, the mice will be confined to one

compartment for 30 min immediately after an injection of naloxone (1 mg/kg, i.p.). On day 6, the mice will be confined to the opposite compartment for 30 min after an injection of saline. The post-conditioning test will be conducted 24 hours after conditioning on day 7. The mice will be allowed to freely explore all three compartments for 15 min and the CPA score will be calculated as the difference between the time spent in the saline-paired compartment and the time spent in the naloxone-paired compartment.

[00249] We expect that KO mice will not develop CPP, relapse or CPA.

[00250] SPECIFIC AIM 2. Investigate the effect that an eEF2K inhibitor has on the analgesic and rewarding responses of morphine. LV1053 will be diluted in vehicle and directly injected into the mouse nucleus accumbens (the area of the brain associated with pain [61] and drug rewarding effects). Stereotaxic injections will be performed on wild type mice under ketamine and xylazine (i.p.100 mg/kg and 10 mg/kg, respectively) anesthesia using a KOPF mouse stereotaxic injection frame with digital reading connected to a KDS Pico pump that has the infusion and withdrawal capabilities with accurate deliveries of picoliter flow. A small volume of the LV1053 solution or vehicle will be injected into the medial AcbSh (AP: +0.98 mm; ML: 0.5 mm; DV: 4.0 mm), at stereotaxic coordinates based on the mouse brain atlas [62]. The compound will be delivered by slow pressure injection lasting 15 min to allow it to diffuse into the brain. After leaving the pipette in the brain for an additional 10 min, the pipette will be slowly retracted. We chose to inject LV1053 into the nucleus accumbens because we observed phosphorylation of eEF2 and an increase in BDNF in the nucleus accumbens after chronic oxycodone administration in rats. After recovery, the mice will be tested with the lowest dose of morphine at which we have observed maximal antinociceptive response in eEF2K KO but not in WT mice identified in Objective 1.1. We will perform several preliminary experiments to estimate the appropriate LV1053 dose to inject into the mouse brain prior to morphine injection and hot plate test. After establishing the appropriate LV1053 dose, we will monitor the effect that the eEF2K inhibitor has on morphine associated behaviors by measuring the cumulative dose curves in Animal group V on day 1 and 9 after chronic 15 mg/kg morphine injections as described in Objective 1.1. To investigate the effect that LV1053 has on the morphine rewarding response, Animal group VI will be evaluated using the CPP and relapse tests and Animal group VII will be evaluated using the CPA test as described in Objective 1.2. After each experiment, brain tissues of mice treated with LV1053 or vehicle will be collected as described in Specific Aim 1.

[00251] We expect that injection of LV1053 into the mouse nucleus accumbens will result in increased sensitivity to morphine (lower AD50) compared to that in vehicle injected mice. We also expect that LV1053 will prevent the development of morphine tolerance, CPP, relapse and CPA. In the event that we do not observe an effect of LV1053 on morphine antinociceptive or rewarding responses, we will investigate the effect that LV1053 injections have on other mesolimbic dopamine structures.

[00252] SPECIFIC AIM 3. Elucidate the effect that eEF2K deletion or inhibition has on pathways associated with opioid use disorders. In this specific aim, we will analyze brain tissues obtained from animals used in Specific Aims 1 and 2.

[00253] Objective 3.1. Mapping morphine-dependent activation of eEF2K signaling in mouse brains. We will validate the correlation between morphine administration and the activation of eEF2K/eEF2-P signaling for the four models (acute morphine, chronic morphine, withdrawal and relapse, FIG.12) in the experiments described above. Brain lysates prepared from mouse cortex, nucleus accumbens and brain stem will be tested for eEF2K/eEF2-P signaling by monitoring the phosphorylation of PKA, AMPK and eEF2 and upregulation of aCamKII, BDNF and Arg3.1/Arc by western blot analysis. To identify specific brain areas associated with morphine-dependent activation of eEF2K/eEF2-P signaling, we will perform immunohistochemical or immuno-fluorescence analysis of P- eEF2, BDNF, and Arg3.1/Arc in paraffin imbedded brain sections containing the cortex, hippocampus, nucleus accumbens, striatum, and brain stem. To identify cell types associated with morphine-dependent activation of eEF2K/eEF2-P signaling, we will perform double immunofluorescence staining of P-eEF2 and neuronal (NeuN), astrocyte (GFAP) and oligodendrocyte (MBP) markers in the brain sections. Special attention will be played to the VTA, nucleus accumbens, and its shell, which are important for the development of opioid addiction.

[00254] In eEF2K KO mice, we expect to observe morphine-induced activation of signaling upstream of eEF2K (phosphorylation of PKA and AMPK) but not downstream (phosphorylation eEF2 and upregulation of BDNF and Arg3.1/Arc) signaling. However, since BDNF and Arg3.1/Arc are also regulated at the gene expression level, we might see an increase in their signals in tissues from morphine treated KO but at a lower level than for the WT mice.

[00255] Objective 3.2. Investigate the effect that eEF2K deletion or inhibition has on MOR and DA signaling.

[00256] MOR expression. MOR is enriched in the cerebral cortex, striatum, hippocampus, locus coeruleus, and spinal cord. To validate whether eEF2K deletion or inhibition affects the distribution or level of MOR expression, we will analyze mouse brain tissues after chronic morphine administration by (1) immuno-fluorescence staining of MOR in paraffin imbedded brain slices; and (2) a [3H]-DAMGO binding assay in frozen brain sections. The [3H]-DAMGO binding assay will also allow us to investigate MOR desensitization.

[00257] Adenylyl cyclase activity. An increase in adenylyl cyclase activity is a hallmark of opioid dependence. The effect that eEF2K deletion or inhibition has on adenylyl cyclase activity after chronic morphine administration will be validated by measuring cAMP levels in striatal lysate samples using the Cyclic AMP Direct EIA Kit (Arbor) as described in [63]. Super-activation of cAMP signaling after prolonged morphine administration leads to neuronal adaptation involved in gene expression (e.g. transcription factors cFos and CREB). We will determine the expression levels for cFos and CREB after chronic morphine administration using western blot analysis of brain lysates.

[00258] Dopamine signaling. We will validate the increase in dopamine transmission in brain sections containing mesolimbic dopamine structures after chronic morphine administration by measuring [3H]raclopride (an antagonist of the D2 receptor) in the frozen brain sections as described in [64].

[00259] Based on our preliminary analyses, we expect that the eEF2K KO will have an elevated basal level of expression for MOR that is not affected by morphine administration (since morphine does not promote MOR internalization). It is possible that there will be an increase in cAMP signaling in the eEF2K KO mice after chronic morphine administration, however, we do not expect to see an increase in dopamine release in KO mice in response to morphine treatment. We expect to observe similar results in WT mice treated with LV1053 in the brain area surrounding the inhibitor injection.

[00260]

EXAMPLE 4

[00261] Characterization of the eEF2K KO mice. To investigate whether eEF2K signaling is involved in opioid-use disorders, we performed experiments using eEF2K knock- out mice. These mice have C57BL/6 background. It was reported that the eEF2K loss does not significantly affect normal biological processes in eEF2K KO mice [1]. According to Heise’s study: Comparison of eEF2K KO and corresponding wild type littermates showed that“gross brain architecture, body weight, food intake, body tone, grip strength, clasping, hanging wire, reflexes, and basic sociability were all normal in the eEF2K-KO mice.

Behavior tests demonstrated that object recognition ability, anxiety-like behavior, locomotor activity, and novel taste memory as well as spatial memory and reversal learning were normal in eEF2K-KO compared with WT mice. Baseline synaptic transmission and LTP formation were also normal in eEF2K-KO mice.” [1].

[00262] We compared eEF2K WT and eEF2K KO mice for their basal nociceptive responses using hot plate (HP) and warm water tail immersion (TIT) tests. We did not observe statistically significant differences in basal nociceptive responses between WT and eEF2K KO mice. To rule out a potential negative effect of eEF2K knock-out on locomotor coordination, we performed the Rotarod and the Open Field tests. WT and eEF2K KO mice demonstrated similar basal locomotor coordination and activity in the Rotarod test. [00263] eEF2K KO mice do not develop hyperalgesia after chronic morphine administration. It was shown that prolong opioid administration is associated with development of hyperalgesia, that is characterized by increased pain sensation due to neuronal plasticity in peripheral and CNS systems [2]. It was shown that microglia-released BDNF promotes development of morphine-induced hyperalgesia in mice [3]. In our experiments we observed increase in BDNF level in brain stem samples of WT mice after 30- days daily 15 mg/kg morphine i.p. injections (see FIG.18). We also observed increase in phospho-eEF2 in the same brain stem samples. Based on our observation that prolong morphine administration does not induce increase in BDNF expression in eEF2K KO mice (FIGs.9 and 16) eEF2K KO mice may not develop or will have delayed opioid-induced hyperalgesia. To test this, we measured nociceptive response in WT and eEF2K KO mice every 24 hours after daily 15 mg/kg morphine i.p. injection for 10 days (FIG.19). Hot plate tests revealed that WT mice started to demonstrate decrease in pain threshold after 5 days of morphine treatment indicating development of hyperalgesia. However, eEF2K KO mice remain the same level of nociception during 11 days of daily morphine treatment indicating lack or delay of opioid-induced hyperalgesia (FIG.19A and B). To tests whether

pharmacological inhibition of eEF2K will produce similar effect, WT mice were injected with the eEF2K inhibitor, 2.5 mg/kg A-484954, or its vehicle 0.5% carboxymethyl cellulose, along with 15 mg/kg morphine daily administration for 10 days. We observed significant decrease in pain threshold in mice treated with vehicle, but not in mice treated with A-484954 after 10 days of morphine administration (FIG.19C). Interestingly, this treatment did not prevent development of morphine tolerance suggesting that A-484954 does not penetrate blood-brain barrier but suppresses eEF2K neuropathic effect in peripheral tissues. To investigate whether eEF2 is phosphorylated under conditions that cause neuropathy, we treated MCF7 cells with Paclitaxel, a chemotherapeutic drug that is known to cause neuropathic pain. Western blot analysis demonstrated increase in phospho-eEF2 in cells grown in the presence of 20 nM paclitaxel for 18 hours (FIG.20). All together, these data indicate that eEF2K/eEF2-P signaling is involved in development of hyperalgesia.

[00264] Withdrawal. We did not observe the behavioral difference between eEF2K KO and WT mice chronically treated with morphine (15 mg/kg every 24 hours for 12 days) after the naloxone-precipitated withdrawal.

[00265] ANIMAL EXPERIMENTS

[00266] Morphine Rewarding and Relapse. The effect of eEF2K deletion on the development of morphine rewarding effect will be tested in WT and eEF2K KO mice by monitoring the conditioned place preference (CPP) as described in [4]. There is a good correlation between self-administration and CPP tests that measure opioids rewarding effect in animal models (reviewed in [5]). In addition, drug-seeking behavior could be measured by the Relapse test, in which animal’s place preference and drug-seeking behavior is tested again after period of abstinence. CPP will be conducted using a three-compartment place- conditioning apparatus. Before the start of morphine CPP training, initial preferences for the chambers will be determined in a 15-min preconditioning baseline session, in which mice are placed in the central compartment and allowed to explore all three compartments (FIG.21, day 0, BL). The time spent in each compartment and number of compartment entrances will be recorded. The morphine-paired compartment will be randomly assigned for each mouse in both the wild type and KO groups, in a counterbalanced unbiased manner. After the baseline assessment, mice will receive eight conditioning sessions during four days (four morning and four evening sessions) (Fig.21, days 1-4, conditioning). During conditioning, mice in all groups will receive an injection of saline (i.p.) in the morning and will immediately be confined to one compartment (the saline-paired compartment) for 30 minutes. Four hours later, in the afternoon, mice in the“morphine” groups will receive an injection of morphine (15 mg/kg, i.p.) and immediately be confined to the other compartment (the morphine-paired compartment) for 30 minutes. Saline controls will be injected with saline only for both the morning and afternoon pairings. On the CPP test day (24 h after the final morphine injection) (Fig.21, day 5, CPP), mice will be placed in the central compartment and allowed to explore all three compartments for 15 min. Place preference scores will be calculated by subtracting the time spent in the drug-paired compartment during baseline testing from the time spent in the drug-paired compartment during the post-conditioning test. The site of saline and morphine/saline injections will be rotated between morning and afternoon sessions. The site of morphine injection will be rotated every day.

[00267] To measure CPP, we will use 80 WT mice [40M: 20M+Saline and

20M+Morph; 40F: 20F+Saline and 20F+Morph] and 80 KO mice [40M: 20M+Saline and 20M+Morph; 40F: 20F+Saline and 20F+Morph]. After the CPP test, 40 WT mice [20M: 10M+Saline and 10M+Morph; 20F: 10F+Saline and 10F+Morph] and 40 KO mice [20M: 10M+Saline and 10M+Morph; 20F: 10F+Saline and 10F+Morph] will be sacrificed for brain tissue analyses: brain tissues from 5 animals in each group/treatment will be harvested for the western blot analysis, another group of 5 animals–– to collect brain that will be used for immunohistochemistry and immunofluorescent analyses. The remaining animals will undergo to the relapse testing (FIG.12).

[00268] The effect of eEF2K deletion or inhibition on relapse after morphine administration will be monitored in the same group of animals tested for CPP. Extinction of morphine CPP will begin 24 h after morphine CPP is completed (FIG.21, day 6). The mice in both groups will receive 10 extinction training sessions during five days (five morning and five evening sessions) (FIG.21, days 6-10, extinction). In the morning, mice will be injected with saline (i.p.) and the mice will be confined to the saline-paired compartment for 30 minutes. During the afternoon extinction training, mice will receive a second injection of saline and will be confined to the morphine-paired compartment for 30 minutes. Side of injections will be rotated between morning and afternoon sessions. Extinction testing will occur 24 h after the final conditioning session (7d after morphine treatment) (FIG.21, day 11, ET); mice will be placed in the central compartment and allowed to explore all three chambers for 15 min. Preference scores will be calculated in the same manner as described above, and extinction of CPP is defined as a preference score that is 15% of the initial preference score. Reinstatement of morphine CPP (relapse) will be tested 24 h after the CPP extinction test (FIG.21, day 12, CPP). Mice that show extinction of morphine CPP and saline controls will be injected with 15 mg/kg morphine. Immediately after morphine

administration, mice will be placed in the central compartment, and allowed to explore all three compartments for 15 minutes. Morphine reinstatement will be calculated in the same manner as in the morphine CPP experiment.

[00269] Opioid dependence. The effect that eEF2K deletion has on the development of morphine dependence will be tested by monitoring the conditioned place aversion (CPA) as described in [6]. This test measures increased avoidance and/or escape behaviors indicative of the aversive effects of a specific stimulus. In our experiment, adverse effects will be produced by the naloxone-induced withdrawal symptoms after mice became addicted to morphine. The CPA test will also be conducted with a three-compartment place-conditioning apparatus. A pre-conditioning baseline test will be given on day 0 (Fig.22, BL). The mice will be placed in the central compartment and allowed to freely explore all three

compartments for 15 min. The naloxone-paired compartment will be randomly assigned for each mouse in both the WT and KO groups, in a counterbalanced unbiased manner.

Morphine dependence in 20 WT mice [10M+Morph and 10F+Morph] and 20 KO mice

[10M+Morph and 10F+Morph] will be induced by twice daily injections of morphine (i.p.), during 6 days, one in the morning and a second injection in the evening, (FIG.22, days 1-6). On day 1, each mouse will receive one morning injection of 5 mg/kg morphine and one evening injection of 5 mg/kg morphine (FIG.22, day 1). The morphine dose will be progressively increased by 5 mg/kg increments from 5 mg/kg on day 1 to 20 mg/kg on day 4, and this dose will be maintained on day 5. Thus, on day 1, each mouse will receive 10 mg/kg morphine in total, on day 2– 20 mg/kg morphine, day 3– 30 mg/kg, and on days 4 and 6– 40 mg/kg morphine in total. Side of injections will be rotated between morning and evening sessions. On day 5, two hours after the evening 20 mg/kg morphine injection, the mice will be injected with naloxone (1 mg/kg, i.p.) and immediately confined to one compartment for 30 min. On day 6, two hours after the evening 20 mg/kg morphine injection, the mice will be injected with saline and confined to the opposite compartment for 30 min. The post- conditioning test will be conducted 24 hours after conditioning on day 7 (FIG.22). The mice will be allowed to freely explore all three compartments for 15 min and the CPA score will be calculated as the difference between the time spent in the saline-paired compartment and the time spent in the naloxone-paired compartment.

[00270] Other drugs Rewarding, Relapse and Dependence. The effect of eEF2K deletion or inhibition on the development of rewarding effect, relapse and dependence induced by other drugs of abuse, such as cocaine, ethanol, amphetamine, cannabinoid, a benzodiazepine, derivatives thereof, or any combination thereof, will be investigated the same way as it is described in the“Morphine Rewarding and Relapse” and“Opioid dependence.” sections above.

[00271]

[00272] REFERENCES CITED IN THIS EXAMPLE

1. Heise C, Taha E, Murru L, Ponzoni L, Cattaneo A, Guarnieri FC, Montani C, Mossa A, Vezzoli E, Ippolito G et al: eEF2K/eEF2 Pathway Controls the Excitation/Inhibition Balance and Susceptibility to Epileptic Seizures. Cerebral cortex (New York, NY : 1991) 2017, 27(3):2226-2248.

2. Lee M, Silverman SM, Hansen H, Patel VB, Manchikanti L: A comprehensive review of opioid-induced hyperalgesia. Pain Physician 2011, 14(2):145-161.

3. Ferrini F, Trang T, Mattioli TA, Laffray S, Del'Guidice T, Lorenzo LE, Castonguay A, Doyon N, Zhang W, Godin AG et al: Morphine hyperalgesia gated through microglia- mediated disruption of neuronal Cl(-) homeostasis. Nat Neurosci 2013, 16(2):183-192. 4. Portugal GS, Al-Hasani R, Fakira AK, Gonzalez-Romero JL, Melyan Z, McCall JG, Bruchas MR, Moron JA: Hippocampal long-term potentiation is disrupted during expression and extinction but is restored after reinstatement of morphine place preference. J Neurosci 2014, 34(2):527-538.

5. Morgan MM, Christie MJ: Analysis of opioid efficacy, tolerance, addiction and dependence from cell culture to human. British journal of pharmacology 2011, 164(4):1322- 1334.

6. Wang J, Li M, Wang P, Zha Y, He Z, Li Z: Inhibition of the lateral habenular CaMK abolishes naloxone-precipitated conditioned place aversion in morphine-dependent mice. Neurosci Lett 2017, 653:64-70.

EXAMPLE 5

[00273] Hyperalgesia. In 1979, the International Association for the Study of Pain referred to pain as“[a]n unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” [1], [2]. For example, pain sensitization can be characterized by the network of neuronal, somatic, emotional and cognitive components. Multiple lines of evidence indicate that modulation of protein synthesis is involved in pain perception in the central and peripheral nervous systems [3]. During acute pain state, activated axonal nociceptors trigger modulation of translation via several routs that leads to neuronal plasticity and microglia activation underlying

development of persistent pain. These newly synthesized proteins are involved in

development of hyperalgesic priming and transition to chronic pain state [4].

[00274] Anxiety-induced hyperalgesia. To measure pain perception, WT and eEF2K KO males mice were tested by the warm water tail immersion (TIT) tests twice a day every other day: before the saline injection (WT-S-0 and KO-S-0) and 30 minutes after saline injection (WT-S-30 and KO-S-30). We did not observe the difference between WT and KO mice in basal response (at the beginning of the experiment). In the experiments when TIT response was measured before the saline injection, WT mice demonstrated decrease in the response time to the painful stimuli from 2nd to 12th day of the experiment (Fig.23A) suggesting development of environment associated anxiety. KO demonstrated lack of experiment environment associated anxiety maintaining their pain response at the same level during 12 days (Fig.23A). Moreover, the difference between WT and KO mice responses to the TIT was statistically significant during days 2-12 (Fig.23A). In the experiments when TIT response was measured after 30 minutes of saline injections, WT mice demonstrated development of hyperalgesia while KO mice had lack of increased sensitivity to pain during 12 days of saline injections (Fig.23B). Considering that saline i.p. injection does not cause pain, these data further suggest that eEF2K KO mice do not develop anxiety-triggered pain perception.

[00275] Morphine-induced hyperalgesia. It was shown that prolong opioid administration is associated with development of hyperalgesia or increased pain sensitivity [5]. To investigate morphine-induced hyperalgesia, WT and eEF2K KO males mice were i.p. injected with 15 mg/kg morphine once a day for 12 days and tested for pain perception by the hot plate (HP) or TIT tests on alternating days. Increased sensitivity to pain stimuli was measured every day, 24 hours after the last morphine administration (WT-M-0 and KO-M-0). WT mice demonstrated faster response to pain stimuli in both HP and TIT tests during starting from day 5. (Fig.24). KO mice demonstrated lack of increased sensitivity to pain during 12 days of morphine injections measured by HP and TIT (Fig.24). The difference between WT and KO mice in their responses to pain stimuli during chronic morphine injection was statistically significant, starting from day 4 in the TIT and day 5 in the HP tests. It was shown that microglia-released BDNF promotes development of morphine-induced hyperalgesia in mice [6]. In our earlier experiments, we observed increase in BDNF level in brain stem samples of WT mice after 30-days daily 15 mg/kg morphine i.p. injections. In addition, we observed increase in BDNF level in hippocampus after 4 days of 25 mg morphine implant in WT but not KO mice. Lower level or delayed increase in morphine- induced BDNF expression in brain may explain lack of hyperalgesia after chronic morphine administration in KO mice.

[00276] Oxycodone-induced hyperalgesia. To investigate whether pharmacological inhibition of eEF2K activity will result in suppression of development of morphine-induced hyperalgesia, WT mice were i.p. injected with the eEF2K inhibitor, 2.5 mg/kg A-484954, or its vehicle 0.5% carboxymethyl cellulose, along with 15 mg/kg oxycodone daily gavaging for 20 days. We observed significant decrease in pain threshold in mice treated with vehicle, by about 60% in both, HP and TIT, tests (Fig.24. C and D). In contrary, mice treated with A- 484954 demonstrated delayed (HP) or lack (TIT) of oxycodone-induced hyperalgesia during 20 days of treatment (Fig.24. C and D).

[00277] Morphine-induced tolerance. Earlier in our pilot experiment, we have demonstrated that mice lacking eEF2K exhibit delayed or no signs of tolerance after morphine administration. To obtain statistical analysis on these data, we measured nociceptive response in WT and eEF2K KO mice every 24 hours after daily 15 mg/kg morphine i.p. injection for 12 days. Both, WT and KO mice demonstrated increase in time of their nociceptive response in the HP and TIT tests after morphine injection (Fig.25A and B, day 2). However, Hot plate tests revealed that WT mice started to demonstrate decrease in pain threshold on 5th day of morphine treatment indicating development of morphine tolerance (Fig.25A). eEF2K KO mice demonstrated statistically higher response to the hot plate compare to the base line (0%) during 8 days of daily morphine treatment indicating lack or delay of morphine-induced tolerance (Fig.25A). In the TIT tests, the difference in morphine response between WT and KO mice were even more drastic. In WT, morphine injections failed to produce nociceptive response starting from 4^th day indicating development of morphine tolerance (Fig.25B). KO mice demonstrated maximum response to morphine in the TIT tests during all 12 days suggesting delayed or lack of morphine tolerance (Fig.25B).

[00278] Morphine-induced Locomotor activity. We have investigated the effect of chronic morphine i.p. injections on locomotor activity. Earlier we have demonstrated that in the Rotarod test, WT and eEF2K KO mice do not differ in their ability to grip and stay the rotating rode during saline or morphine treatments. Next, we have investigated locomotor activity in the open field test (OF) in which a mouse was placed in the plastic box equipped with several sensors (Opto-Varimex 4 system) monitoring its movement. It is documented that acute opioid administration stimulates locomotor activities in animals ([7], [8]). In agreement with that, we observed that morphine treatment increased running distances in both, WT and KO mice (Fig.26A) during 9 days of the experiment. In correlation with that, WT and KO mice demonstrated shorter resting time after first morphine injection (Fig.26B). Interestingly, after 5 days of morphine treatment (day 5), WT mice displayed statistically higher motor activity and lower resting time compare to that of KO mice (Fig.26A and B). These data suggest that morphine has more prolong effect on locomotor activity in WT mice compare to that in KO mice. [00279] Anxiety-like behavior. One of the side effects of opioid consumption is associated with its soothing effect not only on pain but also on emotions reducing anxiety (anxiolytic effect). This property may lead to unnecessary consumption of opioids by people who does not experience pain but seek drugs to relieve anxiety, for example teenagers, promoting development of opioid addiction and overdose. One of the parameters that characterize anxiety-like behavior in mice is rearing breaks. It was demonstrated that morphine administration decreases frequency of rearing in mice [7]. In agreement with that, we observed statistically significant decrease in number of rearing breaks in WT mice during 5 days of morphine administration compare to the base-line (Fig.26C). In contrast, KO mice did not demonstrate statistically significant difference in rearing events between before and during 9 days of morphine treatment (Fig.26C). On day 5 of daily morphine injections, WT mice have statistically less rearing events compare to that of KO mice. These data suggest that acute and chronic morphine treatment does not produce anxiolytic or anxiety-reducing effect in the eEF2K KO mice.

[00280] Rewarding and relapse. We did not observed difference between WT and KO female and male mice in the place preference and drug-seeking behavior tested by the place-conditioning apparatus (data not shown).

[00281] Withdrawal. We did not observed difference between WT and KO mice in the number of jumps after naloxone-induced chronic morphine withdrawal (data not shown). REFERENCES CITED IN THIS EXAMPLE

1.  IASP: Pain terms: a list with definitions and notes on usage. Recommended by the IASP Subcommittee on Taxonomy. Pain 1979, 6(3):249. 2. Merskey H, Bogduk N, Taxonomy IAftSoPTFo: Classification of chronic pain : descriptions of chronic pain syndromes and definitions of pain terms, vol. xvi, 222 p. : ill. United States: Seattle : IASP Press, c1994; 1994.

3. Penas C, Navarro X: Epigenetic Modifications Associated to Neuroinflammation and Neuropathic Pain After Neural Trauma. Frontiers in cellular neuroscience 2018, 12:158.

4. Khoutorsky A, Price TJ: Translational Control Mechanisms in Persistent Pain.

Trends Neurosci 2018, 41(2):100-114.

5. Lee M, Silverman SM, Hansen H, Patel VB, Manchikanti L: A comprehensive review of opioid-induced hyperalgesia. Pain Physician 2011, 14(2):145-161.

6. Ferrini F, Trang T, Mattioli TA, Laffray S, Del'Guidice T, Lorenzo LE, Castonguay A, Doyon N, Zhang W, Godin AG et al: Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl(-) homeostasis. Nat Neurosci 2013, 16(2):183-192.

7. Patti CL, Frussa-Filho R, Silva RH, Carvalho RC, Kameda SR, Takatsu-Coleman AL, Cunha JL, Abílio VC: Behavioral characterization of morphine effects on motor activity in mice. Pharmacology, biochemistry, and behavior 2005, 81(4):923-927. 8. Fujii K, Koshidaka Y, Adachi M, Takao K: Effects of chronic fentanyl administration on behavioral characteristics of mice. Neuropsychopharmacology reports 2019, 39(1):17-35.

***** EQUIVALENTS [00282] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

WHAT IS CLAIMED:

1. A composition comprising a therapeutically effective amount of an analgesic and a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, or a combination thereof.

2. The composition of claim 1, wherein the reducer comprises an eEF2 kinase (eEF2K) inhibitor.

3. The composition of claim 1, wherein the reducer decreases phosphorylation of eEF2 at Thr56.

4. The composition of claim 2, wherein the eEF2K inhibitor comprises a compound of Formula I or derivatives thereof:

5. The composition of claim 1, wherein the analgesic comprises an opioid, opioid peptides, cocaine, ethanol, amphetamine, cannabinoid, a benzodiazepine, derivatives thereof, or any combination thereof.

6. The composition of claim 5, wherein the opioid/opiate comprises morphine, fentanyl, methadone, buprenorphine, hydrocodone, oxycodone, heroin, or derivatives thereof.

7. The method of claim 5, wherein the benzodiazepine comprises midazolam, alprazolam, clobazam, clonazepam, clorazepate, chlordiazepoxide, diazepam, estazolam, lorazepam, oxazepam, temazepam, triazolam, or derivatives thereof.

8. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

9. A method of treating a subject afflicted with pain, the method comprising administering to the subject an analgesic and a therapeutically effective amount of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, or a combination thereof.

10. A method of reducing pain in a subject afflicted with a disease, the method comprising administering to the subject a therapeutically effective amount of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer and an analgesic.

11. A method of increasing the sensitivity of a subject to an analgesic, the method comprising administering to a subject in need thereof a therapeutically effective amount of eukaryotic elongation factor 2 (eEF2) phosphorylation reducer and an analgesic.

12. A method of reducing or preventing the development of hyperalgesia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer and an analgesic.

13. A method of reducing or preventing the abuse potential of an analgesic, the method comprising administering to a subject in need thereof a therapeutically effective amount of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer and an analgesic.

14. A method of reducing or preventing a withdrawal symptom after analgesic cessation, the method comprising administering to a subject in need thereof a therapeutically effective amount of a eukaryotic elongation factor 2 (eEF2) phosphorylation reducer and an analgesic.

15. The method of any one of claims 9-14, wherein the reducer comprises an eEF2K inhibitor.

16. The method of any one of claims 9-14, wherein the analgesic is administered prior to, in conjunction with, or subsequent to administering the eEF2 phosphorylation reducer.

17. The method of any one of claims 9-14, wherein the analgesic comprises an opioid, cocaine, ethanol, amphetamine, cannabinoid, a benzodiazepine, or any combination thereof.

18. The method of claim 17, wherein the opioid comprises morphine, fentanyl, methadone, buprenorphine, hydrocodone, oxycodone, heroin, or derivatives thereof.

19. The method of claim 17, wherein the benzodiazepine comprises midazolam, alprazolam, clobazam, clonazepam, clorazepate, chlordiazepoxide, diazepam, estazolam, lorazepam, oxazepam, temazepam, triazolam, or derivatives thereof.

20. The method of any one of claims 9-14, wherein the reducer decreases phosphorylation of eEF2 at Thr56.

21. The method of any one of claims 9-14, wherein the reducer comprises the composition of claim 1.