Novel therapeutic compositions for treating addictions and methods of use thereof

Abstract

Described herein are novel compounds for use in treating substance addiction and withdrawal induced anxiety and depression, and methods of treating addictions and substance use disorders including alcohol abuse, opioid abuse, and nicotine abuse using compositions comprising MC-100093, wherein the compositions may further comprise ceftriaxone and / or additional therapeutic agents.

Description

[0001] Attorney Docket No.206017-0285-00WO TITLE OF THE INVENTION Novel Therapeutic Compositions for Treating Addictions and Methods of Use Thereof CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority to U.S. Provisional Application No.63 / 771,949, filed March 14, 2025, U.S. Provisional Application No.63 / 685,490, filed August 21, 2024, and U.S. Provisional Application No.63 / 684,038, filed August 16, 2024, each of which are incorporated by reference herein in their entireties. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under R01 AA029674 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION There is a need in the art for novel compositions for treating addiction and the use of addictive agents. The present invention addresses this unmet need in the art. SUMMARY OF THE INVENTION Described herein is a method of treating a substance use disorder in a subject, comprising the step of administering to the subject a therapeutically effective amount of a composition comprising a compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof

[0002] Attorney Docket No.206017-0285-00WO In some examples, the composition further comprises ceftriaxone. In some examples, the substance use disorder comprises abuse of a drug selected from the group consisting of opioids, nicotine, alcohol, fentanyl, marijuana, synthetic cannabinoids, stimulants, barbiturates, benzodiazepines, dextromethorphan (DXM), a sleep medication, khat, synthetic cathinones, cocaine, 3,4- methylenedioxymethamphetamine (MDMA), phencyclidine (PCP), lysergic acid diethylamide (LSD), psilocybin, an inhalant, Rohypnol, gamma-hydroxybutyric acid (GHB), N,N-Dimethyltryptamine (DMT), ayahuasca, mescaline, salvia, and combinations thereof. In some examples, the substance use disorder comprises abuse of opioids. In some examples, the substance use disorder comprises abuse of nicotine. In some examples, the substance use disorder comprises abuse of fentanyl. In some examples, the substance use disorder comprises abuse of a drug which down-regulates a molecule selected from the group consisting of xCT, glutamate transporter 1 (GLT-1), and brain-derived neurotrophic factor (BDNF). In some examples, the substance use disorder comprises abuse of a drug which up- regulates a molecule selected from metabotropic glutamate receptor 1 (mGluR1), tumor necrosis factor alpha (TNF-α), and high mobility group box 1 (HMGB1). In some examples, the substance abuse disorder comprises abuse of hydrocodone (dihydrocodeinone), heroin, codeine, fentanyl, hydromorphone, meperidine, methadone, morphine, oxycodone, or oxymorphone. In some examples, the method reduces a symptom of withdrawal in the subject. In some examples, the method prevents a relapse of the substance use disorder in the subject. In some examples, the method reduces dependence on a substance of the substance use disorder in the subject. In some examples, the composition further comprises an additional therapeutic agent. In some examples, the additional therapeutic agent is selected from the group consisting of an opioid antagonist, a mixed opioid partial agonist / antagonist, an antidepressant, an antiepileptic, an antiemetic, a corticotrophin- releasing factor-1 (CRF-1) receptor antagonist, a selective serotonin-3 (5-HT3) antagonist, a 5-HT2A / 2C antagonist, a cannabinoid-1 (CB1) receptor antagonist, and combinations thereof. In some examples, the method further comprises the step of exposing the subject to an additional pharmacotherapy. In some examples, the additional Attorney Docket No.206017-0285-00WO pharmacotherapy comprises a gradually reducing regimen, a substitution therapy, or a medication assisted treatment. Also described herein are composition for use against overdose from use of a substance comprising ceftriaxone and a compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof In some alcohol, nicotine, fentanyl, or combinations some β-lactam is MC-100093. Also described herein is a method of treating an overdose from an opioid in a subject comprising the step of administering to the subject the composition. BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of illustrating the invention, there are depicted in the drawings certain examples of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the examples depicted in the drawings. Fig.1, comprising Fig.1A through Fig.1C, depicts representative data on the effects of chronic hydrocodone overdose, ceftriaxone, or MC-100093 treatment on locomotion activity in mice. Control (n= 5-6), Hydrocodone (n= 5-6), Hydrocodone-Ceftriaxone (n= 5-6), and Hydrocodone-MC-100093 (n= 5-6). Statistical analyses exhibited a significant increase in activity, in Fig.1A, and ambulatory for the hydrocodone-treated group, in Fig.1B, compared to the control group. Ceftriaxone (200 mg / kg, i.p.) treatment normalized hydrocodone-induced upregulation of activity, in Fig.1A, and ambulatory, in Fig.1B, as compared to the hydrocodone-treated group. Fig.1C depicts statistical analysis that did not demonstrate any significant z-activity differences among all tested groups. The values are expressed as means ± S.E.M. (* p < 0.05, ** p < 0.01). Attorney Docket No.206017-0285-00WO Fig.2, comprising Fig.2A through Fig.2C, depicts representative data on the effects of chronic hydrocodone overdose, ceftriaxone, or MC-100093 treatment on oxygen consumption, carbon dioxide production, and respiratory exchange ratio in mice. Control (n= 5-6), Hydrocodone (n= 5-6), Hydrocodone-Ceftriaxone (n= 5-6), and Hydrocodone-MC-100093 (n= 5-6). Fig.2A depicts one-way ANOVA followed by the Newman-Keuls multiple comparisons test showed that oxygen consumption was higher in hydrocodone treated group as compared to the control group while both ceftriaxone (200 mg / kg, i.p.) and MC-100093 (50 mg / kg, i.p.) treatments attenuated hydrocodone-induced increased in oxygen consumption as compared to the hydrocodone-treated group. Fig.2B depicts one-way ANOVA followed by the Newman-Keuls multiple comparisons test revealed that carbon dioxide production was higher in hydrocodone treated group as compared to the control group while both ceftriaxone (200 mg / kg, i.p.) and MC-100093 (50 mg / kg, i.p.) treatments lowered hydrocodone-induced increased in carbon dioxide production as compared to the hydrocodone-treated group. Fig.2C depicts one-way ANOVA followed by the Newman-Keuls multiple comparisons test demonstrated that the respiratory exchange ratio was downregulated in hydrocodone treated group as compared to the control group while MC-100093 (50 mg / kg, i.p.) treatment upregulates hydrocodone- induced downregulation of respiratory exchange ratio as compared to the hydrocodone-treated group. The values are expressed as mean ± S.E.M. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Fig.3, comprising Fig.3A through Fig.3C, depicts representative data on the effects of chronic hydrocodone overdose, ceftriaxone, or MC-100093 treatment on mGluR1 expression in the nucleus accumbens (NAc), amygdala (AMY), and dorsomedial prefrontal cortex (dmPFC). Control (n= 5-6), Hydrocodone (n= 5-6), Hydrocodone-Ceftriaxone (n= 5-6), and Hydrocodone-MC-100093 (n= 5-6). Representative western blot images for mGluR1 expression from the NAc, AMY, and dmPFC. One-way ANOVA followed by the Newman-Keuls post hoc test revealed a significant increase in the ratio of mGluR1 / β-tubulin in the hydrocodone-treated group for NAc, in Fig.3A, AMY, in Fig.3B, and dmPFC, in Fig.3C, compared to the control group. Treatments with ceftriaxone (200 mg / kg, i.p.) or MC-100093 (50 mg / kg, i.p.) normalized the hydrocodone-induced increased mGluR1 expression in the NAc, in Fig.3A, AMY, in Fig.3B, and dmPFC, in Fig.3C, as compared to the hydrocodone-treated group. Control group data was represented as 100%. The values Attorney Docket No.206017-0285-00WO are expressed as mean ± S.E.M. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). NAc, nucleus accumbens; AMY, amygdala; dmPFC, dorsal medial prefrontal cortex; mGluR1, metabotropic glutamate receptor 1; Hyd, hydrocodone; Cef, ceftriaxone. Fig.4, comprising Fig.4A through Fig.4C, depicts representative data on the effects of chronic hydrocodone overdose, ceftriaxone, or MC-100093 treatment on xCT expression in the NAc, AMY, and dmPFC. Control (n= 5- 6), Hydrocodone (n= 5-6), Hydrocodone-Ceftriaxone (n= 5-6), and Hydrocodone- MC-100093 (n= 5-6). Representative western blot images for xCT expression from the NAc, AMY, and dmPFC. One-way ANOVA followed by the Newman-Keuls post hoc test revealed a significant decrease in the ratio of xCT / β-tubulin in the hydrocodone-treated group for NAc, in Fig.4A, AMY, in Fig.4B, and dmPFC, in Fig.4C, compared to the control group. Treatments with ceftriaxone (200 mg / kg, i.p.) or MC-100093 (50 mg / kg, i.p.) reversed the hydrocodone-induced downregulation of xCT expression in the NAc, in Fig.4A, AMY, in Fig.4B, and dmPFC, in Fig.4C, as compared to the hydrocodone-treated group. Control group data was represented as 100%. The values are expressed as mean ± S.E.M. (* p < 0.05, ** p < 0.01, *** p < 0.001). NAc, nucleus accumbens; AMY, amygdala; dmPFC, dorsal medial prefrontal cortex; xCT, cystine / glutamate antiporter; Hyd, hydrocodone; Cef, ceftriaxone. Fig.5, comprising Fig.5A through Fig.5C, depicts representative data on the effects of chronic hydrocodone overdose, ceftriaxone, or MC-100093 treatment on GLT-1 expression in the NAc, AMY, and dmPFC. Control (n= 5- 6), Hydrocodone (n= 5-6), Hydrocodone-Ceftriaxone (n= 5-6), and Hydrocodone- MC-100093 (n= 5-6). Representative western blot images for GLT-1 expression from the NAc, AMY, and dmPFC. One-way ANOVA followed by the Newman-Keuls post hoc test revealed a significant downregulation in the ratio of GLT-1 / β-tubulin in the hydrocodone-treated group for NAc, in Fig.5A, AMY, in Fig.5B, and dmPFC, in Fig.5C, compared to the control group. Treatments with ceftriaxone (200 mg / kg, i.p.) or MC-100093 (50 mg / kg, i.p.) upregulated the hydrocodone-induced decreasing of GLT-1 expression in the NAc, in Fig.5A, AMY, in Fig.5B, and dmPFC, in Fig.5C, as compared to the hydrocodone-treated group. Control group data was represented as 100%. The values are expressed as mean ± S.E.M. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). NAc, nucleus accumbens; AMY, amygdala; dmPFC, dorsal Attorney Docket No.206017-0285-00WO medial prefrontal cortex; GLT-1, glutamate transporter 1; Hyd, hydrocodone; Cef, ceftriaxone. Fig.6, comprising Fig.6A through Fig.6C, depicts representative data on the effects of chronic hydrocodone overdose, ceftriaxone, or MC-100093 treatment on BDNF expression in the NAc, AMY, and dmPFC. Control (n= 5- 6), Hydrocodone (n= 5-6), Hydrocodone-Ceftriaxone (n= 5-6), and Hydrocodone- MC-100093 (n= 5-6). Representative western blot images for BDNF expression from the NAc, AMY, and dmPFC. One-way ANOVA followed by the Newman-Keuls post hoc test revealed a significant decrease in the ratio of BDNF / β-tubulin in the hydrocodone-treated group for NAc, in Fig.6A, AMY, in Fig.6B, and dmPFC, in Fig.6C, compared to the control group. Treatments with ceftriaxone (200 mg / kg, i.p.) or MC-100093 (50 mg / kg, i.p.) increased the hydrocodone-induced downregulation of BDNF expression in the NAc, in Fig.6A, AMY, in Fig.6B, and dmPFC, in Fig.6C, as compared to the hydrocodone-treated group. Control group data was represented as 100%. The values are expressed as mean ± S.E.M. (* p < 0.05, ** p < 0.01, *** p < 0.001). NAc, nucleus accumbens; AMY, amygdala; dmPFC, dorsal medial prefrontal cortex; BDNF, brain-derived neurotrophic factor; Hyd, hydrocodone; Cef, ceftriaxone. Fig.7, comprising Fig.7A through Fig.7C, depicts representative data on the effects of chronic hydrocodone overdose, ceftriaxone, or MC-100093 treatment on TNF-α expression in the NAc, AMY, and dmPFC. Control (n= 5- 6), Hydrocodone (n= 5-6), Hydrocodone-Ceftriaxone (n= 5-6), and Hydrocodone- MC-100093 (n= 5-6). Representative western blot images for TNF-α expression from the NAc, AMY, and dmPFC. One-way ANOVA followed by the Newman-Keuls post hoc test revealed a significant increase in the ratio of TNF-α / β-tubulin in the hydrocodone-treated group for NAc, in Fig.7A, AMY, in Fig.7B, and dmPFC, in Fig.7C, compared to the control group. Treatments with ceftriaxone (200 mg / kg, i.p.) or MC-100093 (50 mg / kg, i.p.) reversed the hydrocodone-induced upregulation of TNF-α expression in the NAc, in Fig.7A, AMY, in Fig.7B, and dmPFC, in Fig.7C, as compared to the hydrocodone-treated group. Control group data was represented as 100%. The values are expressed as mean ± S.E.M. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). NAc, nucleus accumbens; AMY, amygdala; dmPFC, dorsal medial prefrontal cortex; TNF-α, tumor necrosis factor; Hyd, hydrocodone; Cef, ceftriaxone. Attorney Docket No.206017-0285-00WO Fig.8, comprising Fig.8A through Fig, 8C, depicts representative data on the effects of chronic hydrocodone overdose, ceftriaxone, or MC-100093 treatment on HMGB1 expression in the NAc, AMY, and dmPFC. Control (n= 5- 6), Hydrocodone (n= 5-6), Hydrocodone-Ceftriaxone (n= 5-6), and Hydrocodone- MC-100093 (n= 5-6). Representative western blot images for HMGB1 expression from the NAc, AMY, and dmPFC. One-way ANOVA followed by the Newman- Keuls post hoc test revealed a significant increase in the ratio of HMGB1 / β-tubulin in the hydrocodone-treated group for NAc, in Fig.8A, AMY, in Fig.8B, and dmPFC, in Fig.8C, compared to the control group. Treatments with ceftriaxone (200 mg / kg, i.p.) or MC-100093 (50 mg / kg, i.p.) attenuated the hydrocodone-induced upregulation of HMGB1 expression in the NAc, in Fig.8A, AMY, in Fig.8B, and dmPFC, in Fig. 8C, as compared to the hydrocodone-treated group. Control group data was represented as 100%. The values are expressed as mean ± S.E.M. (* p < 0.05, ** p < 0.01). NAc, nucleus accumbens; AMY, amygdala; dmPFC, dorsal medial prefrontal cortex; HMGB1, high mobility group box 1; Hyd, hydrocodone; Cef, ceftriaxone. Fig.9 depicts a representative study timeline of the present work. Fig.10 depicts excitatory acid transporter 2 (EAAT2) protein 3D visualization. Fig.11, comprising Fig.11A and Fig.11B, depicts 2D and 3D visualization of ceftriaxone, in Fig.11A, and MC-100093, in Fig.11B, from RCSP PDB and biovia. Fig.12 depicts representative data showing the effects of nicotine drinking with / without β-lactams treatments on the average body weight (n = 8-9 / group). Fig.13, comprising Fig.13A and Fig.13B, depicts representative data showing nicotine consumption behaviors. Fig.13A depicts nicotine drinking behaviors in groups treated with normal saline or β-lactam compounds. Fig.13B depicts nicotine preference behaviors in groups treated with normal saline or β-lactam compounds (n = 8-9 / group; *p < 0.05, **p < 0.01). Fig.14, comprising Fig.14A through Fig.14C, depicts effects of nicotine drinking with / without β-lactams treatments on GLT-1, in Fig.14A, xCT, in Fig.14B, and GLAST expression, in Fig.14C, in the NAc (n = 5 / group; *p < 0.05, **p < 0.01). Attorney Docket No.206017-0285-00WO Fig.15 depicts effects of nicotine drinking with / without β-lactam treatments on the glutamate contents in the NAc (n = 5 / group; *p < 0.05, **p < 0.01, ***p < 0.001). Fig.16 depicts effects of nicotine drinking with / without β-lactam treatments on the glutamate contents in the NAc (n = 5 / group; *p < 0.05, **p < 0.01, ***p < 0.001). Fig.17, comprising Fig.17A through Fig.17C, depicts photomicrographs of Nissl’s stained striatum and cortex. Fig.17A depicts photomicrographs of Nissl’s stained striatum and cortex 2 / 3 represent the ameliorative effect of β-lactam compounds on nicotine-induced neurodegeneration. Scale bar = 200 µm at 10x and 50 µm at 40 × magnification. Fig.17B depicts striatal Nissl- positive cells per 400 µm cell. Fig.17C depicts neuronal cell density in the cortex 2 / 3 per 400 µm. Values are presented as mean ± S.E.M. *p < 0.05, **p = 0.01; post hoc Dunnett’s multiple comparison test (n=3). Fig.18 depicts effects of nicotine drinking with / without β-lactam treatments on the serum electrolytes. Fig.19, comprising Fig.19A and Fig.19B, depicts molecular docking models for ceftriaxone and MC-100093. Fig.19A depicts molecular docking for ceftriaxone. The potential molecular interaction between the receptor protein and ligand ceftriaxone. The binding pocket of protein EAAT2 / GLT-1 and the proposed binding conformation of ceftriaxone is represented in the magnified circular image. Fig.19B depicts molecular docking for MC-100093. The molecular interaction between the receptor protein andMC-100093. The binding pocket of protein EAAT2 / GLT-1 and the proposed binding conformation of ligand MC-100093 is represented in the magnified circular image. Fig.20, comprising Fig.20A and Fig.20B, depicts representative 3D- 2D visualizations. Fig.20A depicts 3D and 2D virtualization of complex 1 (ceftriaxone).3D and 2D virtualization of complex 1 from UCFS Chimera and biovia for molecular docking is shown. The 2D visualization of complex 7XR4-Ceftriaxone releases the docking binding pockets (interacting sites). Fig.20B depicts 3D and 2D virtualization of complex 2 (MC-100093).3D and 2D virtualization of complex 2 from UCFS Chimera and biovia for molecular docking is shown in above figure. The 2D visualization of complex 7XR4-MC-100093 releases the docking binding pockets (interacting sites). Attorney Docket No.206017-0285-00WO Fig.21, comprising Fig.21A and Fig.21B, depicts a summary schematic diagram. Fig.21A depicts a glutamatergic system in the NAc in control or nicotine-treated with MC-100093 groups. Fig.21B depicts a glutamatergic system in the NAc in nicotine exposed group. Fig.22 depicts the structure of novel beta-lactam MC-100093. Fig.23 depicts a timeline of experimental procedure. There are three groups: water / saline, ethanol / saline and ethanol / MC-100093. Ethanol concentrations are 15% and 30%, v / v. Fig.24 comprises Fig.24A through Fig.24C. Fig.24A depicts the effect of MC-100093 (100 mg / kg i.p.) on ethanol intake. Statistical analysis using two-way ANOVA followed by Bonferroni post-hoc test revealed that MC-100093 treatment reduced ethanol consumption from Day 1 (24 hours after the first i.p. injection of MC-100093) through Day 5 of Week 6. Fig.24B depicts the effect of MC- 100093 on water intake. Statistical analysis using two-way ANOVA followed by Bonferroni post-hoc test revealed a significant decrease in water consumption in the ethanol / saline group on Day 4. On Day 5, there was a significant increase in water consumption in rats treated with MC-100093. There was also a significant difference found between the water / saline and ethanol / MC-100093 groups on Day 5 of Week 6. Fig.24C depicts the effect of MC-100093 on body weight. Statistical analysis using two-way ANOVA followed by Bonferroni post-hoc test revealed there was no significant change in body weights measured throughout Week 6. Each column is expressed as mean ± S.E.M (Water / Saline, n=5; Ethanol / Saline, n=5; Ethanol / MC- 100093, n=6), (* p < 0.05, ** p < 0.01, and *** p <0.001). Fig.25, comprising Fig.25A through Fig.25D, depicts effects of MC- 100093 and chronic ethanol intake on BDNF expression in the subregions of mPFC (IL and PL) and NAc (shell and core). Fig.25A depicts quantitative analysis using one-way ANOVA followed by Newman Keuls post-hoc test indicated that chronic ethanol intake decreased BDNF expresion as compared to water / saline group, while MC-100093 treatment increased BDNF expression in the IL. Fig.25B depicts quantitative analysis using one-way ANOVA followed by Newman Keuls post-hoc test demonstrated that chronic ethanol intake reduced BDNF expression as compared to water / saline group, while MC-100093 attenuated this effect in the PL. Fig.25C depicts quantitative analysis using one-way ANOVA followed by Newman Keuls post-hoc test revealed that ethanol exposure reduced BDNF expression as compared Attorney Docket No.206017-0285-00WO to water / saline group, while MC-100093 attenuated this effect in the NAc core. Fig. 25D depicts quantitative analysis using one-way ANOVA followed by Newman Keuls post-hoc test showed downregulation of BDNF expression in the ethanol / saline group as compared to the water / saline group, MC-100093 treatment attenuated this effect in the NAc shell. Water / saline control group data were represented as 100%. Each column is expressed as mean ± S.E.M, (n= 5 per group), (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). BDNF, Brain Derived Neurotrophic Factor; IL, Infralimbic cortex; PL, Prelimbic cortex; NAc core, nucleus accumbens core; NAc shell, nucleus accumbens shell. Fig.26, comprising Fig.26A through Fig.26D, depicts effects of chronic ethanol intake and MC-100093 on TNF-α expression in the subregions of mPFC (IL and PL) and NAc (shell and core). Quantitative analysis using one-way ANOVA followed by Newman Keuls post-hoc test indicated a signficant increase in TNF-α expression in the IL (Fig.26A), PL (Fig.26B), NAc core (Fig.26C), and NAc shell (Fig.26D) in ethanol / saline group as compared to water / saline group, while post-treatment with MC-100093 normalized TNF-α expression in all these brain regions. Water / saline control group data were represented as 100%. Each column is expressed as mean ± S.E.M, (n=5 per group), (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). TNF-α, Tumor Necrosis Factor alpha; IL, Infralimbic cortex; PL, Prelimbic cortex; NAc core, nucleus accumbens core; NAc shell, nucleus accumbens shell. Fig.27, comprising Fig.27A through Fig.27D, depicts effects of chronic ethanol intake and MC-100093 on RAGE expression in the subregions of mPFC (IL and PL) and NAc (shell and core). Fig.27A depicts quantitative analysis using one-way ANOVA followed by Newman Keuls post-hoc test revealed that post- treatment with MC-100093 reduced RAGE expression as compared to ethanol / saline group in the IL. There was no significance found between the water / saline and the ethanol / saline groups. Fig.27B depicts quantitative analysis using one-way ANOVA followed by Newman Keuls post-hoc test found a significant increase of RAGE expression in the ethanol / saline group as compared to the water / saline group, while post-treatment with MC-100093 normalized RAGE expression in the PL. Fig.27C depicts quantitative analysis using one-way ANOVA followed by Newman Keuls post-hoc test revealed an upregulation of RAGE expression in the ethanol / saline group as compared to the water / saline group, while post-treament with MC-100093 Attorney Docket No.206017-0285-00WO normalized RAGE expression in the NAc core. Fig.27D depicts quantitative analysis using one-way ANOVA followed by Newman Keuls post-hoc test showed an upregulation of RAGE expresssion in the ethanol / saline group as compared to the water / saline group, while post-treament with MC-100093 normalized RAGE expression in the NAc shell. Water / saline group data were represented as 100%. Each column is expressed as mean ± S.E.M (n=5 per group), (* p < 0.05, ** p < 0.01, and **** p < 0.0001). RAGE, Receptor for Advanced Glycation End Products; IL, Infralimbic cortex; PL, Prelimbic cortex; NAc core, nucleus accumbens core; NAc shell, nucleus accumbens shell. Fig.28, comprising Fig.28A through Fig.28D, depicts effects of chronic ethanol intake and MC-100093 on HMGβ-1 expression in the subregions of the mPFC and NAc. Quantitative analysis using one-way ANOVA followed by Newman Keuls post-hoc test revealed an upregulation of HMGβ-1 in the IL (Fig. 28A), PL (Fig.28B), NAc core (Fig.28C), and NAc shell (Fig.28D) in the ethanol / saline group as compared to the water / saline group, while post-treatment with MC-100093 normalized HMGβ-1 expression in all these brain regions. Water / saline control group data were represented as 100%. Each column is expressed as mean ± S.E.M (n=5 per group), (* p < 0.05, ** p < 0.01, and *** p < 0.001). HMGβ-1, High Mobility Group Box 1; IL, Infralimbic cortex; PL, Prelimbic cortex; NAc core, nucleus accumbens core; NAc shell, nucleus accumbens shell. Fig.29, comprising Fig.29A through Fig.29D, depicts the effects of MC-100093 treatment (100 mg / kg, i.p.) on ethanol intake, ethanol preference, water intake, and body weight in Male P rats for five consecutive days. Fig.29A depicts statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that the average ethanol intake was significantly reduced in ethanol-MC-100093 group as compared to the ethanol-saline group. Fig.29B depicts statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that ethanol preference was significantly decreased in the ethanol-MC-100093 group as compared to the ethanol-saline group. Fig.29C depicts statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons showed that the average water intake was significantly increased in ethanol-MC-100093 group as compared to the ethanol-saline group. Fig.29D depicts statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that there was no significant difference in body weight Attorney Docket No.206017-0285-00WO among all tested groups. Values are expressed as mean ± SEM (n = 5–7 / group), (* p < 0.05 and ** p < 0.01, *** p < 0.001, and **** p < 0.0001). Fig.30, comprising Fig.30A through Fig.30D, depicts effects of MC- 100093 treatment (100 mg / kg, i.p.) on ethanol intake, ethanol preference, water intake, and body weight in Female P rats for five consecutive days. Fig.30A depicts statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that the average ethanol intake was significantly decreased in the ethanol-MC-100093 group as compared to the ethanol-saline group. Fig.30A depicts statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that ethanol preference was significantly reduced in the ethanol-MC-100093 group as compared to the ethanol-saline group. Fig.30C depicts statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that the average water intake was significantly increased in ethanol-MC-100093 group as compared to the ethanol-saline group. Fig.30D depicts statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that there was no significant difference in body weight among all tested groups. Values are expressed as mean ± SEM (n= 5–7 / group), (* p < 0.05 and ** p < 0.01, *** p < 0.001, and **** p < 0.0001). Fig.31, comprising Fig.31A through Fig.31D, depicts effects of MC- 100093 treatment (100 mg / kg, i.p.) for five days on the expression of GLT-1 and xCT in the mPFC-IL and mPFC-PL in Male P rats. Fig.31A depicts immunoblot of GLT-1 and β-tubulin in the mPFC-IL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline and water-saline groups. In addition, a significant downregulation in the expression of GLT-1 was found in the mPFC-IL in the ethanol-saline group as compared to water-saline group. Fig.31B depicts immunoblot of xCT and β-tubulin in the mPFC-IL. Quantitative analysis using one- way ANOVA followed by Newman-Keuls test revealed that MC-100093 significantly upregulated xCT expression as compared to ethanol-saline group. In addition, a significant downregulation in the expression of xCT in the ethanol-saline group as compared to water-saline group in the mPFC-IL. Fig.31C depicts immunoblot of GLT-1 and β-tubulin in the mPFC-PL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in GLT-1 expression in the ethanol-saline group as compared to water-saline group, Attorney Docket No.206017-0285-00WO while post-treatment with MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline group in the mPFC-PL. Fig.31D depicts immunoblot of xCT and β-tubulin in the mPFC-PL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in xCT expression in the ethanol-saline group as compared to water-saline group, while post- treatment with MC-100093 significantly upregulated xCT expression as compared to ethanol-saline group in the mPFC-PL. Water-saline group data were represented as 100% (relative to water-saline). Values are expressed as mean ± SEM (n = 5 / group), (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). Fig.32, comprising Fig.32A through Fig.32D, depicts effects of MC- 100093 treatment (100 mg / kg, i.p.) for five days on the expression of GLT-1 and xCT in the mPFC-IL and mPFC-PL in Female P rats. Fig.32A depicts immunoblot of GLT-1 and β-tubulin in the mPFC-IL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in GLT-1 expression in the ethanol-saline group as compared to water-saline group, while post-treatment with MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline group in the mPFC-IL. Fig.32B depicts immunoblot of xCT and β-tubulin in the mPFC-IL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 significantly upregulated xCT expression as compared to ethanol-saline group. In addition, a significant downregulation in the expression of xCT in the ethanol-saline group as compared to water-saline group in the mPFC-IL. Fig.32C depicts immunoblot of GLT-1 and β- tubulin in the mPFC-PL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline and water-saline groups. Fig.32D depicts immunoblot of xCT and β-tubulin in the mPFC-PL. Quantitative analysis using one- way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in xCT expression in the ethanol-saline group as compared to water-saline group, while post-treatment with MC-100093 significantly upregulated xCT expression as compared to the ethanol-saline group in the mPFC-PL. Water-saline group data were represented as 100% (relative to water-saline). Values are expressed as mean ± SEM (n = 5 / group), (* p < 0.05, ** p < 0.01, and *** p < 0.001). Fig.33, comprising Fig.33A through Fig.33D, depicts effects of MC- 100093 treatment (100 mg / kg, i.p.) for five days on the expression of GLT-1 and xCT Attorney Docket No.206017-0285-00WO in the NAc-shell and NAc-core in Male P rats. Fig.33A depicts immunoblot of GLT- 1 and β-tubulin in the NAc-shell. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline and water-saline groups. In addition, a significant downregulation in the expression of GLT-1 in the ethanol-saline group as compared to water-saline group in the NAc-shell. Fig.33B depicts immunoblot of xCT and β-tubulin in the NAc-shell. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 significantly upregulated xCT expression as compared to ethanol-saline group. In addition, a significant downregulation in the expression of xCT in the ethanol-saline group as compared to water-saline group in the NAc-shell. Fig.33C depicts immunoblot of GLT-1 and β- tubulin in the NAc-core. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test showed that post-treatment with MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline group in the NAc-core. Fig.33D depicts immunoblot of xCT and β-tubulin in the NAc-core. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in xCT expression in the ethanol-saline group as compared to water-saline group, while post-treatment with MC-100093 significantly upregulated xCT expression as compared to the ethanol-saline group in the NAc-core. Water-saline group data were represented as 100% (relative to water-saline). Values are expressed as mean ± SEM (n = 5 / group), (* p < 0.05, ** p < 0.01, and *** p < 0.001). Fig.34, comprising Fig.34A through Fig.34D, depicts effects of MC- 100093 treatment (100 mg / kg, i.p.) for five days on the expression of GLT-1 and xCT in the NAc-shell and NAc-core in Female P rats. Fig.34A depicts an immunoblot of GLT-1 and β-tubulin in the NAc-shell. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test revealed that MC-100093 treatment significantly upregulated GLT-1 expression as compared to the ethanol-saline group. Fig.34B depicts an immunoblot of xCT and β-tubulin in the NAc-shell. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test showed a significant upregulation of xCT expression as compared to ethanol-saline group following treatment with MC-100093. Fig.34C depicts an immunoblot of GLT-1 and β-tubulin in the NAc-core. Quantitative analysis using one-way ANOVA followed by Newman- Keuls test indicated that MC-100093 treatment significantly upregulated GLT-1 Attorney Docket No.206017-0285-00WO expression as compared to the ethanol-saline and water-saline groups. Fig.34D depicts an immunoblot of xCT and β-tubulin in the NAc-core. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test showed that post-treatment with MC-100093 significantly upregulated xCT expression as compared to the ethanol-saline group in the NAc-core. Water-saline group data were represented as 100% (relative to water-saline). Values are expressed as mean ± SEM (n = 5 / group), (* p < 0.05, ** p < 0.01, and *** p < 0.001). Fig.35 depicts a schematic diagram displaying the glutamatergic projections from the mPFC to the NAc, and the effects of chronic ethanol consumption on GLT-1 and xCT in the subregions of mPFC and NAc. Downregulation of the expression of GLT-1 and xCT after chronic ethanol consumption leads to the increase of synaptic glutamate concentration. MC-100093 attenuated ethanol consumption through the upregulation of these transporters. Fig.36 depicts a representative timeline of the experimental procedure. CLAMS, comprehensive laboratory animal monitoring system. Fig.37, comprising Fig.37A through Fig.37C, depicts representative data relating to the effects of exposure to hydrocodone overdose on locomotion activity in mice. Fig.37A depicts statistical analysis which revealed that x activity increased in the hydrocodone-treated group compared to the control group, while treatment with ceftriaxone (200 mg / kg, i.p.) reduced x activity compared to the hydrocodone-treated group. Fig 37B depicts statistical analysis which demonstrated that x ambulatory increased in the hydrocodone-treated group compared to the control group, and there was no significant difference in x ambulatory in the hydrocodone– ceftriaxone group compared to the hydrocodone-treated group. Fig.37C depicts statistical analysis which revealed that z activity increased in the hydrocodone-treated group compared to the control group, and ceftriaxone treatment (200 mg / kg, i.p.) reduced z activity compared to the hydrocodone-treated group. Data from the control group are represented as 100%. Each column is expressed as mean ± S.E.M (n = 7– 8 / group), (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). Hyd, hydrocodone. Fig.38, comprising Fig.38A through Fig.38C, depicts representative data relating to the effects of exposure to hydrocodone overdose on GLT-1, xCT, and mGluR5 expression in the NAc. Fig.38A depicts western blots for GLT-1 and β- tubulin in the NAc. One-way ANOVA followed by the Newman–Keuls multiple Attorney Docket No.206017-0285-00WO comparisons test showed downregulation of GLT-1 expression in the hydrocodone- treated group compared to the control group, and ceftriaxone treatment (200 mg / kg, i.p.) normalized GLT-1 expression in the NAc compared to the hydrocodone-treated group. Fig.38B depicts how one-way ANOVA followed by the Newman–Keuls multiple comparisons test revealed downregulation of xCT expression in the hydrocodone-treated group compared to the control group, and ceftriaxone treatment (200 mg / kg, i.p.) normalized xCT expression in the NAc compared to the hydrocodone-treated group. Fig.38C depicts how one-way ANOVA followed by the Newman–Keuls multiple comparisons test revealed upregulation of mGluR5 expression in the hydrocodone-treated group compared to the control group, and ceftriaxone treatment (200 mg / kg, i.p.) attenuated this effect. There was also a significant difference between the control and hydrocodone–ceftriaxone-treated groups in the expression of mGluR5 in the NAc. Data from the control group are represented as 100%. Each column is expressed as mean ± S.E.M (n = 7–8 / group), (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). GLT-1, glutamate transporter 1; xCT, cystine / glutamate antiporter; mGluR5, metabotropic glutamate receptor subtype 5; Hyd, hydrocodone. Fig.39, comprising Fig.39A and 39B, depict representative data showing the effects of exposure to hydrocodone overdose on the expression of nNOS and ERK in the NAc. Fig.39A depicts western blots for p-nNOS and NOS in the NAc. One-way ANOVA followed by the Newman–Keuls multiple comparisons test revealed that hydrocodone exposure increased nNOS expression compared to the control group, and ceftriaxone treatment (200 mg / kg, i.p.) decreased nNOS expression in the NAc compared to the hydrocodone group. Fig.39B depicts western blots for p- ERK and ERK in the NAc. One-way ANOVA followed by the Newman–Keuls multiple comparisons test revealed that hydrocodone exposure downregulated ERK expression compared to the control group, and ceftriaxone treatment (200 mg / kg, i.p.) upregulated ERK expression in the NAc compared to the hydrocodone group. Data from the control group are represented as 100%. Each column is expressed as mean ± S.E.M (n = 7–8 / group), (* p < 0.05, *** p < 0.001 and **** p < 0.0001). nNOS, neuronal nitric oxide synthase; ERK, extracellular signal-regulated kinases; Hyd, hydrocodone. Fig.40 depicts representative data showing the effects of exposure to hydrocodone overdose on RAGE expression in the NAc. One-way ANOVA followed Attorney Docket No.206017-0285-00WO by the Newman–Keuls multiple comparisons test showed that RAGE expression was upregulated in the hydrocodone group compared to the control group, while ceftriaxone (200 mg / kg) downregulated RAGE expression in the NAc compared to the hydrocodone group. Data from the control group are represented as 100%. Each column is expressed as mean ± S.E.M (n = 7–8 / group), (* p < 0.05). RAGE, receptor for advanced glycation end products; Hyd, hydrocodone. Fig.41 depicts a schematic representation summarizing the effects of exposure to hydrocodone overdose on the mGluR5-nNOS-ERK pathway and GLT-1, xCT, and RAGE expression in the NAc. Exposure to hydrocodone overdose may increase synaptic glutamate release, resulting in an increase in extracellular glutamate concentrations. Under a hyper-glutamatergic state, mGluR5 and NMDAR are overstimulated, thereby increasing intracellular calcium and subsequently upregulating nNOS activity. Activation of nNOS activity can lead to inhibition of the downstream ERK signaling pathway. Additionally, exposure to hydrocodone overdose is associated with an increase in the inflammatory response, such as upregulation of RAGE. Ceftriaxone treatment attenuates hydrocodone-induced mGluR5-nNOS-ERK pathway activation, glutamatergic system dysregulation, and RAGE upregulation. (Blue arrows indicate the downstream pathways; Red arrows indicate upregulation or downregulation of all target proteins or markers). Fig.42 depicts representative average body weight measurements of control and experimental groups overtime. Body weights were measured in the control, fentanyl, fentanyl / ceftriaxone, and fentanyl / MC-100093 groups at three time points: baseline (day 0), during treatment (day 5), and post treatment (day 9). Two- way ANOVA indicated no significant differences in body weight between groups at baseline, during treatment and post treatment (n = 7 / group). Cef; ceftriaxone, MC; MC-100093. Fig.43, comprising Fig.43A and Fig.43B, depicts representative results of the open field (Fig.43A) and Y maze (Fig.43B) behavioral tests to assess locomotion and spatial working memory, respectively. Fig.43A depicts the total distance traveled by mice in 10-minute period as an indication of locomotor activity. Fentanyl alone group showed high locomotor activities compared to control group. Fentanyl / ceftriaxone and fentanyl / MC-100093 groups showed locomotor activities comparable to control group. Fig.43B shows the percentage of spontaneous alteration performance (SAP) as an indication of spatial working memory. There was no Attorney Docket No.206017-0285-00WO significant decrease in SAP between in fentanyl alone exposed group in compared to control group. Data is presented as mean ± SEM. (* p < 0.05, ** p < 0.01, n= 7 / group). Fig.44 depicts a heatmap displaying the relative levels of detected metabolites in the control group, fentanyl group, fentanyl / ceftriaxone group, and fentanyl / MC-100093 group. Each row represents a specific metabolite, and each column corresponds to experimental groups. The color scale ranges from blue (indicating lower metabolite levels) to red (indicating higher metabolite levels). The control group shows a baseline metabolite profile, while the fentanyl group exhibits significant alterations in metabolite levels compared to the control. The fentanyl / ceftriaxone and fentanyl / MC-100093 groups show a slightly different pattern of metabolite changes, suggesting a minimum modulatory effect of β-lactams on fentanyl-induced metabolic alterations (n=5-7 / group). Fig.45 depicts exemplary one-way ANOVA which showed significant changes in the metabolomic profiles of groups exposed to fentanyl overdose compared to control group. Each dot in the graph represents the mean of one metabolite in each group. Data are reported as mean of all metabolites means ± SEM. (* p < 0.05, ** p < 0.01, *** p < 0.0001, n= 5-7 / group). Cef, ceftriaxone; MC, MC- 100093. Fig.46 depicts representative data showing the effect of fentanyl overdose with and without β-lactams on the level of carbohydrates glucose and turanose. The bar graph illustrates the mean ratio of the metabolite peak area (normalized to the control group) across four groups: control, fentanyl overdose, fentanyl overdose with ceftriaxone, and fentanyl overdose with MC-100093, and the error bar represent SEM. The control group is set as the baseline (ratio = 1), while the fentanyl alone group exhibits a significant decrease in the metabolite ratio. No significant difference was found between fentanyl and fentanyl / β-lactams groups (** p < 0.01, n= 5-7 / group). FEN: fentanyl, CEF: ceftriaxone, MC: MC-100093. Fig.47 depicts representative data showing the effect of fentanyl overdose with and without β-lactams on the level of amino acids glycine and valine. The bar graph illustrates the mean ratio of the metabolite peak area (normalized to the control group) across the four groups, and the error bar represent SEM. The control group is set as the baseline (ratio = 1), while the fentanyl overdose group exhibits a Attorney Docket No.206017-0285-00WO significant decrease in the metabolite ratio (* p < 0.05, # p < 0.0001, n= 5-7 / group). FEN: fentanyl, CEF: ceftriaxone, MC: MC-100093. Fig.48 depicts representative data showing the effect of fentanyl overdose with and without β-lactams on the level of arachidonic acid and mono- palmitin. The bar graph illustrates the mean ratio of the metabolite peak area (normalized to the control group) across the four groups, and the error bar represent SEM. The control group is set as the baseline (ratio = 1), while the fentanyl overdose group exhibits a decrease in the metabolites ratio (* p < 0.05, ** p < 0.01, n= 5- 7 / group). FEN: fentanyl, CEF: ceftriaxone, MC: MC-100093. Fig.49 depicts an exemplary network analysis of disrupted metabolites in fentanyl overdose group. Network analysis illustrating the interactions and relationships among significantly disturbed metabolites in the fentanyl overdose group. Each node represents a specific metabolite, while edges (lines) connecting the nodes indicate known biochemical interactions. The size of each node corresponds to the magnitude of change in metabolite level, with larger nodes representing greater disruption. The color of the edges represents the type of correlation between metabolites: red edges denote positive correlations (indicating that as the level of one metabolite increases, the level of the connected metabolite also increases), while blue edges indicate negative correlations (suggesting that as the level of one metabolite increases, the level of the connected metabolite decreases). These correlations were determined based on the Pearson correlation coefficients derived from the metabolomic data. Central hub metabolites, such as arachidonic acid, ribitol and glycine, are prominently affected, indicating their critical role in the response to fentanyl toxicity. The network highlights the complexity of metabolic alterations and the interconnected nature of disrupted pathways, providing insights into the biochemical impact of fentanyl overdose. This analysis underscores the extensive metabolic reprogramming that occurs in response to fentanyl toxicity. Fig.50 depicts an exemplary enrichment analysis highlighting the significantly altered metabolic pathways in the fentanyl overdose group. The analysis was performed to identify which metabolic pathways were significantly impacted due to fentanyl overdose. Each bar represents a different metabolic pathway, with the length of the bar corresponding to the degree of enrichment, measured by the enrichment score. The color of the bars indicates the statistical significance of the Attorney Docket No.206017-0285-00WO enrichment, with darker colors representing lower p-values (higher significance). Pathways shown include those involved in energy metabolism, lipid metabolism, and amino acid metabolism, inflammation processes, and others. The x-axis denotes the enrichment score, while the y-axis lists the specific metabolic pathways. Notably, pathways such as lactose / galactose metabolism, sphingolipid metabolism, and bile acid biosynthesis are significantly enriched, indicating a strong metabolic response to fentanyl toxicity. Fig.51 depicts an exemplary Sparse Partial Least Squares- Discriminant Analysis. sPLS-DA score plot displaying the separation of metabolite profiles between control group and fentanyl overdose groups. Red circles indicating control group samples, green circles indicating fentanyl overdose group samples, purple circles indicating fentanyl / ceftriaxone group samples, and light blue circles indicating fentanyl / MC-100093 group samples. The distinct clustering of samples from the control and fentanyl overdose groups highlights the substantial metabolic differences induced by fentanyl toxicity. There is no clear separation between fentanyl alone group, and both fentanyl / β-lactams groups (n=5-7 / group). Fig.52, comprising Fig.52A through Fig.52C, depicts representative data showing the effect of ceftriaxone and MC-100093 on the expression of GLT-1 (Fig.52A), TLR-4 (Fig.52B), and IL-6 (Fig.52C) in the nucleus accumbens of mouse model of fentanyl overdose (* p < 0.05, ** p < 0.01, *** p < 0.001, n= 5 / group). Cef: ceftriaxone, MC: MC-100093, GLT-1: glutamate transporter 1, IL-6: interleukin 6, TLR-4: toll-like receptor-4. Fig.53 depicts the structures of ceftriaxone and MC-100093. Fig.54 depicts representative overall metabolomics profiles in fentanyl, fentanyl-MC-100093, and fentanyl–ceftriaxone groups. One-way ANOVA showed significant changes in the metabolomic profiles in fentanyl, fentanyl-MC- 100093, and fentanyl–ceftriaxone groups. One-way ANOVA followed by Holm– Sidak’s multiple comparisons test showed significant differences in metabolomics profiles between the fentanyl group and the other three groups. The analysis also found significant differences in metabolomics profiles between fentanyl-MC-100093 and fentanyl–ceftriaxone groups. Each dot in the graph represents the mean of one metabolite in each group. Data are reported as the mean of all metabolites’ means ± Attorney Docket No.206017-0285-00WO SEM. (* p < 0.05, ** p < 0.01, # p < 0.0001, n = 4–6 / group). Cef, ceftriaxone; MC, MC-100093. Fig.55 depicts an exemplary heatmap analysis model of control, fentanyl, fentanyl–ceftriaxone and fentanyl-MC-100093 metabolomic profiles. Cef, ceftriaxone; MC, MC-100093. Fig.56 depicts an exemplary partial least-squares-discriminant analysis (PLS-DA) model control, fentanyl, fentanyl–ceftriaxone and fentanyl-MC-100093 metabolomic profiles. n = 4–6 / group. Cef, ceftriaxone; MC, MC-100093. Fig.57, comprising panels (A)-(S), depicts representative data showing the effects of ceftriaxone and MC-100093 on selected metabolites in liver of fentanyl- overdosed mouse model. (A) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that the MC-100093-fentanyl group showed higher d- glucose compared to fentanyl and the fentanyl-cef groups. (B) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that fentanyl-MC- 100093 had higher xylitol compared to the fentanyl group. (C) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that MC-100093- fentanyl had higher ribitol compared to the fentanyl and fentanyl-cef groups. (D) One- way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that fentanyl- MC-100093 had higher xylitol compared to thew fentanyl group. (E) One- way ANOVA followed by Holm–Sidak’s multiple comparisons test showed lower alanine in the fentanyl group compared to controls. (F) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that octadecanoic acid was higher in fentanyl-MC-100093 compared to the fentanyl group. (G) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that trans-9 octadecanoic acid was higher in fentanyl-MC-100093 compared to the fentanyl group. (H) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that arachidonic acid was lower in the fentanyl group compared to the fentanyl– ceftriaxone and fentanyl-MC-100093 groups. (I) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that palmitic acid was lower in the fentanyl group compared to the fentanyl-MC-100093 group. (J) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed lower lactic acid in the fentanyl group compared to controls; however, lactic acid was higher in the fentanyl- MC-100093 group compared to the fentanyl and fentanyl–ceftriaxone groups. (K) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that Attorney Docket No.206017-0285-00WO succinic acid was lower in the fentanyl and fentanyl–ceftriaxone groups compared to the control group. (L) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that allonic acid was lower in the fentanyl group compared to the fentanyl-MC-100093 group. (M) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that 2-deoxy erythro-pentonic acid was higher in fentanyl-MC-100093 compared to the control and fentanyl groups. (N) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that carbachol was lower in the fentanyl group as compared to the control group. (O) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that biuret was higher in the fentanyl-MC100093 group compared to the fentanyl group. (P) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that beta-prostaglandin was higher in the fentanyl-MC-100093 group compared to the fentanyl and fentanyl–ceftriaxone groups. (Q) One-way ANOVA followed by Holm– Sidak’s multiple comparisons test showed that 2-methylpropanetriol was lower in the fentanyl and fentanyl–ceftriaxone groups compared to the control and fentanyl-MC- 100093 groups. (R) One-way ANOVA followed by Holm–Sidak’s multiple comparisons test showed that gamma lactone was higher in the fentanyl-MC-100093 group compared to the fentanyl group. (S) One-way ANOVA followed by Holm– Sidak’s multiple comparisons test showed that dihydroxyacetone was higher in the fentanyl-MC-100093 group compared to all other groups. The symbol of statistical significance is shown on any group's bar when it was compared to the control group. Data are reported as mean ± SEM. (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 4– 6 / group). Cef, ceftriaxone; MC, MC-100093. Fig.58, comprising panels (A)-(Q), depict representative one-way ANOVA tests followed by Holm-Sidak’s multiple comparisons which showed non- significant differences between methionine (A), glycine (B), l-proline (C), methyl leucine (D), phenylethanolamine (E), phosphoric acid (F), 2-pipecolic acid (G), carbonic acid (H), 2-hexenedioic acid (I), 2-keto isocaproic acid (J), octanedioic acid (K), azelaic acid (L) , pyruvic acid (M), pentaglycerine (N), urea (O), ornithine (P) and 4,5 octanediol (Q). Data are reported as mean ± SEM. Cef, ceftriaxone; MC, MC- 100093. Fig.59 depicts an overview of top 25 enriched metabolite pathways ordered based on p value and enrichment ratio. Attorney Docket No.206017-0285-00WO Fig.60, comprising Fig.60A through 60D, depicts an exemplary histopathology analysis. Fig.60A depicts a section of liver showing normal portal tract and central vein with surrounding unremarkable hepatocytes in the control group. Fig.60B depicts a section of liver obtained from the fentanyl-overdose-treated group showing inflammation in portal tract and central vein. Fig.60C depicts a section of liver showing limited portal tract inflammation in the fentanyl–ceftriaxone group indicating the protective effects of ceftriaxone against fentanyl overdose. Fig. 60D depicts liver tissue showing minimal to mild inflammation around a bile duct in the fentanyl-MC100093 group. Yellow arrows indicate inflammation. H / E stain ×400. Cef, ceftriaxone; MC, MC-100093; PT, Portal tract; CV, Central vein. Fig.61, comprising Fig.61A and Fig.61B, depicts an exemplary protein expression study. Fig.61A depicts IL-6 and CYP3A11 (mouse homolog of human CYP3A4) bands expression in the control, fentanyl, fentanyl–ceftriaxone, and fentanyl-MC100093 groups. Fig.61B depicts one-way ANOVA followed by Holm– Sidak’s multiple comparisons test showed that liver IL-6 expression was increased in the fentanyl and fentanyl-MC100093 groups compared to the control and fentanyl– ceftriaxone groups; moreover, liver CYP3A11 expression was lower in the fentanyl and fentanyl-MC-100093 groups compared to the control and fentanyl–ceftriaxone groups (n = 4 / group). Data are reported as mean ± SEM. (* p < 0.05, ** p < 0.01). Cef, ceftriaxone; MC, MC-100093. Fig.62 depicts a schematic diagram shows the effects of fentanyl and MC-100093 on lactic acid in the gluconeogenesis pathway. ATP, adenosine triphosphate. DETAILED DESCRIPTION OF THE INVENTION It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity, many other elements found in the art related to organic chemistry and methods of treating microbial infections. Those of ordinary skill in the art may recognize that other elements and / or steps are desirable and / or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is Attorney Docket No.206017-0285-00WO directed to all such variations and modifications to such elements and methods known to those skilled in the art. Although any methods, materials and components similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, materials and components similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. As used herein, each of the following terms has the meaning associated with it in this section. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health. A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced. The terms “patient,” “subject,” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in Attorney Docket No.206017-0285-00WO situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human. As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration. A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs. As used herein, the term “treatment” or “treating” is defined as the application or administration of a therapeutic agent, i.e., a compound of the invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein, a sign or symptom of a condition contemplated herein or the potential to develop a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, at least one sign or symptom of a condition contemplated herein or the potential to develop a condition contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. As used herein, the terms “effective amount,” “pharmaceutically effective amount" and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and / or alleviation of a sign, a symptom, or a cause of a disease or disorder, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing an undesirable biological effect or Attorney Docket No.206017-0285-00WO interacting in a deleterious manner with any of the components of the composition in which it is contained. As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, acetic, hexafluorophosphoric, citric, gluconic, benzoic, propionic, butyric, sulfosalicylic, maleic, lauric, malic, fumaric, succinic, tartaric, amsonic, pamoic, p-tolunenesulfonic, and mesylic. Appropriate organic acids may be selected, for example, from aliphatic, aromatic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, camphorsulfonic, citric, fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric, para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic (besylate), stearic, sulfanilic, alginic, galacturonic, and the like. Furthermore, pharmaceutically acceptable salts include, by way of non-limiting example, alkaline earth metal salts (e.g., calcium or magnesium), alkali metal salts (e.g., sodium-dependent or potassium), and ammonium salts. As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as Attorney Docket No.206017-0285-00WO glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference. An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound. As used herein, the term “potency” refers to the dose needed to produce half the maximal response (ED50). As used herein, the term “efficacy” refers to the maximal effect (Emax) achieved within an assay. Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub- ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description Attorney Docket No.206017-0285-00WO This invention includes the unexpected identification of a novel method of treating addiction, or a substance use disorder in a subject in need thereof. Methods of Treating Addiction Described herein is a method of treating a substance use disorder in a subject, comprising the step of administering to the subject a therapeutically effective amount of a composition comprising a compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof The substance use disorder which may be treated by the composition or compounds described herein are defined as abuse of any drug, including, but not limited to, fentanyl, opioids, nicotine, alcohol, marijuana, synthetic cannabinoids, stimulants, barbiturates, benzodiazepines, dextromethorphan (DXM), a sleep medication, khat, synthetic cathinones, cocaine, 3,4- methylenedioxymethamphetamine (MDMA), phencyclidine (PCP), lysergic acid diethylamide (LSD), psilocybin, an inhalant, Rohypnol, gamma-hydroxybutyric acid (GHB), N,N-Dimethyltryptamine (DMT), ayahuasca, mescaline, salvia, or combinations thereof. In some examples, the substance use disorder comprises abuse of opioids. In some examples, the substance use disorder comprises abuse of nicotine. In some examples, the substance use disorder comprises abuse of fentanyl. The substance use disorder may also be or comprise the abuse of a drug which down-regulates a molecule effective in any pathway related to addiction, including, but not limited to, xCT, glutamate transporter 1 (GLT-1), and brain-derived neurotrophic factor (BDNF). In some examples, the substance use disorder comprises abuse of a drug which up-regulates a molecule selected from metabotropic glutamate receptor 1 (mGluR1), tumor necrosis factor alpha (TNF-α), and high mobility group box 1 (HMGB1). Attorney Docket No.206017-0285-00WO In some examples, the method promotes downregulation of mGluR1. In some examples, the method promotes downregulation of TNF-α. In some examples, the method promotes downregulation of HMBG1. In some examples, the method promotes upregulation of xCT. In some examples, the method promotes upregulation of GLT-1. In some examples, the method promotes upregulation of BDNF. In some examples, the substance abuse disorder comprises abuse of hydrocodone (dihydrocodeinone), heroin, codeine, fentanyl, hydromorphone, meperidine, methadone, morphine, oxycodone, or oxymorphone. In some examples, the method reduces a symptom of withdrawal in the subject. In some examples, the method prevents a relapse of the substance use disorder in the subject. In some examples, the method reduces dependence on a substance of the substance use disorder in the subject. In certain examples, the method further comprises the step of exposing the subject to an additional pharmacotherapy. In some examples, the additional pharmacotherapy comprises a gradually reducing regimen, a substitution therapy, or a medication assisted treatment. In some examples, the subject is a mammal. In another embodiment, the mammal is a human. The compounds described herein can be used or found in any composition. Thus, also described herein are compositions comprising a compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof. The compounds and compositions described herein can also be administered in a prodrug form, wherein the compounds or compositions comprise additional moieties which may be converted or split off from the compound or composition to provide the active compound or composition, a pharmaceutically acceptable salt thereof, or a biologically active metabolite thereof. Combination Therapies The compounds and compositions described herein can be used in combination with one or more additional compounds. These additional compounds may comprise compounds of the present invention or therapeutic agents known to treat or reduce the symptoms or effects of addiction and / or impulse-control disorders. Attorney Docket No.206017-0285-00WO The compounds described herein may be combined with ceftriaxone. Thus, also described herein are compositions comprising the compound and ceftriaxone. The compositions may comprise ceftriaxone or any functional alternative, including, but not limited to, ciprofloxacin, doxycycline, clindamycin, cefepime, cefixime, amoxicillin, levofloxacin, cephalexin, gentamicin, and any combination thereof. The compounds and compositions described herein can also be combined with any number of pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants known in the art. In some examples, the composition of the present invention further comprises an additional therapeutic agent. The additional therapeutic agent may be selected from the group consisting of an opioid antagonist, a mixed opioid partial agonist / antagonist, an antidepressant, an antiepileptic, an antiemetic, a corticotrophin- releasing factor-1 (CRF-1) receptor antagonist, a selective serotonin-3 (5-HT3) antagonist, a 5-HT2A / 2C antagonist, and a cannabinoid-1 (CB1) receptor antagonist. Exemplary opioid antagonists include naltrexone and nalmefene. Exemplary antidepressants include fluoxetine, mirtazapine, and bupropion. Exemplary antiepileptics include topiramate, levetiracetam, and gabapentin. Antalarmin is an exemplary CRF-1 receptor antagonist. Ondensetrom is an exemplary selective serotonin-3 (5-HT3) antagonist. Exemplary cannabinoid-1 (CB1) receptor antagonists are rimonabant and tanarabant. Buprenorphine is an exemplary mixed opioid agonist / antagonist. Exemplary opioid agonists include morphine, methadone, fentanyl, sufentanil and heroin. In some examples, administering the compound of the invention to the subject allows for administering a lower dose of the therapeutic agent compared to the dose of the therapeutic agent alone that is required to achieve similar results in treating the subject. In some examples, the compound of the invention enhances the activity of the additional therapeutic compound, thereby allowing for a lower dose of the therapeutic compound to provide the same effect. In some examples, administering the compound of the invention to the subject allows for administering a lower dose of the therapeutic agent compared to the dose of the therapeutic agent Attorney Docket No.206017-0285-00WO alone that is required to achieve similar results in treating or preventing pain or inflammation in the subject. In some examples, the compound of the invention and the therapeutic agent are co-administered to the subject. In another embodiment, the compound of the invention and the therapeutic agent are co-formulated and co-administered to the subject. Administration / Dosage / Formulations The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either before or after the onset of the microbial infection. Further, several divided dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. Administration of the compositions of the present invention to a patient, such as a mammal, (e.g., human), may be carried out using known procedures, at dosages and for periods of time effective to treat a microbial infection in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a microbial infection in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily. In another example, the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 mg / kg to about 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to assess the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular Attorney Docket No.206017-0285-00WO patient, composition, and mode of administration, without generating excessive side effects in the patient. In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts. A medical professional, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start with a dosage of the compound of the invention in the pharmaceutical composition at a level that is lower than the level required to achieve the desired therapeutic effect, and then increase the dosage over time until the desired effect is achieved. In particular examples, it is advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” as used herein refers to a physically discrete unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical vehicle. The dosage unit forms of the invention can be selected based upon (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a microbial infection in a patient. In some examples, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In some examples, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound of the invention and a pharmaceutically acceptable carrier. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), vegetable oils, and suitable mixtures thereof. The proper fluidity may 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 Attorney Docket No.206017-0285-00WO by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some examples, it is useful to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. In some examples, the pharmaceutically acceptable carrier is DMSO, alone or in combination with other carriers. The therapeutically effective amount or dose of a compound of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the severity of the microbial infection in the patient being treated. The skilled artisan is able to determine appropriate doses depending on these and other factors. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses. Doses of the compound of the invention for administration may be in the range of from about 1 μg to about 10,000 mg, from about 20 μg to about 9,500 mg, from about 40 μg to about 9,000 mg, from about 75 μg to about 8,500 mg, from about 150 μg to about 7,500 mg, from about 200 μg to about 7,000 mg, from about 3050 μg to about 6,000 mg, from about 500 μg to about 5,000 mg, from about 750 μg to about 4,000 mg, from about 1 mg to about 3,000 mg, from about 10 mg to about 2,500 mg, from about 20 mg to about 2,000 mg, from about 25 mg to about 1,500 mg, from about 30 mg to about 1,000 mg, from about 40 mg to about 900 mg, from about 50 mg to about 800 mg, from about 60 mg to about 750 mg, from about 70 mg to about 600 mg, from about 80 mg to about 500 mg, and any and all whole or partial increments therebetween. In some examples, the dose of a compound of the invention is from about 1 mg to about 2,500 mg. In some examples, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less Attorney Docket No.206017-0285-00WO than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some examples, the dosage of a second compound as described elsewhere herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof. The compounds for use in the method of the invention may be formulated in unit dosage form. The term “unit dosage form” refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. In some examples, the compositions of the invention are administered to the patient from about one to about five times per day or more. In various examples, the compositions of the invention are administered to the patient, 1-7 times per day, 1-7 times every two days, 1-7 times every 3 days, 1-7 times every week, 1-7 times every two weeks, and 1-7 times per month. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from individual to individual depending on many factors including, but not limited to, age, the disease or disorder to be treated, the severity of the disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosing regime and the precise dosage and composition to be administered to any patient is determined by the medical professional taking all other factors about the patient into account. In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the inhibitor of the invention is optionally given Attorney Docket No.206017-0285-00WO continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Once improvement of the patient’s condition has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced to a level at which the improved disease is retained. In some examples, a patient may require intermittent treatment on a long-term basis, or upon any recurrence of the disease or disorder. Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized. In some examples, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat or prevent a microbial infection in a patient. Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations Attorney Docket No.206017-0285-00WO may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents. Routes of administration of any of the compositions of the invention include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein. Oral Administration For oral administration, suitable forms include tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions formulated for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the Attorney Docket No.206017-0285-00WO release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent. For oral administration, the compounds of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid). Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a “granulation.” For example, solvent-using “wet” granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated. Melt granulation involves the use of materials that are solid or semi- solid at room temperature (i.e., having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the Attorney Docket No.206017-0285-00WO initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e., drug) by forming a solid dispersion or solid solution. U.S. Patent No.5,169,645 discloses directly compressible wax- containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain examples, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) melt. The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the invention, and a further layer providing for the immediate release of a medication for treatment of G-protein receptor-related diseases or disorders. Using a wax / pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release. Parenteral Administration For parenteral administration, the compounds of the invention may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and / or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and / or dispersing agents may be used. Additional Administration Forms Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos.6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO Attorney Docket No.206017-0285-00WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757. Controlled Release Formulations and Drug Delivery Systems In some examples, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations. The term sustained release refers to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a day, a week, or a month or more and should be a release which is longer that the same amount of agent administered in bolus form. The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours. For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation. In some examples of the invention, the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation. The term pulsatile release refers to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration. The term immediate release refers to a drug formulation that provides for release of the drug immediately after drug administration. As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration. Attorney Docket No.206017-0285-00WO As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration. Those skilled in the art recognize, or are able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, examples, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size / volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing / oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application. Preparation of the Compounds of the Invention compositions described herein may be prepared using any synthetic method known by those skilled in the art. The following examples illustrate non-limiting examples of the invention. The compounds of the invention may possess one or more stereocenters, and each stereocenter may exist independently in either the R or S configuration. In some examples, compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In some examples, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In another embodiment, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and / or separation of a mixture of enantiomers and / or diastereomers. Resolution of compounds and isomers thereof is Attorney Docket No.206017-0285-00WO achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography. The methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and / or pharmaceutically acceptable salts of compounds having the structure of any compound of the invention, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like. In some examples, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In another embodiment, the compounds described herein exist in unsolvated form. In some examples, the compounds of the invention may exist as tautomers. All tautomers are included within the scope of the compounds presented herein. In some examples, compounds described herein are prepared as prodrugs. A “prodrug” refers to an agent that is converted into the parent drug in vivo. In some examples, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In another embodiment, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound. In some examples, sites on, for example, the aromatic ring portion of compounds of the invention are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In some examples, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group. Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to2H,3H,11C,13C,14C, Attorney Docket No.206017-0285-00WO36Cl,18F,123I,125I,13N,15N,15O,17O,18O,32P, and35S. In some examples, isotopically-labeled compounds are useful in drug and / or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet another embodiment, substitution with positron emitting isotopes, such as11C,18F,15O and13N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed. In some examples, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels. The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4thEd., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000, 2001), and Green & Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein. Compounds described herein are synthesized using any suitable procedures starting from compounds that are available from commercial sources, or are prepared using procedures described herein. In some examples, reactive functional groups, such as hydroxyl, amino, imino, thio or carboxy groups, are protected in order to avoid their unwanted participation in reactions. Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed. In another embodiment, each protective group Attorney Docket No.206017-0285-00WO is removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal. In some examples, protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and / or oxidative conditions. Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile. Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable. In some examples, carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc. Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively-removable protective groups such as 2,4-dimethoxybenzyl, while co-existing amino groups are blocked with fluoride labile silyl carbamates. Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts. For example, an allyl-blocked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react. Typically blocking / protecting groups may be selected from: Attorney Docket No.206017-0285-00WO Other protecting groups, plus a detailed description of techniques applicable to the creation of protecting groups and their removal are described in Greene & Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, which are incorporated herein by reference for such disclosure. It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. EXAMPLES The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein. The materials and methods employed in the experiments and the results of the experiments presented in this Example are now described. Attorney Docket No.206017-0285-00WO Example 1: Novel beta-lactam, MC-100093, and ceftriaxone attenuate hydrocodone overdose-induced alterations in locomotor activity, respiratory exchange ratio, and glutamatergic system as well as trophic and neuroinflammatory biomarkers in C57BL / 6 mice Chronic exposure and overdose of opioid may cause respiratory depression and neurotoxicity through alteration of several neurotransmitters, including glutamate. Previous studies have shown the importance of modulating glutamate transporters with beta-lactams to attenuate the effects of chronic exposure to drugs of abuse, including opioids. The current study investigates the effects of novel synthetic beta-lactam, MC-100093, and ceftriaxone on locomotor activity, respiratory exchange, and glutamatergic system as well as trophic and neuroinflammatory biomarkers in C57BL / 6 mice exposed to escalated doses of hydrocodone involving overdose. Male C57BL / 6 mice (eight weeks) were injected with hydrocodone (20 mg / kg, intraperitoneal (i.p.)) every other day for 13 days, and on day 15, mice received a higher dose of hydrocodone (40 mg / kg, i.p.). Control group received i.p. injection of saline every other day, and the mice in the treatment groups received MC-100093 (50 mg / kg, i.p.) or ceftriaxone (200 mg / kg, i.p.) during the last five days of the study. This study used comprehensive laboratory animal monitoring systems (CLAMs) to measure mice's respiratory frequency, locomotor activity, oxygen consumption, and carbon dioxide production. Exposure to the overdose of hydrocodone was associated with increased oxygen consumption and carbon dioxide production, which were attenuated after treatment with MC-100093 or ceftriaxone. The respiratory exchange ratio was reduced in the hydrocodone group, and the MC-100093 treatment normalized the hydrocodone-induced decrease in the respiratory exchange ratio. This study demonstrated that ceftriaxone treatment attenuated the hydrocodone overdose-induced increases in oxygen consumption, carbon dioxide production, and locomotor activity with efficacy more than MC100093. Western blotting was performed to determine the expressions of metabotropic glutamate receptor 1 (mGluR1), cystine / glutamate transporter (xCT), glutamate transporter 1 (GLT-1), brain-derived neurotrophic factor (BDNF), tumor necrosis factor-alpha (TNF-α), and high mobility group box 1 (HMGB1) in the nucleus accumbens (NAc), amygdala (AMY), and dorsomedial prefrontal cortex (dmPFC). Hydrocodone overdose is associated with increased mGluR1 expression Attorney Docket No.206017-0285-00WO and decreased GLT-1 and xCT expressions in mesocorticolimbic brain regions. In addition, hydrocodone overdose exposure also upregulated the inflammatory response by upregulating the inflammatory mediators TNF-α and HMGB1. Treatment with β- lactams (ceftriaxone and MC-100093) attenuated hydrocodone-induced downregulation of GLT-1, xCT, and BDNF and hydrocodone-induced upregulation of mGLuR1, TNF-α, and HMGB1. In the current study, ceftriaxone and MC-100093 were found therapeutically efficacious in attenuating hydrocodone-induced alteration of the respiratory exchange, glutamatergic system, neurotrophic factor, and neuroinflammatory biomarkers. These findings suggest that the novel compound, MC100093, might be a therapeutic drug for attenuating hydrocodone overdose- induced dysfunction in biological behavior. Chronic exposure to high doses or overdose of opioids such as morphine, fentanyl, and buprenorphine can lead to fatal side effects, including respiratory depression (Janczewski, W. A. et al., 2022, The Journal of Physiology, 545, 1017; Lalley, P. M., 2003, Journal of Physiology-Regulatory, Integrative, and Comparative Physiology, 285, R1287; Varga, A. G. et al., 2020, The Journal of Physiology, 598, 189). Opioid-induced respiratory depression is characterized by shallow and irregular breathing which leads to hypoventilation and respiratory arrest (Pattinson, K. T., 2008, British Journal of Anaesthesia, 100, 747). The Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments) is equipped with a zirconia-based oxygen sensor and a high-speed single-beam non-dispersive infrared (NDIR) carbon dioxide sensor that provides measurements of oxygen and carbon dioxide flow in each mouse chamber. CLAMS also utilizes IR photocell technology for triple-axis detection of mouse activity. The system can detect mouse movement in length, width, and height (jumping or standing) within the chamber (Columbus-Instruments). Therefore, this current study used CLAMS to determine locomotor activity, oxygen consumption (VO2), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) in mice exposed to a hydrocodone overdose paradigm. CLAMS can be used in metabolomic studies to determine heat production (H), oxygen consumption (VO2), and carbon dioxide production (VCO2) which can then be used to measure energy expenditure via calorimetry (Menzies, C. et al., 2021, Eneuro, 8). A recent report showed that the beta-lactam antibiotic ceftriaxone attenuated the hyperlocomotion activity induced by Attorney Docket No.206017-0285-00WO hydrocodone overdose using the CLAMS (Wong, W., 2024, Brain Sciences, 14, 361). Considering the role of respiratory depression as the most lethal effect of opioid overdose, this study conducts a longer exposure of lower doses of hydrocodone plus a single high dose of hydrocodone to determine the respiratory and locomotion activity using the CLAMS. Emerging evidence suggests that glutamate homeostasis plays a key role in drug-induced presynaptic and glial adaptations and is regulated by glutamatergic systems in the brain. The glutamate transporter (GLT-1) is responsible for the majority uptake of glutamate in the brain, and the cystine / glutamate transporter (xCT) regulates glutamate release in astrocytes; they are expressed in mesocorticolimbic brain regions and play a crucial role in the regulation of extracellular glutamate levels at synapses (Bridges, R. et al., 2012, Pharmacological Reviews, 64, 780; Wong, W., 2023, Toxics, 11, 870). Accumulating evidence also suggested that exposure to substances of abuse such as morphine, methamphetamine, and cocaine often leads to dysregulation of the glutamatergic system in brain reward regions, leading to changes in glutamate receptors and transporters (Aboutalebi, F. et al., 2018, Advanced Biomedical Research, 7, 116; Fujio, M. et al., 2005, European Journal of Neuroscience, 22, 2744; Suto, N. et al., 2010, Psychopharmacology, 211, 267). In addition, previous studies have shown that GLT-1 is downregulated following chronic exposure to cocaine, nicotine, ethanol, and heroin (Gass, J. T. et al., 2011, Addiction Biology, 16, 215; Gipson, C. D. et al., 2013, Proceedings of the National Academy of Sciences, 110, 9124; Knackstedt, L. A. et al., 2010, Biological Psychiatry, 67, 81; LaLumiere, R. T., 2008, Journal of Neuroscience, 28, 3170). Furthermore, others have also shown that increased extracellular glutamate concentrations can lead to neurotoxicity, resulting in increased inflammatory cytokines (Tumor Necrosis Factor-Alpha (TNF-α), High mobility group box 1 (HMG- B1)) and ultimately neuroinflammation (Berrios-Carcamo, P. et al., 2020, Antioxidants, 9, 830; Pan, Y. et al., 2016, Journal of Neuroinflammation, 13, 1; Qian, J. et al., 2020, Neurotherapeutics, 17, 722). In addition to neuroinflammatory cytokines, studies have reported that Brain-derived neurotrophic factor (BDNF) is associated with drug addiction (Russo, S. J. et al., 2009, Neuropharmacology, 56, 73). BDNF is a neurotrophic factor that facilitates neuronal cell growth and plasticity Collectively, these studies suggest that glutamate transporters and neurotrophic factors may be therapeutic targets for opioid use disorder. Attorney Docket No.206017-0285-00WO Ceftriaxone is a beta-lactam antibiotic that has been shown to exert neuroprotective effects by modulating glutamate homeostasis in the brain (Krzyżanowska, W. et al., 2017, PloS One, 12, e0186243; Ramos, K. et al., 2010, Neuroscience, 169, 1888; Thone-Reineke, C. et al., 2008, Journal of Hypertension, 26, 2426). Previous studies have shown that ceftriaxone treatment attenuates ethanol- and hydrocodone-induced downregulation of GLT-1 and xCT expression (Alhaddad, H. et al., 2020, Brain Research Bulletin, 165, 272). However, treatment with the β- lactam antibiotic ceftriaxone may induce antibiotic resistance and adverse side effects, such as colitis caused by Clostridium difficile. In addition, due to its low lipid solubility, ceftriaxone has low brain bioavailability (Granero, L. et al., 1995, Antimicrobial Agents and Chemotherapy, 39, 2728; Nau, R. et al., 1993, Antimicrobial Agents and Chemotherapy, 37, 1518). Therefore, a novel non-antibiotic β-lactam GLT-1 modulator, MC-100093 was synthesized (Abou-Gharbia, M. et al., 2017, Drugs Fut, 42, 489). MC-100093 is a structural modification of the 6-membered ring and carboxylic acid group of ceftriaxone and has been shown to enhance the expression of GLT-1 (Childers, W. E. et al., 2020, ACS Medicinal Chemistry Letters, 11, 1820). Compared with ceftriaxone, MC-100093 exhibits unexpectedly more potent glutamate absorption properties, no antimicrobial effects, high water solubility, high stability in liver microsomes, low binding to plasma proteins, and low partitioning into lipid membranes (Knackstedt, L. A. et al., 2021, Pharmacology and Experimental Therapeutics, 378, 51). In light of the above, it was hypothesized that MC-100093 treatment would be a better candidate for the treatment of opioid use disorder compared with ceftriaxone. Furthermore, it has been previously shown that MC-100093 treatment upregulates GLT-1 level in the brain, attenuates inflammatory markers, and reduces ethanol drinking behavior in animal models exposed to alcohol, morphine, and cocaine (Alhaddad, H. et al., 2022, Journal of Pharmacology and Experimental Therapeutics, 383, 208; Sari, Y. et al., 2024, Saudi Pharmaceutical Journal, 102108; Travaglianti, S. et al., 2024, Brain Research Bulletin, 211, 110935). Therefore, the present study investigates the effects of MC-100093 on the glutamatergic system, neurotrophic factor, and neuroinflammatory markers in the mesocorticolimbic brain regions, as well as the dysregulation of respiratory and locomotion activities induced by longer exposure to low-dose hydrocodone plus single high-dose hydrocodone using CLAMS. Attorney Docket No.206017-0285-00WO Materials and Methods Drugs Eight-week-old male C57BL / 6 mice (25-30g) were purchased from Jackson Laboratory. Mice were housed in a temperature-controlled vivarium (maintained at 21°C) at The University of Toledo. Mice were housed under alternating 12-hour light and 12-hour dark cycles. Mice were acclimated for a minimum of three days prior to starting the experiments. Mice were intraperitoneally (i.p.) injected and handled with care to prevent any distress and minimize any pain. Mice were also monitored every day throughout the study, particularly when hydrocodone treatment was performed. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Toledo, under protocol number 400155 (approved August 2, 2022), and were following the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. MC-100093 was synthesized at Temple University School of Pharmacy’s Moulder Center for Drug Discovery Research in Philadelphia, PA (US patent 9,975,879). MC-100093 was synthesized as a white powder and dissolved in sterile saline at 50 mg / kg (i.p.). Hydrocodone was purchased from Sigma-Aldrich, Inc (St. Louis, MO, USA) and dissolved in sterile saline at 20 mg / kg and 40 mg / kg (i.p.). Ceftriaxone (Pfizer, USA) was dissolved in sterile saline at 200 mg / kg (i.p.). Hydrocodone Overdose Dosing Procedure Mice were divided into four groups: (1) Control group (n = 5-6); (2) Hydrocodone group (n = 5-6); (3) Hydrocodone-ceftriaxone group (n = 5-6); and (4) Hydrocodone-MC-100093 group (n = 5-6). The control group was i.p. injected with sterile saline (vehicle) every other day from day 1 to 15. Groups 2, 3, and 4 were injected with hydrocodone (20 mg / kg, i.p.) on days 1, 3, 5, 7, 9, 11 and 13. On day 15, mice in groups 2, 3, and 4 were challenged with a high dose of hydrocodone (40 mg / kg, i.p.). For the treatment group, group 3 received daily injections of ceftriaxone (200 mg / kg, i.p.) from days 13-17; and group 4 received daily injections of MC- 100093 (50 mg / kg, i.p.) from days 11-17. In addition, groups 1 and 2 were i.p. injected with the same volume of sterile saline (vehicle) from days 13-17. Attorney Docket No.206017-0285-00WO Behavioral Testing Mice were placed in the Minispec NMR, which serves to measure the lean and fat mass of the mice. For behavioral testing, mice were housed individually in a comprehensive laboratory animal monitoring system (CLAMS; Columbus Instruments, Columbus, Ohio) with free access to food and water from day 13 to day 17. Locomotion activity was measured using IR photocell technology. Oxygen consumption, carbon dioxide production, and respiratory exchange rate were calculated and obtained from Oxymax-CLAMS throughout the 5 days. Brain Tissue Extraction Mice were euthanized by CO2 inhalation on Day 18. Brains were isolated and frozen immediately on dry ice and stored at -80°C. NAc (core and shell), dmPFC (cingulate cortex and prelimbic cortex), and AMY (central amygdala, basomedial amygdala, and basolateral amygdala) were extracted using a cryostat machine (Leica CM1950). These brain regions were selected using the Brain Mice Atlas (Paxinos, G. et al., 2020, Atlas of the Developing Mouse Brain). All the samples were stored at -80°C for subsequent Western blot analyses. Western Blot Analyses Brain samples were homogenized using ice-cold lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1% Triton, 0.1% SDS) with phosphatase and protease inhibitors. the homogenate was then centrifuged at 13.2 rpm for 15 minutes at 4°C, and the supernatant was stored at -80°C for Western blotting. Protein concentration in each tissue sample was quantified using a detergent-compatible protein assay (Bio-Rad, Hercules, CA, USA), using bovine serum albumin as the standard. An equal amount of protein from each sample was mixed with laemmili dye, and the mixtures were loaded onto 10% Tris-glycerine gel to separate the protein using electrophoresis. Proteins were then transferred from gels to a polyvinylidene difluoride (PVDF) membrane. Subsequently, the PVDF membranes were blocked with 5% fat-free milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 30 minutes. Membranes were incubated overnight at 4°C with primary antibodies: rabbit anti-mGluR1 (1:1000, Abcam, ab82211), rabbit anti-xCT (1:1000, Abcam ab125186), rabbit anti-GLT-1 (1:5000, Abcam ab205248), rabbit anti-BDNF (1:1000, Abcam ab108319), rabbit anti-TNF-α (1:1000, Abcam Attorney Docket No.206017-0285-00WO ab9739), and rabbit anti-HMGB1 (1:1000, Abcam ab18256). Mouse anti-β-tubulin (1:1000, BioLeagend, San Diego, CA, USA) was used as a control loading protein. On the following day, membranes were washed five times with TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:4000) for 60 minutes. The membranes were then washed with TBST and dried for further analysis. The dried membranes were incubated with chemiluminescent reagents (Super Signal West Pico, Perce Inc., Appleton, WI, USA) for 1-2 minutes. Digitized blot images were developed using the ChemiDoc imaging system (Bio-rad Laboratories). The density of each specific band was quantified using ImageJ software (Version 1.53t 24). The control group was reported as 100% to measure the changes in the expression of the protein of interest in the NAc (core and shell), dmPFC (cingulate cortex and prelimbic cortex), and AMY (central amygdala, basomedial amygdala, and basolateral amygdala) as described in previous studies (Alhaddad, H. et al., 2022, Journal of Pharmacology and Experimental Therapeutics, 383, 208). Statistical Analyses GraphPad Prism (version 10.1.2) was used to analyze behavioral and Western blot data. Behavioral data from Oxymax-CLAMS were compared between groups using one-way ANOVA followed by the Newman-Keuls post hoc test. Data from Western blot studies for mGluR1, xCT, GLT-1, BDNF, TNF-α, and HMGB1 / β- tubulin expression are presented as % of the control, and the results are expressed as means ± S.E.M. The difference between treatments was considered significant at p < 0.05. Results Effects of Hydrocodone Overdose Exposure, Ceftriaxone, and MC-100093 on the Locomotion Activity To evaluate the effects of hydrocodone overdose exposure, ceftriaxone, and MC-100093 the locomotion activity of mice was assessed in the CLAMS. As shown in Fig.1, there was a significant difference in x activity (F3,10= 4.514, p < 0.05, Fig.1A, n= 5-6 / group) and x ambulatory (F3,11 = 8.436, p < 0.01, Fig.1B, n= 5- 6 / group) among all tested group. However, no significant difference was observed in z activity (F3,11 = 2.762, p > 0.05, Fig.1C, n= 5-6 / group). Newman-Keuls post hoc Attorney Docket No.206017-0285-00WO test analyses demonstrated that x activity (p < 0.05, Fig.1A) and x ambulatory (p < 0.01, Fig.1B) were significantly increased in the hydrocodone group compared to the control group. Importantly, treatment with ceftriaxone normalized activity (p < 0.05, Fig.1A) and ambulatory (p < 0.01, Fig.1B) as compared to the hydrocodone group. No significant changes in locomotion activity were detected between the control and hydrocodone groups for both activity and ambulatory (Fig.1A, Fig.1B). Effects of Hydrocodone Overdose Exposure, Ceftriaxone, and MC-100093 on oxygen consumption, carbon dioxide production, and respiratory exchange ratio To determine the effects of hydrocodone overdose exposure, ceftriaxone, and MC-100093 on respiratory frequency, oxygen consumption, carbon dioxide production, and respiratory exchange ratio were measured using CLAMS in mice. One-way ANOVA revealed a significant difference in oxygen consumption (F3,16= 58.09, p < 0.0001, Fig.2A, n= 5-6 / group), carbon dioxide production (F3,12= 127.7, p < 0.0001, Fig.2B, n= 5-6 / group), and respiratory exchange ratio (F3,16 = 17.48, p < 0.0001, Fig.2C, n= 5-6 / group). Newman-Keuls post hoc analyses showed a significant increase in oxygen consumption (p < 0.0001, Fig.2A) and carbon dioxide production (p < 0.0001, Fig.2B) in the hydrocodone group as compared to the control group. Treatment with ceftriaxone (p < 0.0001) and MC-100093 (p < 0.05) significantly attenuated the hydrocodone-induced increased in oxygen consumption as compared to the hydrocodone group (Fig.2A). In addition, the analysis also showed that carbon dioxide production was significantly decreased in groups treated with ceftriaxone (p < 0.0001) and MC-100093 (p < 0.01) as compared to hydrocodone- treated group (Fig.2B). Moreover, the effects of ceftriaxone or MC-100093 on respiratory exchange ratio in mice that were exposed to hydrocodone overdose paradigm were examined by comparing the ratio of oxygen consumption to carbon dioxide production in mice. Post hoc analyses revealed a significant decrease in the respiratory exchange ratio of the hydrocodone group (p < 0.01, Fig.2C) compared to the control group. Hydrocodone-induced downregulation of respiratory exchange ratio was significantly increased in the group treated with MC-100093 (p < 0.001, Fig.2C). However, no significant difference was detected between the hydrocodone and hydrocodone-ceftriaxone group (Fig.2C). Attorney Docket No.206017-0285-00WO Effects of Hydrocodone Overdose Exposure, Ceftriaxone, and MC-100093 on mGluR1 expression in the NAc, AMY, and dmPFC The expression of mGluR1 following hydrocodone overdose exposure was assessed in the NAc, AMY, and dmPFC. One-way ANOVA indicated that there was significant difference in mGluR1 expression in the NAc (F3,18 = 5.258, p < 0.01, Fig.3A, n= 5-6 / group), AMY (F3,18= 6.738, p < 0.01, Fig.3B, n= 5-6 / group), and dmPFC (F3,18 = 25.62, p < 0.0001, Fig.3C, n= 5-6 / group) among all tested groups. Newman-Keuls post-hoc analyses revealed a significant increase in mGluR1 expression in the NAc (p < 0.01, Fig.3A), AMY (p < 0.01, Fig.3B), and dmPFC (p < 0.001, Fig.3C) in the hydrocodone group as compared to the control group, while its expression was significantly decreased in group treated with ceftriaxone in the NAc (p < 0.05, Fig.3A), AMY (p < 0.01, Fig.3B), and dmPFC (p < 0.0001, Fig.3C). The analysis also showed that treatment with MC-100093 significantly decreased mGluR1 expression in the NAc (p < 0.05, Fig.3A), AMY (p < 0.01, Fig.3B), and dmPFC (p < 0.0001, Fig.3C) as compared to the hydrocodone group. However, there were significant differences between the control and hydrocodone-ceftriaxone groups in the dmPFC (p < 0.01, Fig.3C) and between the control and hydrocodone-MC-100093 groups in the dmPFC (p < 0.01, Fig.3C). Effects of Hydrocodone Overdose Exposure, Ceftriaxone, and MC-100093 on xCT expression in the NAc, AMY, and dmPFC Next, the xCT expression in the mesocorticolimbic brain regions following hydrocodone overdose exposure was quantified. It was found that hydrocodone overdose exposure significantly decreased xCT expression in the NAc (F3,19= 8.763, p < 0.001, Fig.4A, n= 5-6 / group), AMY (F3,14= 13.33, p < 0.001, Fig. 4B, n= 5-6 / group), and dmPFC (F3,17 = 9.459, p < 0.001, Fig.4C, n= 5-6 / group). In addition, the posthoc test for multiple comparisons showed a significant decrease in xCT expression in the NAc (p < 0.01, Fig.4A), AMY (p < 0.001, Fig.4B), and dmPFC (p < 0.05, Fig.4C) of the hydrocodone group compared to the control group. Treatment with ceftriaxone significantly attenuated hydrocodone-induced decreased xCT in the NAc (p < 0.001, Fig.4A), AMY (p < 0.01, Fig.4B), and dmPFC (p < 0.01, Fig.4C) compared to the hydrocodone group. Furthermore, the analysis also revealed MC-100093 significantly increased xCT expression in the NAc (p < 0.01, Fig.4A), AMY (p < 0.001, Fig.4B), and dmPFC (p < 0.001, Fig.4C) as compared to Attorney Docket No.206017-0285-00WO the hydrocodone group. No significant differences were detected between the control, hydrocodone, hydrocodone-ceftriaxone, and hydrocodone-MC-100093 in all the mesocorticolimbic brain regions as shown in Fig.4. Effects of Hydrocodone Overdose Exposure, Ceftriaxone, and MC-100093 on GLT-1 expression in the NAc, AMY, and dmPFC The effects of ceftriaxone or MC-100093 on the expression of GLT-1 in the mesocorticolimbic brain regions in mice exposed to hydrocodone overdose were also investigated. One-way ANOVA revealed a significant change in GLT-1 expression in the NAc (F3,20= 21.62, p < 0.0001, Fig.5A, n= 5-6 / group), AMY (F3,16= 18.58, p < 0.0001, Fig.5B, n= 5-6 / group), and dmPFC (F3,17 = 5.118, p < 0.05, Fig. 5C, n= 5-6 / group). Multiple comparison analyses revealed that hydrocodone overdose decreased GLT-1 expression in the NAc (p < 0.0001), AMY (p < 0.05), and dmPFC (p < 0.05) compared to the control group (Fig.5A, B and C respectively). Treatment with ceftriaxone normalized GLT-1 expression in the NAc (p < 0.0001, Fig.5A), AMY (p < 0.001, Fig.5B) and dmPFC (p < 0.05, Fig.5C). In addition, MC-100093 attenuated hydrocodone induced downregulation of GLT-1 in the NAc (p < 0.0001, Fig.5A), AMY (p < 0.0001, Fig.5B), and dmPFC (p < 0.01, Fig.5C). Significant changes were detected between hydrocodone-ceftriaxone and hydrocodone -MC-100093 groups in the NAc (p < 0.05, Fig.5A); between control and hydrocodone-ceftriaxone (p < 0.01), control and hydrocodone-MC-100093 (p < 0.01) in the AMY (Fig.5B); no significant difference was detected in the dmPFC (Fig.5C). Effects of Hydrocodone Overdose Exposure, Ceftriaxone, and MC-100093 on BDNF expression in the NAc, AMY, and dmPFC Data analyses revealed a significant decrease in BDNF protein expression in the NAc among all tested groups (F3,18 = 10.79, p < 0.001, Fig.6A, n= 5-6 / group). Newman-Keuls post-hoc analyses showed a significant decrease in BDNF expression in the NAc in the hydrocodone group as compared to the control group (p < 0.001), and that both the treatments with ceftriaxone (p < 0.001) and MC- 100093 (p <0.001) significantly increased BDNF expression in the NAc as compared to the hydrocodone group (Fig.6A). Besides that, statistical analysis also revealed a significant difference in the expression of BDNF in the AMY (F3,16 = 6.864, p < 0.01, Attorney Docket No.206017-0285-00WO Fig.6B, n= 5-6 / group) and the dmPFC (F3,17 = 5.118, p < 0.05, Fig, 6C, n= 5-6 / group) among all tested groups. Multiple comparison analyses showed a significant decrease in BDNF expression in the AMY (p < 0.05, Fig.6B) and the dmPFC (p < 0.05, Fig. 6C) of the hydrocodone group as compared to the control group. Importantly, ceftriaxone (p < 0.01) and MC-100093 (p < 0.01) treatments significantly increased BDNF expression in both the AMY and dmPFC (Fig.6B and 6C). However, no significant changes were detected between the control, hydrocodone, hydrocodone- ceftriaxone, and hydrocodone-MC-100093 in all three brain regions (Fig.6). Effects of Hydrocodone Overdose Exposure, Ceftriaxone, and MC-100093 on TNF-α expression in the NAc, AMY, and dmPFC The expression of TNF-α in the mesocorticolimbic brain regions following hydrocodone overdose exposure was further quantified. Western blot data analyses showed significant differences in TNF-α expression in the NAc (F3,16= 11.67, p < 0.001, Fig.7A, n= 5-6 / group), AMY (F3, 19= 13.52, p < 0.0001, Fig.7B, n= 5-6 / group), and dmPFC (F3, 20 = 38.77, p < 0.0001, Fig.7C, n= 5-6 / group) among all tested groups. Hydrocodone overdose exposure increased TNF-α expression in the NAc (p < 0.01, Fig.7A), AMY (p < 0.05, Fig.7B), and dmPFC (p < 0.0001, Fig. 7C) compared to the control group. Ceftriaxone treatment attenuated hydrocodone- induced upregulation of TNF-α expression in the NAc (p < 0.001), AMY (p < 0.001), and dmPFC (p < 0.0001) respectively (Fig.7A, 7B, and 7C). Importantly, MC- 100093 also attenuated hydrocodone-induced upregulation of TNF-α expression in the NAc (p < 0.01, Fig.7A), AMY (p < 0.0001, Fig.7B), and dmPFC (p < 0.0001, Fig.7C). No significant difference was detected in the NAc (Fig.7A). However, significant differences were observed between the control and hydrocodone-MC- 100093 groups in the AMY (p < 0.01, Fig.7B) and dmPFC (p < 0.01, Fig.7C); and significant difference was also detected between the control and hydrocodone- ceftriaxone groups in the dmPFC (p < 0.05, Fig.7C). Effects of Hydrocodone Overdose Exposure, Ceftriaxone, and MC-100093 on HMGB1 expression in the NAc, AMY, and dmPFC To evaluate the effects of ceftriaxone and MC-100093 on HMG-β1 expression in the NAc, AMY, and dmPFC, HMGB1 expression in the Attorney Docket No.206017-0285-00WO mesocorticolimbic brain regions following hydrocodone overdose exposure was assessed. One-way ANOVA indicated that there were significant differences in HMGB1 expression in the NAc (F3,16 = 4.747, p < 0.05, Fig.8A, n= 5-6 / group), AMY (F3,20= 6.580, p < 0.01, Fig.8B, n= 5-6 / group), and dmPFC (F3,20= 38.77, p < 0.0001, Fig.8C, n= 5-6 / group). Furthermore, hydrocodone overdose exposure increased HMGB1 expression in the NAc (p < 0.05), AMY (p < 0.05), and dmPFC (p < 0.05) (Fig.8A, 8B, and 8C) respectively compared to the control. In addition, the post-hoc test showed that treatment with ceftriaxone reduced the HMGB1 protein expression significantly in the NAc (p < 0.05, Fig.8A), AMY (p < 0.01, Fig.8B), and dmPFC (p < 0.05, Fig.8C). Notably, MC-100093 attenuated HMGB1 expression in the NAc (p < 0.05, Fig.8A), AMY (p < 0.01, Fig.8B), and dmPFC (p < 0.01, Fig. 8C) compared to the hydrocodone group. No significant differences were observed among all the tested groups in all three brain regions (Fig.8). Discussion In the present study, the major findings are that the novel beta-lactam compound, MC-100093, significantly attenuated dysregulation of the respiratory system associated with the hydrocodone-overdose model. In addition, MC-100093 significantly reversed the hydrocodone overdose-induced decreases in the respiratory exchange ratio, decreased in glutamatergic systems, increased in inflammatory markers, and decreased in neurotropic factors in the mesocorticolimbic brain regions. As opioid overdose deaths rise, there is a growing need to understand better the combined physiological effects of opioids, such as hydrocodone. Here, a series of low-dose intraperitoneal hydrocodone (20 mg / kg) followed by a high-dose intraperitoneal hydrocodone (40 mg / kg) were administered to determine whether hydrocodone exposure causes ventilatory dysfunction. It was found that hydrocodone overdose resulted in increased oxygen consumption and carbon dioxide production in hydrocodone-exposed mice and that these effects were mitigated by MC-100093 (50 mg / kg, intraperitoneally) and ceftriaxone (200 mg / kg). This is consistent with previous studies showing that opioid exposure results in concentration-dependent increases in hypoventilation (rapid and shallow breathing) and respiratory irregularities (Bouillon, T. et al., 2003, European Journal of Anaesthesiology, 20, 127; Pattinson, K. T., 2008, British Journal of Anaesthesia, 100, 747). When the hydrocodone dose is increased, ventilatory depression can lead to opioid-induced Attorney Docket No.206017-0285-00WO respiratory depression, which can be life-threatening. Interestingly, the present study demonstrated that MC-100093, but not ceftriaxone, significantly attenuated the decrease in respiratory exchange ratio induced by hydrocodone overdose. Taken together, the results report that MC-100093 may be a potential pharmacotherapeutic to treat hydrocodone overdose-induced respiratory irregularities other than ceftriaxone. However, the underlying molecular mechanisms regulating respiratory responses to opioids are unclear. Therefore, future studies are needed to focus on brain regions responsible for respiratory control, such as the pre-Bötzinger complex (Gray, P. A. et al., 1999, Science, 286, 1566), Kölliker-Fuse (Varga, A. G. et al., 2021, Journal of Neurochemistry, 156, 16), and parabrachial nuclei of the pons (Dutschmann, M., 2012, Comprehensive Physiology, 2, 2443). In addition, future studies may focus on the arterial blood-gas chemistry in mice exposed to hydrocodone overdose. This study showed that hydrocodone overdose exposure induced behavioral excitation, which is consistent with previous studies that hydrocodone overdose exposure induced hyperlocomotion activity in mice (Wong, W., 2024, Brain Sciences, 14, 361). The results are consistent with previous studies showing that treatment with the antibiotic β-lactam ceftriaxone significantly modulated hydrocodone-induced hyperlocomotion activity. Metabotropic glutamate receptor 1 (mGluR1) is highly expressed in the mesocorticolimbic brain regions in mice (Mitrano, D. et al., 2010, Journal of Comparative Neurology, 518, 1315; Sun, H., 2011, Journal of Neurophysiology, 106, 960). It is typically located at the postsynaptic membrane, interacting with NMDARs and AMPARs to form downstream pathways (Guo, Y. et al., 2009, Neuroscience & Biobehavioral Reviews, 33, 864; Mao, L.-M. et al., 2008, Neuropharmacology, 55, 403). The present study found that mGluR1 expression was upregulated in the mesolimbic region after hydrocodone overdose exposure. This is consistent with previous reports showing that mGluR1 expression is upregulated upon exposure to substances of abuse such as alcohol and nicotine (Alhaddad, H. et al., 2020, Toxics, 8, 95; Cozzoli, D. K. et al., 2014, Neuropsychopharmacology, 39, 435). Therefore, hydrocodone overdose exposure may have similar effects on mGluR1 expression. Furthermore, treatment with MC-100093 or ceftriaxone was associated with the normalization of mGluR1 expression levels in hydrocodone-overexposed mice, which may contribute to the previously observed attenuation of hyperlocomotion behavior Attorney Docket No.206017-0285-00WO by activation of mGluR1 (Nanou, E. et al., 2009, The Journal of Physiology, 587, 3001). Evidence suggested that excessive concentrations of glutamate are responsible for the neurotoxicity associated with opioid dependence (Kofke, W. A. et al., 2000, Neurological Research, 22, 733), and efficient glutamate uptake and transport in synapses is critical to prevent neuronal apoptosis. Notably, both xCT and GLT-1 play important regulatory roles in glutamate homeostasis by removing extracellular glutamate from synapses (Goncalves-Ribeiro, J. et al., 2019, Frontiers in Cellular Neuroscience, 13, 357). Therefore, drugs that can alleviate the brain's hyper- glutamatergic state by regulating GLT-1 expression would be potential therapeutic agents for substance abuse disorders. A recent study has shown that hydrocodone overdose exposure downregulates the expression of xCT and GLT-1 in the nucleus accumbens and that ceftriaxone is able to attenuate the hydrocodone-induced downregulation of xCT and GLT-1 expression (Wong, W., 2024, Brain Sciences, 14, 361). Furthermore, studies have shown that MC-100093 increases GLT-1 expression in animals exposed to alcohol and cocaine (Alhaddad, H. et al., 2022, Journal of Pharmacology and Experimental Therapeutics, 383, 208; Knackstedt, L. A. et al., 2021, Pharmacology and Experimental Therapeutics, 378, 51). The present results showed that both MC-100093 and ceftriaxone treatment normalized hydrocodone overdose-induced downregulation of xCT and GLT-1 in the NAC, AMY, and dmPFC. Together, previous reports and the current findings provide strong evidence that MC-100093 mediates substance-of-abuse–induced dysregulation of glutamate receptors and transporters in mesocorticolimbic brain regions. It is noteworthy that BDNF is closely associated with the regulation of neuronal and behavioral plasticity. BDNF is a neurotrophic factor secreted by neurons and microglia that plays a key role in the proliferation, development, and viability of the peripheral and central nervous systems (Batchelor, P. E. et al., 1999, Journal of Neuroscience, 19, 1708; McAllister, A. K. et al., 1999, Annual Review of Neurosceience, 22, 295). In this study, it was found that BDNF expression is downregulated in NAc, AMY, and dmPFC following hydrocodone overdose exposure. This is consistent with previous studies suggesting that decreased BDNF expression facilitates neuronal impairment caused by chronic morphine exposure (Akbarian, S. et al., 2022, Journal of Neuroscience, 22, 4153; Koo, J. W. et al., 2012, Science, 338, 124; Koo, J. W. et al., 2015, Nature Neuroscience, 18, 415). Furthermore, other studies also have reported that chronic exposure to morphine Attorney Docket No.206017-0285-00WO reduces BDNF expression in the ventral tegmental area (VTA), whereas inhibition of BDNF signaling in the nucleus accumbens (NAc) enhances the rewarding effects of morphine (Alghamdi, B. S., 2021, Frontiers in Behavioral Neuroscience, 15, 762297; Rezamohammadi, F. et al., 2020, Neuroscience Letters, 737, 135332). The present results showed that both MC-100093 and ceftriaxone attenuated the hydrocodone overdose-induced decrease in BDNF expression in the mesocorticolimbic brain regions. Similarly, a recent study has shown that MC-100093 treatment reversed the ethanol-induced downregulation of BDNF expression in the nucleus accumbens and prefrontal cortex (Travaglianti, S. et al., 2024, Brain Research Bulletin, 211, 110935). This is consistent with multiple studies showing that increasing BDNF levels is beneficial for the neurogenesis process. For instance, IL-4 significantly enhanced BDNF secretion from cultured astrocytes, and mice showed increased BDNF expression after water maze training, whereas mice lacking IL-4 (whose performance was impaired) did not show increased BDNF levels (Derecki, N. C. et al., 2010, Journal of Experimental Medicine, 207, 1067); wild-type mice showed significantly increased hippocampal BDNF levels when learning a new environment compared to mice with impaired memory (Spulber, S. et al., 2009, Journal of Neuroimmunology, 208, 46). TNF-α is a pro-inflammatory cytokine and an initiator of downstream inflammatory signaling pathways. Its activation can trigger a series of inflammatory responses in the brain. Overexpression of TNF-α impairs memory, learning (Baune, B. T. et al., 2008, Am. J. Med. Genet. B Neuropsychiatr. Genet., 147B, 1057; Beste, C. Et al., 2010, J. Neurophysiol., 104, 2523), and synaptic plasticity (Beattie, E. C. et al., 2002, Science, 295, 2282). Moreover, elevated levels of TNF-α have been found in Alzheimer's disease (Alvarez, A. et al., 2007, Neurobiology of Aging, 28, 533), Parkinson's disease (Mogi, M. et al., 1994, Neuroscience Letters, 165, 208), traumatic brain injury (Zhou, Y. et al., 2021, Molecular Neurobiology, 58, 2803), alcohol use disorder (Travaglianti, S. et al., 2024, Brain Research Bulletin, 211, 110935), and opioid use disorder (Tsai, R.-Y. et al., 2016, Journal of the Formosan Medical Association, 115, 445). In this study, it has been demonstrated that TNF-α expression was overexpressed after hydrocodone exposure, leading to enhanced neuroinflammatory processes in the mesocorticolimbic brain region. However, treatment with MC-100093 or ceftriaxone was associated with a reversal of TNF-α expression levels in hydrocodone-overexposed mice. The current findings provide Attorney Docket No.206017-0285-00WO strong support that MC-100093 can reduce neuroinflammation caused by exposure to substances of abuse such as alcohol and opioids (Sari, Y. et al., 2004, Saudi Pharmaceutical Journal, 102108). Emerging evidence over the past decade has confirmed the role of HMGB1 as a proinflammatory cytokine released by cells during inflammation and injury (Andersson, U., 2011, Annual Review of Immunology, 29, 139). HMGB1 is an endogenous TLR4 agonist that is considered a key player in mediating neuroinflammatory processes in a variety of pathophysiological conditions, including arthritis, inflammatory diseases, and chronic pain (Agalave, N. M., 2014, Molecular Medicine, 20, 569; Hazlett, L. D. et al., 2021, Pathogens, 10, 1235; Schierbeck, H. et al., 2011, Molecular Medicine, 17, 1039; Zhang, P. et al., 2020, Frontiers in Immunology, 11, 526748). HMGB1 induces inflammation by binding to the receptor for advanced glycation end products (RAGE) or Toll-like receptors 2 and 4 (TLR 2 and 4), activating a signaling cascade leading to the secretion of TNF-α and IL-6 (Agnello, D. et al., 2002, Cytokine, 18, 231; Hori, O. et al., 1995, Journal of Biological Chemistry, 270, 25752; Park, J. S. et al., 2004, Journal of Biological Chemistry, 279, 7370; van Beijnum, J. R. et al., 2008, Angiogenesis, 11, 91). In the current study, it was found that hydrocodone overdose exposure increased the expression of HMGB1 in the NAc, AMY, and dmPFC. Notably, previous studies have shown that the expression of HMGB1 and RAGE is increased in chronic alcohol-exposed rats (Alasmari, F. et al., 2020, Biomolecules, 10, 1030), hydrocodone overdose-exposed mice, chronic morphine-exposed rats (Qian, J. et al., 2020, Neurotherapeutics, 17, 722) and in animal models of neuropathic pain (Grace, P. M. et al., 2018, Brain, Behavior, and Immunity, 72, 45). This implies that opioids are potent stimulators of HMGB1 expression in the mesocorticolimbic brain region. Similar to TNF-α, previous studies have also shown that MC-100093 attenuated ethanol-induced increases in HMGB1 expression in mPFC and NAc subregions of alcohol-preferring rats. In accordance with this, it is shown that both MC-100093 and ceftriaxone attenuate the hydrocodone overdose-induced increase in HMGB1 expression in the mesocorticolimbic brain region. Conclusion The present work provides the unexpected results that MC-100093 can prevent excessive motor activity, respiratory dysregulation, glutamatergic system Attorney Docket No.206017-0285-00WO disruption, and neuroinflammation induced by hydrocodone overdose. This effect is likely mediated by reversing hydrocodone overdose-induced neuroinflammatory activation and upregulating GLT-1 and xCT expression in mesocorticolimbic brain regions. Together, these findings strongly support the notion that MC-100093 could be developed as a potential therapeutic candidate for opioid overdose. Example 2: MC-100093, a Synthetic Beta-Lactam Compound, Modulates Nicotine- Seeking Behavior and Glutamatergic Transporters in a Nicotine Self-Administration Mouse Model Tobacco use disorder is a prevalent condition that can negatively impact public health and economic status. Tobacco companies report that electronic cigarette products were developed to reduce the adverse health effects of conventional cigarettes; however, death cases and impairments of lung, brain, and kidney are reported in subjects exposed to electronic cigarettes. Both electronic and conventional cigarettes contain nicotine in varying amounts, raising concerns about nicotine's toxicity in the body. Although, the pharmacological action of nicotine is involving dopamine, studies suggest that modulating target glutamate transporters with beta- lactam, ceftriaxone, can reduce nicotine-seeking behaviors. In this study, ceftriaxone and MC-100093 were tested as a novel synthetic beta-lactam, which doesn’t have antibiotic action, in mouse model of nicotine self-administration. The results revealed that MC-100093 and ceftriaxone reduced nicotine self-administration. Both compounds attenuated nicotine-induced downregulation of glutamate transporters such as GLT-1, xCT and GLAST, and this effect was associated with attenuation of nicotine-induced in glutamate content in the nucleus accumbens (NAc). Furthermore, MC-100093 attenuated nicotine-induced decrease in cell numbers in the striatum and frontal cortex, and this indicates that MC-100093 has a neuroprotective effect. Additionally, the in silico docking study showed that both MC-100093 and ceftriaxone exhibited acceptable binding properties into GLT-1. Together, these data report potential therapeutic effects of MC-100093 targeting glutamate transporters for treating substance use disorders, particularly tobacco-use disorder. Nicotine is a central nervous system (CNS) stimulant and addictive drug that impacts the brain regions responsible for behavioral and mental functioning. Exposure to nicotine can disrupt the typical progression of brain development and Attorney Docket No.206017-0285-00WO result in long-term effects on cognitive function, mental well-being, and even individual characteristics (Richards, M. et al., 2003, American Journal of Public Health, 93, 994). For instance, nicotine exposure modifies the signaling of cholinergic and glutamate receptors in the prefrontal cortex (Goriounov, N. A. et al., 2012, Cold Spring Harbor Perspectives in Medicine, 2). Nicotine can directly influence the maintenance of self-administration behavior in humans, leading to disorders such as tobacco-use disorder. Nicotine replacement therapies can have therapeutic effects in typical dosages for nicotine cessation, but can also have negative impact on the body, such as increased risk of addiction or heart disease. Moreover, chronic use of nicotine can lead to addictive properties that affect human behavior when it comes to drug- seeking behaviors (Piaseck, M. et al., 2008, Nicotine in Psychiatry: Psychopathology and Emerging Therapeutics). Nicotine's negative effect manifests as increased requirement for higher doses can alleviate withdrawal symptoms, leading to dependence. In addition, withdrawal of nicotine leads to impatience and anxiety, reduced focus, enhanced appetite, thirst, enhanced cognitive function, and weight gain. However, its use has some of the behavioral effects such as mood regulation, stress reduction, and weight loss (Benowitz, N. L., 1998, Nicotine Safety and Toxicity). When nicotine is inhaled or swallowed, it is absorbed into the bloodstream and transported to the brain (Benowitz, N. L., 2009, Annual Review of Pharmacology and Toxicology, 49, 57). Nicotine enters the brain and exerts its effects by binding to specific nicotine acetyl choline receptors (nAchRs). This binding stimulates presynaptic ACh receptors, resulting in an increase in presynaptic dopamine release from the ventral tegmental area into the nucleus accumbens (NAc). Consequently, the dopamine reward pathway is activated (Mansvelder, M. H., 2002, Journal of Neurobiology, 53, 606). In addition, many other important neurotransmitters are also altered, including: γ-aminobutyric acid (GABA), norepinephrine, acetylcholine, glutamate, and serotonin. The release of dopamine is significantly influenced by the release of glutamate and the prolonged suppression of GABA release. Glutamate is the predominant excitatory neurotransmitter in the brain. Significant elevated concentrations of this amino acid in the synapse lead to neuronal loss through excitotoxicity. The glutamate is transported primarily into astrocytes but also into neuronal terminals via excitatory amino acid transporters (EAATs) (Choi, D. Attorney Docket No.206017-0285-00WO W. et al., 1988, Neuron, 1, 623). The EAATs play a crucial role in quickly and efficiently clearing glutamate from the space between neurons (synaptic cleft). When glutamate uptake is reduced or prevented, resulting in elevated levels of extracellular glutamate, the elevated synaptic glutamate can cause neuronal damage or glutamate toxicity (Magi, S. et al., 2019, International Journal of Molecular Sciences, 20). There are five distinct subtypes of glutamate transporters (Fairman, W. A., 1999, American Journal of Physiology – Renal Physiology, 277). EAAT1, which is found in cortex and cerebellum, EAAT2, found in the whole brain and spinal cord, EAAT3, found in cerebellum and hippocampus neurons, EAAT4, in Purkinje cells and EAAT5. EAAT2 is also known as glutamate transporter-1 (GLT-1) which is the predominant transporter responsible for the uptake of synaptic glutamate and the preservation of glutamate stability. Excessive accumulation of glutamate in the synaptic cleft is associated with several neurological disorders, including Parkinson's disease and Alzheimer's disease. Preserving the expression of GLT-1 and Glutamate- aspartate transporter (GLAST) may attenuate these disorders or associated symptoms (Karki, P. et al., 2015, Neurochemistry International, 88, 53; Pajarillo, E. et al., 2019, Neuropharmacology, 161, 107559). The GLT-1 protein in astrocytes and presynaptic area uptake excess glutamate with the assistance of the cystine-glutamate transporter (xCT). xCT exchanges glutamate for cystine and studies found that xCT expression in the NAc was decreased in animals exposed to alcohol, nicotine, and methamphetamine. It is also important to note that xCT might play a negative feedback role in attenuating glutamate toxicity through xCT-presynaptic metabotropic glutamate receptor communications (Hammad, A. M. et al., 2021, Neuroscience, 463, 128; Kalivas, P. W. et al., 2009, Neuropharmacology, 56 Suppl 1, 169). Prior research has demonstrated that prolonged exposure to nicotine leads to a reduction in the expression of GLT-1 and xCT (Bechard, A. R., 2019, Neural Mechanisms of Addiction, 61; Roberts-Wolfe, D., 2015, CNS & Neurological Disorders Drug Targets, 14, 745). GLAST, a different class of glutamate transporters known as EAAT1, is found in the cerebral glial cells in the brain. Research has demonstrated that changes in the amounts of GLAST can impact the regulation of glutamate uptake in these glial cells. However, GLAST expression was not altered in the NAc of animals exposed to drugs of abuse, including nicotine. Attorney Docket No.206017-0285-00WO Ceftriaxone is an antibiotic belonging to the class of broad- spectrum beta (β)-lactam drugs, specifically cephalosporin agents. It is a powerful antibiotic that works by inhibiting the process of bacterial cell wall synthesis. Ceftriaxone has efficacy against both gram-negative and gram-positive bacteria (Rawls, S. M., 2014, Encyclopedia of the Neurological Sciences, 207). As previously reviewed (Rothstein, J. D. et al., 2005, Nature, 433, 73), various beta-lactams, including ceftriaxone, have a potent stimulating effect on GLT-1 expression. These β -lactam drugs enhance the expression of GLT-1 and improve neurobehavioral parameters such as drugs seeking behaviors. Therefore, it might also prevent neurotoxicity through enhancing GLT-1 activities. A prior investigation has demonstrated that following prolonged self-administration of nicotine, there is a down-regulation of GIT-1 expression and an elevation in extracellular glutamate levels. The administration of ceftriaxone demonstrated favorable efficacy in the up- regulation of GLT-1, as well as a notable reduction in both nicotine consumption and seeking behavior (Sari, Y. et al., 2016, Neuroscience, 326, 117). β-lactam compounds also showed the ability to attenuate the seeking behaviors of alcohol, hydrocodone, methamphetamine, and synthetic cannabinoids, indicating that these β-lactam compounds are potential neuroprotective candidates and might be considered for managing substance use disorders. However, prior work reports no past or current clinical studies evaluating ceftriaxone or other β-lactam antibiotics to be under investigation for potential therapeutic effects against substance use disorders. MC-100093 belongs to a novel class of monocyclic β-lactam compounds designed to pose a similar GLT-1 upregulating effects as β-lactam antibiotics, while lacking antibacterial effects. This selectivity is essential to avoid bacterial resistance or antibiotic-related side effects. MC-100093 has superior potency and improved drug-like properties such as oral bioavailability and CNS penetration as compared to ceftriaxone (Knackstedt, L. A. et al., 2010, Biological Psychiatry, 67, 81; Knackstedt, L. A. et al., 2021, The Journal of Pharmacology and Experimental Therapeutics, 378, 51). In previous studies, it was observed that MC-100093 could attenuate cocaine and alcohol seeking behaviors at least in part by increasing GLT-1 and xCT expression in in vivo models (Alhaddad, H. et al., 2022, Journal of Pharmacology and Experimental Therapeutics, 383, 208). Moreover, MC-100093 was found to normalize several liver metabolites in fentanyl overdose mice model, an Attorney Docket No.206017-0285-00WO effect associated with restored liver inflammation (Alasmari, F. et al., 2023, Metabolites, 13). In this study, it was hypothesized that the novel β-lactam, MC-100093, will be able to reduce nicotine seeking behavior and neurodegeneration through modulating glutamatergic system in the NAc in comparison to the positive standard β- lactam compound, ceftriaxone, in nicotine self- administration mouse model. Materials and Methods Materials Nicotine tartrate was bought from Sigma Aldrich (Saint Louis, MO, USA). MC-100093 was provided by Temple University, Pennsylvania, USA. Antibodies for western blotting study were purchased from ABclonal company, Wuhan, China. Glutamate assay kit was purchased from MyBioSource (San Diego, California, United States). Experimental design Forty male C57 mice, aged 6-8 weeks and weighing 25-30 g, were housed and maintained under a 12-hour light / dark cycle. The mice were provided with appropriate water and food. All experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) at King Saud University. The C57 mice were acquired from the Experimental Animal Care Center within the College of Pharmacy at King Saud University. The animals were categorized into four groups. The distribution of groups was based on the goals of the study in the following manner: First group: water-control, saline-treated (0.9%) for 5 days during the last week of the study. The second group was exposed to 100 μg / mL of nicotine in water for five weeks and treated with saline 0.9% during the last 5 days (McCarthy, D. M. et al., 2018, PloS Biology, 16). Third group: nicotine-exposed mice for five weeks and treated with β-lactam antibiotic, ceftriaxone 200 mg / kg for the last 5 days of the study (Matos-Ocasio, F. et al., 2014, Pharmacology Biochemistry and Behavior, 122, 118). Fourth group: nicotine-exposed group for five weeks and treated with the experimental β-lactam compound (MC-100093) (50 mg / kg, i.p) for the last 5 days. The study timeline is shown in Fig.9. Attorney Docket No.206017-0285-00WO Average body weight, nicotine consumption, and preference measurements Male mice were exposed to nicotine via drinking, and it was conducted through 2-bottle free choice continuous access for 5 weeks. This paradigm was adapted from previous paradigms that suggested the use of 100 μg of nicotine in 1 mL of water (Li, X. C. et al., 2005, Behavioral Brain Research, 157, 79; Wilking, J. A. et al., 2012, Behavioral Brain Research, 233, 280). The control group was exposed to 2 bottles of water throughout the study. Groups exposed to nicotine had one bottle of water and one bottle containing nicotine. The two bottles were alternated weekly. The quantity of nicotine intake was determined by calculating the difference between pre- and post- consumption of nicotine level daily to check the amount accurately. Following that, the administration of ceftriaxone and the novel experimental drug (MC-100093) was implemented as treatment measures during the last 5 days of the study. Nicotine and water intake were measured daily from day 1 to day 4 of β-lactam treatments. Euthanasia was carried out early on the following day after last day of injections. Nicotine preference was also calculated based on the total volume of nicotine consumption in the total volume of fluid consumption (Equation 1). Control cages were used to estimate the amount of spilled volume to consider the spilling errors in the calculations. Furthermore, the body weight of the mice was assessed at various time points during the study, including baseline before the commencement of the experiment, after the initiation of treatment, and before the euthanasia of the animals. Nicotine preference =() / (( ( ) ( )) / ) X 100 1) Brain harvesting At the end of the study, the mice were euthanized using isoflurane. Their brains were carefully extracted and immediately stored at -80 °C for molecular studies (Wirths, O., 2017, Bio-protocol, 7). The extracted brains were sectioned and the NAc tissues were free-hand isolated according to the Mouse Brain Atlas (Krebs, B. et al., 2006, The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society, 54, 559). Attorney Docket No.206017-0285-00WO Western blotting assay For the protein counting assay, PierceTMBCA protein assay kit was used to estimate the total protein content in each sample. Both standard BSA and dilution solutions were prepared according to the protocol using the spectrophotometer technique. To prepare brain tissues for the experiment, the collected tissues from each group of animals (consisting of five mice) were subjected to homogenization in a lysis buffer that included protease inhibitors. An equal quantity of protein lysates was loaded to acrylamide gel (8-15% SDS-PAGE gel) and allowed to separate according to their molecular weight, then transferred into a PVDF membrane utilizing a transfer chamber. Membranes were blocked for one hour with 3% non-fat milk in tris-buffered saline with tween (TBST) and incubated overnight at 4°C with primary antibodies, including rabbit-anti xCT primary antibody (1:1000, ABclonal), rabbit-anti GLAST primary antibody (1:1000, ABclonal), and rabbit-anti GLT-1 primary antibody (1:1000, ABclonal). Following a 24-hour incubation period, the samples were blocked for one hour with 3% non-fat milk in TBST and then incubated with a secondary antibody (anti-rabbit antibody) for 90 minutes. The samples were subjected to detection using appropriate reagents and utilizing an imaging system (ChemiDocTM, Bio-Rad). Finally, Image J software was used to quantitatively estimate the expression of the bands of the detected protein. The control group served as the reference point at 100%, and all other groups were normalized based on the control group bands of the same gel. Glutamate content determination For the glutamate assay, a biochemical glutamate assay kitd based on spectrophotometer methodology was employed. Briefly, NAc tissue samples were lysed in extracting solvents following the protocol provided by the manufacturer. To detect the amount of protein in each sample, a PierceTM BCA protein assay was performed, and the obtained data from the glutamate content assay were normalized to the total protein content. The glutamate content data of the treated groups were normalized to the average glutamate content of the control group. Histopathology study Attorney Docket No.206017-0285-00WO For histopathologic analysis, formalin-fixed, paraffin-embedded mouse brain sections were deparaffinized in xylene, rehydrated with alcohol in a series, rinsed with tap and distilled water, and stained with 0.1 % cresyl violet (Alfa Aesar, Thermo Fischer Scientific, USA) at 50°C for 20 min. Sections were then rinsed with distilled water, differentiated in 95% ethanol, washed with absolute alcohol, cleared in xylene, and mounted with DPX medium (Sigma-Aldrich) and a coverslip. For the assessment of nicotine-induced striatal and cortical neurodegenerative changes and the subsequent effect of therapy with β-lactam compounds, prepared brain tissue slides were then visualized, and the region of interest was located using an Olympus CH-2, 838388 microscope (Olympus, Tokyo Japan). Photomicrographs were acquired at 10x and 40x magnification using a digital camera. Using the multi-point tool at 400 µm, cell density was estimated in both striatal and cortical sections using ImageJ 1.53t software (National Institutes of Health, Bethesda, MD, USA), after converting the region of interest into a binary version. Systemic electrolytes quantification To determine the concentrations of electrolytes such as sodium potassium, chloride, and calcium, biochemical assay kits were performed using serum samples of all groups. Protein retrieval The cryo-electron microscopy structure of the human EAAT2 protein was retrieved from the RCSB Protein Database (Fig.10). The selected protein size was large, with a 574aa sequence length, containing three chains: A, B, and C. The protein was refined for docking by removing the chain, already attached ligands and ions using Chimera (UCSF, San Francisco, CA, USA). Only Chain A was used for docking. Ligands retrieval The ligands used were previously reported (Alasmari, F. et al., 2023, Metabolites, 13). Ceftriaxone and MC-100093 were prepared using Chemdraw 3D (PerkinElmer, Waltham, MA, USA). The structures were minimized and prepared for docking (Fig.11). Attorney Docket No.206017-0285-00WO Molecular docking To conduct molecular docking, the appropriately prepared receptor molecule from UCSF Chimera and ligand from Chemdraw 3D Pro were used (Iashia Tur Razia, A. K. et al., 2022, Biomedical Letters). Molecular docking was performed through the PyRx server (Yuan, S. et al., 2017,Wiley Interdisciplinary Reviews: Computational Molecular Science, 7, e1298). The grid size employed along all three dimensions in the x-axis was 79.5, in the y-axis was 79.04, and in the z-axis was 75.89 for molecular docking investigations, respectively. The visualization via Discovery Studio (Los Angeles, CA, USA) and UCSF Chimera was performed for 2D and 3D results (Tian, W. et al., 2018, Nucleic Acids Research, 46, W363). The protein data retrieved from the PDB database provides key details on the atomic positions, bond distances, and angles, as well as the general structure of the protein. Presenting the protein cryo-EM structure in the PDB database provides the scientific community with precise and dependable structural data, which aids in conducting research and making progress in fields like molecular biology, drug discovery, and biochemistry. RCSP PDB fully interfaces with chemical databases like Chemdraw, ChemSpider and PubChem, allowing users to search and access chemical information directly in the software. This integration simplifies the process of obtaining chemical data and aids in research and analysis. Therefore, this software was used for the modeling of ligands. Statistical analysis GraphPad Prism software (Boston, MA, USA) was used to analyze the obtained data. Two-way ANOVA was used to investigate whether there were any changes in the average body weight. One-way ANOVA followed by Fisher's least significant difference (LSD) was used to analyze nicotine consumption, water intake, and nicotine preference in all studied groups. One-way ANOVA followed by Tukey's post hoc multi-comparison test was used to compare the glutamate content, protein expression data, and histopathology study between the four groups. Results Effects of nicotine drinking with / without β-lactam treatments on the average body weight Attorney Docket No.206017-0285-00WO The effect of nicotine drinking with / without β-lactam treatments on the average body weight was determined. Statistical analysis indicates significant main effect of treatments (F (3, 145) = 12.16, p < 0.0001) and days (F (4, 145) = 2.581, p = 0.0397) but not their interactions. However, Tukey's multiple comparisons test did not reveal any significant changes in the average body weight either between the four groups at all phases of the study or between the treatment phases of the same group throughout the study (Fig.12). Effects of nicotine drinking with / without β-lactam treatments on the water intake The effect of nicotine drinking with / without β-lactam treatment on water intake (mL / gram of body weight) was determined. One-way ANOVA did not reveal any significant changes of water intake among baseline and days of treatments in control, nicotine, and nicotine-ceftriaxone groups. However, the analysis showed significant alterations of water consumption among baseline and days of treatments in nicotine-MC-100093 group F (1.907707, 13.35395) = 4.190899, p = 0.0401). Fisher's least significant difference (LSD) showed an increase of water intake in day 1 as compared to baseline of nicotine-MC-100093 group (Fig.13). Nicotine drinking and preference in groups treated with normal saline or β-lactam compounds The effect of nicotine drinking with / without β-lactam treatments on nicotine intake (mL / gram of body weight) was investigated. One-way ANOVA did not reveal any significant changes of nicotine intake among baseline and days of treatments in nicotine group. However, the analysis showed significant alterations of nicotine consumption among baseline and days of treatments in nicotine groups treated with ceftriaxone (F (2.381236, 16.66865) = 4.096073, p = 0.0302) or MC- 100093 group (F (2.612345, 18.28642) = 5.942322, p = 0.0066). Fisher's LSD showed a decrease of nicotine intake in days 3 and 4 in nicotine-ceftriaxone and nicotine-MC-100093 groups as compared to the baseline phase of the same groups (Fig.14A). The effects of nicotine drinking with / without β-lactam treatments on nicotine preference (volume %) were investigated. One-way ANOVA did not reveal any significant changes of nicotine preference among baseline and days of treatments in nicotine group. However, the analysis showed significant alterations of nicotine Attorney Docket No.206017-0285-00WO preference among baseline and days of treatments in nicotine groups treated with ceftriaxone (p = 0.0223) or MC-100093 group (p = 0.0407). Fisher's LSD showed a decrease in nicotine preference on days 3 and 4 in nicotine-ceftriaxone and nicotine- MC-100093 groups as compared to the baseline phase within the same groups (Fig. 14B). Effects of nicotine drinking with / without β-lactam treatments on GLT-1, xCT and GLAST expression in the NAc One-way ANOVA showed significant changes in GLT-1 expression in the NAc among the four studied groups (p = 0.0129). Statistical analysis showed that nicotine drinking reduced the GLT-1 expression. The β-lactams (ceftriaxone and MC- 100093) normalized the GLT-1 expression in the NAc in nicotine exposed groups (Fig.15A). Statistical analysis showed significant changes on xCT expression in the NAc among the four studied groups (p = 0.0057). The analysis showed that nicotine drinking reduced the xCT expression. The β-lactams (ceftriaxone and MC-100093) normalized the xCT expression in the NAc in nicotine exposed groups (Fig.15B). Statistical analysis did not show significant changes in GLAST expression in the NAc among the four studied groups (p = 0.9756) (Fig.15C). Effects of nicotine drinking with / without β-lactam treatments on the glutamate content in the NAc Statistical analysis showed significant changes in the glutamate content in the NAc among the four studied groups (p = 0.0005). The analysis showed that nicotine drinking increased the glutamate content in the NAc, and that the β -lactams (ceftriaxone and MC-100093) restored the glutamate content in NAc of nicotine exposed groups (Fig.16). Histopathology data Chronic alterations in striatal and cortical cellular pathology, i.e. neurodegeneration and cell loss, after exposure of mice to nicotine and ameliorative impact of subsequent therapy with β-lactam compounds was assessed using Nissl’s staining (Fig.17). One-way ANOVA revealed significant intergroup differences in striatal neuronal density (F (3, 8) = 10.18; p = 0.0042). Brain sections of the control group showed intact striatal neuronal morphology and normal cytoarchitecture (835 ± Attorney Docket No.206017-0285-00WO 58.53). However, nicotine exposure significantly induced tissue disruption, nuclei fragmentation, cytoplasmic vacuolation, pyknosis, and increased neuronal depletion (391 ± 89.20; p = 0.0018) compared to saline-treated mice. Ceftriaxone treatment to some extent presented non-significant reduction in striatal neuronal degeneration (541 ± 42.80; p = 0.2408) compared to the brains exposed to nicotine only. Therapy with MC-100093 markedly prevented pathologic abnormalities, as evidenced by the reduced number of necrotizing and apoptotic neurons (655 ± 24.26; p = 0.0323), supporting its potential role in the modulation of adaptive and goal-directed behaviors such as habit formation, reward, and aversion in the ventral division of the striatum. Furthermore, morphological aspects and neuronal density in cortex 2 / 3 were assessed (F (3, 8) = 9.432; p = 0.0053). In the control group, rare areas of neuronal loss were detected, and the visual field showed clear and intact neurons without apoptotic bodies (728 ± 49.64). However, a greater proportion of cortical cells in the nicotine group exhibited extensive degenerative alterations, including loss of integrity, damaged cytoarchitectonics in layers 2 / 3, oval-shaped condensed nuclei appearing as darkly stained cells, and a shrunken cytoplasm (423 ± 52.04; p = 0.0054). Furthermore, ceftriaxone administration did not prevent cortical neuronal loss and pyknosis (459 ± 45.00). Serum Electrolyte Concentrations One-way ANOVA did not reveal any significant changes on serum sodium, potassium, calcium or chloride among the four studied groups (Fig.18). Molecular docking data Ceftriaxone and MC-100093 were used for docking against the A- chain of the human GLT-1 protein with PDB ID: 7XR4. The interaction having the highest score for energy between the protein and ligand is considered the most favorable. Through the analysis of docking binding affinities, it was observed that Ceftriaxone showed a DG value of -7.2 kcal / mol whereas DG = -5.6 kcal / mol was obtained for MC100093 (Fig.19A, Fig.19B). The docking results from PyRx and top selected binding affinity generated are shown in Table 1. The model predicted that ceftriaxone displays a more favorable binding energy than that of MC-100093. Attorney Docket No.206017-0285-00WO Table 1. Results generated from PyRx for best binding structures. Binding Ligand Affinity Rmsd / ub Rmsd / lb Rmsd / ub - Rmsd / lb MC-100093 -5.6 33.847 32.114 1.733 Ceftriaxone -7.2 40.225 38.868 1.357 The docking model predicted interactions between ceftriaxone and several residues, including ALA 349, PHE 345, GLY 329, GLY 328, VAL 484, LEU 324, ILE 325 PHE 352, PHE 342, SER 480, TRP and TRP 355 and others. FMC- 100093 the model predicted interactions with MET 415, LYS 299, LYS 304, VAL 308, ASP 305, ASP 463, LYS 148 and others. The minimum differences in energy score between ceftriaxone and MC-100093 ceftriaxone and MC100093 suggest that both should be effective chemicals for targeted receptor activity. The 2D and 3D visualization is shown in Fig.20A and Fig.20B. Discussion The present work focused on nicotine, a psychoactive compound that is known to induce addictive behaviors. The expression of GLT-1 and xCT proteins were reduced in nucleus accumbens in nicotine self-administration models. The exposure to nicotine was suggested to be linked to the down-regulation of GLT-1 and xCT expression as conducted in previous studies (Alajaji, M. et al., 2013, Psychopharmacology, 228, 419; Knackstedt, L. A. et al., 2009, Biological Psychiatry, 65, 841; Namba, M. D. et al., 2020, Addiction Biology, 25, e12797). These effects were also observed in this study in nicotine treated group. According to reports, ceftriaxone, a beta-lactam antibiotic, has demonstrated the capacity to lessen nicotine dependency in mouse models. Additionally, it has been shown to suppress the proliferation of glutamate exchanger xCT and glutamate transporters GLT-1 in the nucleus accumbens. Ceftriaxone demonstrated higher safety and efficacy in the treatment of various neurological symptoms by effectively regulating extracellular glutamate levels when treated for a duration of 5 to 7 days compared to numerous other beta-lactam drugs. Nevertheless, there is concern associated with using this compound for nicotine addiction due to the bacterial resistance that arises out of widespread use (Abulseoud, O. A. et al., 2022, Frontiers in Neuroscience, 16, 841036). This study demonstrates Attorney Docket No.206017-0285-00WO that the beta-lactams ceftriaxone and MC-100093 showed ability to increase the expression of GLT-1 and xCT in the NAc leading to a decrease in nicotine seeking behavior as compared to nicotine only treated group MC-100093 is a newly developed β-lactam that does not possess antibacterial properties, recently proven effective in enhancing the up regulation of GLT-1 and xCT (León, B. E. et al., 2023, Neuropharmacology, 232, 109515). Interestingly, as shown in previous studies (Alhaddad, H. et al., 2022, Journal of Pharmacology and Experimental Therapeutics, 383, 208). MC-100093 could reduce alcohol and cocaine seeking at least in part through upregulating glutamatergic transporters. In this study, this compound was also found to increase the expression of the GLT-1 and xCT in the NAc and attenuate the nicotine seeking and preference behaviors. Taken together, these data indicate that MC-100093 is able to modulate glutamatergic transporters in animals exposed to drugs of abuse. The GLAST expression was also investigated in the NAc in the nicotine group, as well as in groups treated with ceftriaxone and MC-100093, in comparison to the control group. The findings indicated that there were no statistically significant differences observed among all the groups. Furthermore, consistent with previous research, the GLAST expression in the NAc was not significantly changed after exposure to nicotine and other drugs, such as ethanol and hydrocodone (Alhaddad, H. et al., 2014, Psychopharmacology, 231, 4049; Alshehri, F. S. et al., 2018, Behavioral Brain Research, 347, 368). To summarize, GLT-1 and xCT in the NAc exhibit greater sensitivity to alteration when exposed to nicotine as compared to GLAST. This may be attributed to the higher distribution and functioning of GLT-1 and xCT in the forebrain. The nicotine group showed a higher content of glutamate in the NAc compared to the treated and control groups. β-lactam compounds normalized NAc glutamate content after exposure to nicotine for five weeks. This difference in glutamate levels might be attributed to the effectiveness of the β-lactam at up- regulating GLT-1 and xCT. In addition, the higher glutamate content in the NAc of the nicotine group might be due to the reduction of GLT-1 and xCT expression in the NAc, since the NAc receives glutamatergic projections from multiple brain regions such as frontal cortex, amygdala and hippocampus. The pattern of nicotine seeking behavior and nicotine preference has been reported to be linked to the downregulation of GLT-1, in mice, as a consequence Attorney Docket No.206017-0285-00WO of ethanol drinking (Qrunfleh, A. M. et al., 2013, British Association for Psychopharmacology, 27, 541). This study revealed that all three groups administered nicotine exhibited a tendency and desire for nicotine. However, the groups treated with ceftriaxone and MC-100093 demonstrated a significant decrease in both nicotine intake and preference on days 3 and 4 of treatment, as compared to the initial baseline. Furthermore, the group administered MC –100093 exhibited a noticeable enhancement in water consumption from on day 1 of therapy. The observed result can be attributed to the restoration of normal functioning of glutamate transporters GLT-1 and xCT in the NAc. The effects of ceftriaxone and MC–100093 on nicotine-induced cellular pathologies, neurodegeneration, and cell loss in the frontal cortex and striatum were assessed in this study. There were no notable alterations observed following ceftriaxone administration in comparison to the nicotine group. This may be attributed to several factors: 1) ceftriaxone might require a longer term of treatment (more than five days, as observed in this study), 2) a higher dosage maybe required to achieve the desired effect, and 3) the mouse strain may exhibit greater sensitivity to MC – 100093 compared to ceftriaxone. Compared to ceftriaxone, treatment with MC– 100093 attenuated nicotine-induced neuronal loss and reduced necrotizing and apoptotic neurons in the frontal cortex and striatum. This also suggests that MC- 100093 might have fast positive effects on cellular impairments in the forebrain in animals exposed to nicotine as compared to ceftriaxone. Recent research indicates that using nicotine can have a direct impact on the kidneys, resulting in renal dysfunction and potentially causing an indirect increase in serum electrolyte content (Nwaji, A. R. et al., 2022, Nigerian Journal of Physiological Sciences, 37, 153). In this investigation, there were no significant differences observed in the electrolyte content between all studied groups. This may indicate the drinking pattern of nicotine in this study does not have acute effects on the kidneys. Therefore, it is possible that a higher dosage or longer duration of nicotine exposure is necessary to induce acute effects on electrolytes concentrations in the blood. The results of the docking test indicate that both ceftriaxone and MC- 100093 could possess the ability to directly interact with GLT-1, which could have direct impact on the concentration of glutamate which then affects other pathways. Attorney Docket No.206017-0285-00WO The minimum differences in energy score between ceftriaxone and MC-100093 suggest that both could be effective chemicals for targeted receptor activity. Conclusion The present study supports the hypothesis that the beta-lactam drug, ceftriaxone, possesses the capability to modulate the expression of glutamate transporters (GLT-1 and xCT) and glutamate contents in the NAc, resulting in neutralizing the behavioral alterations associated with their seeking pattern of nicotine self-administration. These effects were also observed with using a novel synthetic beta-lactam drug, MC-100093, which yields similar outcomes compared to ceftriaxone (Fig.21). In addition, MC-100093 lacks the antibacterial activity seen with ceftriaxone, which avoids the resistance risk and other side effects associated with antibiotic use. The findings of this study indicate that the two beta-lactam compounds have a significant ability to reverse the alterations of the glutamatergic system induced by nicotine drinking. Further, these drugs demonstrate a noticeable attenuation in nicotine seeking and preference behaviors. Example 3: Beta-lactams for treatment of alcohol withdrawal-induced anxiety and Anxiety is a prominent and common symptom of withdrawal from chronic ethanol in humans. Downregulation of the inhibitory GABA neurotransmitter system and an upregulation in excitatory glutamatergic neurotransmission is thought to be a major player in the anxiety seen in human alcohol withdrawal syndrome. One of the key causes of glutamate dysregulation during alcohol withdrawal is a downregulation in astrocytic glutamate reuptake through the Glutamate Transporter-1 (GLT-1), also known as the Excitatory Amino Acid Tranporter-2 (EAAT-2). The GLT-1 modulator ceftriaxone has been shown to reduce anxiety-like behavior in a number of animal models of ethanol withdrawal induced anxiety. These studies suggest that glutamate transporters are a potential target for the treatment of ethanol withdrawal-induced anxiety. In addition, reduced GLT-1 levels have been associated with depression-like symptoms both in conjunction with ethanol withdrawal and in the absence of ethanol withdrawal. Ceftriaxone has been shown to alleviate depression-like behavior in animal models of depression. Thus, GLT-1 transporters may also represent a potential target for the treatment of both ethanol withdrawal Attorney Docket No.206017-0285-00WO associated and non-ethanol withdrawal-associated depression. This proprietary series of drug-like small molecule GLT-1 uptake enhancers, exemplified by MC-100093, reduced both anxiety-like behaviors and depression-like behaviors in rats withdrawn from chronic ethanol consumption. In addition, these compounds reduced depression- like behavior in naïve rats not exposed to ethanol. Around 8 to 10 percent of Americans drink heavily. In 2019, according to the NSDUH, nearly 15 million Americans age 12 and older had Alcohol Use Disorder (AUD). Alcohol use disorder (AUD) is a complex chronically relapsing disorder that, with its progression, induces long lasting neuroadaptations as a result of alterations in normal brain circuitry. Dysregulation of the glutamatergic system in key brain reward regions such as Nucleus Accumbens (NAc), basal lateral amygdala (BLA), prefrontal cortex (PFC) and hippocampus is a common consequence of chronic ethanol exposure, resulting in altered glutamate receptors and transporters (Goodwani et al., 2017, Neurosci, Biobehav. Rev., 77, 14). Chronic exposure to ethanol in alcohol preferring (P) rats resulted in reduced expression of Glutamate Transporter-1 (GLT-1) and the Glutamate-Cystine Antiporter (xCT) and, as a result, increased extracellular glutamate concentration in NAc (Alhaddad et al., 2014; Das et al., 2015). Moreover, treatment with GLT-1 modulators (e.g. Ceftriaxone and MS- 153) upregulated GLT-1 and xCT expressions in NAc and attenuated ethanol drinking behaviors (Alhaddad et al., 2014; Alhaddad et al., 2014). These studies suggested the glutamate transporters as potential target for the treatment of ethanol dependence. These results suggest that manipulation of glutamate transporters / receptors may serve as therapeutic target for the treatment of ethanol dependence. Anxiety is thought to be one of the symptoms experienced during ethanol withdrawal (Gonzales and Jaworski, 1997, Alcohol Health Res. World, 21, 120; Koob and Le Moel, 1997, Science, 278, 52; Meyer, 1986, J. Stud. Alcohol, 47, 269) as well as one of the precipitating factors in relapse (Driessen et al., 2001, 36, 249; Sinha, 2001, Psychopharmacol., 158, 343). Chronic consumption of ethanol induces excessive signaling through the inhibitory neurotransmitter gamma amino butyric acid (GABA) (De Witte, 2004, Addict. Behav., 29, 1325). To balance the inhibitory signals of ethanol, neuroadaptation occurs and excitatory glutamatergic neurotransmission is increased (Rao and Sari, 2012, Curr. Med. Chem., 19, 5148). When ethanol consumption is discontinued, this neurotransmitter imbalance is behaviorally expressed as anxiety (Maciel and Kerr-Correa, 2004, Rev. Bras. Attorney Docket No.206017-0285-00WO Psiquiatr., 26, 47). One of the key causes of increased glutamatergic neurotransmission during withdrawal from chronic ethanol consumption is down regulation of the astrocytic Glutamate Transporter-1 (GLT-1), also known as Excitatory Amino Acid Transporter-2 (EAAT-2) (Alhaddad et al., 2020, Brain Res. Bull., 165, 272). Ceftriaxone, a known enhancer of glutamate uptake through the GLT-1 transporter, has been shown to attenuate ethanol withdrawal-induced anxiety- like behavior in rats (Abulseoud et al., 2014, Neuropsychopharmacol., 39, 1674; Kang et al., 2018, Neuropharmacol., 129, 47) and zebrafish (Agostini et al., 2020, Neurochem. Res., 45, 1526). Similar effects were observed with other glutamate uptake enhancers such as MS-153 and GPI-1046 (Sari et al., 2012, Neuroscience, 227, 327; Rao et al., 2015, Front. Neurosci., 9, 144). Currently, benzodiazepines are the drugs of choice for treating the anxiety associated with alcohol withdrawal syndrome (Amato et al., 2011, Cochrane Database Syst. Rev., CD008537). However, the use of benzodiazepines in alcohol withdrawal suffers from its own risks and side effects, including sedation, synergy with ethanol, addiction and a host of benzodiazepine- associated withdrawal symptoms. Other non-benzodiazepine anticonvulsants and muscle relaxants such as baclofen, gabapentin, topiramate, carbamazepine and valproic acid have also been used but demonstrate only limited clinical efficacy (Leggio et al., 2008, Biol. Psychiatry, 32, 1106). What is needed is a safe, efficacious drug for treating the anxiety associated with alcohol withdrawal syndrome. Ethanol consumption and withdrawal is also associated with depression (Briere et al., 2014, Compr. Psychiatry, 55, 526-533). Depression can have deleterious effects on alcohol withdrawal and abstinence, increasing the likelihood of relapse (Greenfield et al., 1998, Arch. Gen. Psychiatry, 55, 259-265; Hasin et al., 2002, Arch. Gen. Psychiatry, 59, 375-380). Depression is associated with increases in glutamate in several brain regions (Lowy et al., 1995, J. Neurochem., 65, 268-274; Moghaddam et al., 1993, J. Neurochem., 60, 1650-1657.), and the brain normally compensates by increasing levels of GLT-1 (Reagan et al, 2004; Wood et al., 2004). However, under conditions of ethanol withdrawal following chronic consumption where GLT-1 levels are decreased it would be expected that glutamate-associated depression would be exacerbated. Support for this hypothesis comes from the observation that the GLT-1 enhancer ceftriaxone demonstrated efficacy in a number of animal models of depression (Mineur et al., 2007, Proc. Nat. Acad. Sci. USA, 101, 2179). Some studies have shown that standard antidepressant agents such as fluoxetine reduce the Attorney Docket No.206017-0285-00WO depressive symptoms in alcohol-dependent subjects (Ostacher, 2007, Psychiatr. Clin. North Am.2007, 30, 69). However, animal studies indicate that antidepressant treatment promotes alcohol consumption (Alen et al., 2013, Int. J. Neuropsychopharmacol.2013, 8, 1809), and clinical results indicate that superior outcomes in alcohol withdrawal are obtained when antidepressants are combined with anti-addiction drugs such as naltrexone (Pettinati et al., 2010, Am. J. Psychiatry, 167, 668) or the glutamate antagonist memantine (Muhonen et al., 2008, J. Clin. Psychiatry, 69, 392). Thus, a drug that both prevents alcohol reinstatement and treats the depression associated with alcohol consumption and withdrawal would be a significant advancement in the field. Described herein are a proprietary series of GLT-1 uptake enhancers and exemplified by MC-100093 which possess novel undescribed uses, namely, to treat ethanol withdrawal-induced anxiety and ethanol withdrawal induced depression. New uses have been discovered for compounds of Formula (I): including hydrates, salts, prodrugs and complexes thereof, wherein: A is selected from the group consisting of R is selected from the group consisting of hydrogen, C1-6 linear alkyl, C1-6 branched alkyl, optionally substituted aryl, C(O)R2, C(O)OR3, C(O)NR4a4b, SO2R5, and SO2NH2; R1a, R1b, R1c, R1d, R1e, R1f, R1g, and R1hare each independently selected from the group consisting of hydrogen, C1-6 linear alkyl, and C1-6 branched alkyl. In some examples, R1band R1gare joined together with the atoms to which they are bound to form a ring containing 5, 6 or 7 atoms. In some examples, R1b and R1f are joined together with the atoms to which they are bound to form a ring containing 5, 6 or 7 atoms. In some examples, R1d and R1f are joined together with the atoms to which they are bound to form a ring containing 5, 6 or 7 atoms; In some examples, R1band R1cjoined together with the atoms to which they are bound to form a ring containing 5 or 6 atoms; R2 is selected from the group consisting of C1-6linear alkyl, C1-6branched alkyl and optionally substituted aryl; R3 is selected from the group consisting of C1-6 linear Attorney Docket No.206017-0285-00WO alkyl, C1-6 branched alkyl, and optionally substituted aryl; R4a is selected from the group consisting of C1-6linear alkyl, C1-6branched alkyl, and optionally substituted aryl; R4b is selected from the group consisting of C1-6 linear alkyl, C1-6 branched alkyl, and optionally substituted aryl; R5is selected from the group consisting of C1-6linear alkyl, C1-6 branched alkyl, and optionally substituted aryl; R6 is selected from the group consisting of C1-6linear alkyl, C1-6branched alkyl, and optionally substituted aryl; R7a, R7b, R7c, and R7d are each independently selected from the group consisting of hydrogen, halogen, OH, C1-6linear alkyl, C1-6branched alkyl, C1-6alkoxy, C1-6haloalkyl, C1-6 haloalkoxy, cyano, NH(C1-6 alkyl), N(C1-6 alkyl)2, CHC(O)Ry, C(O)NHR8, C(O)NR8, SH, SC1-6alkyl, SO2NH2, SO2NHR8, SO2R8, and NHSO2R8; R8 is independently selected at each occurrence from a group consisting of hydrogen, C1-6linear alkyl, C1-6branched alkyl, and C3-7cycloalkyl. Also described herein are compositions comprising: An effective amount of one or more compounds according to the present invention and an excipient. The present invention further relates to a method for treating alcoholism, alcohol withdrawal, alcohol relapse and the associated fatty liver disease resulting from alcohol consumption involving administering to a subject an effective amount of a compound or composition according to the present invention. Synthesis of (3S,4R)-3-((R)-(1-hydroxy-ethyl)-4-((R)-[1-methyl-2-(4-methyl- piperazin-1-yl)-2-oxo-ethyl]-azetidin-2-one (MC-100093) Step 1: Synthesis of (3S,4R)-3-((R)-[1-(tert-butyl-dimethylsilanyloxy)- ethyl]-4-((R)-[1-methyl-2-(4-ethylpiperazin-1-yl)-2-oxo-ethyl]azetidin-2-one: To a solution of (R)-2-((2S,3S)-{3-((R)-[1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-4- oxoazetidin-2-yl}-propionic acie (10.0 g, 33.17 mmol) in dimethylformamide (200 ml) was added N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)uranium hexafluorophosphate (15.1 g, 39.8 mmol). After stirring at room temperature for 30 min, N-methyl piperazine (3.98 g, 39.8 mmol) was added and stirred for 18 h. The solvent was removed under vacuum. The oil remaining in the flask was dissolved in 100 ml ethyl acetate and extracted with saturated aqueous NaHCO3 and saturated aqueous NH4Cl. The organic layer was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated to oil under reduced pressure. The crude oil was purified by flash chromatography using MeOH / CH2Cl2 as eluent to afford (3S,4R)-3- Attorney Docket No.206017-0285-00WO ((R)-[1-(tert-butyldimethyl-silanyloxy)-ethyl]-4-((R)-[1-methyl-2-(4-ethylpiperazin-1- yl)-2-oxoethyl]azetidin-2-one as a light yellow solid. 1H-NMR (400 MHz, DMSO-D6) 8.01 (s, 1H),4.05 (m, 1H), 3.58 (dd, J=2.0 Hz, J=5.3 Hz, 1H), 3.48-3.49 (m, 4H), 2.94 (quint, J=7.0 Hz 1h), 2.72 (m, 1H), 2.28 (m, 4H), 2.17 (s, 3H), 1.03 (t, J=7.0 Hz, 6H), 0.83 (s, 9H), 0.02 (s, 3H), 0.01 (s, 3H). LC / MS; M+1=384.1. Step 2: Synthesis of (3S,4R)-3-((R)-(1-hydroxy-ethyl)_4- ((R)-[1-methyl-2-(4-methyl-piperazin-1-yl)-2-oxo-ethyl]-azetidin-2-one (MC- 100093): To a solution of (3S,4R)-3-((R)-[1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-4- ((R)-[1-methyl-2-(4-ethylpiperazin-1-yl)-2-oxo-ethyl]azetidin-2-one (3.5 g, 9.12 mmol) in 20 ml THF at 0oC, 2 mL HF / Pyridine (70%) was added and stirred for 10 minutes. The reaction was stirred at room temperature for 18 h. The reaction was cooled in an ice bath and quenched with Conc. NH4OH to pH 7. The resulting suspension was filtered and the filtrate was purified on reverse phase 80 g C18 column using H2O / ACN as eluent to afford the desire product (0.94 g, 37%) as an off white solid. 1H-NMR (400 MHz, Methanol-d4) ^ 4.01 (q, J=6.5 Hz, 1H), 3.73 (dd, J=6.8 Hz, J=2.1 Hz, 1H), 3.63 (m, 4H), 3.04 (q, J=7.1 Hz, 1H), 2.81 (dd, J=6.7 Hz, J=12.1 Hz, 1H), 2.49 (m 2H), 2.42 (t, J=5.1 Hz, 2H), 1.22 (d, J=6.4 Hz, 3H), 1.19 (d, J=6.8 Hz, 3H. LC / MS; M+1=270. Methods of Testing Ethanol drinking, withdrawal and anti-anxiety / antidepressant effects of MC-100093 Ethanol drinking was established in adult Long-Evans Rats under the intermittent access 2 bottle free choice (IA2BC) paradigm (Kang et al., 2017). Rats were divided into the following groups: Naïve with saline, Naïve with MC-100093, EtOH withdrawal-with saline and EtOH withdrawal with MC-100093. Rats in the ethanol groups drank 20% (v / v) ethanol in the IA2BC paradigm for 3 months. MC- 100093 (100 mg / kg) in saline, or the saline only (in the same volume as the MC- 100093 group) was administered by intraperitoneal injection (i.p.) once daily for 5 days prior to ethanol withdrawal. Anxiety-like behavior or depression-like behavior was then examined at 24 – 30 hours following ethanol withdrawal and 0.5 – 5 hours after the last saline or MC-100093 injection. Two-way ANOVA was used to detect any main and interactive effects of the group (naïve vs. EtOH withdrawal) and treatment (saline vs. MC-100093) on anxiety-like behaviors. Post hoc comparisons Attorney Docket No.206017-0285-00WO between the groups were performed using the Tukey test, when the main or interactive ANOVA effects were significant. The elevated plus maze is a widely used behavioral assay for rodents to assess the anti-anxiety effects of pharmacological agents as described (Kang et al., 2017, Neuropharmacol., 129, 47-; Walf and Frye, 2007, Nature Protocols, 2, 322). Briefly, the maze has four arms, two of which are open, without walls and two of which are enclosed by 30 cm walls. Animals are placed at the junction of the arms and allowed to explore the maze. The normal behavior is for animals to explore both open and closed arms. A decrease in number of entries into open arms and time spent in open arms represents an anxiety-like behavior. An increase in the number of entries into open arms and time spent in open arms following introduction of a pharmacological agent reflects anti-anxiety behavior. Groups of female Long-Evans rats were trained to consume ethanol as described above. Ethanol was withdrawn and the animals were treated with either saline of MC-100093 as described above. Animals were then placed in the elevated plus maze 0.5 – 5 hours following the last injection of saline or MC-100093 and observed for 5 minutes. Withdrawal from chronic alcohol exposure induces an increase in anxiety-like behaviors in the EPM that are decreased in animals injected with MC- 093. Female rats during alcohol withdrawal displayed a significant reduction in open arms entries compared to naïve rats. MC-093 increased open arm entries of the EPM in female rats. Female rats during alcohol withdrawal displayed a significant reduction in time spent in open arms. MC-100093 significantly increased the time spent in the open arms during alcohol withdrawal. The total distance traveled by female rats was not affected by ethanol withdrawal or treatment with MC-100093 or saline. ***p<0.001 Saline vs MC-093, ^p<0.05 vs. relative naïve rats. Two-way ANOVA. Withdrawal from chronic ethanol exposure induced an increase in anxiety- like behavior in female Long-Evans rats, expressed as a reduction in the number of entries into open arms and time spent in the open arms of the maze. Treatment with MC-100093 increased the number of entries into open arms and the time spent in open arms following ethanol withdrawal, indicating an anti-anxiety effect. No effect on number of entries into open arms or time spent in open arms was seen following treatment with saline. There was no change in the total distance traveled by the rats under any of the conditions examined. Thus, MC-100093 demonstrated efficacy against ethanol withdrawal-induced anxiety in the elevated plus maze. Attorney Docket No.206017-0285-00WO The open field test is commonly used to assess anti-anxiety behavior in rodents (Crusio 2001). The apparatus consists of four fixed walls around a plastic square floor (80 x 120 x 60 cm). Test animals are placed in the center of the open field and allowed to explore for 30 minutes. Their behavior is recorded by computerized video tracking software (SMART System, San Diego Instruments, San Diego, CA). Parameters measured were total distance traveled and distance / time spent in the center. Results were analyzed using 2-way ANOVA. A decrease in exploratory behavior, as indicated by reduced travel time and reduced time spent outside of the center of the apparatus, is reflective of anxiety-like behavior. An increase in exploratory behavior, as indicated by increased distance travel and increased time spent outside of the center of the apparatus, following introduction of a pharmacological agent represents anti-anxiety behavior. Animals were treated with either saline of MC-100093 as described above. Animals were then placed in the open field apparatus 0.5 – 5 hours following the last injection of saline or MC-100093 and observed for 30 minutes. Withdrawal from ethanol induced an increase in anxiety-like behavior in adult female Long-Evans rats, expressed as a decrease in percent of total distance traveled in the central area and percent of total time spent in the central area. Treatment with MC-100093 increased the percent of total distance traveled in the central area and percent of total time spent in the central area, indicating an anti- anxiety effect. No effect on ethanol withdrawal-induced decrease in distance traveled and time spent in the central area was seen with saline treatment. There was no change in total distance traveled by the rats under these conditions. Thus, MC-100093 demonstrated efficacy against ethanol withdrawal-induced anxiety in the open field test. MC-100093 treatment attenuates the anxiety-like behaviors in the Open Field Test in adult female Long-Evans rats withdrawn from chronic alcohol administration. The percentage of distance traveled within the central area was decreased following ethanol withdrawal and increased following treatment with MC- 100093 but not saline. The percent of total time spent in the central area was decreased following ethanol withdrawal and increased following treatment with MC- 100093 but not saline. The total distance traveled was not affected by ethanol withdrawal or treatment with MC-100093 or saline. ***p<0.001, ^p<0.05, ^^p<0.01, ^^^p<0.001. Anxiety and depression - Marble Burying Marble burying is a commonly Attorney Docket No.206017-0285-00WO used model for assessing anti-anxiety and anti-obsessive compulsive activity in rodents (Broekkamp et al., 1986. Eur. J. Pharmacol., 126, 223) and has also been used to identify antidepressant agents (Dekeyne, 2005, Therapie, 60, 477; Dourish et al., 1996; Harasawa et al., 2006, Behav. Pharmacol., 17, 637). Rodents will naturally bury foreign objects such as marbles. An increase in the number buried marbles reflects anxiety-like or depression-like behavior, and a reduction in the number of buried marbles in response to administration of a pharmacological agent represents anti- anxiety or anti-depression activity. Female Long-Evans rats withdrawn from chronic ethanol consumption, as described above, buried more marbles than naive rats, indicating an anxiety-like or depression-like behavior. Treatment of ethanol-withdrawn rats with MC-100093 reversed the increase in marble burying behavior, indicating an anti-anxiety or anti- depressant activity for the compound. Treatment with saline has no effect on ethanol withdrawal-induced increases in marble burying behavior. Thus, MC-100093 demonstrated efficacy against ethanol withdrawal-induced anxiety or depression in the marble burying test. MC-100093 attenuated the anxiety or depression behavior in rats induced by withdrawal of ethanol. Female Long-Evans rats withdrawn from chronic ethanol consumption buried significantly more marbles in 30 minutes than naïve rats. Treatment with MC-100093 reversed the increase in marble burying induced by ethanol withdrawal. ***p<0.001, &p<0.05. Benzodiazepines are the drug class of choice for treating ethanol withdrawal-induced anxiety. However, benzodiazepines suffer from a host of side effects which include sedation, dizziness, confusion, muscle weakness, memory problems, slurred speech, constipation, dry mouth and blurred vision. In addition, patients taking benzodiazepines can become dependent on and addicted to them. Symptoms of benzodiazepine withdrawal include sleep disturbances, irritability, rebound anxiety, panic attacks, muscle tremors, headache, heart palpitations, muscular pain, stiffness seizures and a variety of perceptual changes. Other non-benzodiazepine anticonvulsants and muscle relaxants such as baclofen, gabapentin, topiramate, carbamazepine and valproic acid have also been used to treat ethanol withdrawal- induced anxiety but demonstrate only limited clinical efficacy. Ceftriaxone is a clinically approved antibiotic but is unsuitable for sub-chronic use in treating alcohol withdrawal-induced anxiety due to the fact that it is given intravenously, displays Attorney Docket No.206017-0285-00WO poor brain penetration, requires extremely high doses to affect GLT-1 levels and carries the risk of the development of antibiotic resistance. Alcohol withdrawal- induced depression is usually treated with standard antidepressant agents such as SSRIs, mixed reuptake inhibitors (venlafaxine, milnacipran, etc.) or tricyclic antidepressants. These drugs possess a plethora of side effects such as sexual dysfunction, sleep disturbances, dry mouth, loss of appetite, agitation, dizziness and headache. Patients taking these drugs can also experience withdrawal symptoms upon discontinuation. Additionally, animal data suggest that SSRIs may promote ethanol consumption, which would complicate attempts to discontinue drinking. There is no drug currently approved for specifically treating ethanol withdrawal-induced depression. Example 4: Effects of novel GLT-1 modulator, MC-100093, on neuroinflammatory and neurotrophic biomarkers in mesocorticolimbic brain regions of male alcohol preferring rats exposed chronically to ethanol This example is related to the role of MC-100093 in attenuating the chronic ethanol exposure in inducing reduction on the expression of major glutamate transporters in the brain. MC-100093 prevents ethanol-induced increases in neuroinflammation and ethanol-induced decrease in trophic factor in the brain. MC- 100093 is a unique drug, which can reduce excess of glutamate as well as reducing inflammation and increasing trophic factor in animal model of chronic exposure to alcohol. MC-100093 will be a therapeutic drug to help reduce the side effects of chronic exposure to alcohol as well can help reduce craving to alcohol. Chronic ethanol consumption can lead to increased extracellular glutamate concentrations in key reward brain regions, such as medial prefrontal cortex (mPFC) and nucleus accumbens (NAc), and consequently leading to oxidative stress and neuroinflammation. Previous studies tested β-lactam antibiotics and novel beta- lactam non-antibiotic, MC-100093, and showed these β-lactam upregulated the major astrocytic glutamate transporter, GLT-1, and consequently reduced ethanol intake and normalized glutamate homeostasis. This present study tested the effects of novel synthetic β-lactam non-antibiotic drug, MC-100093, in chronic ethanol intake and neuroinflammatory and trophic factors in subregions of the NAc (NAc core and shell) and mPFC (Prelimbic, PL; and Infralimbic, IL) of male P rats. MC-100093 treatment reduced ethanol intake after 5-week drinking regimen. Importantly, MC-100093 Attorney Docket No.206017-0285-00WO attenuated ethanol-induced downregulation of brain derived neurotrophic factor (BDNF) expression in these brain regions. In addition, MC-100093 attenuated ethanol-induced upregulation of pro-inflammatory cytokines such as TNF-a and HMGB1 in all these brain regions. Furthermore, MC-100093 treatment attenuated ethanol-induced increase in RAGE in these brain regions. MC-100093 prevented neuroinflammation caused by ethanol intake as well as increased neurotrophic factor in mesocorticolimbic brain regions. MC-100093 treatment reduced ethanol intake and this behavioral effect was associated with attenuation of reduced trophic factors and increased pro-inflammatory factors. MC-100093 is considered a small molecule that may have potential therapeutic effects for the treatment of the effects of chronic exposure to ethanol. Chronic alcohol consumption alters several neurotransmitters, including glutamate. This may cause an increase in extracellular glutamate concentrations in key reward brain regions, including the dorsomedial prefrontal cortex (dmPFC) and nucleus accumbens (NAc), which can lead to oxidative stress and neuroinflammation. Chronic ethanol intake induced inflammation and consequently apoptosis of neurons and glial cells (Vall´es et al., 2004, Brain Pathol. 14, 365–371). The NAc has been found to be a mediator of the reward system in animal models and contributes to the mechanism of ethanol drinking (Alasmari et al., 2020, Biomolecules 10, 1030; Neasta et al., 2011, Biol. Psychiatry 70, 575). Studies performed previously have determined that beta-lactam antibiotics such as ceftriaxone and others are beneficial in decreasing ethanol consumption in alcohol preferring (P) rats (Goodwani et al., 2015, Brain Res 1622, 397; Rao et al., 2015, Neuroscience 295, 164; Sari et al., 2011, Alcohol, 46, 239). Among beta-lactams, ceftriaxone has been one of the drugs to reduce ethanol intake and this effect was associated with normalization and upregulation of GLT-1. Several studies from others and ours revealed the efficacy of beta-lactams, including ceftriaxone, in attenuating drug seeking and the effects of drugs of abuse such as cocaine, methamphetamine, opioids, and nicotine. These effects are associated with normalization and upregulation of GLT-1 (Abulseoud et al., 2012, Brain Res.1456, 14; Alasmari et al., 2018, Pharmacol. Biochem. Behav.170, 44; Alshehri et al., 2018, Behav. Brain Res, 347, 368; Althobaiti et al., 2019, J. Mol. Neurosci.67, 1; Knackstedt et al., 2010, Biol. Psychiatry 67, 81; Sari et al., 2009, J. Neurosci.29, 9239). Attorney Docket No.206017-0285-00WO Recent study tested a novel synthetic beta-lactam, MC-100093, which does not have any antibiotic action, and the drug was found to reduce ethanol intake (Alhaddad et al., 2022, J. Pharm. Exp. Ther.383, 208). The decrease in ethanol intake was found to be associated with the normalization of GLT-1 in the NAc. It is important to note that the pharmacokinetics of MC-100093 have been studied using in vitro and in vivo paradigms as reported in previous study (Knackstedt et al., 2021, J. Pharmacol. Exp. Ther.378, 51). This latter study showed that MC-100093 has high aqueous solubility as well as displayed a high stability in rodent and human liver microsomes in vitro. The drug has low binding to plasma protein and low partitioning in lipid membranes. In vivo pharmacokinetic studies found that MC-100093 displayed some bioavailability following oral administration. There is no oxidative metabolism, which points to the fact that the bioavailability following intraperitoneal administration is similar to the bioavailability of oral dosing. In vivo half-life was also found to be 4.2 hours via the intraperitoneal route and 5.26 hours via the oral route. The present example investigated, in part, whether MC-100093 would attenuate ethanol-induced increases in neuroinflammatory factors such as tumor necrosis factor (TNF-α), receptor for advanced glycation end products (RAGE) and high mobility group box-1 (HMGβ-1), and alteration of neurotrophic factors such as brain derived neurotrophic factor (BDNF). This study focused on, in part, investigating potential changes in the expression of BDNF in the subregions of the NAc (NAc core and shell) and mPFC (Prelimbic, PL; and Infralimbic, IL) as there is association between BDNF signaling and glutamate release (Cutuli and Sampedro- Piquero, 2022, Curr. Neuropharmacol., 20, 2202). BDNF is a trophic factor involved in neuronal survival and growth, and substances of abuse can alter its expression. The NAc shell mediates ethanol extinction and is involved in signaling satiety (Barker et al., 2015, Brain Res., 1628, 68). BDNF overexpression in the NAc has been found to reduce withdrawal symptoms and craving behavior in a rat model (Xu et al., 2020, Front. Neural Circuits 14, 37). Next, the present example focused on investigating HMGβ-1 as inflammatory marker, which stimulates several receptors, and RAGE is one of these target receptors (Paudel et al., 2018, Front Neurosci.12, 628). HMGβ-1 is a pro-inflammatory-like cytokine that is released due to increased oxidative stress. It has been observed to be overexpressed in several neuroinflammatory conditions, such as Alzheimer’s disease and Parkinson’s disease (Paudel et al., 2020, Int. J. Mol. Sci.21, 4609). RAGE is a multi-ligand receptor that binds to several different ligands, Attorney Docket No.206017-0285-00WO including HMGβ-1. Interaction between RAGE and its ligands activates several cellular processes, including neuroinflammation (Lee and Park, 2013, Genom. Inform. 11, 224). The tumor necrosis superfamily of cytokines plays a role in many chronic inflammatory and neurodegenerative conditions, and TNF-α is a key mediator of neuroinflammation (Jayaraman et al., 2021, Acta Neuropathol. Commun.9, 1). Ethanol-induced neuroinflammation is mediated by pro-inflammatory cytokines such as TNF-α (Lippai et al., 2013, PloS One 8, e70945). HMGβ-1 and TNF-α have been suggested to be involved in glutamate neurotoxicity (Crews and Vetreno, 2014, Int Rev. Neurobiol.118, 315; Zou and Crews, 2005, Brain Res 1034, 11). Thus, this study focused on these signaling inflammatory pathways in association with BDNF as trophic factor in the subregions of the NAc (NAc core and shell) and mPFC (Prelimbic, PL; and Infralimbic, IL) of male P rats exposed to free choice of water and ethanol (15% and 30%, v / v) for 6 weeks and treated with either vehicle saline or MC-100093. Materials and methods Animal Model Male P rats were obtained from Indiana University School of Medicine (Indianapolis, IN, USA). P rats were ~90 days old at the beginning of the drinking paradigms. P rats were used in this study as they fit the necessary criteria of ethanol preference. P rats may consume freely ethanol (15% and 30%, v / v) and achieve a pharmacologically blood alcohol concentration (BACs) of 50–200 mg% as shown in previous studies from Indiana University (Bell et al., 2006, Addict. Biol.11, 270; Li et al., 1987, Alcohol Alcohol Suppl.1, 91; McBride et al., 2014, PloS One 8, e53793; Murphy et al., 1986, Alcohol 3, 331). Rats were housed in a room kept at 21◦C on a 12 / 12-hour light / dark cycle and had free access to water and food. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Toledo under protocol #400160, in accordance with all guidelines of IACUC of the National Institute of Health, and the Guide for the Care and Use of Laboratory Animals. Ethanol-intake model Rats were assigned to three groups prior to the start of the experiments based on their weights, and the average weights of each group were similar between Attorney Docket No.206017-0285-00WO all groups. Male P rats were divided into three groups: a) Water / saline control group was exposed to water for five weeks and received saline vehicle injection (i.p.) every 24 hours during Week 6 for a total of five days; b) Ethanol / saline group was exposed to free choice of ethanol (15% and 30%,v / v) as well as water for 5 weeks and received saline vehicle (i.p.) every 24 hours during Week 6 for a total of five days; and c) Ethanol / MC-100093 group was exposed to free choice of ethanol (15% and 30%, v / v) as well as water for 5 weeks and received MC-100093 (100 mg / kg, i.p.) every 24 hours during Week 6 for 5 days. The positioning of the three bottles were randomly interchanged at the beginning of each week to avoid preference amongst the animals. Measurements of ethanol intake occurred by taking the weight in grams. After Week 3, measurements of ethanol intake occurred every other day through Week 4. Ethanol / saline (n=5 per group) and ethanol / MC-100093 (n=6 per group) groups were exposed to free choice water, 15% ethanol and 30% ethanol (v / v) for 24 hours over 5 weeks. The baseline was calculated by taking the average of the prior 5 weeks of water and ethanol consumption as rat weight. The water / saline control group and the Ethanol / saline groups received saline for 5 days starting on Day 1 to Day 5 on week 6. Ethanol / MC- 100093 group received an equivolume i.p. injection of MC- 100093 (100 mg / kg) for 5 days starting on Day 1 to Day 5 of Week 6. As weights of the bottles were recorded, the measurements were converted using g of ethanol intake / kg of body weight / day formula to determine ethanol consumption in g / kg of body weight. Rats with an average ethanol intake less than 4 g / kg / day were excluded from the study as this measurement did not follow the criteria for the development of ethanol dependence (Sari and Sreemantula, 2012, Neuroscience 227, 327). Rats were then euthanized by CO2 inhalation followed by decapitation on Day 6 of Week 6. MC-100093 was synthesized and characterized at Temple University, Moulder Center for Drug Discovery Research, Philadelphia, PA (Fig, 22, Fig.23). Brain tissue extraction All P rats were euthanized by CO2 inhalation and decapitated with a guillotine 24 hours after the final i.p. injection was administered on Day 6 of Week 6. Brains were removed and immediately frozen on dry ice and stored at -80◦C. The mPFC subregions (infralimbic cortex and prelimbic cortex) and NAc subregions (core and shell) were dissected using a cryostat machine kept at -20◦C. Surgical blades were used to dissect all brain regions following visualized landmarks using the stereotaxic Attorney Docket No.206017-0285-00WO coordinates provided by Paxinos and colleague’s rat brain stereotactic atlas (Paxinos et al., 2021). All brain regions were stored in -80◦C for Western blot analyses. Western Blot procedure Western blot was performed to determine the expression of BDNF, TNF-α, RAGE, HMGβ-1, and β-tubulin in the subregions of mPFC and NAc as described in previous studies. Brain samples were lysed with a lysing buffer containing protease inhibitors. The amount of protein in each sample was quantified using a detergent compatible protein assay (Bio-Rad, Hercules, CA, USA). Polyacrylamide gels (10%) were loaded with the lysate mixture, and proteins were separated using gel electrophoresis for 1 hour. Proteins were then transferred from the gels onto Polyvinylidene difluoride (PVDF) membranes (BioRad). After transfer, membranes were incubated for 30 minutes in a mixture of nonfat milk in 1x Tris- buffered saline with Tween-20 (TBST) at room temperature. Membranes were then incubated at 4◦C for 24 hours with primary antibodies: anti-rabbit BDNF (1:1000, Abcam, #ab108319), anti-rabbit TNF-α (1:500, Abcam, #ab9739), anti-rabbit HMGβ- 1 (1:500, Abcam, #ab18256), and anti-rabbit RAGE (1:1000, Abcam, #ab37407). Anti-mouse-β-tubulin (1:1000, Cell Signaling Technology, #D71G9) was used as a loading control antibody. After incubating for 24 hours, secondary antibody such as donkey anti-rabbit (1:4000, Invitrogen, #31458) or anti-mouse (1:4000, Cell Signaling, #7076 S) was added to the membrane for 1 hour at room temperature. The membranes were then washed with 1x TBST three times for 5 minutes each and then dried. Membranes were then incubated in chemiluminescent reagents (Super Signal West Pico, Pierce Inc.) for 2 minutes. Blot images were developed using ChemiDoc. Imaging System and ImageJ software version 1.53a were used to quantify and analyze the expression of BDNF, TNF-α, RAGE, HMGβ-1, and β-tubulin. The data for the water / saline control group were expressed as 100% based on the values corresponding to the ethanol / saline and ethanol / MC-100093 groups for each set of blotting. Thus, the expression of selected proteins for each group (ethanol / saline group or ethanol / MC- 100093 group) was calculated relative to the water / saline control group being set to 100% as was previously done (Devoto et al., 2013, Neuroscience 252, 190; Koehler et al., 2019, Neurochem. Int.122, 31; Li et al., 2003, Neuroscience 118, 45; Logan et al., 2020, Psychopharmacology 237, 2007). Attorney Docket No.206017-0285-00WO Statistical analysis Statistical analyses were performed using GraphPad Prism Version 10. Two-way repeated measures ANOVA followed by Bonferroni post-hoc tests were used to compare water and ethanol intake, and body weight, amongst water group, ethanol / saline group and ethanol / MC- 100093 group. A one-way ANOVA test was performed to compare the expression of the studied proteins in selected brain regions in water / saline, ethanol / saline and ethanol / MC-100093 groups. Statistical significance was determined by a p-value of or less than 0.05. Results Effects of MC-100093 on ethanol and water consumption as well as body weight in P rats Two-way repeated measures ANOVA analysis revealed a significant main effect of Day on ethanol intake, [F (1,61) = 68.81, p<0.0001] and a significant Treatment by Day interaction, [F 5,61) = 3.312. p<0.0104] (Fig.24A). Bonferroni post-hoc test revealed a significant decrease in ethanol intake on days 1 and 2 in rats treated with MC-100093 as compared to rats treated with saline vehicle (p<0.01). There was also a significant decrease in ethanol consumption in rats treated with MC- 100093 as compared to rats treated with saline vehicle (p<0.001) on days 3 and 4, and a significance decrease in ethanol intake on day 5 in rats treated with MC-100093 as compared to rats treated with saline vehicle (p<0.01). Two-way repeated measures ANOVA analysis revealed no significant main effect of Day on water intake [F(5,83) = 0.3422, p=0.6549] as well as no significant Treatment Dose by Day interaction on water intake [F (10,83) = 0.7722, p=0.8858] (Fig.24B). Bonferroni post-hoc test revealed a significant decrease in water intake on day 4 (p<0.05) and day 5 (p<0.01) in rats treated with saline vehicle as compared to control group exposed to water only and treated with saline vehicle. There was a significant increase in water intake in rats treated with MC-100093 and exposed to ethanol on day 5 (p<0.01) (Fig.24B). Two- way repeated measures ANOVA followed by Bonferroni post-hoc tests did not reveal any main effect of Day on average body weight [F(5,96) = 0.2161, p=0.9549], and there was no significant difference found between the Day and average body weight (Fig.24C). There was no significant effect of Treatment by Day interaction on body weight [F (10,96) = 0.1016, p=0.9998]. Attorney Docket No.206017-0285-00WO Effects of MC-100093 and chronic ethanol consumption on BDNF expression in the subregions of mPFC and NAc One-way ANOVA revealed a significant difference in BDNF expression in the subregion of mPFC, IL, among all tested groups [F(2,11) = 57.01, p<0.0001]. Newman-Keuls test showed a significant increase (40%) in BDNF expression in the ethanol / MC-100093 group as compared to water / saline and ethanol / saline groups (p<0.0001) as well as a significant decrease (13%) in BDNF expression in the ethanol / saline group as compared to water / saline group (p<0.05) (Fig.25A). In the subregion of mPFC, PL, there was a significant difference in BDNF expression among all tested groups [F(2,10) =41.96, p<0.0001]. Newman-Keuls test showed a significant increase (27%) in BNDF expression in the ethanol / MC-100093 group as compared to the water / saline group (p<0.001) and the ethanol / saline group (p<0.0001) (Fig.25B). There was a significant decrease (18%) in BDNF expression in the PL in the ethanol / saline group as compared to the water / saline group (p<0.01) (Fig.25B). Regarding the subregions of the NAc, there was significance difference in the expression of BDNF in the NAc core among all tested groups [F(2,9) = 55.86, p<0.0001]. Newman-Keuls test showed a significant increase (11%) in BDNF expression in the NAc core of rats treated with MC-1000093 as compared to the water / saline group (p<0.01) and ethanol / saline group (p<0.0001), as well as a significant decrease (21%) in BDNF expression in the ethanol / saline group compared to the water / saline group (Fig.25C). There was also a significant decrease in BDNF expression in the ethanol / saline group as compared to the water / saline group (p<0.0001). Furthermore, there was also significance difference in the expression of BDNF in NAc shell among all tested groups [F(2,10) = 11.78, p<0.05]. Newman- Keuls test showed a significant increase in BDNF expression in the ethanol / MC- 100093 group as compared to the ethanol / saline group (p<0.01) (Fig.25D). There was also a significant decrease (18%) in BDNF expression in the NAc shell in the ethanol / saline group as compared to the water / saline group (p<0.01) (Fig.25D). Effects of MC-100093 and chronic ethanol consumption on TNF-α expression in the subregions of mPFC and NAc One-way ANOVA revealed a significant difference in TNF-α expression in the mPFC subregion, IL, among all tested groups [F(2,9) = 8.732, p<0.01]. Newman-Keuls test showed a significant increase (21%) in TNF-α Attorney Docket No.206017-0285-00WO expression in the IL in the ethanol / saline group as compared to the water / saline group (p<0.05), and MC-100093 treatment decreased significantly TNF-α expression in the IL as compared to ethanol / saline group (p<0.01) (Fig.26A). Regarding the mPFC subregion, PL, there was also a significant difference in the TNF-α expression among all groups [F (2,10) = 36.26, p<0.0001]. Newman-Keuls test revealed a significant increase (28%) in TNF-α expression in the PL in the ethanol / saline group as compared to the water / saline group (p<0.0001) (Fig.26B), and MC- 100093 treatment significantly decreased the expression of TNF-α as compared to the ethanol / saline group (p<0.0001) (Fig.26B). Regarding the subregions of the NAc, one-way ANOVA revealed a significant difference in the expression of TNF-α in the NAc core among all groups [F (2,11) = 13.62, p<0.01]. Newman-Keuls test showed a significant increase (20%) in the TNF-α expression in the NAc core in the ethanol / saline group as compared to the water / saline group (p<0.01) (Fig.26C). MC-100093 treatment decreased significantly TNF-α expression in the NAc core as compared to the ethanol / saline group (p<0.001). In the NAc shell, there was a significant difference in the TNF-α expression among all groups [F(2,11) =11.88, p<0.01]. Newman-Keuls test showed a significant increase (23%) in the TNF-α expression in the NAc shell in the ethanol / saline group as compared to the water / saline group (p<0.01) (Fig.26D). Similarly, MC-100093 treatment decreased TNF-α expression in the NAc shell as compared to the ethanol / saline group (p<0.01) (Fig.26D). Effects of MC-100093 and chronic ethanol consumption on RAGE expression in the subregions of mPFC and NAc In the IL subregion of mPFC, there was a significant difference in the expression of RAGE among all groups [F(2,9) = 5.558, p<0.05]. Newman-Keuls test revealed that MC-100093 treatment decreased (14%) the expression of RAGE in the IL as compared to the ethanol / saline group (p<0.05) (Fig.27A). In the PL, one-way ANOVA revealed significant difference among all groups [F(2,9) = 71.40, p<0.0001]. Newman-Keuls test revealed a significant increase (30%) in the RAGE expression in the PL in the ethanol / saline group as compared to the water / saline group (p<0.0001) (Fig.27B). MC-100093 treatment decreased RAGE expression in the PL as compared to the ethanol / saline group (p<0.0001) (Fig.27B). In the NAc core, there was a significant difference among all groups [F(2,11) = 9.679, p<0.01]. Neuman-Keuls test revealed a significant increase (14%) in the RAGE expression in the NAc core in the Attorney Docket No.206017-0285-00WO ethanol / saline group as compared to the water / saline group (p<0.05) (Fig.27C). MC- 100093 treatment decreased (12%) RAGE expression as compared to the ethanol / saline group (p<0.01) (Fig.27C). In the NAc shell, there was a significant difference among all groups [F (2,9) =13.90, p<0.01)]. Newman-Keuls test revealed a significant increase (30%) in RAGE expression in the NAc shell in the ethanol / saline group as compared to the water / saline group (p<0.01) (Fig.27D). MC- 100093 treatment decreased RAGE expression as compared to the ethanol / saline group (p<0.01) (Fig.27D). Effects of MC-100093 and chronic ethanol consumption on HMGβ-1 expression in the subregions of mPFC and NAc One-way ANOVA analysis revealed a significant difference in the HMGβ-1 expression in the mPFC subregion, IL, among all groups [F(2,8) =18.48, p<0.01)]. There was a significant increase (35%) in HMGβ-1 expression in the ethanol / saline group as compared to the water / saline group (p<0.01) (Fig.28A). MC- 100093 treatment decreased HMGβ-1 expression in the IL as compared to the ethanol / saline group (p<0.01) (Fig.28A). Furthermore, there was a significant difference in the expression of HMGβ-1 in PL among all groups [F (2,9) = 9.358, p<0.01]. There was a significant increase (27%) in HMGβ-1 expression in the ethanol / saline group as compared to the water / saline group (p<0.01) (Fig.28B). MC- 100093 treatment decreased the expression of HMGβ-1 in the PL as compared to the ethanol / saline group (p<0.01) (Fig.28B). Regarding the subregions of the NAc, there was a significant difference in the expression of HMGβ-1 in the NAc core among all groups [F (2,8) = 8.929, p<0.01)]. Neuman-Keuls test showed a significant increase (15%) in the expression of HMGβ-1 in the NAc core in the ethanol / saline group as compared to the water / saline group (p<0.05) (Fig.28C). MC- 100093 treatment decreased the expression of HMGβ-1 in the NAc core as compared to the ethanol / saline group (p<0.01) (Fig.28C). One-way ANOVA revealed a significant difference in the expression of HMGβ-1 in the NAc Shell among all groups [F(2,9) = 11.79, p<0.01]. Neuman-Keuls tested revealed a significant increase (38%) in the expression of HMGβ-1 in the NAc shell as compared to the water / saline group (p<0.01) (Fig.28D). MC-100093 treatment decreased the expression of HMGβ-1 in the NAc shell as compared to the ethanol group (p<0.01) (Fig.28D). Attorney Docket No.206017-0285-00WO Discussion This study revealed that treatment with novel beta-lactam, MC- 100093, attenuated the effects of ethanol-induced alterations in several neuroinflammatory and trophic factors in brains of male P rats. This study tested male P rats to determine the effects of the novel synthetic small molecule beta-lactam, MC- 100093, in ethanol intake and neuroinflammatory and neurotrophic biomarkers. This study was focused on identifying MC-100093 as potential drug target for potential therapeutic effects in ethanol dependence animal model. Further studies are ongoing for testing the effects of MC-100093 in female P rats to determine whether this drug has an effect in ethanol intake and neuroinflammatory and trophic factors. In the present study, using male P rats, it was found that MC-100093 treatment reduced ethanol intake in P rats staring 24 hours after the first i.p. injection of the drug through the five days regimen. In addition, MC-100093 showed an effect on water consumption. There was a trend of an increase in water consumption throughout the five days of treatment. On the fifth day, the ethanol / MC-100093 group drank significantly more water than the ethanol / saline group. It is important to note that MC-100093 at a higher dose (100 mg / kg, i.p.) was more effective as compared to the dose of MC-100093 (50 mg / kg, i.p.) tested in recent studies. Thus, MC-100093 (100 mg / kg, i.p.) showed a robust effect in ethanol intake in this present study. Chronic ethanol consumption for six weeks decreased the expression of BDNF in the subregions of mPFC (IL and PL) and NAc (core and shell). In addition, chronic exposure to ethanol induced upregulation of TNF-α, RAGE and HMGβ1 in the IL, NAc core, and NAc shell. Importantly, MC- 100093 treatment during Week 6 reduced ethanol intake and this effect was associated with attenuation of ethanol-induced decrease in BDNF expression and increase in TNF-α, RAGE and HMGβ1 expression. MC- 100093 reduced ethanol intake in P rats at higher dose (100 mg / kg), and this in accordance with a previous study that showed similar effect on ethanol intake. This latter study showed also that reduction in ethanol intake with MC-100093 was associated with upregulation of GLT-1 and xCT in the NAc. In this present study, MC-100093 treatment attenuated ethanol-induced downregulation of BDNF expression in the subregions of NAc and mPFC. It is important to note that BDNF is critical in modulating vesicular glutamate transporters and glutamate itself may alter BDNF expression, which suggest the interactive role between glutamate and BDNF in the regulation of synaptic transmission (Kojima et al., 2002, Neurosci. Res 43, 193; Attorney Docket No.206017-0285-00WO Martin and Finsterwald, 2011, Commun. Integr. Biol.4, 14; Mattson, 2008, Ann. N. Y Acad. Sci.1144, 97; Melo et al., 2013, PloS One 8, e53793). MC-100093 is known to attenuate ethanol-induced downregulation of GLT-1 expression in central reward brain region such as NAc, thus, normalizing glutamate homeostasis is an effect that is suggested to be associated with normalization of BDNF expression. It is important to note that the effect of ethanol exposure on the expression of BDNF depends on the ethanol exposure regimen, timing, age of animals and the targeted brain regions. For example, acute ethanol exposure increased BDNF expression; however, withdrawal after long term exposure to ethanol induced downregulation of BDNF in the central amygdala and medial amygdala (Pandey et al., 2008, J. Neurosci.28, 2589). This is in accordance with the present finding and others suggesting the downregulatory effect of BDNF with exposure to ethanol (Martín-Gonzalez et al., 2022). However, studies from previous works showed that chronic ethanol intake increased BDNF expression only in the NAc shell. This differential effect of ethanol exposure in BDNF expression in this brain region is probably due to the fact that P rats consumed a higher amount of ethanol (~10 g / kg / 24 hr) during the five days of i.p. injection of MC-100093 performed on Week 6 of either saline vehicle or ceftriaxone as compared to the present study where rats drank less than 6 g / kg / 24 hr over the five days i.p. injection of saline vehicle or MC-100093 on Week 6. The effect of high consumption of ethanol may lead to a neuroadaptive mechanism involving BDNF expression. In addition, acute exposure to ethanol increased BDNF expression in the dorsal striatum; however, exposure to ethanol for six weeks decreased BDNF expression in the cortex of mice (Logrip et al., 2009, J. Neurochem 109, 1459) an effect similar to the present finding. It is important to note that BDNF polymorphism might be associated with increase in ethanol consumption and development of ethanol dependence in mice (Warnault et al., 2016, Biol. Psychiatry 79, 463). Additionally, study demonstrated that microRNA-induced reduction of BDNF expression in the mPFC was associated with escalated ethanol intake in Wistar rats (Tapocik et al., 2014, J. Neurosci.34, 4581). Further studies are warranted to investigate the amount consumed of ethanol and duration of ethanol exposure on the expression of BDNF in several reward brain regions. This study further investigated the effects of chronic exposure to ethanol and MC-100093, a beta-lactam known to upregulate GLT-1, in neuroinflammatory factor such as TNF-α. This study revealed that chronic ethanol exposure increased TNF-α expression in the subregions of mPFC and NAc. This is in accordance with previous Attorney Docket No.206017-0285-00WO studies that demonstrated that chronic exposure to ethanol for six weeks increased the expression of TNF-α in the NAc shell of male high alcohol drinking (HAD1) rats. Additionally, binge ethanol consumption for only ten days followed by one day withdrawal period increased TNF-α in the brain (Qin and Crews, 2012, J. Neuroinflamm.9, 130). Other studies revealed that chronic ethanol exposure increased TNF-α expression in the cerebral cortex of both mouse genders (Alfonso- Loeches et al., 2013, Toxicology 311, 27). Importantly, from success in attenuating the effect of chronic ethanol-induced increase on TNF-α expression in the NAc of HAD1 with beta-lactams ampicillin / sulbactam, known to upregulate GLT-1, the novel drug, MC-100093, known to upregulate GLT-1 expression in the brain was tested. Indeed, MC-100093 was effective in normalizing TNF-α expression against the effect of chronic ethanol consumption, and this is in accordance with a previous study using beta-lactam antibiotics, ampicillin / sulbactam. Furthermore, this present study revealed that chronic ethanol exposure increased the expression of HMGβ-1 and RAGE in the subregions of mPFC and NAc. It is important to note that HMGβ-1 can activate RAGE, which in turn activates the NF-kB signaling pathway to increase TNF-α (You et al., 2013, PLoS One 8, e60290). In postmortem human brains from AUD patients, RAGE was found to be increased (Orio et al., 2019, Front. Mol. Neurosci.490). In addition, studies demonstrated that chronic ethanol consumption resulted in an increase of NF-kB / pathway, which was associated with increase in several cytokines in the rat brain, including HMGβ-1 (Crews et al., 2008, Biol. Psychiatry). Furthermore, binge ethanol drinking for ten days increased HMGβ-1 expression in the brain of mice. Other studies reported that chronic ethanol exposure for five weeks increased HMGβ-1 as well as its receptor, RAGE, in the cerebellum (Lippai et al., 2013, PloS One 8, e70945). Additionally, previous studies demonstrated that chronic ethanol intake for six weeks increased both HMGβ-1 and RAGE in the NAc of HAD1 rats. Similar to TNF-α, this latter study revealed that beta-lactams, ampicillin / sulbactam known to upregulate GLT-1, attenuated the effect of ethanol- induced increase in both HMGβ-1 and RAGE in the NAc of HAD1 rats. This is in accordance with the present study demonstrating that MC-100093, GLT-1 upregulator, attenuated ethanol-induced increase in both HMGβ-1 and RAGE in the subregions of mPFC and NAc of P rats. It is important to note that increase in the expression of HMGβ-1 is critical in the glutamate neurotoxicity as suggested by other studies. Thus, ethanol-induced downregulation of GLT-1 is associated with increased Attorney Docket No.206017-0285-00WO extracellular glutamate concentration, which might be associated with increase in HMGβ-1 expression in target brain regions of the reward circuit. Thus, modulating GLT-1 expression with MC-100093 can lead to regulation of glutamate homeostasis and consequently attenuation of ethanol-induced neuroinflammation. Conclusion MC-100093 treatment attenuated chronic ethanol consumption in male P rats. Furthermore, chronic ethanol consumption reduced BDNF expression in the subregions of the mPFC and NAc, increased pro-inflammatory cytokines such as TNF-α and HMGB1. In addition, chronic ethanol intake increased the expression of RAGE as a mediator protein of inflammatory response. The attenuating effects of MC-100093 in the expression of BDNF and pro-inflammatory cytokines and mediator protein of inflammation might be associated with normalization of glutamate homeostasis as the drug is known to normalize the expression of astrocytic glutamate transporters such as GLT-1. This study presents a novel beta-lactam, MC-100093, that has the potential to attenuate ethanol intake in chronic ethanol regimen, and this attenuating effect of ethanol consumption was associated with attenuation of both increase in neuroinflammatory factors and decrease in trophic factors. This suggests that MC-100093 has the potential to prevent the effects of chronic ethanol consumption involving inflammation and oxidative stress. Example 5: Effects of Hydrocodone Overdose and Ceftriaxone on Astrocytic Glutamate Transporters and Glutamate Receptors, and Associated Signaling in Nucleus Accumbens as well as Locomotor Activity in C57 / BL Mice Chronic opioid treatments dysregulate the glutamatergic system, inducing a hyperglutamatergic state in mesocorticolimbic brain regions. This study investigated the effects of exposure to hydrocodone overdose on locomotor activity, expression of target proteins related to the glutamatergic system, signaling kinases, and neuroinflammatory factors in the nucleus accumbens. The locomotor activity of mice was measured using the Comprehensive Laboratory Animal Monitoring System (CLAMS). CLAMS data showed that exposure to hydrocodone overdose increased locomotion activity in mice. This study tested ceftriaxone, known to upregulate major glutamate transporter 1 (GLT-1), in mice exposed to an overdose of hydrocodone. Thus, ceftriaxone normalized hydrocodone-induced hyperlocomotion activity in mice. Attorney Docket No.206017-0285-00WO Furthermore, exposure to hydrocodone overdose downregulated GLT-1, cystine / glutamate antiporter (xCT), and extracellular signal-regulated kinase activity (p-ERK / ERK) expression in the nucleus accumbens. However, exposure to an overdose of hydrocodone increased metabotropic glutamate receptor 5 (mGluR5), neuronal nitric oxide synthase activity (p-nNOS / nNOS), and receptor for advanced glycation end products (RAGE) expression in the nucleus accumbens. Importantly, ceftriaxone treatment attenuated hydrocodone-induced upregulation of mGluR5, p- nNOS / nNOS, and RAGE, as well as hydrocodone-induced downregulation of GLT-1, xCT, and p-ERK / ERK expression. These data demonstrated that exposure to hydrocodone overdose can cause dysregulation of the glutamatergic system, neuroinflammation, hyperlocomotion activity, and the potential therapeutic role of ceftriaxone in attenuating these effects. Chronic treatment with opioids alters several neurotransmitters, particularly glutamate, in the central and peripheral systems. Many opioids are used for the management of pain. Among these opioids, hydrocodone is a semisynthetic and potent opioid agonist with a high abuse potential similar to fentanyl and morphine. Along with other opioids, hydrocodone is among the most prescribed opioids in the United States to treat moderate to severe pain (Cicero et al., 2013, PAIN, 154, 2639). The current study focused on, in part, investigating the effects of exposure to hydrocodone overdose on locomotor activity and the expression of target glutamate transporters and signaling in one of the brain regions of the reward circuit, such as the nucleus accumbens (NAc). Repeated exposure to drugs of abuse is known to increase motor activity, leading to behavioral hypersensitivity. For example, cocaine, amphetamines, morphine, ethanol, and nicotine have been reported to cause behavioral hypersensitivity (Fish et al., 2002, Psychopharmacology, 160, 39; Hirabayashi, 1981, Pharmacol. Biochem. Behav., 15, 925; Kalivas, 1991, Brain Res. Rev., 16, 223; Kita et al., 1992, Jpn. J. Psychopharmacol., 12, 17). Behavioral hypersensitivity induced by drugs of abuse relies on neural sensitivity and neuroplasticity within the reward circuit of the brain (Steketee, 2011, Pharmacol. Rev., 63, 348). Other studies have shown that exposure to opioids, including hydrocodone, induces locomotor sensitization (Emery et al., 2015, Behav. Brain Res., 284, 37). Although a previous study showed that ceftriaxone, a beta- lactam antibiotic known to upregulate the major glutamate transporter GLT-1, attenuated reinstatement to hydrocodone using conditioned place preference (Alshehri Attorney Docket No.206017-0285-00WO et al., 2018, Behav. Brain Res., 347, 368), the effect of ceftriaxone in attenuating hydrocodone-induced locomotor sensitization is less studied. Thus, in this study, the Comprehensive Laboratory Animal Monitoring System (CLAMS) was used to determine locomotion activity in mice exposed to repeated doses of hydrocodone with a challenge at a higher dose, and ceftriaxone treatment by breaking the infrared beam using infrared photocell technology. Glutamate function is regulated by several transporters and receptors, including the metabotropic glutamate receptor 5 (mGluR5), glutamate transporter 1 (GLT-1), known to regulate the majority of extracellular glutamate, and cystine / glutamate antiporter (xCT), which regulates glutamate output from astrocytes (Bridges et al., 2012, Br. J. Pharmacol., 165, 20). mGluR5 is highly expressed in the NAc (Tallaksen-Greene, 1998, Brain Res., 780, 210), and it is an important mediator of synaptic plasticity and excitatory neurotransmission (She et al., 2009, Eur. J. Neurosci., 29, 1379; Drouin-Ouellet, 2011, Glia, 59, 188). Exposure to substances of abuse downregulates GLT-1 and xCT expression in several brain regions, and beta- lactams (e.g., ceftriaxone) attenuate these effects (Abulseoud et al., 2022, Front. Neurosci., 16, 841036). Indeed, a study indicated that ceftriaxone treatment attenuates hydrocodone-induced downregulation of GLT-1 and xCT expression in mesocorticolimbic brain regions, as well as hydrocodone-induced upregulation of mGluR5 expression in chronic hydrocodone-exposed mice (Wong, 2023, Toxics, 11, 870). Increased GLT-1 expression by beta-lactams can lead to a sustained reduction in extracellular glutamate concentrations in the NAc (Rasmussen et al., 2011, Amino Acids, 40, 761) and consequently reduce the activation of mGluR5 downstream pathways, thereby reducing neuroexcitotoxicity. Therefore, this study tested whether the beta-lactam ceftriaxone attenuates the effects of exposure to hydrocodone overdose on the expression of GLT-1, xCT, and mGluR5 in the NAc. Further, the effects of exposure to hydrocodone overdose in the signaling pathways involved in neuronal nitric oxide synthase (nNOS) activity, the extracellular signal-regulated kinases (ERK) signaling pathway, and the receptor for advanced glycation end products (RAGE) as a signaling inflammatory marker, and whether ceftriaxone attenuates these effects were investigated. Additionally, this study explored the modulatory effects of ceftriaxone against hydrocodone-induced hyperlocomotion activity using CLAMS. Attorney Docket No.206017-0285-00WO Materials and Methods Animal Use Approval All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Toledo, protocol number 400155 (approved 2 August 2022). This protocol follows the guidelines for the use of animals in research, as described in the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. Mice were intraperitoneal (i.p.) injected and handled with care to prevent any distress and minimize any pain. The mice were monitored every day throughout the study, particularly when hydrocodone treatment was performed, and this was to determine any potential health issues. The mice were euthanized using CO2inhalation and decapitated at the end of the experiment. Animal and Study Design C57BL / 6 mice were used in this study. These mice were reported to show robust differences in drug dependence-relevant behaviors, including locomotor sensitization to substance abuse (Orsini et al., 2005, Psychopharmacology, 181, 327). Eight-week-old male C57BL / 6 mice (Jackson Laboratory, Bar Harbor, ME, USA, 25– 30 g) were grouped into three groups: (1) control group (n = 7–8); (2) hydrocodone group (n = 7–8); and (3) hydrocodone–ceftriaxone group (n = 7–8). The animals were housed in a room maintained at 21 °C with a 12 / 12 h light / dark cycle. Hydrocodone (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in saline at 20 mg / kg and 40 mg / kg, and ceftriaxone (Pfizer, New York, NY, USA) was dissolved in saline at 200 mg / kg. For acclimatation purposes, mice were handled for three days prior to the start of the experiment. The control mice received an i.p. injection of saline (vehicle) every other day from Day 1 to Day 9. Groups 2 and 3 received hydrocodone (20 mg / kg, i.p.) on Days 1, 3, 5, and 7. On Day 9, mice received an overdose of hydrocodone (i.p.) at 40 mg / kg. In addition, group 3 received ceftriaxone (200 mg / kg, i.p.) on Days 5–9, and groups 1 and 2 received equivalent volume of vehicle saline (i.p.) on Days 5–9. On Day 7 of the drug treatment, the mice were placed in Minispec NMR, which is used to measure the lean and fat mass of mice (Fig. 36). It is important to note that 40 mg / kg (i.p.) of hydrocodone was considered a sublethal dose since it was found that 45 mg / kg Attorney Docket No.206017-0285-00WO (i.p.) of hydrocodone was lethal in the mice (n = 3). Thus, the present example tested 40 mg / kg (i.p.) of hydrocodone, which was considered as a higher and sublethal dose. Comprehensive Laboratory Animal Monitoring System (CLAMS) Mice were placed individually in a Comprehensive Laboratory Animal Monitoring system (CLAMS; Columbus Instruments, Columbus, OH, USA) and had access to free food and water from day 7 to day 11 (Fig. 36). The mice were placed at room temperature under alternating 12 h light and 12 h dark cycles. After adaptation for one day, individual locomotor activity was detected using IR photocell technology. Brain Tissue Extraction The mice were sacrificed using CO2 inhalation euthanasia procedure on Day 12 (Fig.36). The brains were dissected out and frozen on dry ice and further stored at −80 °C. NAc (core and shell) was extracted using a cryostat machine (Leica CM1950, Leica, Deer Park, IL, USA). The NAc was selected using the Brain Mouse Atlas (Paxinos et al., 2020, Atlas of the Developing Mouse Brain). NAc samples were stored at −80 °C for determination of target proteins using a Western blot assay. Western Blot Analyses Protein expression of phospho-nNOS, nNOS, RAGE, phospho-ERK, ERK, xCT, GLT-1, mGluR5, and β-tubulin was determined in the NAc (core and shell) using a Western blot assay. NAc tissues from all groups were lysed using lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1% Triton, 0.1% SDS) with phosphatase and protease inhibitors. Quantification of the amount of protein was performed using a detergent-compatible protein assay (Bio-Rad, Hercules, CA, USA). Protein from each sample with equal amounts was mixed with laemmili dye and further loaded onto 10% Tris-glycerine gel for separation of loaded proteins using an electrophoresis apparatus. Separated proteins were transferred from the gels into a polyvinylidene difluoride (PVDF) membrane. The PVDF membranes were incubated in 5% fat-free milk in Tris-buffered saline with Tween 20 (TBST) for 30 min at room temperature. The membranes were further incubated overnight at 4 °C with primary antibodies: rabbit anti-phospho-ERK (1:1000, Abcam, Waltham, MA, USA, Attorney Docket No.206017-0285-00WO ab201015), rabbit anti-ERK (1:1000, Abcam, Waltham, MA, USA, ab17942), rabbit anti-Phospho-nNOS (1:1000, Abcam, Waltham, MA, USA, ab16650), rabbit anti- nNOS (1:1000, Abcam, Waltham, MA, USA, ab76067), rabbit anti-RAGE (1:1000, Abcam, Waltham, MA, USA, ab37647), rabbit anti-GLT-1 (1:5000, Abcam, Waltham, MA, USA, ab205248), rabbit anti-xCT (1:1000, Abcam, Waltham, MA, USA, ab125186), and rabbit anti-mGluR5 (1:1000, Abcam, Waltham, MA, USA, ab76316). Mouse anti-β-tubulin (1:1000, BioLeagend, San Diego, CA, USA) was used as a control loading protein. The next day, the membranes were washed with TBST five times and incubated with the corresponding secondary antibody (1:4000) for 60 min. The membranes were washed with TBST and dried for further analysis. The membranes were then incubated in chemiluminescent reagents (Super Signal West Pico, Perce Inc., Appleton, WI, USA) for 1–2 min. The GeneSys imaging system (Syngene, Frederick, MD, USA) was used for blot development and digitization. The expression of phospho- nNOS, nNOS, RAGE, phospho-ERK, ERK, xCT, GLT-1, mGluR5, and β-tubulin blots were quantified and analyzed using ImageJ software (Version 1.53t 24). The control vehicle group was reported as 100% for determination of changes in the expression of selected target proteins in the NAc, as described previously (Alasmari et al., 2020, Biomolecules, 10, 1030; Alhaddad et al., 2022, J. Pharmacol. Exp. Ther., 383208). Statistical Analyses GraphPad Prism software (Version 10) was used to perform statistical analyses of the expression of the studied proteins. The analyses of Western blot data were conducted using one-way ANOVA followed by the Newman–Keuls post hoc multiple comparison test. The data were presented and analyzed as a percentage (relative to control values) ratio to the loading control protein, β-tubulin. The data are reported for a significance level of p < 0.05. Results Effects of Exposure to Hydrocodone Overdose and Ceftriaxone Treatment on Locomotion Activity First evaluated were the effects of exposure to hydrocodone overdose and ceftriaxone on locomotion activity. There was a significant difference in x activity Attorney Docket No.206017-0285-00WO (n = 7–8 mice per group, F2,9 = 12.97, p < 0.01, Fig.37A), x ambulatory (n = 7–8 mice per group, F2,11= 8.394, p < 0.01, Fig. 37B), and z activity (n = 7–8 mice per group, F2,11 = 37.13, p < 0.0001, Fig.37C) among all tested groups. The Newman–Keuls post hoc test analysis demonstrated that x activity (p < 0.01, Fig. 37A), x ambulatory (p < 0.01, Fig. 37B), and z activity (p < 0.0001, Fig. 37C) significantly increased in the hydrocodone group compared to the control group. Importantly, treatment with ceftriaxone normalized x activity (p < 0.05, Fig. 37A) and z activity (p < 0.001, Fig. 37C) in the mice. Significant changes in locomotion activity were found between the control group and the hydrocodone–ceftriaxone group in x activity (p < 0.05), x ambulatory (p < 0.05), and z activity (p < 0.05) (Fig. 37). However, no significant change was detected in x ambulatory between the hydrocodone and hydrocodone– ceftriaxone groups (Fig.37B). Effects of Exposure to Hydrocodone Overdose and Ceftriaxone on GLT-1, xCT, and mGluR5 Protein Expressions in the NAc The effects of exposure to hydrocodone overdose on GLT-1, xCT, and mGluR5 expression were determined in the NAc. Immunoblot analyses revealed significant differences in the expression of GLT-1 (F2,14= 7.837, p < 0.01, Fig. 38A), xCT (F2,15 =15.90, p <0.001, Fig. 38B), and mGluR5 (F2,13 = 66.11, p < 0.0001, Fig. 38C) in the NAc among all groups. Statistical analyses demonstrated downregulation of the expression of GLT-1 (p < 0.01, Fig. 38A) and xCT (p < 0.001, Fig. 38B) in the hydrocodone-treated group compared to the control group. Furthermore, exposure to hydrocodone overdose significantly increased mGluR5 expression in the NAc (p < 0.05, Fig. 38C) in the hydrocodone group compared to the control group. Ceftriaxone treatment normalized hydrocodone-induced downregulation in GLT-1 (p < 0.05, Fig. 38A) and xCT (p < 0.001, Fig. 38B) expression and attenuated the effect of hydrocodone exposure on mGluR5 expression (p < 0.0001, Fig. 38C). There were no changes in the expression of GLT-1 (Fig.38A) and xCT (Fig.38B) between the control saline and hydrocodone–ceftriaxone groups. However, there were significant changes in mGluR5 expression (p < 0.0001, Fig. 38C) between the control and hydrocodone– ceftriaxone groups. Attorney Docket No.206017-0285-00WO Effects of Exposure to Hydrocodone Overdose and Ceftriaxone on nNOS and ERK Protein Expression in the NAc Next explored were the effects of exposure to hydrocodone overdose and ceftriaxone on the protein expression of nNOS and ERK in the NAc. One-way ANOVA showed a significant difference in nNOS (F2,15= 42.44, p < 0.0001, Fig.39A) and ERK (F2,15 = 14.10, p < 0.001, Fig.39B) expression among all tested groups in the NAc. Newman–Keuls post hoc analyses revealed that hydrocodone exposure upregulated nNOS expression in the NAc compared to the control group (p < 0.05, Fig. 39A). The analysis also revealed that hydrocodone exposure downregulated ERK expression compared to the control group (p < 0.001, Fig.39B). Importantly, treatment with ceftriaxone significantly attenuated hydrocodone-induced upregulation of nNOS (p < 0.0001, Fig. 39A) and hydrocodone-induced downregulation of ERK (p < 0.05, Fig.39B) compared to the hydrocodone group. In addition, significant differences were observed when comparing the control group with the hydrocodone–ceftriaxone group in both nNOS (p < 0.0001, Fig.39A) and ERK (p < 0.05, Fig.39B). Effects of Exposure to Hydrocodone Overdose and Ceftriaxone on RAGE Protein Expression in the NAc Lastly, the effects of hydrocodone, and hydrocodone–ceftriaxone on RAGE expression were tested. One-way ANOVA analysis demonstrated a significant difference in the expression of RAGE in the NAc among all tested groups (F2,15= 4.277, p < 0.05, Fig.40). Newman–Keuls post hoc analysis revealed that hydrocodone exposure increased RAGE expression in the NAc compared to the control group (p < 0.05), and ceftriaxone treatment normalized this effect (p < 0.05) (Fig.40). Discussion The present example relates to the investigation of the effects of exposure to hydrocodone overdose on locomotion activity and whether ceftriaxone could modulate changes in locomotion activity in mice. Using CLAMs, it was found that exposure to hydrocodone overdose increases locomotion activity in mice. An aim of this study was to establish a hydrocodone overdose mouse model using CLAMS.20 mg / kg (i.p.) of hydrocodone were used every other day (four i.p. injections) and then Attorney Docket No.206017-0285-00WO challenged the mice with a sublethal dose of hydrocodone (40 mg / kg, i.p.) since 45 mg / kg (i.p.) of hydrocodone was lethal. Further investigated was whether ceftriaxone attenuates the effects of sublethal hydrocodone dose in locomotor activity and the changes in the expression of target proteins. Hydrocodone-induced hyperlocomotion activity is consistent with studies demonstrating that exposure to morphine and fentanyl increases locomotor activity in rats (Berrios-Carcamo et al., 2022, Int. J. Mol. Sci., 23, 3874; Gaulden et al., Drug Alcohol Depend., 229, 109101). Hyperlocomotion activity caused by drugs of abuse is known as locomotion sensitization. This behavioral sensitization is thought to underlie some aspects of drug dependence and is related to dopaminergic systems, which are implicated in motor function and reward (Allouche et al., 2013, Prog. Neuro-Psychopharmacol. Biol. Psychiatry, 40, 286; Delage et al., 2023, Prog. Neuro-Psychophamacol. Biol. Psychiatry, 127, 110824). Importantly, the present results revealed that treatment with the beta-lactam ceftriaxone, which is known to upregulate GLT-1 expression, significantly reduces hydrocodone-induced hyperlocomotion activity in mice. This is consistent with other studies showing that ceftriaxone attenuates the development of behavioral sensitization produced by chronic cocaine and amphetamine exposures (Rasmussen et al., 2011, Drug Alcohol Depend., 118, 484). These latter studies supported the finding that ceftriaxone is associated with normalizing behavioral sensitization upon exposure to hydrocodone overdose in mice (Fischer et al., 2018, Psychopharmacology, 235, 1371). In this present study, only male mice were studied as the aim was to establish a model of hydrocodone overdose using CLAMS and to determine whether ceftriaxone attenuates the effect of hydrocodone exposure, particularly with a higher dose. Further studies are warranted to investigate the effects of extended duration of exposure to opioids in the brains of male as well as female mice for determination of sex difference. In addition, further studies are warranted to determine the effects of different doses of hydrocodone and ceftriaxone, as well as to test a novel beta-lactam non-antibiotic such as MC-100093, which has been shown to be protective in the brain of rats exposed to ethanol. Increased extracellular glutamate concentrations at the synaptic cleft can lead to glutamate neuroexcitotoxicity, which might be associated with certain neuroinflammatory and neurodegenerative diseases (Fischer et al., 2018, Psychopharmacology, 235, 1371; Iovino et al., 2020, J. Pharmacol. Sci., 144, 151; Pitt et al., 2000, Nat. Med., 6, 67; Arundine, 2003, Cell Calcium, 34, 325). Therefore, maintaining glutamate homeostasis is very important. GLT-1 and xCT are highly Attorney Docket No.206017-0285-00WO expressed in astrocytes and help remove excess extracellular glutamate concentrations from the synaptic cleft. Hydrocodone exposure has been associated with reduced GLT- 1 and xCT, resulting in an elevation of extracellular glutamate concentrations in the NAc. Previous studies have demonstrated that a reduction in GLT-1 expression in the brain is associated with chronic exposure to substances of abuse (Sari et al., 2009, J. Neurosci., 29, 9239). Furthermore, it was recently reported that chronic hydrocodone exposure induces downregulation of GLT-1 and xCT expression in the mesocorticolimbic brain region, and that ceftriaxone treatment attenuates hydrocodone- induced downregulation of GLT-1 and xCT expression (Wong, 2023, Toxics, 11, 870). These findings are consistent with the current results showing downregulation of GLT- 1 and xCT in the NAc following exposure to hydrocodone overdose. Importantly, ceftriaxone treatment restored GLT-1 and xCT expression in the NAc. Therefore, the current and previous findings suggest that chronic exposure to hydrocodone overdose may lead to dysregulation of glutamate homeostasis in the brain and that this effect can be attenuated by ceftriaxone treatment. Alternatively, the effects of hydrocodone and ceftriaxone on the expression of mGluR5 in the NAc were investigated. mGluR5 is highly expressed in the brain and is involved in mediating the potentiating effects of opioids (Hubert et al., 2001, J. Neurosci., 21, 1838; Romano et al., 1995, J. Comp. Neurol., 355, 455; Abe et al., 1992, J. Biol. Chem., 267, 13361). Previous studies have revealed an increase in mGluR5 expression in the mesocorticolimbic brain regions during chronic exposure to hydrocodone and in morphine place preference paradigms (Qi et al., 2015, Addict. Biol., 20, 927). The results showed that mGluR5 increased in the NAc following exposure to hydrocodone overdose. Hydrocodone-induced upregulation of mGluR5 expression was attenuated with ceftriaxone treatment. Glutamate acts on mGluR, which is coupled to intracellular second messengers via G proteins, guanine nucleotide regulatory, or phosphorylation of MAP kinase (Niswender, 2010, Annu. Rev. Pharmacol. Toxicol., 50, 295; Ribeiro et al., 2017, Pharmacol. Res., 115, 179). Activation of mGluR5 may result in cellular depolarization and increased neuronal excitability. mGluR5 is positively coupled to phosphatidylinositol (PI) hydrolysis, leading to the activation of protein kinase C and increasing intracellular calcium ions Ca2+(Biber et al., 1999, J. Neurochem., 72, 1671; Pasti et al., 1997, J. Neurosci., 17, 7817; Bruno et al., 1995, Neuropharmacology, 34, 1089). The increase in intracellular calcium ions may induce the production of nitric oxide (NO) through Ca2+ / calmodulin activation of nNOS, and high NO concentration Attorney Docket No.206017-0285-00WO can trigger numerous downstream neurotoxic cascades. It has been shown in studies by others and us that nNOS activity (phosphorylated nNOS resulting in a higher p- nNOS / nNOS ratio) increases following cue-induced reinstatement of amphetamine, cocaine seeking, and chronic ethanol exposure in the NAc of mice and rats (Smith, 2017, J. Neurosci., 37, 742; Narita et al., 2005, J. Neurochem., 94, 1297; Alhaddad et al., 2020, Brain Res. Bull., 165, 272). These studies support the finding that the p- nNOS / nNOS ratio increased in the NAc after exposure to hydrocodone overdose. Importantly, ceftriaxone treatment reversed the effects of hydrocodone-induced upregulation of the p-nNOS / nNOS ratio. This study demonstrated that exposure to hydrocodone overdose is accompanied by a decreased phosphorylation of ERK in the NAc and that this effect was attenuated with ceftriaxone treatment. This is consistent with previous findings showing that ceftriaxone attenuates hydrocodone-induced downregulation of p-ERK expression in the mesocorticolimbic brain regions. ERK is involved in the regulation of GLT-1 transcription through the initiation of nuclear transcription factor-κβ (NF-κβ) and cAMP response element-binding protein (CREB). Notably, nNOS-derived NO can also regulate synaptic plasticity by inducing the ERK signaling pathway (Zhang et al., 2018, Aging Cell, 17, e12754). A prior study indicated that the inhibition of the ERK signaling pathway is due to the generation of free radicals upon the activation of nNOS in vitro (Raines et al., 2004, J. Biol. Chem., 279, 3933). These studies supported the findings that downregulation of GLT-1 expression is associated with increased nNOS activity and decreased ERK expression in the NAc of mice exposed to hydrocodone overdose. Furthermore, this study investigated the RAGE signaling pathways with exposure to hydrocodone overdose and ceftriaxone treatment. The RAGE is known to induce neuroinflammation through activation of the NF-κB signaling pathway (Lin et al., 2009, Front. Biosci. A J. Virtual Libr., 14, 1403; Hudson, 2018, Annu. Rev. Med., 69, 349; Gasparotto et al., 2018, J. Biol. Chem., 293, 226). Current analysis showed that exposure to hydrocodone overdose increased RAGE expression in the NAc, indicating the role of inflammatory factors in opioid overdose events and further validating the induction of brain inflammation. Interestingly, ceftriaxone attenuated hydrocodone-induced increases in RAGE expression. Studies from other laboratories confirmed these findings, demonstrating that pharmacological inhibition of RAGE attenuated neuroinflammation in the brain (Li et al., 2017, Mol. Neurobiol., 54, 755; Attorney Docket No.206017-0285-00WO Shen et al., 2017, Neurochem. Res., 42, 2902; Wang et al., 2020, Front. Neurosci., 14, 353). The present example suggests that activation of the mGluR5-nNOS- ERK pathway reduces GLT-1 expression, leading to excessive extracellular glutamate concentrations in the brain, thereby increasing neuroexcitotoxicity (Fig.41). Glutamate also binds to the N-methyl-d-aspartate receptor (NMDAR) and activates the enzyme nNOS to produce NO. Increased NO expression inhibits ERK production and inactivates the downstream signaling pathway of ERK (Fig. 41). Furthermore, hydrocodone upregulates RAGE expression, leading to neuroinflammation. Exposure to hydrocodone overdose induces hyperlocomotion activity in mice. Ceftriaxone treatment successfully attenuates hydrocodone-induced hyperlocomotion activity. In addition, exposure to hydrocodone overdose decreases GLT-1 and xCT expression in the NAc, thereby disrupting glutamate homeostasis. Increased extracellular glutamate concentrations at the synaptic cleft may overstimulate mGluR5 and increase nNOS activity. As observed in this study, activation of nNOS activity can lead to inhibition of the ERK signaling pathway. Furthermore, exposure to hydrocodone overdose increases RAGE expression, thereby inducing neuroinflammation in the brain. However, ceftriaxone treatment attenuates hydrocodone-induced upregulation of mGluR5, NOS activity, and RAGE, as well as hydrocodone-induced downregulation of GLT-1, xCT, and ERK expression. Future studies are warranted to investigate the beneficial effects of other novel synthetic beta-lactams (non-antibiotics), and longer exposure of doses of hydrocodone on opioid-induced hyperlocomotion activity, dysregulation of glutamatergic systems, and neuroinflammation. Example 6: Effects of MC-100093 on Ethanol Drinking and the Expression of Astrocytic Glutamate Transporters in the Mesocorticolimbic Brain Regions of Male and Female Alcohol-Preferring Rats Chronic ethanol consumption increased extracellular glutamate concentrations in several reward brain regions. Glutamate homeostasis is regulated in majority by astrocytic glutamate transporter 1 (GLT‐1) as well as the interactive role of cystine / glutamate antiporter (xCT). In part, this example aimed to determine the attenuating effects of a novel beta‐lactam MC‐100093, lacking the antibacterial properties, on ethanol consumption and GLT‐1 and xCT expression in the subregions Attorney Docket No.206017-0285-00WO of nucleus accumbens (NAc core and NAc shell) and medial prefrontal cortex (Infralimbic, mPFC‐IL and Prelimbic, mPFC‐PL) in male and female alcohol‐ preferring (P) rats. Female and male rats were exposed to free access to ethanol (15% v / v) and (30% v / v) and water for five weeks, and on Week 6, rats were administered 100 mg / kg (i.p) of MC‐100093 or saline for five days. MC‐100093 reduced ethanol consumption in both male and female P rats from Day 1–5. Additionally, MC‐100093 upregulated GLT‐1 and xCT expression in the mPFC and NAc subregions as compared to ethanolsaline groups in female and male rats. Chronic ethanol intake reduced GLT‐1 and xCT expression in the IL and PL in female and male rats, except there was no reduction in GLT‐1 expression in the mPFC‐PL in female rats. Although, MC‐100093 upregulated GLT‐1 and xCT expression in the subregions of NAc, there was no observation of any reduction in GLT‐1 and xCT expression with chronic ethanol intake in female rats. These findings strongly suggest that MC‐ 100093 treatment effectively reduced ethanol intake and upregulated GLT‐1 and xCT expression in the mPFC and NAc subregions in male and female P rats. Changes in glutamate homeostasis is associated with the development of drug dependence (Kalivas et al., 2009, Nat. Rev. Neurosci.10, 561). Glutamate projections in the mesocorticolimbic pathways are critical in mediating response to drug rewards and drugs of abuse. Among many projecting glutamatergic pathways, there are glutamatergic signals that project from the medial prefrontal cortex (mPFC), amygdala, and hippocampus to the nucleus accumbens (NAc) (McFarland et al., 2003, J. Neurosci.23, 3531; LaLumiere and Kalivas, 2008, J. Neurosci.28, 3170; Parent et al., 2009, Cereb. Cortex 20, 393; Mitrano et al., 2010, J. Comp. Neurol.518, 1315; Papp et al., 2012, Brain Struct. Funct.217, 37). The NAc is divided into two distinct sub-regions termed the nucleus accumbens shell (NAc-shell) and the nucleus accumbens core (NAc‐core). Glutamatergic projections from the mPFC to the NAc are suggested to be involved in regulating reward‐related behaviors and drug dependence (Torregrossa et al., 2008, Neurosci. Lett.438, 142). The mPFC regulates reward and motivation, and its disruption triggers the loss of control over compulsive drug‐seeking behaviors in drug dependence (Goldstein and Volkow, 2011, Nat. Rev. Neurosci.12, 652). There are at least four subregions in the mPFC, two of which, mPFC‐infralimbic (mPFC‐IL) and mPFCprelimbic (mPFC‐PL), have been shown to regulate drug‐seeking behavior differently. The mPFC‐PL appears to drive drug‐ Attorney Docket No.206017-0285-00WO seeking behavior and reinstatement of drug‐seeking after extinction or abstinence, while the mPFC‐IL is involved in the inhibition of drugseeking behavior and suppresses the reinstatement of drug‐seeking (Zavala et al., 2003, Brain Res.990, 157; Pelloux et al., 2013, Eur. J. Neurosci.38, 3018; West et al., 2014, Eur. J. Neurosci.39, 1891; Moorman et al., 2015, Brain Res.1628, 130; Gutman et al., 2017, Addict. Biol.22, 1719). It is important to note that disruption of mPFC‐NAc glutamatergic pathways might contribute to the development of dependence to drugs of abuse, including ethanol (Kalivas and Volkow, 2005, Am. J. Psychiatry 162, 1403; Moussawi and Kalivas, 2010, Eur. J. Pharmacol.639, 115; Goodwani et al., 2017, Neurosci. Biobehav. Rev.77, 14). Glutamate homeostasis is disrupted with ethanol exposure and dependence, leading to increased extracellular glutamate concentrations in mesocorticolimbic brain regions (Das et al., 2015, Neuropharmacology 97, 67; Alasmari et al., 2018, Prog. Neurobiol.171, 32). Several glutamate transporters mediate the regulation of extracellular glutamate concentration. Among these glutamate transporters, glutamate transporter 1 (GLT‐1, its human homolog is excitatory amino acid transporter 2, EAAT2) regulates the uptake of the majority of extracellular glutamate concentrations in the brain. Chronic exposure or self‐ administration to drugs of abuse can lead to downregulation of GLT‐1, an effect that is associated with increased extracellular glutamate concentrations at the synaptic cleft (Rao and Sari, 2012; Das, et al., 2015; Roberts‐Wolfe and Kalivas, 2015; Alasmari, et al., 2018; Kim et al., 2018; Abulseoud et al., 2022). Extensive studies demonstrated clearly that chronic ethanol consumption downregulated the expression of GLT‐1 in mesocorticolimbic brain regions, and this effect was associated with increased extracellular glutamate concentration in the brain of alcohol‐preferring (P) rats (Rao and Sari, 2012, Curr. Med. Chem.19, 5148; Sari et al., 2013, OA Alcohol 1, 6; Alhaddad et al., 2014, Psychopharmacology 231, 4049; Aal‐Aaboda et al., 2015, J. Neurosci. Res.93, 930; Rao et al., 2015, Neuroscience 295, 164; Rao et al., 2015, Psychopharmacology (Berl) 232, 2333). Alternatively, cystine‐glutamate exchanger (xCT) is another glutamate transporter that is responsible for the release of astrocytic glutamate in exchange for cystine (Baker et al., 2002, J. Neurosci.22, 9134; Moran et al., 2005, J. Neurosci.25, 6389). Several studies showed that chronic ethanol exposure decreased xCT expression in several brain reward regions of P rats Attorney Docket No.206017-0285-00WO (Alhaddad, et al., 2014, Psychopharmacology 231, 4049; Hakami et al., 2016, Front. Neurosci.10, 171). This lab has been studying the effects of GLT‐1 upregulators on attenuating the effects of chronic exposure to drugs of abuse, including ethanol. Thus, these studies have clearly demonstrated that ceftriaxone, a beta‐lactam antibiotic known to upregulate GLT‐1 (Rothstein et al., 2005, Nature 433, 73), decreased ethanol consumption in rats, and this effect was associated with the attenuation of downregulation of GLT‐1 and xCT expression and consequently normalizing the extracellular glutamate concentration in the brain reward regions such as the NAc. In addition to ceftriaxone, other beta‐lactam antibiotics are effective on attenuating ethanol consumption and relapse behaviors, in part through upregulation of GLT‐1 and xCT expression in brain reward regions such as NAc and mPFC (Alasmari et al., 2015, Neurosci. Lett.600, 148; Alasmari et al., 2016, Brain Res.1634, 150; Hakami et al., 2016, Front. Neurosci.10, 17). Importantly, a recent study revealed the efficacy of a novel beta‐lactam, MC‐100093, in reducing ethanol intake in P rats, and this effect was associated with upregulation of GLT‐1 in NAc. It is important to note that the pharmacokinetics of MC‐100093 were performed in a previous study, which showed that MC‐100093 displayed 28% oral bioavailability (F = 28%) in rats, and the drug was associated with a 14% brain / plasma ratio after intraperitoneal (i.p) injection. This latter study revealed that MC‐100093 was associated with a 23.5% enhancement of glutamate uptake with an IC50 of 0.1 μM in an astrocyte‐neuron co‐culture model. In addition, MC‐100093 lacks antimicrobial activity against gram‐positive and gram‐ negative bacteria. This study examined the effects of MC‐100093 on attenuating ethanol consumption in both male and female P rats. Additionally, this study explored the neurocircuits involving the mPFC subregions (mPFC‐IL and mPFC‐PL) and the NAc subregions (NAc‐shell and NAc‐core) to determine any changes in the expression of GLT‐1 and xCT in P rats of both sexes exposed to ethanol and MC‐ 100093. Experimental procedures Animals Male and female alcohol‐preferring (P) rats were acquired from Indiana University School of Medicine, Indianapolis, IN at the age of 80–85 days. All animals were single housed in a room with temperature at 22 °C and 50% humidity in Attorney Docket No.206017-0285-00WO a 12 h light / dark cycle. Rats had free access to food and water throughout the experimental procedures. All the experimental procedures were approved by the University of Toledo Institutional Animal Care and Use Committee (IACUC) under IACUC protocol# 400160. Ethanol intake measurements At the age of 90–100 days, 5–7 male and female rats were assigned randomly to three groups: (1) Water‐drinking (Water‐Saline) group with no access to ethanol and served as a control group; (2) Ethanol‐Saline group, which was exposed to continuous free‐choice access to ethanol (15% and 30%, v / v, concurrently), and water for five weeks, and saline i.p. injections were performed from Days 1–5 on Week 6; and (3) Ethanol‐MC‐100093 group, which had continuous freechoice access to ethanol (15% and 30%, v / v), and water for five weeks, and MC‐100093 i.p. injections were performed from Days 1–5 on Week 6. Ethanol and water intake were measured as g / kg / day. Rats were required to meet the criterion of an average ethanol consumption of 4 g / kg / day to be included in the study, for at least 2 weeks before saline vehicle or MC‐100093 i.p. injections, as adopted in previous studies (Alhaddad, et al., 2022;Sari, et al., 2011;Sari and Sreemantula, 2012). Average ethanol and water consumptions throughout Week 5 served as a baseline. At Week 6 of the experiment, ethanol‐MC‐100093 group received MC‐100093 at a dose of 100 mg / kg (i.p.) once daily for five days, and ethanol‐saline and water‐saline groups received saline vehicle as an i.p injection for five consecutive days. Water and ethanol intakes were measured daily 24 h after the first i.p. injections of saline or MC‐100093 and 24 h after the last i.p. injections of saline or MC‐100093. Brain tissue harvesting After 24 h of receiving the last i.p. injection of either saline or MC‐ 100093, rats were euthanized by CO2 inhalation followed by decapitation with a guillotine. Brains were harvested and immediately frozen on dry ice and stored at −80 °C. Cryostat (Leica) was used to isolate the mPFC subregions (mPFC‐IL and mPFC‐ PL), and subregions of the NAc (NAc‐core and NAc‐shell). Brains were micro‐ punctured using the stereotaxic coordinates following the Rat Brain Atlas (Paxinos Attorney Docket No.206017-0285-00WO and Watson, 2006). The isolated brain regions were stored at −80 °C for subsequent protein detection using Western Blot assay. Western Blot analyses Immunoblot assays were performed to determine the expression of GLT‐1, xCT and β‐tubulin in mPFC‐IL, mPFC‐PL, NAc‐core, and NAcshell of all groups as described previously. Briefly, brain samples were homogenized in lysis buffer supplemented with protease inhibitor and total protein was quantified using protein assay (Bio‐Rad, Hercules, CA, USA). Equal amount of the samples was loaded on polyacrylamide gel (10–20%). Subsequently, proteins were transferred on a PVDF membrane and blocked with 5% milk in Tris‐buffered saline Tween‐20 (1x TBST) for 30 min at room temperature. Membranes were then incubated overnight at 4 °C with one of the following antibodies: rabbit anti‐GLT‐1 (1:1000, Abcam, AB41621), and / or rabbit anti‐xCT (1:1000, Abcam, AB175186). Mouse anti‐β‐ tubulin (1:1000; Cell Signaling D71G5) was used as a loading control antibody. On the following day, membranes were washed five times with 1x TBST followed by incubation with appropriate secondary antibody (1:4000) for 60 min at room temperature. Chemiluminescent reagents (Super Signal West Pico, Pierce Inc.) were used to detect proteins using a ChemiDoc imaging system (BioRad, USA). GLT‐1 and xCT expression were normalized against β‐tubulin, a control loading protein. Imaging System and ImageJ software version 1.53a were used to quantify and analyze the expression of GLT‐1 and xCT in the subregions of the mPFC and NAc. Data from water‐control group were represented as 100% and all other values were expressed relative to this control to evaluate the changes in protein expression. The ratios for treated animals in the ethanol / saline and ethanol / MC‐100093 groups were normalized to the mean ratios for the control group (water / saline group). The control ratio was set at 100, and the results from each of the treated groups were expressed as a percentage relative to the water / saline group value of 100%. The ethanol / saline and ethanol / MC‐ 100093 groups data were normalized to water / saline group to reduce any differences of contrast with Western blots for each set of groups (control / water / saline, ethanol / saline, and ethanol / MC‐100093 groups) as it was performed in previous studies (Hammad et al., 2021, Alcohol Alcohol.56, 210). Statistical analyses Attorney Docket No.206017-0285-00WO All statistical analyses were conducted using GraphPad Prism (10). Two‐way (mixed) ANOVA followed by Bonferroni multiple comparison post‐hoc test was performed to analyze daily ethanol intake, average daily water intake and body weight. One‐way ANOVA followed by Newman‐Keuls post hoc tests were used to analyze Western blot data. All statistical analyses data were reported as a p < 0.05 of significance. Results Effect of MC-100093 treatment on ethanol consumption, ethanol preference and body weight in male P rats Statistical analysis using two‐way ANOVA revealed significant main effects of Day [F(5, 61) =10.04, p < 0.0001], and Treatment [F(1, 61)= 88.97, p < 0.0001] as well as a significant Treatment × Day interaction [F(5, 61)= 4.754, p = 0.0010]. Bonferroni multiple comparison test showed a significant decrease in ethanol consumption from Day 1 through Day 5 of treatment in the ethanol‐MC‐100093 group compared to the ethanol‐saline group (Fig.29A). Ethanol preference was calculated as total ethanol consumption / total fluid consumption×100 from daily ethanol and water consumption (Goodwani et al., 2015, Prog. Neurobiol.171, 32). Two‐way ANOVA revealed a significant main effect of Day [F(5, 71)= 9.958, p < 0.0001] and Treatment [F(1, 71)= 122.8, p < 0.0001] as well as a significant Treatment × Day interaction [F(5, 71)= 5.815, p = 0.0001]. Bonferroni multiple comparison test showed a significant decrease in ethanol preference (%) in ethanol‐ MC‐100093 group as compared to the ethanol‐saline group starting on Day 1 through Day 5 (Fig.29B). Statistical analysis of water consumption data revealed a significant Treatment × Day interaction [F(10, 95)= 2.219, p = 0.0228] and Treatment [F(2, 95)= 70.46, p < 0.0001]. Bonferroni multiple comparison test showed a significant increase in water consumption from treatment Day 1 through Day 5 in the ethanol‐MC‐100093 group compared to the ethanol‐saline group (Fig.29C). Body weights (g) of rats were monitored throughout the study. MC‐100093 treatment had no significant effect on body weights in male P rats (Fig.29D). Effect of MC-100093 treatment on ethanol consumption, ethanol preference and body weight in female P rats Attorney Docket No.206017-0285-00WO Two‐way ANOVA revealed significant main effects of Day [F(5, 54) = 3.014, p = 0.0180], and Treatment [F(1, 54)= 49.92, p < 0.0001] as well as a significant Treatment×Day interaction [F(5, 54)= 2.477, p=0.0431]. Bonferroni multiple comparison test showed a significant decrease in ethanol consumption from Day 1 through Day 5 in the ethanol‐MC‐100093 group as compared to the ethanol‐ saline group (Fig.30A). Furthermore, ethanol drinking between male and female groups was analyzed. A Two‐way ANOVA analysis showed a significant main effect of Day [F(5, 66)= 20.53, p < 0.0001], and Sex [F(1,66)= 17.68, p < 0.0001] between male and female groups. A posthoc analysis using Bonferroni multiple comparison test showed a significant decrease in ethanol consumption from Day 3 through Day 5 in the ethanol‐MC‐100093 male group compared to the ethanol‐MC‐100093 female group (Fig S1.B). Two‐way ANOVA revealed a significant main effect of Day [F(5, 55)= 5.999, p=0.0002] and Treatment [F(1, 55)= 67.73, p < 0.0001] as well as a significant Treatment × Day interaction [F(5, 55)= 3.608, p = 0.0068]. Bonferroni multiple comparison test showed a significant decrease in ethanol preference (%) in ethanol‐MC‐100093 group as compared to the ethanol‐saline group starting on Day 1 through Day 5 (Fig.30B). Statistical analysis of water consumption data revealed a significant a significant main effect of Day [F(5, 72)= 2.725, p=0.0260] and Treatment [F(2,72)= 128.3, p < 0.0001] and a significant Treatment × Day interaction [F(10, 72)= 3.709, p=0.0005]. Bonferroni multiple comparison test showed a significant increase in water consumption from Day 1 through Day 5 in the ethanol‐ MC‐100093 group as compared to the ethanol‐saline group (Fig.30C). Body weights (g) of rats were monitored throughout the study. MC‐100093 treatment had no significant effect on body weights in female P rats (Fig.30D)., Effect of MC-100093 on the expression of GLT-1 in the mPFC-IL of male and female P rats One‐way ANOVA showed a significant difference in GLT‐1 expression in the mPFC‐IL [F(2, 12)= 34.30, p < 0.0001] in male P rats (Fig.31A). Newman‐Keuls post‐hoc analysis revealed a significant decrease in GLT‐1 expression in the mPFC‐IL of the ethanol‐saline group as compared to the water‐saline group (p < 0.01), while MC‐100093 attenuated ethanol‐induced downregulation of GLT‐1 expression (p < 0.0001, p < 0.001 respectively) (Fig.31A). Alternatively, there was a significant difference in GLT‐1 expression among the three groups in the mPFC‐IL of Attorney Docket No.206017-0285-00WO female P rats [F(2, 12)= 12.47, p < 0.0012] (Fig.32A). Newman‐Keuls post‐hoc analysis showed MC‐100093 upregulated GLT‐1 expression in the mPFC‐IL of female rats as compared to ethanol‐saline group (p < 0.001) and water‐saline group (p < 0.05), whereas a significant reduction in the expression of GLT‐1 was found in the ethanol‐saline group as compared to the water‐saline group (p < 0.05) (Fig.32A). Effect of MC-100093 on the expression of xCT in the mPFC-IL of male and female P rats One‐way ANOVA revealed a significant difference in the expression of xCT in the mPFC‐IL of male P rats among all tested groups [F(2, 12) = 13.81, p < 0.0008] (n = 5 / group) (Fig.31B). Newman‐Keuls revealed a significant decrease in the expression of xCT in the mPFCIL of the ethanol‐saline group as compared to the water‐saline group (p < 0.05), while treatment with MC‐100093 reversed the effect of ethanol and significantly increased the expression of xCT (p < 0.001) (Fig.31B). In female P rats, there was a significant difference in xCT expression among the three groups in the mPFC‐IL [F(2, 12)= 14.04, p < 0.0007] (Fig.32B). Newman‐Keuls post‐hoc analysis showed a significant upregulation in the expression of xCT in MC‐ 100093‐treated female rats as compared to ethanol‐saline and water‐saline groups (p < 0.001 and p < 0.01 respectively), whereas a significant reduction in the expression of xCT was found in the ethanol‐saline group as compared to the water‐saline group (p < 0.05) (Fig.32B). Effect of MC-100093 on the expression of GLT-1 in the mPFC-PL of male and female P rats In male P rats, one‐way ANOVA showed a significant difference in GLT‐1 expression in the mPFC‐PL among all tested groups [F(2, 12) = 22.65, p < 0.0001] (Fig.31C). Newman‐Keuls post‐hoc analysis revealed a significant decrease in GLT‐1 expression in the mPFC‐PL of the ethanol‐saline group as compared to the water‐saline group (p < 0.05), while its expression was significantly increased in group treated with MC‐100093 (p < 0.001) as compared to the ethanolsaline group and water‐saline group (p < 0.01) (Fig.31C). Furthermore, there was a significant difference in GLT‐1 expression among the three groups in the mPFC‐PL of female P rats [F(2, 12)= 8.886, p = 0.0043] (Fig.32C). Newman‐Keuls post‐hoc analysis showed a significant upregulation of GLT‐1 expression in the mPFC‐PL of MC‐ Attorney Docket No.206017-0285-00WO 100093‐treated group as compared to ethanol‐saline and water‐saline groups (p < 0.01 and p < 0.05 respectively) (Fig.32C). No significant changes were detected between ethanol‐saline and water‐saline groups in the expression of GLT‐1 in the mPFC‐PL of female P rats. Effect of MC-100093 on the expression of xCT in the mPFC-PL of male and female P rats One‐way ANOVA revealed a significant difference in the expression of xCT in the mPFC‐PL of male P rats among all tested groups [F(2, 12) = 11.79, p = 0.0015] (n = 5 / group) (Fig.31D). Newman‐Keuls test revealed a significant decrease in the expression of xCT in the mPFC‐PL of the ethanol‐saline group as compared to the water‐saline group (p<0.05), while treatment with MC‐100093 reversed the effect of ethanol and significantly increased the expression of xCT (p<0.01) (Fig.31D). In female P rats, there was a significant difference in xCT expression among the three groups in the mPFC‐PL [F(2, 11) = 13.31, p = 0.0009] (Fig.32D). Newman‐Keuls post‐hoc analysis showed a significant upregulation of the expression of xCT in MC‐ 100093‐treated female rats as compared to ethanol‐saline and watersaline groups (p < 0.001 and p < 0.05 respectively), whereas a significant reduction in the expression of xCT was found in the ethanolsaline group as compared to the water‐saline group (p < 0.05) (Fig.32D). Effect of MC-100093 on the expression of GLT-1 in the NAc-shell of male and female P rats In male P rats, one‐way ANOVA showed a significant difference in GLT‐1 expression in the NAc‐shell among all tested groups [F(2, 12) = 16.68, p = 0.0003] (Fig.33A). A significant decrease in GLT‐1 expression in the NAc‐shell of the ethanol‐saline group as compared to the water‐saline group was revealed by Newman‐Keuls post‐hoc analysis (p < 0.05), while its expression was significantly increased in group treated with MC‐100093 as compared to the ethanol‐saline and water‐saline groups (p < 0.001, p < 0.01 respectively) (Fig.33A). Moreover, there was a significant difference in GLT‐1 expression among the three groups in the NAc‐ shell of female P rats [F(2, 12) = 10.41, p = 0.0024] (Fig.34A). Newman‐Keuls post‐ hoc analysis showed a significant upregulation of GLT‐1 expression in the NAcshell of MC‐100093 treated female rats (p < 0.01), however, there was no significant Attorney Docket No.206017-0285-00WO reduction in the expression of GLT‐1 in the ethanol‐saline group compared to the water‐saline goup (Fig.34A). Effect of MC-100093 on the expression of xCT in the NAc-shell of male and female P rats One‐way ANOVA revealed a significant difference in the expression of xCT in the NAc‐shell of male P rats among all tested groups [F(2, 12) = 40.16, p = 0.0006] (Fig.33B). Newman‐Keuls revealed a significant decrease in the expression of xCT in the NAc‐shell of the ethanol‐saline group as compared to the water‐saline group (p < 0.05), while treatment with MC‐100093 increased the expression of xCT (p < 0.001) (Fig.33A). In female P rats, there was a significant difference in xCT expression among the three groups in the NAc‐shell [F(2, 12) = 8.526, p = 0.0050] (Fig.34B). Newman‐Keuls post‐hoc analysis showed a significant upregulation in the expression of xCT in the MC‐100093 treated group as compared to ethanol‐saline and watersaline groups (p<0.01), whereas no significant changes in the expression of xCT was found in the ethanol‐saline group as compared to the water‐saline group (Fig. 34B). Effect of MC-100093 on the expression of GLT-1 in the NAc-core of male and female P rats In male P rats, one‐way ANOVA showed a significant difference in GLT‐1 expression in the NAc‐core among all tested groups [F(2, 12) = 10.12, p = 0.0027] (Fig.33C). Newman‐Keuls post‐hoc analysis revealed that GLT‐1 expression was significantly increased in group treated with MC‐100093 as compared to the ethanol‐saline and water‐saline groups (p < 0.01 and p < 0.05 respectively) (Fig. 33C). There was no difference in the expression of GLT‐1 in ethanol‐saline group compared to water group. Furthermore, there was a significant difference in GLT‐1 expression among the three groups in the NAc‐core of female P rats [F(2, 12)= 5.176, p = 0.0239] (Fig.34C). Newman‐Keuls post‐hoc analysis showed a significant upregulation of GLT‐1 expression in the NAc‐core of MC‐100093‐treated female rats (p < 0.05) (Fig.34C). No significant changes were detected between ethanol‐saline and water‐saline groups in the expression of GLT‐1 in NAc‐core of female P rats. Attorney Docket No.206017-0285-00WO Effect of MC-100093 on the expression of xCT in the NAc-core of male and female P rats One‐way ANOVA revealed a significant difference in the expression of xCT in the NAc‐core of male P rats among all tested groups [F(2, 12) = 14.52, p < 0.0006] (Fig.33D). Newman‐Keuls revealed a significant decrease in the expression of xCT in the NAc‐core of the ethanol‐saline group as compared to the water‐control group (p < 0.05), while treatment with MC‐100093 reversed the effect of ethanol, and significantly increased the expression of xCT (p < 0.001). In female P rats, there was a significant difference in xCT expression among the three groups in the NAc‐core [F(2, 12)= 13.87, p < 0.0008] (Fig.34D). Newman‐Keuls post‐hoc analysis showed a significant upregulation of xCT expression in ethanol‐MC‐100093 group as compared to ethanolsaline and water‐saline groups (p < 0.001 and p < 0.01 respectively), whereas no significant changes in the expression of xCT was found in the ethanol‐ saline group as compared to the water‐saline group. Discussion The present findings demonstrate that treatment with MC‐100093 at a dose of 100 mg / kg (i.p.) reduced ethanol intake in male P rats, and for the first time it is reported that MC‐100093 is effective in reducing ethanol consumption in female P rats. These results are in parallel with a previous study that showed MC‐100093 reduced ethanol intake in male P rats with a dose of 50 mg / kg (i.p.), which compared the effect of MC‐100093 at a dose of 50 mg / kg in attenuating ethanol drinking to ceftriaxone. The study also compared the effect of MC‐100093 and ceftriaxone on GLT‐1 and xCT expression in the subregion of NAc shell in male P rats. MC‐100093 (50 mg / kg, i.p.) attenuated moderately ethanol intake as compared to ceftriaxone at a dose of 200 mg / kg, i.p. In part, this study focused on testing higher dose of MC‐ 100093 (100 mg / kg, i.p.) as well as on the neurocircuits of PFC and NAc subregions regarding the expression of GLT‐1 and xCT. Importantly, the effects of ethanol consumption and MC‐100093 at dose of 100 mg / kg were investigated in these neurocircuits to determine for any potential sex difference. Thus, this study included female P rats for the first time and examined the effects of ethanol and MC‐100093 in the expression of GLT‐1 and xCT in the neurocircuits involving the subregions of mPFC and NAc for comparison with male P rats. MC‐100093‐induced reduction in ethanol intake was associated with upregulation of GLT‐1 expression in the Attorney Docket No.206017-0285-00WO subregions of the mPFC and NAc. The reduction of ethanol drinking was associated with a decrease in ethanol preference starting from Day 1 throughout Day 5 in the ethanol‐MC‐100093 group as compared to the ethanol‐saline group. It is important to note that the reduction of ethanol consumption was associated with a significant increase in water intake in the ethanol‐MC‐100093 group. This suggests that ethanol‐ MC‐100093 group tend to have less preference to ethanol (Fig.29B and Fig.30B), and rats tend to consume more water as compared to ethanol‐saline group (Fig.29C and Fig.30C). Moreover, MC‐100093 treatment showed no effect on the body weight of male and female P rats, which indicates its specificity in reducing ethanol consumption. This is in line with a previous study, showing that MC‐100093 has no effect on body weight and sucrose intake. In addition, other beta lactam antibiotic, ceftriaxone was proven not to influence body weight or sucrose intake (Sari et al., 2011, Alcohol Alcohol.46, 239; Qrunfleh et al., 2013, J. Psychopharmacol.27, 541). This indicates the MC‐100093 specificity in reducing ethanol consumption without affecting body weight. Nevertheless, it is noteworthy that MC-100093 was associated with a higher attenuation effect on ethanol consumption in male P rats exceeding female P rats. This effect was apparent and continued to increase daily in reducing ethanol intake through the end of the experiment. Whereas MC‐100093 attenuation effect in female remained by some means equal across the last three days. Furthermore, a previous study investigated ethanol consumption between male and female P rats, showed that female P rats tend to consume more ethanol than male P rats (Bell et al., 2011, Pharmacol. Biochem. Behav 100, 90). The mesocorticolimbic pathway plays an important role in mediating the rewarding effects of drugs of abuse, including ethanol. The mPFC sends and receives glutamatergic projections into the NAc and other brain regions (Mcdonald et al., 1996, Neuroscience 71, 55). Therefore, consumption of ethanol is linked to the dysregulation of the glutamatergic pathways in the mesocorticolimbic system, specifically in the mPFC and NAc. (Kapasova and Szumlinski, 2008, Alcohol. Clin. Exp. Res.32, 617; Ding et al., 2012, Alcohol. Clin. Exp. Res.36, 633; Ding et al., 2013, Addict. Biol.18, 297). Microdialysis studies in rats revealed increased of extracellular glutamate concentrations following chronic ethanol consumption in the NAc. Furthermore, magnetic resonance spectroscopy (MRS) studies in rats showed an increase in glutamate levels in the mPFC (Hermann et al., 2012, Biol. Psychiatry 71, 1015). MRS studies in clinical settings also showed that ethanol dependent Attorney Docket No.206017-0285-00WO individuals had an increased glutamate levels in the mPFC (Frye et al., 2016, J. Clin. Psychopharmacol.36, 669) and NAc (Bauer et al., 2013, Neuropsychopharmacology 38, 1401) compared to healthy individuals. Thus, NAc and mPFC were chosen as target brain reward regions. Several astroglial glutamate transporters, GLT‐1 and xCT, which regulate extracellul...

Claims

Attorney Docket No.206017-0285-00WO CLAIMS What is claimed is:

1. A method of treating a substance use disorder in a subject, comprising the step of administering to the subject a therapeutically effective amount of a composition comprising a compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof2. The method of claim 1, wherein the composition further comprises ceftriaxone.

3. The method of claim 1, wherein the substance use disorder comprises abuse of a drug selected from the group consisting of opioids, nicotine, alcohol, fentanyl, marijuana, synthetic cannabinoids, stimulants, barbiturates, benzodiazepines, dextromethorphan (DXM), a sleep medication, khat, synthetic cathinones, cocaine, 3,4-methylenedioxymethamphetamine (MDMA), phencyclidine (PCP), lysergic acid diethylamide (LSD), psilocybin, an inhalant, Rohypnol, gamma-hydroxybutyric acid (GHB), N,N-Dimethyltryptamine (DMT), ayahuasca, mescaline, salvia, and combinations thereof.

4. The method of claim 1, wherein the substance use disorder comprises abuse of opioids.

5. The method of claim 1, wherein the substance use disorder comprises abuse of nicotine.Attorney Docket No.206017-0285-00WO 6. The method of claim 1, wherein the substance use disorder comprises abuse of fentanyl.

7. The method of claim 1, wherein the substance use disorder comprises abuse of a drug which down-regulates a molecule selected from the group consisting of xCT, glutamate transporter 1 (GLT-1), and brain-derived neurotrophic factor (BDNF).

8. The method of claim 1, wherein the substance use disorder comprises abuse of a drug which up-regulates a molecule selected from metabotropic glutamate receptor 1 (mGluR1), tumor necrosis factor alpha (TNF-α), and high mobility group box 1 (HMGB1).

9. The method of claim 1, wherein the substance abuse disorder comprises abuse of hydrocodone (dihydrocodeinone), heroin, codeine, fentanyl, hydromorphone, meperidine, methadone, morphine, oxycodone, or oxymorphone.

10. The method of claim 1, wherein the method reduces a symptom of withdrawal in the subject.

11. The method of claim 1, wherein the method prevents a relapse of the substance use disorder in the subject.

12. The method of claim 1, wherein the method reduces dependence on a substance of the substance use disorder in the subject.

13. The method of claim 1, wherein the composition further comprises an additional therapeutic agent.

14. The method of claim 13, wherein the additional therapeutic agent is selected from the group consisting of an opioid antagonist, a mixed opioid partial agonist / antagonist, an antidepressant, an antiepileptic, an antiemetic, a corticotrophin- releasing factor-1 (CRF-1) receptor antagonist, a selective serotonin-3 (5-HT3) antagonist, a 5-HT2A / 2C antagonist, a cannabinoid-1 (CB1) receptor antagonist, and combinations thereof.Attorney Docket No.206017-0285-00WO 15. The method of claim 1, wherein the method further comprises the step of exposing the subject to an additional pharmacotherapy.

16. The method of claim 14, wherein the additional pharmacotherapy comprises a gradually reducing regimen, a substitution therapy, or a medication assisted treatment.

17. A composition for use against overdose from use of a substance comprising ceftriaxone and a compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof18. The composition of claim 17, wherein the substance is an opioid, alcohol, nicotine, fentanyl, or combinations thereof.

19. The composition of claim 17, wherein the β-lactam is MC-100093.

20. A method of treating an overdose from an opioid in a subject comprising the step of administering to the subject the composition of claim 17.

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