Troriluzole for the treatment of substance use disorder

Abstract

A method of treating a substance use disorder in a patient in need thereof includes administering to the patient a therapeutically effective amount of troriluzole, wherein the substance use disorder is opioid use disorder or nicotine use disorder. Administration of troriluzole can reduce substance seeking behavior, relapse, alleviate substance withdrawal symptoms, or a combination thereof.

Description

TREATMENT OF SUBSTANCE USE DISORDERCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims priority to U.S. Provisional Application No. 63 / 790,822, filed April 18, 2025, and all the benefits accruing therefrom under 35 U.S.C. § 119, the content of which is incorporated herein by reference in its entirety.GOVERNMENT SUPPORTThis invention was made with government support under grants R01DA046526, R21DA044479, R03DA045881, R21DA049130, R01DA051205, R01DA045499, and P30DA013429 awarded by the National Institute on Drug Abuse. The government has certain rights in the invention.TECHNICAL FIELD

[0001] The present disclosure relates to methods for treating substance use disorder. More particularly, the disclosure relates to methods of treating opioid use disorder and nicotine use disorder.BACKGROUND

[0002] Substance use disorders, including opioid use disorder and nicotine use disorder, represent major public health challenges. Since the 1990s, increased prescribing of opioids, together with misuse of illicit fentanyl and its analogs, has contributed to a widespread opioid epidemic. In recent years, drug overdose deaths in the United States have exceeded 100,000 annually, with opioids accounting for the majority of such fatalities. Nicotine use disorder similarly remains a leading cause of preventable morbidity and mortality worldwide, with high rates of relapse despite the availability of smoking cessation therapies.

[0003] Current pharmacotherapies for opioid use disorder primarily target opioid receptors and include opioid agonists and partial agonists. While effective in some patients, these approaches are associated with limitations, including development of tolerance and physical dependence, respiratory depression, and abuse liability. Likewise, pharmacotherapies for smoking cessation, including nicotine replacement therapies and centrally acting agents such as varenicline and bupropion, demonstrate only modest efficacy and may be associated with adverse effects or sex dependent variability in treatment response.

[0004] Accordingly, there remains a need for improved methods for treating substance use disorders. It would be advantageous to provide therapies that reduce substance seeking, relapse, and associated adverse effects without introducing reinforcing or abuse related properties.120321-WO-PCT / 103241-023PCTSUMMARY

[0005] In an aspect, a method of treating a substance use disorder in a patient in need thereof includes administering to the patient a therapeutically effective amount of troriluzole, wherein the substance use disorder is opioid use disorder or nicotine use disorder.

[0006] In another aspect, a method of treating pain in a patient in need thereof includes administering to the patient a therapeutically effective amount of troriluzole and an opioid analgesic.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] These and / or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings.

[0008] FIGS. 1A-1E illustrate the effects of troriluzole (TRLZ) on oxycodone selfadministration (SA) under fixed-ratio 1 (FR1) and progressive ratio (PR) schedules of reinforcement.

[0009] FIG. 1A shows the average number of oxycodone self-administration under an FR1 reinforcement schedule in the presence or absence of TRLZ.

[0010] FIG. IB shows representative event records of oxycodone self-administration, wherein animals treated with vehicle or a lower dose of TRLZ maintained a regulated pattern of oxycodone self-administration, while a higher dose of TRLZ disrupted this pattern.

[0011] FIG. 1C shows the average number of active and inactive lever presses during oxycodone self-administration following treatment with TRLZ or a vehicle.

[0012] FIG. ID shows the number of oxycodone infusions obtained under a PR schedule of reinforcement in the presence or absence of TRLZ treatment.

[0013] FIG. IE shows breakpoints obtained under a PR schedule of reinforcement in the presence or absence of TRLZ treatment.

[0014] FIGS. 2A-2D illustrate the effects of TRLZ on cue-induced reinstatement of oxycodone seeking.

[0015] FIGS. 2A and 2B show the average numbers of active and inactive lever presses during 20 extinction sessions, respectively.

[0016] FIGS. 2C and 2D show the average numbers of active and inactive lever presses, respectively, during cue-induced reinstatement following presentation of drug-associated light and tone cues in the presence or absence of TRLZ.

[0017] FIGS. 3A-3C illustrate the effects of TRLZ on morphine-induced changes in respiratory parameters.220321-WO-PCT / 103241-023PCT

[0018] FIG.3A shows peripheral capillary oxygen saturation (percent, %) over time (minute, min) following acute morphine administration, with or without pretreatment with TRLZ.

[0019] FIG.3B shows respiratory rate measurements (percent, %) following administration of morphine alone or in combination with TRLZ.

[0020] FIG.3C shows the area under the curve for peripheral capillary oxygen saturation (AUC) following treatment with vehicle plus morphine or TRLZ plus morphine.

[0021] FIGS.4A-4D illustrate the effects of TRLZ on antinociceptive tolerance during repeated morphine exposure.

[0022] FIGS.4A and 4B show the time course of morphine's antinociceptive effect (percent of maximum possible effect, % MPE) in the presence of TRLZ for days 1 and 5, respectively.

[0023] FIGS.4C and 4D show antinociceptive responses (% MPE) measured at 30 minutes and 60 minutes, respectively, on day 1 and day 5 following treatment with vehicle or morphine, with or without TRLZ.

[0024] FIGS. 5A and 5B illustrate the effect of TRLZ on naloxone precipitated withdrawal signs in morphine-dependent animals.

[0025] FIG.5A shows changes in body weight (gram, g) following naloxone-precipitated withdrawal in animals with a history of morphine exposure who were pretreated with vehicle or TRLZ.

[0026] FIG.5B shows global withdrawal scores following naloxone administration in morphine-dependent rats pretreated with vehicle or TRLZ.

[0027] FIGS. 6A-6D illustrate the effects of TRLZ treatment on nicotine self-administration.

[0028] FIG.6A shows the experimental timeline for the nicotine self-administration study.

[0029] FIG.6B shows lever presses as a function of future treatment group across selfadministration sessions.

[0030] FIG.6C shows the percentage of active lever presses per group across the 15 selfadministration sessions, wherein the dotted line represents the 2:1 active:inactive lever press criterion, or 66.67% active lever pressing.

[0031] FIG.6D shows nicotine consumption (milligram per kilogram, mg / kg) as a function of future treatment group during self-administration sessions.

[0032] FIGS. 7A and 7B illustrate the effects of chronic TRLZ treatment on extinction and reinstatement of nicotine-seeking behavior. #p<0.0001 main effect of lever; ****p<0.0001 main effect of session; shown below x axis; §p<0.05 treatment x lever; ¥p<0.0001 session x lever.320321-WO-PCT / 103241-023PCT

[0033] FIG. 7A shows active and inactive lever presses as a function of treatment group throughout the 14-day extinction phase, wherein the dotted line represents a 40 active lever press criterion.

[0034] FIG. 7B shows lever presses during cue-induced reinstatement testing as a function of treatment group, compared with responding on the final day of extinction.

[0035] FIGS. 8A-8C illustrate the effects of TRLZ on responding for saline-paired cues following non-contingent saline administration. **p<0.01 main effect of session, shown below x axis; #p<0.001 main effect of lever; ¥p<0.05 session x lever.

[0036] FIG. 8A shows active and inactive lever presses during non-contingent saline administration.

[0037] FIG. 8B shows active and inactive lever presses during "extinction" from saline.

[0038] FIG. 8C shows active and inactive lever presses during a contingent saline-paired cue test in saline control animals.

[0039] FIGS. 9A-9D illustrate the effects of chronic TRLZ on protein expression of glutamate biomarkers in the nucleus accumbens (NAc) and prefrontal cortex (PFC) of rats following nicotine reinstatement. ***p<0.001, **p<0.01 or *p<0.05 versus specific treatment group as determined by Tukey's post-hoc analysis.

[0040] FIGS. 9A and 9C show relative protein expression of Navi.6, GLT1 and glutaminase (GLS1, GLS2) in the NAc and PFC, relatively, normalized to -actin.

[0041] FIG. 9B and 9D show representative Western blots of glutamate biomarkers in the NAc and PFC, respectively.

[0042] FIGS. 10A and 10B illustrate the effects of chronic troriluzole (TRLZ) treatment on levels of multiple cytokines, including TNFa, IFNy, CXCL1, IL-ip, IL-4, IL-5, IL-6, IL-10, and IL-13, in the NAc (FIG. 10A) and PFC (FIG. 10B) of rats following nicotine reinstatement, with data expressed as protein concentration (pg / mg). ***p<0.001, **p<0.01 or *p<0.05 versus specific treatment group as determined by Tukey's post-hoc analysis.DETAILED DESCRIPTION

[0043] The present methods, compounds, compositions, or disorders are not limited to specific methods, compounds, compositions, or disorders described or exemplified herein. It is also to be understood that the terminology used herein is for the purpose of describing and is not intended to be limiting.420321-WO-PCT / 103241-023PCTTERMINOLOGY

[0044] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this application belongs. However, if a term in the present application contradicts or conflicts with a term in the incorporated reference, the term from the present application takes precedence over the conflicting term from the incorporated reference.

[0045] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other.

[0046] The terms "a" and "an" and "the" do not denote a limitation of quantity and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0047] "Or" means "and / or" unless clearly stated otherwise.

[0048] A "combination thereof" is open and includes any combination comprising at least one of the listed components or conditions optionally together with a like or equivalent component or condition not listed.

[0049] Unless otherwise indicated, any reference to a compound (e.g., troriluzole) herein by structure, name, or any other means includes pharmaceutically acceptable salts; alternate solid forms, such as polymorphs, solvates, hydrates, or a combination thereof.

[0050] If stereochemistry is not indicated, a name or structural representation includes any stereoisomer or any mixture of stereoisomers. Also encompassed are all isotopic variants having natural or enriched isotopic content such as deuterated compounds or other isotopically enriched forms.

[0051] The term "carrier" applied to pharmaceutical compositions of the disclosure refers to a diluent, excipient, or vehicle with which an active compound (e.g., troriluzole) is provided. A "pharmaceutically acceptable carrier" means a substance, e.g., excipient, diluent, or vehicle, which may be administered to a patient, together with troriluzole, and which does not destroy the pharmacological activity of troriluzole and is non-toxic when administered in doses sufficient to deliver a therapeutic amount of troriluzole. A carrier can include one or more than one such carrier.

[0052] A "patient" or a "subject" is a human or non-human animal, preferably a human.

[0053] "Treatment" or "treating" means alleviating, inhibiting, ameliorating, relieving, reducing, and / or slowing a condition or a disease in a subject.

[0054] The term "therapeutically effective amount" or "therapeutically effective dose" is the amount or dose sufficient to effect treatment, as defined herein, when administered to a patient or a subject in need of such treatment.520321-WO-PCT / 103241-023PCT

[0055] "Administering" or "administration" means giving, providing, applying, or dispensing by any suitable route.

[0056] "Substance use disorder" refers to a condition characterized by compulsive use of a substance, loss of control over intake, continued use despite adverse consequences, craving or substance seeking, relapse, physical dependence, tolerance, withdrawal, or a combination thereof, and includes opioid use disorder and nicotine use disorder as described herein.

[0057] "Opioid use disorder" refers to a disorder associated with use of one or more opioid compounds, including prescription opioids and non-prescription opioids, and encompasses behaviors and physiological states including drug seeking, relapse, physical dependence, tolerance, and withdrawal.

[0058] "Nicotine use disorder" refers to dependence on nicotine arising from use of nicotine-containing products, including tobacco products, electronic nicotine delivery systems, or other nicotine sources, and includes relapse-like behavior, craving, and withdrawal.

[0059] A "period of non-use of the substance" refers to a time during which the subject is not actively consuming the substance, including periods following cessation, detoxification, withdrawal, or abstinence.

[0060] "Following cessation of substance intake" includes administration after voluntary or involuntary discontinuation of the substance and encompasses early abstinence, post-withdrawal, and recovery phases.

[0061] "Concurrent with opioid exposure" refers to administration of troriluzole before, during, or overlapping with opioid use, including during active opioid consumption or maintenance therapy.

[0062] "Non-prescription opioid" refers to opioids not lawfully prescribed for the subject.

[0063] "Prescription opioid" refers to an opioid compound approved for medical use and prescribed for pain management or other therapeutic indications.

[0064] "Alleviate substance withdrawal symptoms" refers to a reduction in the severity, frequency, or duration of one or more physiological or behavioral signs associated with discontinuation or sudden decrease in intake of the substance.

[0065] "Relapse" refers to the resumption of substance use or drug-seeking behavior following a period of non-use, including cue-induced or context-induced reinstatement.620321-WO-PCT / 103241-023PCTSUBSTANCE USE DISORDER

[0066] The disclosure provides a method of treating a substance use disorder in a patient in need thereof by administering a therapeutically effective amount of troriluzole. The substance use disorder includes opioid use disorder or nicotine use disorder.

[0067] Administration of troriluzole can reduce substance seeking behavior, relapse, and / or associated adverse effects, alleviate substance withdrawal symptoms, or a combination thereof, without introducing reinforcing or abuse related properties.

[0068] In certain embodiments, administration of troriluzole alleviates one or more withdrawal symptoms associated with discontinuation or a sudden decrease in intake of the substance. Such withdrawal symptoms may include nausea, vomiting, diarrhea, fever, sweating, headaches, insomnia, muscle cramping, abdominal cramping, running nose, increased tearing, dilated pupils, mood changes, anxiety, agitation, tremors, or a combination thereof.

[0069] Such withdrawal symptoms can be uncomfortable and distressing and, when unmanaged, may increase the risk of returning to substance use. In particular, unassisted withdrawal may lead to relapse. After withdrawal, reduced tolerance to the substance can increase the risk of adverse outcomes if the substance is used again. Administration of troriluzole may reduce relapse, which may in turn reduce the risk of adverse outcomes.

[0070] Troriluzole may be administered during a period of non-use of the substance. For example, troriluzole can be administered following cessation of substance intake to reduce relapse or cue-induced reinstatement. Troriluzole may also be administered concurrently with substance exposure.

[0071] As described herein, the method treats opioid use disorder, which is associated with the use of opioids. Opioids can include naturally derived compounds from the poppy plant as well as semi synthetic and synthetic opioid compounds, including, for example, opium, morphine, heroin, oxycodone, hydromorphone, methadone, codeine, and related substances. Although many opioids are commonly prescribed for therapeutic purposes such as pain management, cough suppression, or treatment of diarrhea, opioid exposure may also produce sedative, anxiolytic, or euphoric effects. These effects can promote continued use despite the development of adverse consequences. Such consequences may include dose escalation to maintain desired effects, which increases the risk of respiratory depression and may result in fatal overdose.

[0072] Administration of troriluzole can reduce one or more of the following: opioid selfadministration, reinforcing efficacy of the opioid, cue-induced reinstatement of opioid seeking, opioid physical dependence, development of opioid tolerance, or opioid-induced respiratory720321-WO-PCT / 103241-023PCTdepression. In an embodiment, troriluzole prevents or reduces opioid-induced decrease in blood oxygen saturation.

[0073] In some embodiments, administration of troriluzole reduces opioid selfadministration. Reduced self-administration indicates a reduction in the rewarding or reinforcing effects of opioids. Such reduction can decrease voluntary opioid intake and motivation to use the opioid, consistent with therapeutic objectives in the treatment of opioid use disorder.

[0074] Administration of troriluzole may also reduce cue-induced reinstatement of opioid seeking. Cue-induced reinstatement is a relapse-like opioid seeking behavior that occurs when a subject is exposed to environmental or contextual cues previously associated with opioid use. A decrease in cue-induced reinstatement may lead to reduced relapse-like behavior following periods of non-use.

[0075] In addition, administration of troriluzole may reduce opioid physical dependence. Physical dependence is a condition in which the body adapts to the presence of an opioid and requires continued opioid exposure to maintain normal physiological function. Abrupt reduction or discontinuation of the opioid may result in withdrawal symptoms. Opioid physical dependence can arise from physiological changes caused by repeated or ongoing opioid use and may develop over a relatively short period of time, including during medically prescribed opioid treatment. By reducing the development of opioid physical dependence, troriluzole may limit these physiological adaptations, thereby reducing the onset or severity of withdrawal symptoms following opioid discontinuation.

[0076] Administration of troriluzole may further reduce or prevent the development of opioid tolerance. Opioid tolerance occurs when the body becomes less responsive to an opioid over time, such that higher doses are needed to produce the same effect. Reducing tolerance may decrease the need for dose increases, which are associated with higher risks of adverse effects, physical dependence, and overdose. Limitation of tolerance development may therefore reduce escalation of opioid use and slow progression of opioid use disorder.

[0077] Administration of troriluzole may also reduce or prevent opioid induced respiratory depression. Respiratory depression, characterized by decreased blood oxygen saturation, is a primary cause of death in opioid overdose. By reducing or preventing opioid induced respiratory depression, troriluzole may lower the risk of fatal outcomes associated with opioid misuse and overdose.

[0078] Opioid use disorder can be associated with use of a non-prescription opioid, a prescription opioid, or a combination thereof. Exemplary prescription opioids include oxycodone,820321-WO-PCT / 103241-023PCTmorphine, hydrocodone, fentanyl, oxymorphone, hydromorphone, codeine, tramadol, and combinations thereof. In particular embodiments, the prescription opioid is oxycodone or morphine.

[0079] When the opioid use disorder is associated with use of oxycodone, administration of troriluzole can reduce one or more of: oxycodone self-administration, reinforcing efficacy of oxycodone, cue-induced reinstatement of oxycodone seeking, oxycodone physical dependence, development of oxycodone analgesic or antinociceptive tolerance, or oxycodone-induced respiratory depression.

[0080] When the opioid use disorder is associated with use of morphine, administration of troriluzole can reduce one or more of: morphine-induced respiratory depression, development of analgesic or antinociceptive tolerance to morphine, or development of morphine physical dependence.

[0081] In other embodiments, the method treats nicotine use disorder. Advantageously, administration of troriluzole can reduce relapse to nicotine use. Administration of troriluzole can also reduce cue-induced reinstatement of nicotine seeking. Troriluzole can be administered following cessation of nicotine intake.

[0082] In the method disclosed herein for treating nicotine use disorder, administration of troriluzole may modulate neuroinflammatory signaling associated with substance seeking behavior. Alternatively or in addition, administration of troriluzole may normalize nicotine-induced dysfunction of corticolimbic cytokine signaling.TREATMENT OF PAIN

[0083] In an aspect, the present disclosure provides a method for treating pain in a patient in need thereof. The method comprises administering to the patient a therapeutically effective amount of troriluzole and an opioid analgesic. The opioid analgesic can comprise morphine, oxycodone, hydrocodone, fentanyl, hydromorphone, tramadol, or a combination thereof.

[0084] Troriluzole can be administered prior to opioid analgesic administration, simultaneously with opioid administration, or following initiation of opioid therapy. The patient may or may not have opioid use disorder. In some embodiments, the patient does not exhibit opioid dependence, opioid use disorder, or compulsive drug-seeking behavior prior to treatment.

[0085] It has been discovered that administration of troriluzole may attenuate the development of opioid analgesic tolerance, preserves opioid antinociceptive efficacy during repeated dosing, reduces opioid-induced respiratory depression, or a combination thereof. These effects support the use of troriluzole as an adjunct or co-therapy for pain management.

[0086] The pain that can be treated may comprise acute pain, chronic pain, post-operative920321-WO-PCT / 103241-023PCTpain, inflammatory pain, neuropathic pain, cancer-related pain, pain associated with medical procedures, or a combination thereof.

[0087] Oxycodone is one example of an opioid analgesic that may be co administered with troriluzole for pain treatment. Exemplary oxycodone formulations include immediate release oral tablets, capsules, extended release tablets or capsules, and oral solutions. Oxycodone may be administered in daily doses within ranges commonly used for pain management, such as from 5 mg to 240 mg per day, including divided doses or extended release formulations. Initial dosing may include administrations of 5 mg to 10 mg per dose, with dosing intervals selected based on patient response. Oxycodone can be delivered orally. Other delivery methods include intravenous, intramuscular, intranasal, rectal, and epidural administration.

[0088] Hydrocodone represents another opioid analgesic suitable for use in combination with troriluzole. Hydrocodone may be administered in amounts commonly used for pain treatment, including initial doses of 5 mg to 10 mg per administration, with subsequent titration based on analgesic response. Hydrocodone may be provided alone or in combination with acetaminophen, with total daily acetaminophen exposure selected within acceptable limits. Hydrocodone can be administered orally.

[0089] Hydromorphone may also be used as the opioid analgesic. Exemplary dosing includes parenteral administration in amounts of 0.2 mg to 0.6 mg per dose, as well as oral administration in amounts of 2 mg per dose. Dosing intervals may be selected based on clinical response and patient tolerance. Other known dosage amounts and interval may also be used. Hydromorphone may be delivered through oral, rectal, and injectable routes.

[0090] Methadone may likewise be employed as the opioid analgesic in combination with troriluzole. Methadone dosing may be selected based on prior opioid exposure or morphine equivalent dosing, with conversion ratios applied as appropriate. Calculated methadone doses may be reduced relative to morphine equivalent amounts to account for methadone potency and accumulation. Methadone may be primarily delivered orally as a liquid concentrate, syrup, or dispersible tablet. It can also be administered via injection, rectal, or other methods.

[0091] Morphine may also be used as the opioid analgesic in combination with troriluzole for the treatment of pain. Suitable morphine formulations include oral extended release capsules, extended release tablets, immediate release tablets, and oral solutions. Morphine dosing may vary depending on factors such as formulation, prior opioid exposure, dosing frequency, and the condition being treated. For example, extended release oral morphine formulations may be administered at dosing intervals of about every 8, 12, or 24 hours, with total daily doses selected within ranges commonly used for moderate to severe pain management. Immediate release oral1020321-WO-PCT / 103241-023PCTmorphine formulations may be administered in divided doses, such as doses of about 10 mg to about 30 mg per administration at dosing intervals selected based on patient response. The total daily amount of morphine, dosing frequency, and treatment duration may be adjusted as appropriate to achieve analgesia while limiting adverse effects. Morphine may be delivered through various routes depending on the pain severity and patient needs, including oral (tablets, capsules, solutions), injection (intravenous, subcutaneous, epidural, intrathecal), sublingual, and rectal suppository.

[0092] The administration and dosage of troriluzole are described in detail below.

[0093] In certain embodiments, troriluzole is administered concurrently with an opioid analgesic to reduce development of analgesic tolerance, thereby maintaining effective pain control at lower or stable opioid doses over time.

[0094] Administration of troriluzole may also reduce the dose escalation of an opioid analgesic required to achieve analgesia. In certain embodiments, administration of troriluzole may reduce opioid-induced respiratory depression while preserving antinociceptive efficacy.TRORILUZOLE

[0095] Troriluzole is a prodrug of riluzole and can be converted to riluzole or metabolized into an active form of riluzole by the body. Troriluzole has the following structure:Troriluzole is described, for example, in U.S. Patent No. 10,485,791. As used herein, any reference to troriluzole herein includes pharmaceutically acceptable salts; alternate solid forms, such as polymorphs, solvates, hydrates, or a combination thereof. For example, troriluzole can include troriluzole dihydrochloride, troriluzole dihydrochloride monohydrate, troriluzole hydrochloride (one HCI), troriluzole hydrochloride monohydrate, or a combination thereof, specifically troriluzole dihydrochloride monohydrate.ADMINISTRATION AND DOSAGE

[0096] In certain embodiments, administration of troriluzole comprises administering a pharmaceutical composition comprising troriluzole and one or more pharmaceutically acceptable carriers.

[0097] The pharmaceutical composition can be in the form of a dosage form that includes troriluzole in an amount effective to treat a substance use disorder or pain in a patient in need1120321-WO-PCT / 103241-023PCTthereof. The amount of troriluzole in the pharmaceutical composition may be 50 mg to 450 mg, 100 mg to 450 mg, 120 mg to 450 mg, 140 mg to 450 mg 160 mg to 450 mg, 180 mg to 450 mg, 60 mg to 400 mg, 70 mg to 350 mg, 70 mg to 300 mg, 100 mg to 200 mg, 200 mg to 280 mg, 100 mg, 125 mg, 140 mg, 200 mg, or 280 mg.

[0098] The pharmaceutical composition may further include a pharmaceutically acceptable carrier. Examples of such carriers are well known to those skilled in the art and can be prepared in accordance with acceptable pharmaceutical procedures, such as, for example, those described in Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is incorporated by reference herein for all purposes.

[0099] Pharmaceutically acceptable carriers that may be used in the pharmaceutical composition can include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as a-tocopherol, polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as a-, P-, and y-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-p-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of troriluzole.

[0100] The pharmaceutical composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The pharmaceutical compositions may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated troriluzole or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0101] The pharmaceutical composition may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension1220321-WO-PCT / 103241-023PCTmay be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as those described in Pharmacopeia Helvetica or a similar alcohol, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and / or suspensions. Other commonly used surfactants such as Tweens or Spans and / or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

[0102] The pharmaceutical composition may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents may be added.

[0103] The pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., troriluzole) that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of troriluzole that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range in some embodiments from 1 percent to ninety-nine percent of active ingredient, in some embodiments from 5 percent to 70 percent, and in some embodiments from 10 percent to about 30 percent. Such unit dosage form can contain from 50 mg to 450 mg, 100 mg to 450 mg, 120 mg to 450 mg, 140 mg to 450 mg 160 mg to 450 mg, 180 mg to1320321-WO-PCT / 103241-023PCT450 mg, 60 mg to 400 mg, 70 mg to 350 mg, 70 mg to 300 mg, 100 mg to 200 mg, 200 mg to 280 mg, 100 mg, 125 mg, 140 mg, 200 mg, or 280 mg of troriluzole.

[0104] The selected dosage level will depend upon a variety of factors including the activity of troriluzole, the route of administration, the time of administration, the rate of excretion of troriluzole, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, gender, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0105] A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of troriluzole employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0106] In general, a suitable daily dose of troriluzole will be the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0107] If desired, the effective daily dose of troriluzole may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, troriluzole may be administered two or three times daily. In some embodiments, troriluzole will be administered once daily.

[0108] Optionally, troriluzole can be administered alone or co-administered with another therapeutic agent. As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic compounds such that the desired effect is obtained. For example, the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). The different therapeutic compounds may be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. Thus, an individual who receives such treatment may benefit from a combined effect of different therapeutic compounds. Co-administration includes simultaneous or sequential administration of two or more compounds.

[0109] To treat opioid use disorder, in certain embodiments, troriluzole is co-administered with a partial opioid agonist, opioid agonist, a non-opioid medication, or a combination thereof. The co-administered drug can include buprenorphine, methadone, naltrexone, lofexidine, or clonidine,1420321-WO-PCT / 103241-023PCTfor example. To treat nicotine use disorder, troriluzole may be used together with a nicotine replacement therapy which provides low doses of nicotine without the harmful chemicals in tobacco to ease withdrawal, non-nicotine medications, such as varenicline or bupropion.

[0110] The invention is further illustrated by the following non-limiting examples.EXAMPLES EFFECTS OF TRORILUZOLE ON OPIOID USE DISORDERMethodsAnimals

[0111] Male Long-Evans rats (Charles River, Frederick, MD) (initially weighing 250-300 g) were used in self-administration (SA) experiments. During experimentation, rats were housed individually in a climate-controlled animal colony room with 21-239C temperature and 40-60% humidity, on a reversed 12-h light-dark cycle (lights on at 7am) with access to food and water. Experiments were conducted during the active period (dark cycle).

[0112] For physical dependence, respiratory depression, and tolerance studies, male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) (initially weighing 250-300 g) were used. Housing conditions and animal care were consistent with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Protocols used experiments were approved by the Institutional Animal Care and Use Committees at the National Institute on Drug Abuse Intramural Research Program and Temple University.Surgery for SA studies

[0113] Under standard aseptic conditions and xylazine / ketamine (90 / 10 mg kg1, ip) anesthesia, rats were prepared for oxycodone SA, as described (Galaj et al, Neuropsychopharmacology, 2022, 47, 2309-2318; and Galaj et al., Journal of Neuroscience, 2020, 40 (36), 8853-8869). Briefly, a microrenathane catheter (Braintree Scientific Inc., Braintree, MA, US) was implanted in the right jugular vein and its free end was fed subcutaneously along the scapula and connected to a 22-gauge stainless connector that was mounted to the rat's skull using four stainless steel screws and dental acrylic. To prevent clogging, catheters were flushed daily with a gentamicin-heparin-saline solution (30 lU / ml heparin; ICN Biochemicals, Cleveland, OH, USA) immediately after surgery and on subsequent days.

[0114] Intravenous oxycodone SA sessions were conducted in standard operant conditioning chambers (Med-Associates, VT, US), each housed in a sound-attenuating box. Each1520321-WO-PCT / 103241-023PCTchamber was equipped with two retractable levers, a white light and tone above the active lever and a drug line connected to a syringe pump (PHM-100, 3.33 rpm; Med-Associates, VT, US).Materials

[0115] Oxycodone hydrochloride (provided by NIDA IRP Pharmacy) was dissolved in 0.9% saline to achieve final doses of 0.05 mg / kg / infusion. Morphine was purchased from Sigma Aldrich (St. Louis, MO, US) and dissolved in saline to achieve a final dose of 5 mg / kg. Troriluzole monohydrochloride (TRLZ) was designed and synthesized by Fox Chase Chemical Diversity Center (Doylestown, PA, US) (Pelletier et al., 2018). TRLZ was dissolved in distilled water to obtain doses of 8 and 16 mg / kg that were administered (ip) 30 min before testing.Experiment 1: Effects of TRLZ on oxycodone SA under a FR1 schedule of reinforcement

[0116] The experiment investigated whether TRLZ can reduce oxycodone reward in rats selfadministering oxycodone under a fixed ratio 1 (FR1) schedule of reinforcement. Animals were trained to self-administer oxycodone (0.05 mg / kg / infusion) under an FR1 schedule of reinforcement during daily 3-h sessions. Responding on the active lever activated the syringe pump for 4.2 s, causing a delivery of oxycodone and activation of the light and tone stimuli above the active lever. Responses on the inactive lever were counted but had no consequences. During the infusion period, additional responses on the active lever were recorded, but did not lead to additional infusions. Once animals demonstrated a pattern of stable responding, defined as less than 20 % variability in daily oxycodone intake across three consecutive sessions, and an active / inactive lever pressing ratio exceeding 2:1, they were tested with TRLZ at 0, 8 or 16 mg / kg (n=8 in each group). Animals then received an additional 5-7 days of oxycodone self-administration until a baseline response rate was reestablished prior to being tested with another dose of TRLZ.Experiment 2: Effects of TRLZ on oxycodone SA under a PR schedule of reinforcement

[0117] Next, the effects of TRLZ on motivation to seek oxycodone were investigated. Rats were initially trained under an FR1 schedule of reinforcement as outlined in Experiment 1. Rats with stable responding were then allowed to self-administer oxycodone (0.05 mg / kg / infusion) under a progressive ratio (PR) schedule of reinforcement. The number of lever presses required to receive a single infusion of oxycodone increased according to the following schedule: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50 and so on. Under this PR schedule the animals eventually stopped responding and reached a break point (BP) with final ratio, operationally defined as a number of lever presses for the last oxycodone infusion before a one-hour period during which no oxycodone reward was obtained.1620321-WO-PCT / 103241-023PCTOnce responding stabilized with day-to-day variability in BP within 1 or 2 ratio increments for 3 consecutive days, animals were tested with TRLZ. Thirty min prior to a test session, rats were injected with TRLZ [0 (n=8), 8 (n=7) or 16 mg / kg (n=7)] and later allowed to press the lever for oxycodone under the same conditions. In the following days behavior was re-stabilized and 5-6 days later rats were tested with a different, randomly chosen, dose of TRLZ.Experiment 3: Effects of TRLZ on cue-induced reinstatement of oxycodone seeking

[0118] The experiment assessed whether TRLZ reduces reinstatement of oxycodone seeking. After oxycodone SA under FR1 and PR schedules of reinforcement, the extinction phase began during which responding on either lever produced no consequences; oxycodone was not delivered, and drug cues were not presented. This phase continued for 20 days. The cue-induced reinstatement test (3-hour long) occurred next day. Rats were injected with TRLZ (0, 8 or 16 mg / kg; n=7 in each group) 30 min before the test session. Non-contingent oxycodone cues (4.2s- light / tone and 4.2-s empty syringe pump) were presented twice, 2 min apart, at the beginning of a reinstatement session. Active lever presses were reinforced with the oxycodone cue (light / tone / pump) but oxycodone was not delivered. Responding on the inactive lever produced no consequences. Presses on active and inactive levers were then analyzed.Experiment 4: Effects of TRLZ on morphine-induced respiratory depression

[0119] Oxygen saturation (SpO2) and respiratory rate (RR) were measured using a MOUSEOX Plus - pulse oximeter (mouse and rat pulse oximeter, Starr Life Sciences Corp., Oakmont, PA, USA) in freely moving rats. Rats were acclimated individually in observation boxes for 30 min. Then, a collar with a sensor was placed to the neck of the rats and without any recording, rats were acclimated for another 30 min. To test if pretreatment with TRLZ had an effect on morphine-induced respiratory depression, rats were pretreated with vehicle (n=5) or TRLZ (8 mg / kg, n=6) and recording was started to determine whether TRLZ has any effect on respiration. Fifteen min later, all rats received morphine (5 mg / kg, sc) and recording was continued for another 60 min. Respiration and SpO2were recorded every second and averaged over 1-minute periods for total of 75 minutes. The dose of morphine was chosen based on previous studies showing that 5 mg / kg reduces oxygen saturation (Inan et al., Life Sci., 2021, 285, 120014; Bongiovanni et al., Drug Alcohol Depend, 2022, 238:109556).1720321-WO-PCT / 103241-023PCTExperiment 5: Effects of TRLZ on development of antinociceptive tolerance to morphine

[0120] The effect of TRLZ on the antinociceptive efficacy of morphine was measured using the hot plate assay of thermal nociception. The hot plate (Ugo Basile Sri 7280, Gemonio, Italy) with a pre-set plate temperature of 52°C was used. The assay was performed on day 1 after the first morphine injection, and on day 5 following chronic morphine administration. Two doses of morphine, 5 and 7.5 mg / kg, were studied, and were chosen based on previous studies (Morgan et al., The Journal of Pain, 2006, 7, 358-366; Paul et al., Molecules, 2021, 26 (14), 4355). On both experimental days, rats were brought to the testing room and acclimated for 1 h. Rats were assigned randomly to the following groups (n=7-8 / group): vehicle (ip) + vehicle (sc) (n=7); TRLZ (8 mg / kg, ip) + vehicle (n=7); vehicle + morphine (5 mg / kg, sc) (n=7); TRLZ + morphine (5 mg / kg) (n=7); vehicle + morphine (7.5 mg / kg) (n=8); or TRLZ + morphine (7.5 mg / kg) (n=8). TRLZ or vehicle (for TRLZ) was administered 15 min before saline or morphine. On day 1, following acclimation, baseline response latency was measured two times (1 h apart) and averaged as pre-drug latency. The response latency was the time between placing the animal on the heated plate and its reaction (licking, shaking or stepping of hind paws or jumping). Then, rats were injected with TRLZ or vehicle, and then administered saline or morphine 15 min later. Latencies were measured at 30, 60, 90, and 120 min following saline or morphine administration. The cut-off time was 60 s to prevent tissue damage. Drugs were administered 2 times (8 AM and 6 PM) daily for 4 days. On day 5, the hot plate test was repeated following administration of the morning doses. Latencies on days 1 and 5 were converted to percent of maximum possible effect (% MPE) according to the formula:% MPE = ((post drug latency - pre drug latency) / (cut-off latency - pre drug latency)) *100 Rats were euthanized at the end of the testing on day 5.Experiment 6: Effects of TRLZ on development of morphine physical dependence

[0121] Rats were implanted (subcutaneous (sc), flank area) with 75 mg morphine pellets that remained intact for 7 d. Beginning on the first day of pellet implantation and continuing throughout the 7-d implantation period, rats received daily injections of TRLZ (4, 8, or 12 mg / kg, ip, delivered in a ml kg1volume, n=8 / group). On test day, rats were transported to the testing room and allowed to acclimate for 60 min before naloxone hydrochloride (10 mg / kg, ip, delivered in a 1 ml kg1volume) was injected to precipitate withdrawal signs. Immediately after naloxone administration, rats were placed into individual observation boxes for 60 min and observed for 30 min for withdrawal signs including wet dog shakes, paw tremors, jumping, teeth chattering, and eye blinking. Signs were counted and then scored as follows: 0 = none; 1 = 1-5 times; 2 = 6-10 times; and 3 = >10 times. Global withdrawal score was calculated as sum of these scores for each group1820321-WO-PCT / 103241-023PCT(Inan et al., Brain Behav Immun., 2024, 117, 100-111). Weights were measured before and 2 h after naloxone injection. Each treatment group included eight animals, except that the highest-dose TRLZ group included seven animals in the body-weight data set.Statistical analysis

[0122] In the oxycodone intravenous self-administration (IVSA) experiment, the total numbers of infusions were averaged and compared across groups with a one-way Analysis of Variance (ANOVA). This was followed by a Dunnett's test. Active and inactive lever presses were averaged and compared across groups using a two-way ANOVA (lever x treatment). Infusions earned under a PR schedule were analyzed with a two-way ANOVA (phase x treatment). Significant interactions were followed by a Dunnett's test. For respiratory depression data, comparisons between effects produced by TRLZ and vehicle were done using a two-way ANOVA (treatment x time) followed by Sidak's multiple comparisons test. Area under the curve analysis between the 2 groups was performed using an unpaired Student's t-test. For tolerance studies, two-way ANOVA (treatment x time) followed by Tukey's multiple comparisons test were used. For physical dependence studies, one-way ANOVA was used to analyze weight gain and global withdrawal scores followed by Dunnett's test. Graph Pad Prism (version 10, La Jolla, CA, USA) was used for creation of graphs and statistical analysis.ResultsTRLZ attenuated oxycodone SA under FR1 reinforcement schedule

[0123] FIG. 1A shows the average number of oxycodone infusions self-administered by LE rats within 3-h-long sessions. As shown in FIG. 1A, TRLZ significantly reduced oxycodone SA under a FR1 reinforcement schedule (p*<0.05 as compared to vehicle). A one-way ANOVA revealed a significant treatment effect. Dunnett's post hoc test revealed a significant difference between the 16 mg / kg dose and vehicle groups, but not between the 8 mg / kg and vehicle groups. To further understand the observed TRLZ-induced reduction in oxycodone self-administration, individual event records were analyzed.

[0124] FIG. IB shows representative event records of oxycodone self-administration in the presence or absence of the TRLZ treatment. Animals treated with vehicle or a low dose of TRLZ showed a stable pattern of drug self-administration with similar inter-infusion intervals. However, rats treated with a high dose (16 mg kg1) of TRLZ displayed an extinction-like pattern of behavior, characterized by an initial high rate of response, followed by irregular drug taking or cessation of self-administration. This suggests a reduction in the rewarding effects of oxycodone.1920321-WO-PCT / 103241-023PCT

[0125] In FIG. 1C, animals treated with TRLZ show a significant and dose-dependent reduction in active, but not inactive, lever pressing, suggesting an oxycodone-specific, rather than a non-specific locomotor, effect for TRLZ (*p<0.05, **<0.01 as compared to the vehicle). A two-way ANOVA revealed a significant dose x lever interaction, which was driven by the TRLZ effect on the active lever, but not inactive lever. Post-hoc Tukey tests reveal significant differences in active lever presses between the vehicle and TRLZ-treatment groups.TRLZ attenuated oxycodone reinforcing efficacy under PR reinforcement schedule

[0126] FIG. ID shows an average number of oxycodone infusions under a PR schedule in the presence or absence of TRLZ treatment, indicating a significant reduction in oxycodone infusions after 16 mg / kg of TRLZ administration (*p<0.05 as compared to vehicle). A two-way ANOVA revealed a significant dose x phase interaction. Tests of simple effects of TRLZ treatment at each level of the phase revealed no group differences at the baseline and significant group differences at the test session. Dunnett's test revealed a significant reduction in infusions after 16 mg / kg TRLZ, but not after 8 mg / kg TRLZ.

[0127] FIG. IE shows that TRLZ resulted in a significant reduction in break points, defined as the number of lever presses required for the last infusion, under the PR reinforcement schedule (*p<0.05 as compared to the vehicle). A one-way ANOVA revealed a significant treatment effect and Dunnett's test showed a significant difference between 16 mg / kg dose and vehicle, but not between 8 mg / kg and vehicle. These data suggest TRLZ dose-dependently reduces motivation to seek oxycodone.TRLZ reduced cue-induced reinstatement of oxycodone seeking

[0128] FIGS 2A and 2B show the average numbers of active and inactive lever presses during 20 extinction sessions, respectively. Rats in all groups showed a reduction in active lever pressing over time as shown in FIG. 2A. A two-way ANOVA revealed a significant main effect of time, but no treatment effect, and no session x treatment interaction. Similarly, as indicated in FIG. 2B, rats in all groups showed a reduction in responding on the inactive lever over time. A two-way ANOVA revealed a significant main effect of time, but no treatment effect and no session x treatment interaction.

[0129] FIG.2C shows that presentation of oxycodone-associated cues induced robust reinstatement of oxycodone seeking, which was dose-dependently reduced by TRLZ. In particular, TRLZ caused a reduction in active lever pressing. A one-way ANOVA revealed a significant TRLZ2020321-WO-PCT / 103241-023PCTtreatment effect, which was driven by 8 mg / kg and 16 mg / kg doses of TRLZ (**p=0.003 and ***p=0.0003, respectively).

[0130] FIG.2D shows that TRLZ did not affect inactive lever pressing, indicative of the absence of TRLZ non-specific effects. A one-way ANOVA on the inactive lever pressing revealed no significant effect of TRLZ. These findings suggest that TRLZ causes a dose-dependent reduction in cue-induced reinstatement of drug seeking.Pretreatment with TRLZ prevented morphine-induced decrease in blood oxygen saturation

[0131] FIGS. 3A-3B show recordings for peripheral capillary oxygen saturation (SpO2) and respiratory rate, respectively. Systemic administration of morphine (5 mg kg1) significantly decreased SpO2with a peak reduction to ~80% at 65 min after morphine injection, which is indicative of hypoxia. Pretreatment with TRLZ (8 mg kg1) prevented morphine-induced reduction in SpO2. A two-way ANOVA revealed significant main effects of TRLZ treatment and time. There was no significant treatment x time interaction. Post hoc analysis revealed a significant difference between the vehicle-morphine and TRLZ-morphine groups between the 50 and 70 min time points following morphine treatment. Neither morphine nor TRLZ pretreatment significantly altered respiratory rates as shown in FIG.3B. Two-way ANOVA revealed a significant main effect of time, but no treatment effect nor time x treatment interaction.

[0132] FIG.3C shows the calculated area under the curves (AUC) of SpO2in the (vehicle + morphine) group was significantly lower than the AUC of SpO2in the (TRLZ + morphine) group(***p<0.001). TRLZ itself did not produce any effects on SpO2during the first 15 min recording as seen in FIG. 3A. For riluzole, pretreatment with riluzole (8 mg kg1) did not affect the morphine-induced reduction in SpO2.Pretreatment with TRLZ reduced development of tolerance to morphine antinociception

[0133] FIGS.4A-4D show that pretreatment with TRLZ reduced antinociceptive tolerance during repeated morphine exposure. FIGS.4A and 4B show the time course of morphine's antinociceptive effect (% MPE) in the presence of TRLZ for days 1 and 5. Over the course of 5 d, a significant tolerance developed to morphine antinociception. Since the antinociceptive effect of morphine peaked at 30-60 min and diminished at 90 and 120 min, the 30- and 60-min responses were analyzed separately.

[0134] For the 30-min response (FIG.4C), a two-way ANOVA revealed significant effects of time, and treatment, and a significant interaction. Tukey's multiple comparison analysis showed that morphine, at 5 and 7.5 mg / kg by itself and in combination with TRLZ, produced significant2120321-WO-PCT / 103241-023PCTantinociception effect on day 1 at 30 min compared to vehicle control (&&&&p<0.0001). TRLZ by itself did not produce antinociception. On day 1, there was no statistically significant difference in antinociceptive effects of morphine (5 and 7.5 mg kg1) by itself or in combination with TRLZ. On day 5, morphine at 5 mg / kg still produced a significant antinociceptive effect (&&p<0.01), but the effect was significantly lower compared to day 1 (*p<0.05). On day 5, rats treated with a combination of TRLZ and 5 mg / kg or 7.5 mg / kg morphine also still displayed an antinociceptive effect compared to drug-naive rats0.0001 and&<p 0.05, respectively), but the antinociceptive effect in both groups was diminished compared to day 10.0001 and&<p 0.05, respectively).

[0135] For the 60-min response (FIG.4D), a two-way ANOVA revealed effects of time , treatment, and a significant interaction. Post hoc analysis showed that on day 1, morphine alone at 5 and 7.5 mg / kg induced an antinociceptive effect (&p<0.05,&&&&p<0.0001, respectively), but pretreatment with TRLZ prior to morphine at 5 mg / kg produced greater antinociception compared to morphine alone (***p<0.001). On day 5, morphine at 5 and 7.5 mg / kg alone did not induce an antinociceptive effect compared to vehicle. However, on day 5, rats treated with a combination of morphine (5 mg kg1) and TRLZ displayed greater antinociception compared to drug-naive vehicle rats (&&&&p<0.0001) and rats treated with only morphine (5 mg kg1) (*p<0.05) (FIG.4D). Similarly, for the higher dose of morphine (7.5 mg kg1) on day 5, greater antinociception was produced by a combination of TRLZ and morphine compared to drug-naive rats (&&&&p<0.0001) and rats treated only with morphine (7.5 mg kg1) (****p<0.0001).TRLZ reduced development of morphine physical dependence

[0136] FIGS. 5A-5B show effects of TRLZ on naloxone-precipitated morphine withdrawal signs. Weight loss (FIG. 5A) and global withdrawal score (FIG. 5B) were lower in rats treated with TRLZ compared to rats treated with vehicle. In particular, rats with a history of morphine exposure (75 mg pellet x 7d) that were pretreated daily with TRLZ displayed less weight loss (FIG. 5A) and a lower global withdrawal score (FIG. 5B) than TRLZ-naive rats pretreated with vehicle. *p<0.05 or ****<0.0001 compared to TRLZ-naive group (0 mg kg1). Separate one-way ANOVAs revealed group differences on those measures. Rats treated with 12 mg / kg of TRLZ also showed no jumping behaviors, unlike those treated with vehicle. For riluzole, neither weight loss nor global withdrawal score following naloxone-precipitated withdrawal were significantly different in rats treated with riluzole (12 mg-kg-1) or vehicle.Discussion2220321-WO-PCT / 103241-023PCT

[0137] The effects of TRLZ, a dual glutamate release inhibitor and glutamate reuptake enhancer, on opioid-induced behaviors were evaluated in rats. TRLZ reduced oxycodone selfadministration under fixed-ratio and progressive ratio reinforcement schedules, indicating a reduction in opioid reward and motivation to seek opioids. TRLZ also reduced cue-induced reinstatement of oxycodone seeking, attenuated morphine physical dependence, and reduced development of morphine antinociceptive tolerance. In addition, TRLZ prevented opioid-induced respiratory depression, demonstrating a favorable safety profile. Collectively, the results demonstrate that TRLZ reduces opioid intake, reinforcing efficacy, and relapse-like behaviors in selfadministration assays.

[0138] Since TRLZ enhances glutamate reuptake, it is believed that TRLZ reduced oxycodone seeking behaviors by normalizing the dysregulation of glutamate signaling in the nucleus accumbens (NAC) that typically occurs during reinstatement. TRLZ further inhibit glutamate release from presynaptic terminals by blocking voltage-gated sodium channels or by opening voltage-gated calcium channels (Urbani et al., The European Journal of Neuroscience, 2000, 12, 3567-3574; Wang et al., Neuroscience, 2004, 125(1), 191-201). Accordingly, in the presence of TRLZ, excessive neuronal glutamate release during reinstatement may have also been reduced, thereby contributing to diminished oxycodone seeking.

[0139] Oxycodone acquisition and reinforcing efficacy were also reduced by TRLZ. Glutamate systems contribute to opioid reward and reinforcement. Acute rewarding effects of opioids are facilitated by increased dopamine release in the NAC. During chronic opioid exposure, however, glutamatergic input onto neurons in the NAC is enhanced, leading to a progressive increase in the opioid rewarding effect. TRLZ may have reduced oxycodone acquisition and reinforcing efficacy by attenuating glutamatergic drive onto NAC neurons during oxycodone self administration. The interpretation is consistent with evidence that disruption of glutamatergic transmission can reduce opioid self administration. Contributions from non-glutamatergic mechanisms, including direct or indirect inhibition of dopamine transmission in the NAC, or modulation of gamma-aminobutyric acid (GABA) transmission, cannot be excluded.

[0140] The data showed that TRLZ prevents development of antinociceptive tolerance to morphine. Without wishing to be bound by theory, it is believed that TRLZ preserved morphine's antinociceptive efficacy by normalizing enhanced glutamate transmission at inotropic and metabotropic glutamate receptors that facilitates development of antinociceptive tolerance. TRLZ itself was not antinociceptive in the experiments.2320321-WO-PCT / 103241-023PCT

[0141] TRLZ also reduced the severity of morphine physical dependence. TRLZ may have dampened activation of glutamate systems during chronic morphine exposure, thereby reducing naloxone-precipitated withdrawal behaviors.

[0142] TRLZ prevented the reduction in oxygen saturation after acute morphine injection, demonstrating a favorable safety profile. TRLZ may have prevented morphine-induced hypoxia by normalizing deficits in glutamate reuptake that contributed to morphine-induced decreases in oxygen saturation. Glutamate and opioid systems overlap extensively in brainstem regions that regulate breathing. Vesicular glutamate transporter 2 (VGLUT2), one of three glutamate transporters that medicate vesicular glutamate uptake, is highly expressed in brainstem cells that are vulnerable to opioid regulation (Furdui, et al., Journal of Neurophysiology, 2024, 132(1), 108-129). A substantial proportion of these VGLUT2-expressing neurons also express p-opioid receptors, indicating potential opioid-glutamate crosstalk within respiratory control circuits. Modulation of glutamatergic signaling by TRLZ within these regions may therefore contribute to the observed protection against opioid-induced respiratory depression.

[0143] In conclusion, the disclosed results demonstrate that TRLZ reduces multiple opioid derived adverse effects, including drug reward, reinforcing efficacy, relapse-like behaviors, physical dependence, analgesic tolerance, and opioid-induced respiratory depression. TRLZ simultaneously enhances glutamate reuptake and inhibits neuronal glutamate release, distinguishing it from pharmacologically-related glutamate drugs that act primarily through one mechanism, such as ceftriaxone (CTX), clavulanic acid, N-acetylcysteine. TRLZ is a prodrug of riluzole with optimized pharmacokinetic and metabolic profiles, including enhanced gastrointestinal absorption, metabolic cleavage after or during absorption in plasma, and reduced first-pass metabolism, active transport by the PepTl transporter, and a favorable safety profile. These properties address pharmacokinetic limitations associated with riluzole. Accordingly, TRLZ is suitable for use as an effective therapeutic agent for the treatment of opioid use disorder and pain, without exhibiting reinforcing or abuse-related properties.EFFECTS OF TRORILUZOLE ON NICOTINE USE DISORDER

[0144] In the current study, it is examined if repeated administration of TRLZ during abstinence (with extinction training) from nicotine self-administration (SA) and during reinstatement inhibited cue-induced nicotine seeking behavior in male rats. Specifically, the following were examined: (1) the ability of TRLZ to facilitate extinction learning , (2) the effects of TRLZ on reinstatement of nicotine seeking behavior, and (3) the ability of TRLZ to reverse nicotine-induced glutamatergic and neuroinflammatory dysregulations. It is believed that compared to vehicle-treated2420321-WO-PCT / 103241-023PCTmales, chronic treatment with TRLZ would show enhanced extinction learning and decreased nicotine seeking behavior. Further, it is believed that nicotine-induced neuroinflammatory signaling as measured by increases in proinflammatory cytokine and chemokine levels within key nodes of the mesocorticolimbic reward pathway, including the prefrontal cortex (PFC) and NAc, would be reversed by TRLZ.MATERIALS AND METHODSAnimals and Materials

[0145] Twenty-seven male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 250 g were single housed and maintained at a constant temperature (23 ± 1°C) and on a 12:12 reverse light-dark cycle (lights on at 6:00AM) with ad libitum access to water and standard laboratory chow (Envigo Teklad #2018). Experiments were conducted during the dark cycle. All experimental procedures were approved by the Arizona State University Animal Care and Use Committee and were performed in accordance with NIH guidelines. Due to catheter patency issues, 7 rats were excluded from experimentation, and 3 were excluded from analysis due to not meeting extinction criteria. An experimental timeline can be seen in FIG. 6A. TRLZ (troriluzole mono hydrochloride mono hydrate) was synthesized as described previously and provided by Fox Chase Chemical Diversity Center (Doylestown, PA). TRLZ was dissolved in water and injected intraperitoneally in a volume of 1 mL / kg.Surgery

[0146] Rats were allowed one week of acclimation to the animal facility with daily handling prior to surgery, where they received intravenous jugular vein catheters. Briefly, rats were anesthetized with ketamine (87.5 mg / kg, i.m.) and xylazine (5 mg / kg, i.m.) and were implanted with a silastic catheter 2.5 mm into the right jugular vein and secured to a cannula within a backpack-style harness (PLASTICSONE, Roanoke, VA). Rats received cefazolin (100 mg / kg, i.v.) and heparinized saline (10 usp / infusion, i.v.) once per day for one week, as well as meloxicam (1 mg / kg, s.c.) for three days, postoperatively. This was followed by daily heparinized saline each day thereafter to maintain catheter patency during nicotine self-administration.Behavioral testing and TRLZ administration

[0147] Following recovery from surgery, rats received a 15-h food training session in operant conditioning chambers (Med Associates, Inc., Fairfax, Vermont), which contained two levers (active and inactive), cue lights above each lever, a food receptacle, and an auditory tone generator. Active2520321-WO-PCT / 103241-023PCTlever presses delivered a food pellet (45 mg, Bio-Serv, Flemington, NJ) into a food receptacle. The delivery of food was not paired with discrete cues. Next, rats began nicotine self-administration (2-hs / day, 7 days / week), beginning on a fixed-ratio 1 (FR-1) schedule of reinforcement, where one lever press resulted in the delivery of a nicotine infusion (0.02 mg / kg / infusion across 5.9 s) paired with a discrete compound stimulus comprised of a cue light above the active lever and an auditory tone. The auditory tone persisted for the duration of the infusion. Nicotine delivery was followed by a 20-s timeout period, where the cue light remained illuminated throughout the duration of the timeout. Inactive lever presses resulted in no programmed consequences but were still recorded. Rats remained on a FR-1 schedule for 5 days. After completing training on a FR-1 schedule, rats were then moved to a FR-2 schedule for 10 days or until criteria were met (>10 infusions / session and a 2:1 active lever press ratio). All rats met criteria within 10 days, and thus all rats had a total of 15 sessions of nicotine SA. Catheter patency was checked with brevital (3-4 mg / kg) prior to rats entering extinction training. Following nicotine SA, rats entered extinction training for 14 sessions, where active lever presses no longer resulted in delivery of nicotine or associated cues. Rats received TRLZ (8 mg / kg, i.p.) or vehicle (1 mL / kg saline, i.p.) 30 min prior to each extinction session similar to another study (Sepulveda-Orengo et al., 2018). After extinction training, rats were tested for a 2-h cue-induced reinstatement test, where active lever presses resulted in delivery of nicotine-associated cues but not nicotine. Total active and inactive lever presses were recorded.

[0148] A non-contingent saline administration group was included as a control. Saline rats received identical surgical and food training procedures to the nicotine SA group. Following recovery, rats underwent non-contingent saline delivery in a 2-h session, with saline infusions paired with a light + tone compound stimulus occurring every 9 minutes. Lever presses were recorded, but led to no programmed consequences. For simplicity, levers are termed "active" and "inactive" as in the nicotine group, however, these terms do not reflect that a programmed consequence followed "active" lever presses during the saline or "extinction" phases. Following 10 sessions, rats entered an "extinction" phase in which no saline infusions were delivered, and lever presses were recorded from both levers separately as in the nicotine groups. Following 14 sessions, rats were placed in a contingent cue test session, identical to the reinstatement test session in the nicotine groups. For simplicity, this session is termed "reinstatement" for the saline groups, but reflects contingent delivery of previously non-contingent saline-paired cues.Tissue collection and regional dissection

[0149] Immediately after reinstatement testing, rats were rapidly decapitated for brain tissue collection. Whole brains were removed from the skull, wrapped in the foil and stored at -80°C.2620321-WO-PCT / 103241-023PCTFor the regional dissection, brains were removed from -80°C, allowed to partially thaw on ice, placed dorsoventrally in the brain matrix, and sectioned coronally (2 mm). NAc tissue was collected using a pre-chilled stainless-steel cannula with an inner diameter of 1.1 mm on 1 mm rostral to bregma coronal brain section. Prefrontal cortices were extracted using a scalpel. Tissue samples were placed in pre-chilled 1.5 mL Eppendorf tubes, immediately weighed, and processed for tissue homogenization.Tissue Homogenization

[0150] For tissue homogenization, radioimmunoprecipitation assay lysis buffer (150 mM NaCI, 50 mM Tris-HCI pH 7.4, 1 mM EDTA, 0.25% deoxycholic acid, and 1% NP-40) supplemented with 2x Halt Protease and Phosphatase Inhibitor Cocktail (Cat No 78440) was added to weighed tissue at a ratio of 100 pL of buffer per 20 pg of brain tissue. Brain samples were homogenized using a handheld pestle homogenizer, followed by sonication (40% amplitude, 4 cycles with 15 sec pulse with 30 sec rest on ice). Sonicated tissue samples were rotated for 30 minutes at 4°C, followed by centrifugation at 20,000 x g for 10 min at 4°C. Cleared lysates containing soluble protein fractions were removed and stored at -80°C for cytokine analysis and immunoblotting. Separate aliquots of cleared lysates were processed for protein quantification using Pierce BCA Protein Assay Kit (Pierce Biotechnology, Cat No 23225). Samples were run in triplicates to determine average protein concentrations.Cytokine expression assay

[0151] To examine the concentration of proinflammatory cytokines, cleared lysates were analyzed using V-PLEX Proinflammatory Panel 2 Rat kit (MesoScale Diagnostics Cat No K15059D). This panel allows for simultaneous detection of IL-1R, IL-2, IL-4, IL-6, IL-10, IL-13, IFN-y, KC / GRO (CXCL1), and TNF-a. Samples were diluted to final lmg / mL (100 pg of total protein was loaded per well). The assay was performed as described in the manufacturer's protocol. Tissue cytokine levels are reported as pg of cytokine / mg of protein.Western blot analysis

[0152] Tissue obtained from rats undergoing SA and behavioral experimentation were run though a 10% Bis-Tris polyacrylamide gel using gel electrophoresis in MOPS buffer under denaturing conditions. Proteins were transferred to a 0.45 pm PVDF membrane. Membranes were blocked with Odyssey blocking buffer in phosphate-buffered saline (Li-Cor Biosciences, Cat No 927-40000) for 1 h at room temperature. Membranes were incubated with affinity-purified rabbit anti-Navl.6 / SCN8AT120321-WO-PCT / 103241-023PCT(1:1000, abeam, ab65166), rabbit anti-GLSl (1:1000, Novus, NBP1-89766), rabbit anti-GLS2 (1:1000, Novus, NBP1-76544), guinea pig-anti GLT1 (1:1000, Sigma Millipore, AB1783), and mouse-anti P-actin (1:5000, Sigma Cat No A2228) in Odyssey blocking buffer supplemented with 0.05% Tween-20 at 4°C overnight. Membranes were then incubated in IRDye 800CW goat anti-rabbit or IRDye 800CW goat anti-rabbit and IRDye 680RD goat anti-mouse secondary antibodies (Li-Cor) diluted 1:10,000 in Odyssey blocking buffer supplemented with 0.05% Tween-20 and 0.01% SDS at room temperature for 45 min. Protein blots were visualized with the Odyssey Imaging System (Li-Cor). Band intensities were quantified using ImageJ software (NIH, Bethesda). Data are presented as the relative intensity of bands normalized to the b-actin.Statistical analyses

[0153] Behavioral data were analyzed using repeated measures and mixed model analyses of variance (ANOVAs) with post hoc Tukey multiple comparison tests where appropriate.Greenhouse-Geisser corrections of degrees of freedom were utilized for violations of sphericity, where necessary. Two-way repeated measures ANOVA was used to analyze self-administration data. For self-administration, the independent variable included session, and dependent variables included lever presses and infusions. Linear trend analyses (also referred to as linear regression; see (Leyrer-Jackson et al., 2020. Strain and sex matters: Differences in nicotine self-administration between outbred and recombinase-driver transgenic rat lines. Exp Clin Psychopharmacol) were also conducted on SA data to confirm nicotine acquisition. Lever press data from the extinction phase was analyzed via a 3-way ANOVA. Independent variables included session and treatment and dependent variables included lever presses. Reinstatement data were analyzed by mean of each treatment group compared with the previous extinction session prior to reinstatement. Rats with more than 40 lever presses on the last day of extinction were excluded from reinstatement data analysis. Thus, due to unequal animals in both groups a mixed-effect analysis of reinstatement was conducted with post hoc Tukey comparisons. Lever press data during saline administration, "extinction", and "reinstatement" from saline control groups were analyzed in an identical fashion to the nicotine SA groups.

[0154] Cytokine and Western blot data were analyzed by two-way ANOVA (TRLZ pretreatment x nicotine treatment). In case of significant main effects, Tukey's post-hoc analysis was applied to identify differences between individual groups. Results were expressed as mean ± SEM with differences considered significant at p < 0.05. Data were analyzed using Prism v6.0 (GraphPad Software, San Diego, CA).2820321-WO-PCT / 103241-023PCTRESULTSAcquisition of nicotine SA

[0155] Nicotine SA acquisition was analyzed according to future treatment group. A two-way ANOVA analysis of effect of TRLZ or vehicle on active and inactive lever presses in nicotine selfadministration showed no main effect of treatment or session X treatment interaction (FIG. 6B), indicating that TRLZ and vehicle groups did not differ in active or inactive lever pressing prior to group assignment. A significant effect of session was found, indicating that all rats readily selfadministered nicotine. Next, proportion of lever pressing (active / active + inactive) was analyzed using a repeated measures two-way ANOVA. This analysis further showed no main effect of future treatment group or group x session interaction in the proportion of active lever pressing prior to group assignment (FIG. 6C). Again, a significant main effect of session was found. Next, nicotine consumption was analyzed (FIG. 6D). A repeated measures two-way ANOVA showed no significant effect of TRLZ or vehicle treatment or interaction of session and treatment, although there was main effect of session. In addition, linear regression analyses of active and inactive lever presses showed no significant difference in slopes of future treatment groups, but was significantly different from zero in both groups. Active and inactive lever presses were significantly different within both treatment TRLZ and vehicle. Taken together, these results indicate that all rats acquired nicotine SA.Effects of chronic TRLZ treatment on extinction and cue-induced nicotine seeking

[0156] Extinction data was next analyzed using an omnibus three-way ANOVA. A significant main effect of session, lever and treatment group was found (FIG. 7A). Further, lever x session and lever x treatment interactions were found. However, there were no session x treatment or session x lever x treatment interactions, thus no post-hoc comparisons were conducted.

[0157] A mixed-effect analysis was next conducted on data from the last day of extinction compared to a reinstatement test session (FIG.7B). Significant main effects of session (last day of extinction vs reinstatement) and lever (active vs inactive), as well as session x lever and treatment x lever interactions were found. These results indicate that lever pressing during reinstatement was significantly higher compared to extinction. No significant effects of treatment, session x treatment, or session x treatment x lever were found, thus no post-hoc comparisons were conducted.

[0158] Next, data from non-contingent saline control animals were analyzed. A two-way ANOVA analysis on active and inactive lever presses during saline administration as a function of future treatment group revealed no significant main effects of treatment or session x treatment interaction (FIG.8A). However, there was main effect of session. Following removal of saline infusions or cues, "extinction" was analyzed similarly to the nicotine SA animals above. A two-way2920321-WO-PCT / 103241-023PCTANOVA revealed no significant effects of session, treatment, or session x treatment interaction (FIG.8B). Finally, "reinstatement" to contingent saline-paired cues was analyzed using a three-way ANOVA on the last day of "extinction" compared to "reinstatement". There was a significant main effect of session, lever and a session x lever interaction. There was no significant effect of treatment, or session x treatment, treatment x lever, or session x treatment x lever interactions (FIG. 8C), thus no post-hoc comparisons were conducted. These data show that chronic TRLZ treatment does not reduce responding for saline-paired cues. No significant differences were found between treatment groups during "extinction" versus "reinstatement".Effects of TRLZ and nicotine on glutamate biomarkers

[0159] Effects of TRLZ and nicotine exposure on protein levels of glutamate biomarkers in the NAc and PFC are presented in FIG. 9A-9D. In the NAc, for GLT-1 protein levels, two-way ANOVA revealed effects of nicotine treatment and a significant interaction (FIG. 9A). Post-hoc analysis showed that TRLZ by itself (TRLZ + saline) enhanced GLT-1 protein expression compared to drug-naive rats (vehicle + saline). GLT-1 protein levels in rats exposed to TRLZ by itself (TRLZ + saline) were also enhanced relative to rats exposed to nicotine alone (vehicle + nicotine) and to TRLZ + nicotine (FIG. 9A). In the PFC, for GLT-1 protein levels, two-way ANOVA revealed effects of nicotine treatment(FIG. 9C). Post-hoc analysis showed that GLT-1 protein levels were significantly lower in rats exposed to TRLZ + nicotine compared to drug-naive rats (vehicle + saline). Two-way ANOVA did not reveal significant effects for GLS1, GLS2 or NAVI.6 in the NAc or PFC(FIGS. 9A and 9B).Effects of TRLZ and nicotine on cytokine levels

[0160] FIG. 10A presents effects of TRLZ and nicotine treatment on cytokine expression in the NAc. For TNFa, two-way ANOVA revealed effects of nicotine treatment and a significant interaction (FIG. 10A). Post-hoc analysis showed that TNFa levels in rats that self-administered nicotine were significantly lower compared to drug-naive rats (vehicle + saline). The lower TNFa levels in the NAc of rats that self-administered nicotine were normalized by pretreatment with TRLZ ( / .e., TNFa levels following nicotine SA lower in vehicle-pretreated rats (vehicle + nicotine) versus TRLZ-pretreated rats (TRLZ + nicotine). TRLZ by itself (TRLZ + saline) did not significantly affect TNFa levels compared to drug-naive rats (vehicle + saline). For I FNy, two-way ANOVA revealed effects of nicotine treatment (FIG. 10A). Post-hoc analysis showed that for rats pretreated with TRLZ, TNFa levels were significantly reduced in rats that self-administered nicotine compared to nicotine-naive rats (FIG. 10A). For CXCL1, two-way ANOVA revealed significant effects of TRLZ pretreatment and nicotine treatment (FIG. 10A). Post-hoc analysis showed that CXCL1 levels in the NAc of rats that3020321-WO-PCT / 103241-023PCTwere pretreated with TRLZ and self-administered nicotine (TRLZ + nicotine) were significantly enhanced compared to drug-naive rats (vehicle + saline), nicotine-naive rats pretreated with TRLZ (TRLZ + saline), and TRLZ-naive rats that self-administered nicotine (vehicle + nicotine). For IL13, two-way ANOVA revealed significant effects of TRLZ pretreatment and nicotine treatment (FIG. 10A). Post-hoc analysis showed that TN Fa levels in the NAc of TRLZ-naive rats that self-administered nicotine (vehicle + nicotine) were significantly lower compared to nicotine-naive rats pretreated with TRLZ. Two-way ANOVA did not reveal significant effects for I Lip, IL4, IL5, IL6 and I LIO.

[0161] FIG. 10B presents effects of TRLZ and nicotine treatment on cytokine expression in the PFC. For IL5, two-way ANOVA identified a significant interaction between factors(FIG. 10B). For rats that self-administered nicotine, post-hoc analysis showed that IL5 levels were significantly enhanced by TRLZ pretreatment (TRLZ + nicotine) compared to TRLZ-naive rats pretreated with vehicle (vehicle + nicotine). For the remaining cytokines analyzed in the PFC (TNFa, I FNy, CXCL1, I Lip, IL4, IL6, I LIO, and IL13), significant effects were not detected by two-way ANOVA.Discussion

[0162] The data showed that chronic treatment with TRLZ attenuated cue-induced reinstatement of nicotine seeking behavior in male rats. Reinstatement active lever presses in the TRLZ group decreased by almost half (109 versus 66 presses in the vehicle and TRLZ groups, respectively). Prior to treatment group assignment, no differences were detected in nicotine SA between TRLZ and vehicle-treated rats, indicating that differences in extinction and reinstatement behavior were not due to baseline differences in nicotine consumption or acquisition of lever press behavior. Further, the results showed that TRLZ did not alter lever press behavior during a contingent cue test session in saline control rats, demonstrating that general responding for cues was not affected by TRLZ treatment.

[0163] At the cellular level, reinstatement of nicotine seeking coincided with deficits in TNFa levels in the NAc that were restored by chronic TRLZ exposure. The effects of nicotine on TNFa levels were brain region specific as changes in the PFC were not detected. Functionally, TNFa is known to be a pro-inflammatory cytokine that exerts neuroprotective action against glutamate-induced cytotoxicity mediated by NMDA receptor activation. TNFa also regulates homeostatic synaptic plasticity, where small changes in local concentrations within the brain parenchyma alter synaptic plasticity. Nicotine exposure has been reported to reduce TNFa production in vitro and within serum and the hippocampus of rats. TNFa levels in THP-1 monocytes also decrease when treated with nicotine and cigarette smoke extract (Ween et al., Physiological Reports, 2017, 5 (16): el3370). Thus, for the study, one explanation is that reduced TNFa levels following nicotine reinstatement3120321-WO-PCT / 103241-023PCTenhanced glutamate-mediated postsynaptic excitability within the NAc, leading to enhanced nicotine seeking behaviors, which were reduced by chronic TRLZ exposure.

[0164] TNFa was quantified 2 h after cue-induced reinstatement of nicotine seeking, rather than earlier (e.g., 15 min) during the reinstatement session. In the present study, the reduction in TNFa levels at the delayed time point (2 h after reinstatement) may have reflected dissipation of TNFa levels. TNFa is produced initially as a transmembrane molecule (tmTNFa) but then released from cells as a soluble cytokine (sTNFa) through cleavage of tmTNFa by TNFa converting enzyme (TACE) (Brambilla et al., Psychiatry Research, 2001, 103 (2-3), 107-14). Without being bound by theory, it is possible that nicotine reinstatement may have triggered cleavage of tmTNFa into sTNFa, through TACE activation, leading to elevated levels of sTNFa earlier in the reinstatement session followed by the lower levels of sTNFa that was detected 2 h following reinstatement.

[0165] Chronic administration of TRLZ normalized TNFa levels in the NAc 2 h following nicotine reinstatement. One possible mechanism is that TRLZ normalized TNFa levels by interfering with protein kinase C (PKC) signaling to attenuate nicotine reinstatement. Given that TRLZ restored TNFa levels in the NAc 2 h following nicotine reinstatement in the study, and RLZ is a PKC inhibitor, one interpretation is that TRLZ inhibited PKC to reduce downstream activation of TACE, thereby normalizing nicotine-induced changes in sTNFa levels that occurred over the duration of the reinstatement session.

[0166] Another possibility is that TRLZ disrupted PKC-glutamate interactions to reduce nicotine reinstatement. In the study, TRLZ, through PKC- and channel-dependent mechanisms, may have inhibited the enhancement in corticolimbic glutamate release that facilitates reinstatement of drug-seeking behaviors.

[0167] TNFa can also induce downstream nuclear factor kappa B (NF-KB) signaling. In this study, nicotine induced a downregulation of TNFa and prevented TRLZ from increasing total levels of GLT-1. Thus, it is possible that nicotine-induced dysregulation of cytokine signaling and subsequent NF-KB signaling could impair GLT-1 expression, leading to increased cue-motivated nicotine seeking.

[0168] In conclusion, the disclosed results demonstrate that TRLZ reduces relapse to nicotine use and attenuates pro-inflammatory signaling induced by nicotine seeking behavior. These findings support the use of TRLZ as a therapeutic agent for the treatment of nicotine use disorder and related substance use disorders. Given the limited efficacy of existing pharmacotherapies for smoking cessation, TRLZ provides an alternative approach for treating nicotine use disorder through reversing nicotine seeking-induced neuroinflammatory signaling.

[0169] The composition and method can alternatively comprise, consist of, or consist essentially of, any appropriate materials or steps herein disclosed. The composition and method can3220321-WO-PCT / 103241-023PCTadditionally, or alternatively, be formulated so as to be devoid, or substantially free, of any materials (or species), or steps, which are otherwise not necessary to the achievement of the function or objectives of the composition and method.

[0170] Various possible mechanisms are described herein; however, the Applicant does not wish to be bound by any particular theory.

[0171] All cited patents, patent applications, and other references are incorporated herein by reference in their entirety.

[0172] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or may be presently unforeseen may arise to applicants or others skilled in the art.3320321-WO-PCT / 103241-023PCT

Claims

CLAIMS1. A method of treating a substance use disorder in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of troriluzole, wherein the substance use disorder is opioid use disorder or nicotine use disorder.

2. The method of Claim 1, wherein administration of troriluzole alleviates one or more substance withdrawal symptoms.

3. The method of Claim 1 or Claim 1, wherein administration of troriluzole reduces relapse or cue-induced reinstatement.

4. The method of any of Claims 1 to 3, wherein administration of troriluzole reduces substance-seeking behavior.

5. The method of any of Claims 1 to 4, wherein troriluzole is administered concurrently with substance exposure.

6. The method of any of Claims 1 to 4, wherein troriluzole is administered during a period of non-use of the substance.

7. The method of any of Claims 1 to 6, wherein the substance use disorder is opioid use disorder.

8. The method of Claim 7 , wherein administration of troriluzole reduces one or more of: (i) opioid self-administration, (ii) reinforcing efficacy of an opioid, (iii) cue-induced reinstatement of opioid seeking, (iv) opioid physical dependence, (v) development of opioid tolerance, or (vi) opioid-induced respiratory depression.

9. The method of Claim 7 or Claim 8, wherein the opioid use disorder is associated with use of a non-prescription opioid, a prescription opioid, or a combination thereof.3420321-WO-PCT / 103241-023PCT10. The method of Claim 7 or Claim 8, wherein the opioid use disorder is associated with use of a prescription opioid selected from oxycodone, morphine, hydrocodone, fentanyl, oxymorphone, hydromorphone, codeine, tramadol, or a combination thereof.

11. The method of Claim 10, wherein the prescription opioid is oxycodone.

12. The method of Claim 11, wherein administration of troriluzole reduces one or more of: (i) oxycodone self-administration, (ii) reinforcing efficacy of oxycodone, (iii) cue-induced reinstatement of oxycodone seeking, (iv) oxycodone physical dependence, (v) development of oxycodone analgesic or antinociceptive tolerance, or (vi) oxycodone-induced respiratory depression.

13. The method of Claim 10, wherein the prescription opioid is morphine.

14. The method of Claim 13, wherein administration of troriluzole reduces one or more of: (i) morphine-induced respiratory depression, (ii) development of analgesic or antinociceptive tolerance to morphine, or (iii) development of morphine physical dependence.

15. The method of any of Claims 1 to 6, wherein the substance use disorder is nicotine use disorder.

16. The method of Claim 15, wherein troriluzole is administered following cessation of nicotine intake.

17. The method of Claim 15 or Claim 16, wherein administration of troriluzole reduces relapse to nicotine use, cue-induced reinstatement of nicotine seeking, or a combination thereof.

18. A method of treating pain in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of troriluzole and an opioid analgesic.

19. The method of Claim 18, wherein the patient does not have opioid use disorder.

20. The method of Claim 18 or Claim 19, wherein the opioid analgesic comprises oxycodone, morphine, hydrocodone, fentanyl, hydromorphone, tramadol, or a combination thereof.3520321-WO-PCT / 103241-023PCT21. The method of any of Claims 18 to 20, wherein administration of troriluzole reduces development of opioid analgesic tolerance.

22. The method of any of Claims 18 to 21, wherein administration of troriluzole preserves antinociceptive efficacy of the opioid analgesic during repeated dosing.

23. The method of any of Claims 18 to 22, wherein administration of troriluzole reduces opioid-induced respiratory depression.

24. The method of any of Claims 18 to 23, wherein administration of troriluzole reduces escalation of an opioid dose required to achieve analgesia.

25. The method of any of Claims 1 to 24, wherein troriluzole is administered as a pharmaceutical composition comprising troriluzole.

26. The method of Claim 25, wherein the pharmaceutical composition comprises 50 to 450 mg of troriluzole.

27. The method of Claim 25 or Claim 26, wherein the pharmaceutical composition is in the form of a solid oral dosage form.3620321-WO-PCT / 103241-023PCT

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