WO2017139382A1 - Combination of adjuvant drugs esketamine and brimonidine for medical treatments - Google Patents

Abstract

The invention disclosed herein generally relates to compositions and methods related to a combination of adjuvant drugs for medical treatments. The present invention relates to combining the adjuvant drugs esketamine and brimonidine to treat medical conditions such as pain, depression and hearing loss.

Description

COMBINATION OF ADJUVANT DRUGS ESKETAMINE AND BRIMONIDINE FOR

MEDICAL TREATMENTS

CROSS REFERENCE TO RELATED APPLICATIONS

[ 0001 ] This application claims the benefit of U.S. Provisional Application No. 62/292,652, filed on February 8, 2016, entitled Combination of Adjuvant Drugs Esketamine and Brimonidine for Medical Treatments. The entire content of the foregoing is hereby incorporated by reference.

FIELD OF THE INVENTION

[ 0002 ] The invention disclosed herein generally relates to use of a combination of adjuvant drugs for medical treatments. The present invention relates to combining the adjuvant drugs esketamine and brimonidine to treat medical conditions such as pain, depression and hearing loss.

BACKGROUND

[ 0003 ] Inadequate pain control remains a major problem across the world. More than 25% of the total population suffers from clinically significant pain. Because pain affects day-today activities to a significant extent, its management presents a considerable challenge for physicians and their patients. There is an urgent and unmet need to manage effectively pain related to a variety of diseases and conditions. Currently, the management of pain is not sufficiently effective due to inaccurate diagnoses by physicians and the relative absence of efficacious treatments. Neuropathic pain is a relatively common complication of various diseases (e.g., diabetes mellitus, shingles), conditions (e.g., nerve injury, vascular lesions) and chemotherapy (cancer). In many cases, such as pain related to diabetes mellitus and cancer, 50% of the people taking medicines are not satisfied with the results. Although glycemic control is the ultimate goal in slowing the progression of diabetic neuropathy, often patients need analgesic agents to palliate painful diabetic neuropathy. Treatment (chemotherapy and/or surgery) results in significant pain for patients afflicted with cancer. Pharmacologic agents, administered orally and / or topically, that are used to treat neuropathic pain from various causes include: antidepressants (amitriptyline, duloxetine), antiepileptics (pregabalin, gabapentin), local anesthetics (lidocaine), capsaicin and opioids. Unfortunately, these pharmacotherapeutic agents often provide only partial relief of symptoms, when used individually, and many have some dose-limiting adverse effects. Most likely the previous drugs are limited in efficacy because multiple mechanisms are involved in pain perception / generation.

[ 0004 ] Persistent (chronic) pain interferes with the quality of life, decreases the productivity and effectiveness of the military forces, and utilizes healthcare resources excessively (Jonas and Schoomaker, 2014). Transition from acute to chronic pain occurs in discrete, but complex, pathophysiological steps involving multiple pathways. It has been suggested that this evolution is related to the duration and intensity of the pain stimulus and involves both peripheral and central sensitization mechanisms that act synergistically to exacerbate perception of pain. Because orally administered analgesics (often opioids) do not suppress pain adequately and produce significant adverse effects, the identification of appropriate analgesic adjuvants, administered by efficient and convenient routes of administration, represents an important unmet medical challenge. Ideally, drugs should be utilized that will treat not only pain but as many co-morbidities (e.g., depression) as possible, looking for the best fit with the total clinical picture of a patient.

[ 0005 ] Pain management of the wounded warrior continues to be challenging from point of injury on the battlefield through MEDEVAC and even in tertiary care facilities. Beyond the obvious need and desire to reduce the unnecessary pain and suffering of our nation's heroes, there are long-term sequelae in failing to do so. These may include post traumatic pain disorder (PTSD), depression, sleep disturbances and, chronic pain syndrome. Previous information suggests that failure to appropriately treat acute pain can result if the increased incidence of chronic pain and predispose to PTSD (Otis et al., 2003). The same holds true for civilian patients, especially victims of polytrauma. However, the tactical considerations of battlefield and far-forward care, as well as the unique challenges of aeromedical evacuation complicate and perhaps limit the analgesic modalities utilized for our combat casualties.

[ 0006 ] Morphine is one of the most commonly used and most effective analgesics for the treatment of pain on the battlefield. Conventional pain management techniques often are staid and need both improvement and enhancement. It is easy to see why, considering the most common pre-hospital pain medication: intramuscular (i.m.) or intravenous (IV) morphine. Intramuscular administration does not lend itself to easy titration, if IV titration is feasible. However, both routes of administration require needles and requisite disposal of sharps. These routes also imply a need for exposure of the casualty; a problem in hypothermic trauma victims or soldiers in a tactical and potentially chemical environment. Morphine induced respiratory and cardiovascular depression can also be especially challenging in this patient cohort, which is prone to shock and hemorrhage; using morphine to treat pain associated with a fractured femur may be lethal if the casualty also has internal bleeding and/or a pulmonary injury that cannot be identified on the field. A well-documented adverse effect of opioids is cardiorespiratory depression. Combat medics do not have feasible alternatives for the treatment of pain.

[0007 ] Another common medical condition on the battlefield is hearing loss. Hearing loss is a very common disorder and a significant disability in civilian and military personnel. It is estimated that about 48 million Americans have significant hearing loss in one ear; another 30 million have hearing loss in both ears. In armed service personnel, there is a direct connection between traumatic brain injury, PTSD and tinnitus ('ringing in the ears'). Tinnitus is the perception of sound without external acoustic stimulation. Data collected by the Veterans Administration concludes that the amount of disability compensation paid for tinnitus was $1.5 billion, and the amount of compensation is expected to exceed $3 billion by 2017. Currently, there is a lack of: 1) a universal standard of care for tinnitus and 2) an approved pharmacotherapy for tinnitus. Currently, there is no FDA approved drug available to treat tinnitus / hearing loss.

SUMMARY

[0008] The invention relates to a composition including esketamine and brimonidine. Esketamine and brimonidine can act synergistically in a subject to treat or prevent one or more medical conditions in the subject, or to facilitate an analysis in the subject.

[0009] In some embodiments, the one or more medical conditions can be pain, depression, hearing loss, and/or the like.

[0010] In some embodiments, esketamine and brimonidine can be encapsulated in chitosan microspheres.

[0011 ] In some embodiments, the composition can be delivered intranasally.

[0012] In some embodiments, the composition can be in the form of a gel for topical application.

[0013] Some embodiments of the invention relate to a method of treatment, prevention, or analysis involving a subject, including: delivering the composition to the subject; where esketamine and brimonidine can act synergistically in the subject to achieve the treatment or prevention, or to facilitate the analysis.

[0014] In some embodiments, the treatment can be treating pain, treating depression, treating hearing loss, alleviating the adverse effects of opioid use in a subject treated with a first dosage of opioids, or the like, and/or any combination thereof.

[0015] In some embodiments, alleviating the adverse effects of opioid use in a subject treated with a first dosage of opioids further includes: after delivering the composition to the subject, delivering to the subject a second, lower dosage of opioids, wherein the esketamine and brimonidine can act synergistically to alleviate the adverse effects of opioid use.

[0016] In some embodiments, the adverse effects can be hyperalgesia, allodynia, dependence on opioids, constipation, respiratory depression, nausea, vomiting, or the like, and/or any combination thereof.

[0017] In some embodiments, the prevention can be preventing hearing loss, preventing the conversion of acute pain to chronic pain, or the like, and/or a combination thereof.

[0018] In some embodiments, the esketamine and brimonidine can act synergistically to prevent hearing loss by protecting SCG neurons in response to injury.

[0019] In some embodiments, preventing the conversion of acute pain to chronic pain includes delivering the composition to the subject within 0 to 9 days of the onset of acute pain.

[0020] In some embodiments, the analysis can be determination of a therapeutic target for treatment of pain, and where changes in miRNA in the subject are measured, and where a change in miRNA can indicate that the corresponding gene is a therapeutic target for treatment of pain.

[0021] In some embodiments, the composition can be applied topically. In some embodiments, the composition can be injected. In some embodiments, the composition can be delivered intranasally.

[0022] In some embodiments, the composition can be encapsulated in chitosan microspheres. BRIEF DESCRIPTION OF THE DRAWINGS

[ 0023 ] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[ 0024 ] Figure 1 is a general depiction of the invention. The transformation of pain medicine can be achieved more efficiently by repurposing existing drugs while continuing to perform discovery research for new chemical entities and/or biological products that can be developed into novel drugs for pain management. Pharmacogenomics can be used to individualize pain management regimens and this approach can lead to greater precision in controlling pain and its co-morbidities.

[ 0025 ] Figure 2 is a general depiction of the invention: Esketamine and brimonidine can be delivered by the intranasal and intramuscular routes to act synergistically to: a) suppress acute pain and its conversion to chronic pain (peripheral/central sensitization) and b) evoke changes in miRNA(s) that can be used as a biomarker(s) to predict the analgesic response and/or therapeutic targets.

[ 0026 ] Figure 3 illustrates an example treatment protocol. Subjects are treated with escalating doses of S-ketamine.

[ 0027 ] Figure 4 illustrates an example treatment protocol. Racemic (R, S) ketamine hydrochloride (HCI), esketamine (S-ketamine) hydrochloride plus brimonidine tartrate solutions are prepared in isotonic saline for intramuscular (i.m.) injections and in 0.5 ug chitosan - HCI / 0. lmL for intranasal (i.n.) administration.

[ 0028 ] Figure 5 illustrates an example experimental timeline for behavioral testing in rats. OF=open field test, SP=sucrose preference test, FST=forced swim test.

DETAILED DESCRIPTION OF THE INVENTION

[ 0029 ] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[ 0030 ] In the past 50 years, only four analgesics have been introduced that offered novel mechanisms of action: capsaicin, dronabinol, zoconotide, ketamine (Kissin, 2010). As recently as 2012, the Defense Health Board approved the use of racemic ketamine as a prehospital analgesic based on its ease of utility and efficacy under the battlefield conditions. Low- dose ketamine can have several advantages as an adjunctive analgesic when used in combination with opioids and other analgesics because it has: 1) analgesic actions of its own, part of which is mediated at the local level and 2) anti-hyperalgesic / - allodynic effects when coadministered with opioids. (Schmid et al., 1999). Embodiments of the invention can relate to a third dose range of ketamine, where it has no significant analgesic activity of its own, but it interacts synergistically with other analgesic agents such as opioids, yielding a 'sparing effect' and rendering superior pain relief than either drug alone.

[ 0031 ] Intramuscular (i.m.) ketamine alone (0.5-1.0 mg/kg) produces significant analgesia and has a rapid onset (within 5 minutes) with a duration of action of 0.5 -2.0 hrs. At low doses, administered i.m., ketamine has minimal hemodynamic, respiratory or cognitive effects. Others have demonstrated that esketamine (S - ketamine), by the intranasal (i.n.) route, has a rapid onset in terms of analgesic actions (Yeaman et al., 2014) as well as producing relatively rapid effect in refractory depression (Andrade, 2015). The advantages of esketamine are presumed to be those of racemic ketamine albeit at lower doses; these advantages are: 1) broad range of clinical applicability, 2) exceptional therapeutic index, 3) versatility with respect to routes of administration in austere environments and 4) ability to maintain support of cardiorespiratory function.

[ 0032 ] Embodiments of the invention relate to the utility of esketamine, combined with brimonidine (an alpha-2 adrenoceptor [A2a-R] / imidazoline [I₂-R]) agonist, as a means of enhancing efficacy and safety in the adjunctive treatment of pain. Like other A2a-R / I₂-R agonists, brimonidine can have: 1) analgesic actions that are synergistic with those of esketamine and opioids (Stone et al, 1997), 2) neuroprotective and anti-inflammatory actions (WoldeMussie et al., 2001; Guo, et al., 2015) and 3) the ability to lower pressure in the eye and brain by inhibiting formation of aqueous humor (Burke and Potter, 1986) and cerebrospinal fluid. Peripheral, spinal and supraspinal A2a-Rs are neuronal sites of action for brimonidine. At these sites, brimonidine inhibits transmitter release from primary afferent fibers; in addition, this action allows K+ efflux from Gi protein-gated channels resulting in hyperpolarization and suppression of neuronal firing.

[ 0033 ] Because the failure to appropriately treat acute pain can result in the increased incidence of chronic pain alternative/adjunctive pharmacotherapeutic agents are a high priority. The advantages of esketamine include: 1) safe and effective analgesia in sub-anesthetic doses, 2) minimal interference with spontaneous respiration with stable airway/pharyngeal reflexes, 3) enabling sympathomimetic effects: keeping blood pressure stable, even in cases of hypovolemia, 4) wide therapeutic range / safety margin, 5) useful amnestic and hypnotic effects and 6) relatively short-acting adverse effects. Embodiments of the invention relate to the combination of esketamine with brimonidine, which can improve overall efficacy (synergism) and safety as well as minimize potential adverse effects of esketamine. If it becomes necessary to include opioids as part of the pain treatment regimen, the combination of esketamine and brimonidine can be synergistic with the analgesic actions of opioids. The judicious use of esketamine and brimonidine can permit smaller doses of opioids and adverse effects can be attenuated.

[ 0034 ] Based on these pharmacologic criteria, embodiments of the invention relate to the combination of esketamine and brimonidine having advantages in casualties with pain associated with traumatic brain injury (controlled intracranial pressure) or penetrating eye injuries (stability of intraocular pressure) as identified by the Defense Health Board. An additional benefit is that the combination (esketamine / brimonidine) can have utility in treating / preventing hearing loss (tinnitus) due to excessive noise levels (Bing, et al., 2015; Cai, et al., 2013). Moreover, several of the adverse effects attributable to effects of S-ketamine can be ameliorated by the presence of an A2a/ I₂-R agonist, such as brimonidine (Kanner and Tsai, 2006).

[ 0035 ] Current evidence suggests that glutamate (NMDA-/AMPA-Rs) and A2a-R/ I₂- Rs, located on central and peripheral nerves, can modulate pain-signaling mechanisms at multiple levels. Although the primary evidence is indicative of ketamine's ability to suppress pain by antagonizing NMDA-Rs non-competitively on sensory nerve endings, there is also evidence that implicates NO/GMP pathways as well as ATP- sensitive K+ channels in the process. At the spinal level, antagonists of NMDAR and A2a-R/ I₂-R agonists can significantly attenuate the development of tolerance to opioids as well as mitigate the changes in nociceptive thresholds (hyperalgesia, allodynia) induced by chronic opioid therapy. A2a-Rs participate in inhibiting primary afferent nerve terminals as well as suppressing the firing of projection (descending) neurons. Nociceptor activity on sensory nerves is suppressed by both ketamine and brimonidine. For example, activating A2a-Rs with brimonidine can inhibit the expression / release of CGRP, a nociceptive neuropeptide (Supowit, et al., 1998). It is noteworthy that keratinocytes in the epidermis also express NMDA and A2a-Rs that can modulate release of glutamate and inflammatory cytokines; therefore, keratinocytes can also play a role in modulating pain signaling in the periphery as well. In addition to anti-nociceptive effects, brimonidine promotes the production of nerve growth factors that preserve nerve function. Because both efficacy (peripheral / central) and adverse effect profiles of A2a-R/I₂ agonists and NMDAR antagonists are effected substantively by the route of administration, embodiments of the invention relate to intramuscular and intranasal routes. Embodiments of the invention relate to establishing a paradigm where combined drugs can be given systemically and locally (including, for example, transdermally) to improve analgesia (and diminish hyperalgesia and allodynia)— a laudable achievement in battlefield / pre-hospital analgesia. Importantly, a complementary analgesic effect can be achieved if the drug combinations are administered with facility and have different modes / sites of action.

[ 0036 ] Peripherally and centrally mediated pain and hyperalgesia are mediated, in part, by pro -inflammatory cytokines (TNF-a, IL-1, IL-6, IL-8) released by nitric oxide, free radicals, prostaglandins, peptides and excitatory amino acids, like glutamate (Watkins et al., 2003). Brimonidine has anti-inflammatory activities that are beneficial in cases where neuronal function has been compromised such as in the compressed spinal cord (Tanabe et al., 2011). Sequestosome (p62) plays a crucial role in tumor necrosis factor (TNF) - induced neurodegenerative disease (Zatloukal et al., 2002). In addition to protective effects on ganglion cell bodies (Lafuente et al., 2001), brimonidine can obviate the crucial role of TNF-induced axonal degeneration (Kitaoka et al., 2015) which involves upregulation of p62 leading to nerve degeneration. After TNF injection, brimonidine can exert a substantial protective effect by decreasing p62 levels in a dose-related manner. Likewise, post-traumatic neuronal degeneration can be benefitted by the ability of S - ketamine to suppress: 1) pro-inflammatory cytokine (TNF, IL-6, IL-8) production (Welters, et al., 2011) and 2) free radical-evoked lipid peroxidation after spinal cord injury (Kose et al., 2012).

[ 0037 ] Evidence has shown that imidazoline ligands, including brimonidine, interact with binding sites characterized as imidazoline-2 (I₂) 'receptors' (Burke et al., 1995). However, I₂ receptors have not been cloned, and their signaling pathways have not been fully characterized. Ligands (agonists) acting at I₂ receptors effectively suppress tonic inflammatory and neuropathic pain but are much less effective for acute phasic pain (Li and Zhang, 2011). Interestingly, I₂ receptor agonists enhance the analgesic effects of opioids in both acute phasic pain and chronic tonic pain (Bektas at al., 2015). In this regard, brimonidine is not only active at A2a-Rs but also at I₂ receptors as shown by binding and biologic activity studies using the I₂ receptor antagonist, idazoxan (Burke et al., 1996). This invention relates to the synergistic actions of brimonidine with esketamine to induce analgesia in a neuropathic / inflammatory pain states. In addition, A2a-R / I₂-R agonists, such as brimonidine, can: 1) enhance the antinociceptive actions of opioids, 2) reduce development of opioid tolerance and 3) minimize the predisposition to addiction. Thus, A2a-R / I₂-R agonists are valuable individually or in combination as analgesics and can serve as therapeutic co-adjuvants of opioids (Guo et al., 2003).

[ 0038 ] Noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators of neuronal functions (Bali and Kuner, 2014). The ability to influence gene expression via epigenetic mechanisms can provide an innovative approach to new therapies for pain. For example, miRNAs are gene regulators that can represent viable therapeutic targets for developing novel treatments for neurologic diseases and conditions related to pain. In this regard, miRNA expression can be part of the mechanism by which the antidepressant / analgesic action of ketamine regulates various central nervous system regions (O'Connor et al, 2013; Strickland et al., 2014; Camkurt et al., 2015). Moreover, the ability of ketamine and other antidepressants (e.g., imipramine) to alter histone deacetylase (HDAC) in the nucleus accumbens, simultaneously with alleviation of pain and stress-induced depression (e.g., PTSD), suggests a role for epigenetics in these conditions (Duman and Voleti, 2011). It is of interest that miR-206 is a critical novel gene for the expression of BDNF induced by ketamine (Yang et al., 2014). Coadministration of clonidine and ketamine countered the mechanical hyperalgesia and inhibited the normalization of NMDARI MRNA expression in mice (Ohnesorge et al., 2013). These data indicate the possibility that epigenetic molecular events, induced by ketamine, with or without clonidine, are necessary in reversing specific events in conversion of acute to chronic pain and countering opioid-induced hyperalgesia (see Fig. 1). These epigenetic mechanisms can also account for the sustained therapeutic effects of ketamine; thus, miRNAs can be used as biomarkers to identify pain phenotypes, i.e., distinct groups of responders vs non-responders (Borsook and Kalso, 2013) and serve as a rational basis for selecting the most effective pharmacotherapy for painful conditions.

[ 0039 ] In summary, two drugs, used in combination, can be used as adjuvant analgesics, esketamine (non-competitive NMDA antagonist) and brimonidine a relatively selective alpha-2 adrenoceptor agonist). After appropriate compounding using nanotechnology, this combination of drugs can be used by two topical routes (skin, nose) in order to suppress pain that accompanies neuropathy. The therapeutic roles of adjuvant analgesics can: 1) provide unique, efficacious and long-acting analgesic actions in opioid resistant pain, 2) lessen the burden of adverse effects when administered to the skin and nose and 3) increase the therapeutic index of concomitantly administered opioids by a dose-sparing effect resulting in decreased liability to addiction and opioid-induced adverse effects (constipation, hyperalgesia, aliodynia).

[0040] Embodiments of the invention relate to multimodal analgesia, induced by esketamine and brimonidine, administered intranasally or intramuscularly, optimizes pain relief through multiple mechanisms (NMDA-R / A2/ I₂-R respectively) along multiple sites of the nociceptive pathway, both peripheral and central. Important in the multimodal approach is that pharmacogenetic influences among patients in response to analgesic agents can be minimized. The novel combination (esketamine/brimonidine) can have several benefits: 1) lowering doses of both agents can minimize adverse effects, 2) mitigating the adverse effects of esketamine in penetrating eye injury or significant traumatic brain injury because brimonidine lowers intraocular pressure and intracranial pressure and 3) minimizing doses of opioids used chronically for pain suppression thereby preventing opioid-induced hyperalgesia and aliodynia as well as obtunding central sensitization to acute pain. There is the additional advantage that esketamine can negate the development of post-traumatic stress disorder (PTSD) following injuries because of its rapid onset anti-depressant effect.

[0041] This invention is unique in that it offers a combination of adjuvant drugs (esketamine, brimonidine) that can: 1) be more efficacious in ameliorating neuropathic pain, 2) lower the dose requirement for opioids as well as lessening the adverse effects of opioids and 3) reduce dose requirements for regional anesthesia (brachial, tibial) with local anesthetics. The unique combination can have advantages in that efficacy will be optimized and adverse effects can be minimized by both pharmacodynamic (synergism) and pharmacokinetic (duration) means. Interactions of opioid receptors with other families of receptors can be utilized to improve the quality of analgesia and reduce the adverse effects (respiratory depression, nausea, vomiting) of opioids. Thus, the novel combination can be utilized to decrease the number of prescriptions written for opioids in the treatment of chronic pain. The choice of multiple routes of administration can provide the convenience of treatment of localized (topical cream) pain as well as more generalized, systemic pain (intranasal spray) scenarios.

[0042] The combination can be more efficacious than existing products because they act by multimodal mechanisms and offer the utility of multiple routes of delivery that are 'user friendly'. Moreover, the use of this combination can lessen the requirement for systemic opioids and minimize opioid-induced adverse effects. Both of the drugs in this combination have been demonstrated to elevate neurotrophic factors that can delay or prevent further neuropathy along with subsequent pain and discomfort. Moreover, the pharmacologic characteristics of these drugs can be effective in pain caused by either neuritis (inflammation) or injury.

[ 0043 ] Embodiments of the invention relate to the use of esketamine (S [+] ketamine) because it is more potent and selective than the racemic (R [-], S [+] -ketamine) mixture that is normally used alone or in combination with other drugs. In addition, embodiments of the invention relate to the use of nanotechnology for drug delivery to demonstrate better regulation of bioavailability of the combined drugs when administered by the proposed routes of administration. The innovative combination capitalizes on repurposing drugs that have been successful individually for other conditions but, in combination, are uniquely synergistic in alleviating neuropathic pain. Moreover, the adverse effects can be reduced by: 1) allowing lower concentrations of the individual drugs to be used and 2) utilizing appropriate functional antagonism (e.g., esketamine tending to raise blood pressure whereas brimonidine tends to lower blood pressure). The vasoconstrictive effects of brimonidine in the blood vessels of the nose can retard the absorption of esketamine thereby prolonging the duration of effect.

[ 0044 ] This drug combination can be prescribed by primary care and pain management physicians to be used alone or in combination with systemic opioids. This combination of drugs (esketamine /brimonidine) can also be used to treat the co-morbid conditions of pain and depression because of the unique properties of ketamine as a rapidly acting antidepressant.

[ 0045 ] Embodiments of the invention relate to two drugs that will provide a multimodal approach to alleviating a variety of painful conditions and co-morbidities. Moreover, an additional aim will identify an epigenetic means of identifying those individuals that are responders (vs. non-responders) so that the path to successful pharmacotherapy will be more achievable. Recent evidence suggests that clonidine can minimize the hemodynamic and psychological effects of s-ketamine as shown during a study for dressing changes in patients with major burns; this suggest that the combination of brimonidine, a clonidine-like compound with greater selectivity for A2a-Rs, plus S-ketamine can perform in a similar, but more efficacious, manner (Pretto et al., 2014). Hearing

[0046] Phantom sensory perceptions, such as phantom pain and phantom sound (tinnitus), share similarities in terms of: 1) initiating mechanisms ,e.g., pathophysiological activation of the NMDA - operated system in the same brain areas and 2) modulation by the NMDA receptor antagonist e.g., ketamine. Recent evidence has identified cochlear NMDA receptors and alpha-2 adrenoceptors (A2-ARs) as therapeutic targets for prevention of synaptopathic cochlear tinnitus. NMDA receptor inhibition has been proposed as a pharmacologic approach for treatment of damage to the inner hair cell synapse. Likewise, activation of A2-ARs in the spiral ganglion neurons (SCG) can mediate protective effects against glutamate- and hydrogen peroxide-induced toxicity to SCG neurons. It is proposed that when applied topically to the inner ear, a combination of esketamine (or norketamine) can be used in combination with brimonidine to prevent and protect SCG neurons in response to injury (chemical, noise, drugs) and subsequent hearing loss / tinnitus.

[0047] The combination can be used to treat existing auditory dysfunction or be used prophylactically to prevent /protect against hearing loss due to noise, chemicals (drugs) and /or disease.

[0048] This combination can represent a multi-modal approach to treating tinnitus / hearing loss. Esketamine (norketamine) can prevent further damage and brimonidine can not only prevent but can also protect existing, normal hair cells / SCG neurons from potential damage.

[0049] The combination of drugs can be applied topically in a gel into the auditory canal or injected through a micro-incision in the round window via a delivery tube (catheter).

[0050] Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

[0051] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

PROOF OF CONCEPT

[ 0052 ] To achieve proof of concept, a randomized, enriched, double-masked, flexible dose, crossover clinical trial is performed in patients afflicted with neuropathy comparing the short-term efficacy and safety of the combination of esketamine and brimonidine (topically administered as a cream or intranasal spray). Using application to the skin and nasal mucous membrane can provide evidence of local and systemic efficacy, respectively. Subsequently, another trial is conducted with an identified concentration of esketamine and brimonidine in patients who are also receiving systemic opioids. Achieving proof of concept can document the rationale of combined, topically applied agents as effective analgesic adjuvants and establish the validity of the combined adjuvants in permitting the use of lower doses of systemic opioids thereby obviating their adverse effects. Achieving this goal can improve pain management overall and reduce prescriptions written for opioids in the management of chronic pain.

EXAMPLE 2

RAT MODEL OF PAIN

[ 0053 ] The method for evaluating the combination of esketamine and brimonidine is a rat model of neuropathic / inflammatory pain initiated by spinal contusion. This model not only allows the assessment of analgesic activity but also behavioral indices that are associated with depression. Given that pain and depressive syndromes, e.g., PTSD, often coexist in injured soldiers, this is an appropriate model for testing efficacy of the proposed combination. The routes of administration used for the evaluation, intramuscular and intranasal, were selected because they have been shown to be convenient routes to utilize when under duress in battlefield conditions. Of the two routes proposed, the predicted onsets of action are: 7-10 minutes (i.m.) and 3-6 minutes (i.n.), respectively, for esketamine. However, in the presence of brimonidine, the onset is predicted to be slower, and the duration of action is extended. Useful pharmacokinetic and pharmacodynamic information is generated by this study and is the basis for a future IND application and clinical trials with the combination therapy. An additional outcome of this study is more confirmatory information about the value of esketamine as an analgesic agent alone and in combination. These data could enhance the probability of approval for clinical use in the U.S. The design of the study involves racemic ketamine as the comparator because it has been recommended by the TCCC guidelines and the Defense Health Board as recommended pharmacotherapy for acute and chronic pain at the point of injury. This approach is in contrast to recent data that showed that oral transmucosal fentanyl citrate is the most commonly administered medication pre- and post-release (Black and McManus, 2009; Litwack, 2015). Convincing dose-response data can provide a substantial preclinical rationale not only for additional clinical studies involving racemic ketamine but also provide a rationale for the combination of esketamine and brimonidine.

[ 0054 ] The rodent spinal contusion injury (SCI) is an excellent model system that is particularly relevant for the military population. It serves as a model for testing agents that suppress neuropathic and inflammatory pain. The primary aim of the proposed studies is to identify effective doses of esketamine and brimonidine for the treatment of pain after SCI. We will simultaneously evaluate the effects of these medications on long-term physical (locomotor and sensory function), psychological (anxiety, depression) recovery and changes in miRNA after injury and drug administration.

EXAMPLE 3

DETERMINE THE EFFECTIVE ANALGESIC DOSES OF ESKETAMINE AND BRIMONIDINE BY THE INTRANASAL (I.N.) ROUTE

[ 0055 ] Evaluate an expeditious (intranasal) route of drug administration, to enable the direct delivery of esketamine and brimonidine to the central nervous system (CNS). Avoiding the gastrointestinal and hepatic pre-systemic metabolism will enhance CNS drug bioavailability in comparison with that obtained after intramuscular absorption, and the intranasal route provides the ease of administration that is requisite in the battlefield.

[ 0056 ] This phase of the project will focus on the nasal delivery aspects of the combination of esketamine and brimonidine using a permeation enhancer (chitosan). Nasal delivery of ketamine is well tolerated and results in a rapid and greater bioavailability (30-50%) as compared to oral administration; a small portion is transported into the brain via the olfactory nerve as well as the epithelial region of the nose. Based on the preliminary pharmacokinetic interaction data between esketamine and brimonidine, a mucoadhesive nano/microparticle formulation of the combination drugs will be developed. Considerations will include the limited nasal capacity and mucociliary clearance necessary to further enhance its nasal bioavailability. Aqueous solutions of ketamine (racemic) and esketamine plus brimonidine will be prepared for intramuscular administration and similar analyses will be conducted regarding ADME.

EXAMPLE 4

DISTINGUISH THE EFFECTIVE ANALGESIC DOSES OF ESKETAMINE AND BRIMONIDINE BY THE INTRAMUSCULAR (I.M.) ROUTE.

[ 0057 ] Determine the effective dose(s) of racemic ketamine (comparator) and esketamine plus brimonidine administered intramuscularly. The analgesic efficacy of the drugs alone and in combination is determined long-term in SCI rats. The invention posits that esketamine can provide analgesia and alleviate psychological (depressive) symptoms. Brimonidine can maintain act synergistically to enhance analgesia as well as maintain cognitive function and minimize the dissociative effects of esketamine.

EXAMPLE 5

ESTABLISH THE UTILITY OF MIRNAS AS BIOMARKERS AND / OR THERAPEUTIC

TARGETS IN PREDICTING / MEDIATING THE ANALGESIC RESPONSE TO

ESKETAMINE PLUS BRIMONIDINE.

[ 0058 ] Changes in miRNA levels are determined in blood samples and spinal cord tissue at predetermined intervals. MiRNAs can represent biomarkers that can predict efficacy of drugs in subsets of patients and can also represent therapeutic targets.

[ 0059 ] Elucidate whether epigenetic biomarkers (e.g., miRNAs) are used to predict individual responsiveness to efficacious doses of esketamine plus brimonidine. This study can monitor miRNA levels and determine if racemic ketamine and/or esketamine plus brimonidine can elicit a change in miRNA levels that are therapeutic targets and/or biomarkers to predict efficacy of these drugs in subsets of patients.

[ 0060 ] It has been suggested that targeting well-defined patients for clinical trials can play a crucial role in developing analgesic drugs in the future because of epigenetically based variability in patient response. Therefore, a method in this study can monitor miRNA levels and determine if racemic ketamine and/or esketamine plus brimonidine can elicit a change in miRNA levels that are a either a therapeutic target and/or biomarker to predict efficacy of these drugs in subsets of patients.

EXAMPLE 6

PHARMACOKINETIC INTERACTION BETWEEN ESKET AMINE AND

BRIMONIDINE

[0061] The nasal mucosa is highly vascularized. Nasal lumen is two cell layers thick and offers a rapid absorption. Nasal drug delivery is an attractive alternative to parenteral injections which are linked with a risk of infection and needle-stick accident risks. Exclusively, transmucosally absorbed drugs are not subject to gastrointestinal degradation and circumvent the hepatic first-pass metabolism. This is the main advantage for drugs like esketamine which are prone to hepatic first-pass after oral application. In vitro studies of supraclinical doses of ketamine on rat tracheal epithelial cells showed no signs of cilia toxicity, indicating that the nasal mucosa is an appropriate absorption target. The reported bioavailability after nasal application ranged from 33% to about 50% (Yanagihara, et al., 2003, Malinovsky, et al., 1996, Christensen, et al., 2007) as compared to 17% by oral route (Grant et al., 1981).

[0062] Pharmacokinetics (PK) of esketamine and brimonidine and their interaction following concomitant use are carried out in healthy male rats. Single dose PK is performed to estimate the preliminary PK parameters, and PK interaction studies can determine any significant alteration in PK parameters on concomitant administration of these drugs. Both esketamine and brimonidine are p-glycoprotein substrates, and since esketamine is a substrate for CYP3A4 and brimonidine is an inhibitor of CYP3A4, alteration in PK parameters when used in combination are expected [Kwan et al., 2011, Greig et al., 2015]. Mass spectrometric detection is performed on LC-MS/MS mass spectrometer (Waters) with Analyst software.

[0063] Concentrations of esketamine and brimonidine are measured in plasma samples by use of an API 3000 liquid chromatography-tandem mass spectrometry system (Freireich et al., 1966). Pharmacokinetic interaction between esketamine and brimonidine is performed with i.m. and intranasal routes of administration at two doses. For i.m. route, two sets of 42 male Sprague-Dawley rats (8-10 weeks old) are used to determine PK parameters of esketamine with two different doses. Rats are housed in well ventilated plastic cages in standard laboratory conditions with regular 12 h light-dark cycle ad libitum. The animals are acclimatized for a minimum period of 7 days prior to the experiment. The next day, (i) esketamine solution in PBS is administered i.m. at two different doses. Blood samples (0.1-0.2 ml) are collected via the tail vein at different time intervals from 0.25 h up to 48 h. Blood samples is collected in dried heparinized Eppendorf tubes, centrifuged at 4 000 rpm, plasma is collected and stored at -20 °C until analysis. The plasma drug concentration data obtained after animal treatments is analyzed by HPLC method for assessment PK parameters Tmax, Area under the curve (AUC), and mean residence time (MRT)). In a similar experiment as the above two other sets of each 42 rats are administered with brimonidine solution at two different doses two separate sets. PK interaction between esketamine and brimonidine following i.m. injection is investigated at two fixed combination doses of these drugs. Two sets of each 42 rats are given a predetermined combination of doses. Blood samples are collected at predetermined time intervals (based on PK parameters of the individual drug) and drug concentration is analyzed by a LC-MS/MS method.

[ 0064 ] Pharmacokinetics of esketamine, brimonidine and their combination following nasal administration are assessed to determine their relative bioavailability and potential pharmacokinetic interaction when administered through nasal route. In a study similar to the above, esketamine/brimonidine or a fixed combination of these drugs solution are administered separately to two sets of 42 male Sprague-Dawley rats at two different doses using the intranasal Mucosal Atomization Device (Teleflex Medical, Morrisville, NC). Blood samples are collected via the tail vein at different time intervals up to 48 h and drug(s) concentration are determined as above. Pharmacokinetic parameters are calculated using the WinNonlin® software (Pharsight Corporation Ltd.). Relative bioavailability of the drugs by intranasal route is calculated. All experimental protocols involving animals are to be prior approved by the Institute of Biosciences and Technology, Texas A&M Health Science Center Institutional Animal Care and Use Committee (IACUC).

EXAMPLE 7

PREPARATION OF CHITOSAN MICROSPHERES ENCAPSULATING ESKETAMINE AND BRIMONIDINE

[ 0065 ] Chitosan has the generally recognized as safe (GRAS) status (Shahidi and Abuzaytoun 2011), and is a safe and effective permeation enhancer due to interaction with mucosal membranes and transient opening of the tight junctions which enhances paracellular absorption (Schipper et al., 1997, Smith et al., 2004). Absorption enhancing properties for transmucosal nasal drug delivery was reported in in vitro and in vivo studies (Hinchcliffe et al., 2005, Yu et al., 2004, Pavis et al., 2002, Martinac et al., 2005, Van der Lubben et al., 2001). Chitosan microspheres are prepared by water-in-oil (W/O) emulsification method using paraffin oil as the external phase (Casettari and Ilium, 2014, Abdel et al., 2014). Briefly, chitosan is dissolved in 2% aqueous acetic acid solution and drug (esketamine/brimonidine in PBS) is dispersed. The dispersion is added slowly through a syringe to 100 mL liquid paraffin containing 0.2% w/v of dioctyl sodium sulfosuccinate (DOSS) as stabilizer under constant stirring for a predetermined time using a high speed stirrer to make an emulsion. The emulsion glutaraldehyde (25% solution as the crosslinking agent) is added slowly and stirred for further 2 h. The microspheres formed are separated by vacuum filtration and washed several times with hexane to remove paraffin oil, followed by water to remove glutaraldehyde. The microspheres are air dried for 24 h and stored in vacuum desiccator until further use.

EXAMPLE 8

CHARACTERIZATION OF THE DRUG-LOADED CHITOSAN MICROSPHERES

[ 0066 ] Particle size, distribution and their zeta potential have a significant influence on the rate of drug release and absorption and clearance and stability of the microspheres from the nasal cavity. To ensure the batch-to-batch variation in size and to determine the uniformity in vitro and in vivo performance, size, polydispersity and zeta potential of the microsphere are determined by dynamic light scattering using ZetaPALS zeta potential analyzer (Brookhaven Instruments Corporation). Physicochemical properties of free drug and the drug loaded in microspheres are characterized by techniques like differential scanning calorimetry (DSC), X-ray diffraction (XRD), Fourier Transform Infrared (FTIR) Analysis. In DSC, the alterations or shifts in the exo- and endothermic peaks of drug and drug-polymer inclusion complexes are studied, whereas XRD analysis is used to analyze crystalline and amorphous nature of the same. IR spectroscopy analysis gives unique fingerprint pattern of peaks with each type of the molecule. Following optimization of the process parameters to obtain a stable microspheres with desired drug loading, the physical state of esketamine and brimonidine associated with the microspheres is recorded using differential scanning calorimetry (DSC) thermograms. Drug loading is determined by crushing a known amount of the microspheres and dissolving in a mixture of methanol and water (1: 1) under stirring. The dispersion is filtered through 0.42 um filter and the drug content in the filtrate is measured by HPLC method. [ 0067 ] In vitro mucoadhesive property of the microspheres is assessed using sheep nasal mucosa (Patil et al., 2009, Patil et al., 2010). The microspheres are fixed to the polyethylene support and then placed on the nasal mucosa. The mucosa is placed in a desiccator at >80% RH at room temperature for 30 min to allow the polymer to hydrate and to prevent drying of the mucus. The mucosa is observed under microscope and the number of particles attached to the mucosa are counted. After 30 min, the polyethylene support is placed in plastic tube. The mucosa is washed with PBS pH 6.2 for 5 min and the mucosa is observed again under the microscope to find the number particles retained on the mucosa. The adhesion number is determined by Na= N/No where Na is the adhesion number, N is the total number of particles in the area and N is the number of particles attached to the mucosa after washing.

[ 0068 ] The drug-release profile of esketamine and brimonidine loaded chitosan microspheres are tested using a Franz diffusion cell apparatus with a 12000 MWCO dialysis membrane between the donor and receptor compartments. The microspheres are dispersed in PBS pH 6.2 (normal pH of the nasal cavity) in the donor compartment and dialyzed against PBS pH 6.2 buffer maintained at 37+1 ⁰ C. The receptor compartment is stirred on a magnetic stirrer. At predetermined time intervals aliquots are withdrawn from the receptor compartment and replaced with same volume of fresh medium. Drug concentration in the samples is determined using a HPLC method.

EXAMPLE 9

PHARMACOKINETIC STUDY OF THE DRUG LOADED CHITOSAN

MICROSPHERES

[ 0069 ] Relative bioavailability of the drug (esketamine, brimonidine and their combination)-loaded chitosan microspheres with reference to their nasal solution formulation will be assessed in male Sprague-Dawley rats following nasal administration using an intranasal Mucosal Atomization Device as mentioned above. In a study similar to the above, esketamine microsphers, brimonidine microspheres or microspheres loaded with a combination of these drugs are administered at a predetermined dose separately to three sets of 42 male Sprague- Dawley rats. Blood samples are collected via the tail vein at different time intervals up to 48 h and drug(s) concentration is determined as above. Pharmacokinetic parameters are calculated using the WinNonlin® software and relative bioavailability (RA) of each formulation is determined. EXAMPLE 10

DRUGS, DOSES AND ROUTES OF ADMINISTRATION

[0070] Racemic (R, S) ketamine hydrochloride (HCI), esketamine (S-ketamine) hydrochloride plus brimonidine tartrate solutions is prepared in isotonic saline for intramuscular (i.m.) injections and in 0.5 ug chitosan - HCI / O. lmL for intranasal (i.n.) administration (see Fig. 4). Intranasal administration of s-ketamine has been utilized as a safe and effective route for short-term sedation and restraint in children. It is less demanding technically, avoids pain and anxiety associated with parenteral injections, and circumvents hepatic 1st pass metabolism (Nielsen et al., 2013). Chitosan can enhance the permeability of the nasal mucosa in a reversible manner. Drug concentrations / doses (mg/mL) are calculated as the base compound. Water and feed are withheld 8 hrs. before dosing of animals.

EXAMPLE 11

EXPERIMENTAL TREATMENTS /ANALGESIC AND BEHAVIORAL

ASSESSMENTS

[0071] The subjects are male Sprague-Dawley rats housed individually with food and water ad libitum. Contusion surgeries are performed while subjects are anesthetized using isoflurane. Anesthesia is induced using a 5% isoflurane gas in an induction chamber and a stable plane of anesthesia is maintained using 2% isoflurane gas during contusion surgery. Contusion injuries are made using the Infinite Horizons impactor, specifically designed for medical research using rats and mice. Contused subjects receive two injections of Penicillin G (100,000 units/kg, i.p.) and are given saline injections to maintain Hydration.

[0072] On the day following the spinal contusion injury, baseline locomotor and pain reactivity thresholds are assessed as described below. Subjects are assigned to experimental conditions, so that baseline locomotor and pain reactivity thresholds are balanced across groups. Each group of subjects is treated with a prescribed dose of either racemic ketamine or esketamine and brimonidine (intramuscular or intranasal). To assess the analgesic efficacy of the drugs, pain reactivity is re-assessed 30 min., lhr., 3hr., 6hr., 24 hr., 72 hr., and 7 days following the administration of the drugs. Blood (serum) samples are taken at 1, 2 and 3 hours for miRNA analysis. A second group of animals is subjected to the treatment protocol to assess changes in the miRNA signatures in spinal cord tissues. [ 0073 ] Recovery of motor and sensory functions can be monitored for 28 days post injury. Details on the behavioral tests used to monitor recovery (Hook et al., 2011). Briefly, locomotor recovery is assessed using the Basso, Beattie and Bresnahan (BBB) scale. This 21- point scale is used as an index of hind limb functioning after a spinal injury. Pain, or sensory reactivity, can be assessed using both mechanical (tactile stimulation of the hind paws and girdle regions using von Frey monofilaments) and thermal (tail-flick test) stimuli. Psychological well- being is assessed using tests established to measure depression and anxiety-like symptoms in rats, including the sucrose preference test, social exploration tests, and the forced swim test. Subjects are euthanized using a lethal dose of pentobarbital (100 mg/kg, i.p.), according to the recommendations of the Panel of Euthanasia of the American Veterinary Association.

[ 0074 ] Subjects can undergo a moderate spinal contusion at T12 (T12 vertebral level, IH impactor, 150 kdynes with 1 sec dwell time). On the day following injury, and following the assessment of baseline locomotor (BBB scale) function and pain reactivity (mechanical and thermal) thresholds, subjects are treated with racemic ketamine (comparator) and escalating doses of S-ketamine plus brimonidine (see Fig. 3). Racemic ketamine has been demonstrated to decrease anhedonia and immobility on the forced swim test, without increases in locomotor activity, for up to 8 days (Wang et al. 2011; Ma et al., 2013). The remaining subjects in each cohort can receive an injection of an equivalent volume of 0.9% saline. BBB scores are balanced across treatment groups.

[ 0075 ] Pain reactivity thresholds are assessed prior to S-ketamine treatment and then 30 min., 1 hr., 3 hr., 6 hr., 24 hr., 72 hr., and 7 days following administration. Thermal reactivity is assessed using radiant heat and the tail flick test (see Hook et al. 2011 for procedural details). Subjects are placed in clear Plexiglas tubes with their tail positioned in a 0.5 cm deep groove, cut into an aluminum block, and allowed to acclimate to the apparatus (IITC Inc., Life Science, CA) and testing room for 15 min. The testing room is maintained at 26.5°C. Thermal thresholds can then be assessed, using a halogen light that is focused onto the rat's tail. Prior to testing the temperature of the light, focused on the tail, is set to elicit a baseline tail flick response in 3-4 sec (average) in intact rats. This pre-set temperature is maintained across all subjects. In testing, the latency to flick the tail away from the radiant heat source (light) is recorded. If a subject fails to respond, the test trial is automatically terminated after 8 sec. of heat exposure, to avoid tissue damage. Mechanical reactivity is assessed using von Frey stimuli (Semmes-Weinstein Anesthesiometer; Stoelting Co., Chicago, IL) applied to the plantar surface of the hind paws. Subjects are placed into clear Plexiglas tubes with both hind limbs outside the tube and hanging freely. After a 15-min acclimation period, the von Frey stimuli are applied sequentially at approximately 2 sec. intervals until the subjects withdraw their paw and vocalize. If no response is observed, testing is terminated at a force of 300 g. Stimulus intensity is reported using the formula provided by: Semmes-Weinstein: Intensity= log 10 (10,000*g force).

[ 0076 ] For all subjects, depression is assessed with a comprehensive battery of tests (see Table 1) prior to SCI and on days 9-11 and 19-21-post injury (see figure previous page). Details of the basic methods and clinical significance of each of the behavioral tests comprising the depression ethogram, including sucrose preference, open field activity, social exploration, burrowing, and forced swim tests, are detailed in Table 1 (see Luedtke et al. 2014, Maldonado et al. 2015 for more details). Appetite deviation (AD), another clinical symptom of depression, can also be derived by subtracting average food consumption in the acute phase (days 9-11) or chronic phase (19-21) of injury from baseline average daily food consumption (derived over the 5 days immediately prior to injury). Comparisons are made across treatment groups for each of the behavioral measures using repeated measures analysis of variance (ANOVA) tests. Baseline scores are used as a covariate when significant (p<0.05).

[ 0077 ] To detect co-morbid anxiety (and characterize any additional subject clusters derived in hierarchical clustering), the shock-probe burying task is conducted on Days 5 and 20 post injury. For this test, rats are placed in a plastic box with a small probe (5 cm) placed 2 cm above the bedding level at one far end of the box. The probe is connected to a source of mild electrical stimulation (0.08 mA). As soon as the subject comes into contact with the probe (nose or forepaws touch), the electrical stimulation is turned off. The time that the rats spend engaged in burying and freezing behavior (indicative of anxiety) is recorded from post hoc video analyses. A novel object test can also be conducted at the end of the recovery period to assess cognitive function. This test compares time spent exploring a novel, versus a familiar, object. As rats inherently prefer to explore novel objects, more time than chance spent exploring the novel object indicates intact memory for the familiar object.

[ 0078 ] Data Analysis: The results are analyzed using an analysis of variance (ANOVA) and trend analyses. In experiments with a continuous independent variable (e.g., recovery period, rostra) / caudal histological sections), mixed— design ANOVAs are used. In cases where significant between-subjects differences are obtained (main effect of a single variable), group means are compared using a Duncan's New Multiple Range Test (p < 0.05). Group differences on dichotomous variables (e.g., mortality) are evaluated using X2 probability tests. [0079] MicroRNA Extraction: MicroRNAs are isolated using the miRNAeasy kit (Qiagen) according to manufacturer's protocol with a few modified steps. Total RNA with microRNA is eluted into 40 microliter RNAase- and DNAase-free water. The purity and concentration of all RNA samples are quantified spectrophotometrically using the NanoDrop ND-1000 system (Fisher), and RNA quality is assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Samples with RIN numbers higher than 9 are used for qRT-PCR.

[0080] MicroRNA screening: After micro-RNA extraction, a total of 381 specific rodent miRNAs are profiled using TaqMan® Array Rodent MicroRNA Cards (run on 7900 ABI) and are analyzed using DataAssistTM Software.

[0081] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described are achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[0082] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[0083] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

[0084] In some embodiments, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the application are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[ 0085 ] In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) are construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[ 0086 ] Variations on preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[ 0087 ] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

[ 0088 ] In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

Claims

CLAIMS What is claimed is:

1. A composition comprising esketamine and brimonidine, wherein esketamine and brimonidine act synergistically in a subject to treat or prevent one or more medical conditions in the subject, or to facilitate an analysis in the subject.

2. The composition of Claim 1, wherein the one or more medical conditions are selected from the group consisting of pain, depression, and hearing loss.

3. The composition of Claim 1, wherein esketamine and brimonidine are encapsulated in chitosan microspheres.

4. The composition of Claim 1, wherein the composition is delivered intranasally.

5. The composition of Claim 1, wherein the composition is in the form of a gel for topical application.

6. A method of treatment, prevention, or analysis involving a subject, comprising, delivering the composition of Claim 1 to the subject;

wherein esketamine and brimonidine act synergistically in the subject to achieve the treatment or prevention, or to facilitate the analysis.

7. The method of Claim 6, wherein the treatment is selected from the group consisting of: treating pain, treating depression, treating hearing loss, alleviating the adverse effects of opioid use in a subject treated with a first dosage of opioids, and any combination thereof.

8. The method of Claim 7, wherein alleviating the adverse effects of opioid use in a subject treated with a first dosage of opioids further comprises, after delivering the composition to the subject, delivering to the subject a second, lower dosage of opioids, wherein the esketamine and brimonidine act synergistically to alleviate the adverse effects of opioid use.

9. The method of Claim 8, where the adverse effects are hyperalgesia, allodynia, dependence on opioids, constipation, respiratory depression, nausea and/or vomiting.

10. The method of Claim 6, wherein the prevention is selected from: preventing hearing loss, preventing the conversion of acute pain to chronic pain, and a combination thereof.

11. The method of Claim 10, wherein the esketamine and brimonidine act synergistically to prevent hearing loss by protecting SCG neurons in response to injury.

12. The method of Claim 10, wherein preventing the conversion of acute pain to chronic pain comprises delivering the composition to the subject within 0 to 9 days of the onset of acute pain.

13. The method of Claim 6 wherein the analysis is determination of a therapeutic target for treatment of pain, and wherein changes in miRNA in the subject are measured, and wherein a change in miRNA indicates that the corresponding gene is a therapeutic target for treatment of pain.

14. The method of Claim 6, wherein the composition is applied topically.

15. The method of Claim 6, wherein the composition is encapsulated in chitosan microspheres.

16. The method of Claim 6, wherein the composition is injected.

17. The method of Claim 6, wherein the composition is delivered intranasally.