Glutathione trisulfide (GSSSG) in neuroprotection

Abstract

Methods for the use of glutathione trisulfide (GSSSG) in neuroprotection, for example in neurodegenerative diseases, and to reduce the risk of ischemic injury. For example, the methods can be used to reduce the risk of injury to the brain, spinal cord, and peripheral nerves due to ischemia or low blood flow conditions that can occur due to surgery, trauma, and other conditions that reduce / impair blood flow and / or oxygen delivery to the nervous system.

Description

Technical Field

[0001] Claims of Priority This application claims the benefit of U.S. Provisional Patent Application No. 63 / 023,686, filed May 12, 2020. The entire contents of the foregoing application are incorporated herein by reference.

[0002] This specification describes the use of glutathione trisulfide (GSSSG) for, for example, neuroprotection in neurodegenerative diseases and for reducing the risk of ischemic injury. For example, the methods can be used to reduce the risk of injury to the brain, spinal cord, and peripheral nerves due to ischemic or low blood flow conditions that can occur due to surgeries, trauma, and other conditions that reduce or harm blood flow and / or oxygen delivery to the nervous system.

Background Art

[0003] Delayed paraplegia is a devastating complication of ischemic spinal cord injury (SCI) that can occur after thoracic and / or abdominal aorta surgery and trauma to the spinal cord. While the incidence of ischemic SCI has been reported to be approximately 3%, over 80% present with delayed onset of symptoms (Ullery et al., 2011). Although the mechanism of delayed paraplegia is poorly understood, studies suggest that apoptosis of motor neurons (Kakinohana et al., 2011), as well as the recruitment of microglia and bone marrow-derived macrophages (BMDMs) in ischemic stroke (Denes et al., 2008) and SCI (Bell et al., 2013; Donnelly et al., 2011; Kigerl et al., 2007), play a significant role.

Summary of the Invention

[0004] This specification provides methods for treating or reducing the risk of neurodegenerative disorders in subjects. These methods involve administering a therapeutically or prophylactically effective amount of a composition prepared using glutathione trisulfide (GSSSG) crystals to a subject in need. In some embodiments, the method includes preparing a GSSG-containing composition by dissolving the crystalline form of GSSG in saline at pH 3–6. Furthermore, this specification provides GSSG-containing compositions for use in methods for treating or reducing the risk of neurodegenerative disorders in subjects, such as compositions prepared by dissolving the crystalline form of GSSG in saline at pH 3–6.

[0005] In some embodiments, the impairment is post-ischemic neuronal cell death.

[0006] In some embodiments, the disorder is a chronic neurodegenerative disease, such as multiple stroke dementia, Alzheimer's disease, Parkinson's disease, or Lewy body dementia.

[0007] In some embodiments, the method involves administering an effective amount of a composition containing GSSSG within a few minutes to a few hours after a traumatic injury occurs.

[0008] In some embodiments, the method involves administering an effective amount of a composition containing GSSSG prior to a planned thoracic and / or abdominal aortic surgical procedure.

[0009] In some embodiments, the method involves administering an effective amount of the composition containing GSSSG several hours to several days before a scheduled thoracic and / or abdominal aortic surgical procedure.

[0010] In some embodiments, the method involves administering an effective amount of the composition containing GSSSG 2 to 24 hours and / or 1, 2, 3, 4, 5, 6, and / or 7 days prior to a scheduled thoracic and / or abdominal aortic surgical procedure.

[0011] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. Methods and materials are described herein for use in this invention; other preferred methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and not intended to limit. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of any conflict, this specification shall prevail, including definitions.

[0012] Other features and advantages of the present invention will become apparent from the following detailed description and figures, and from the claims. [Brief explanation of the drawing]

[0013] [Figure 1] [Figure 1A-B] Graphs showing BMS (A) and survival rate (B) of mice that underwent pre-treatment with GSSSG or DMSO alone before being converted to SCI. [Figure 2] This graph shows that polysulfides, rather than Na2S, protected SH-SY5Y cells from MPP+-induced cell death. N=4 for each group. *, **, ***, ****P<0.05, 0.01, 0.001, 0.0001 vs vehicle; P<0.01 control vs MPP+ in each treatment. [Modes for carrying out the invention]

[0014] Persulfides (RS-SH) and polysulfides (RS-Sn-SR) are molecules containing sulfen sulfur, which is a sulfur atom with six valence electrons but no charge, and have a protective effect against oxidative stress (Akaike et al., 2017; Ida et al., 2014). These molecules can release H2S, and therefore, the antioxidant or protective effect of these molecules is thought to be mediated by both H2S and sulfen sulfur. Glutathione trisulfide (GSSSG) is one of the major polysulfide species in mammalian tissues, consisting of GSSG, a metabolite of glutathione, and one additional sulfen sulfur atom.

[0015] Until recently, methods for producing GSSG compounds involved the use of or the generation of toxic gases, resulting in compounds that were unstable or unsuitable for pharmaceutically acceptable use. European Patent Application Publication No. 3560947 (EP3560947) describes a method for producing GSSSG in a stable crystalline form. However, the efficacy of this crystalline form of GSSSG in vivo for neuroprotection is not described.

[0016] This study investigated the beneficial effects of crystalline GSSSG in neuroprotection, including its effects on neurological dysfunction after SCI in mice. Specifically, the effects of pre-treatment with GSSSG prior to the onset of SCI were examined. Patients often undergo aortic surgery after a certain diagnostic period (e.g., one week), depending on their condition, which provides an opportunity to use the treatments described herein to reduce the risk of postoperative complications. In addition, the results confirmed the protective effect of GSSSG against 1-methyl-4-phenylpyridinium (MPP+)-induced neuronal cell (SH-SY5Y cell) death. Since MPP+ poisoning is an in vitro model of Parkinson's disease, this demonstrates that crystalline GSSSG can be similarly used in neurodegenerative diseases.

[0017] The results described herein demonstrate the beneficial capacity of GSSSG crystals in in vivo neuroprotection. These results indicate the effect of GSSSG pre-modification on maintaining neuronal function after SCI and suggest that the drug can be administered after the onset of ischemia due to its antioxidant effects.

[0018] Treatment method The methods described herein include methods for treating or reducing the risk of neurodegenerative disorders in subjects, e.g., mammalian subjects, e.g., human or non-human veterinary subjects. In some embodiments, the disease is post-ischemic neuronal cell death. In some embodiments, the disease is a chronic neurodegenerative disease (e.g., multiple stroke dementia, Alzheimer's disease, Parkinson's disease, or Lewy body dementia). Generally, the methods include administering a therapeutically effective amount of a composition comprising a crystalline form of GSSSG as described herein to a subject that requires or is judged to require such treatment.

[0019] In this context, “to treat” means to improve at least one symptom of a neurodegenerative disorder. Conditions that can be treated using the methods described herein may be associated with loss of motor control, paralysis, or paraplegia. Administration of therapeutically effective doses of the compounds described herein may result in improved motor control, reduction of paralysis, or paraplegia.

[0020] In addition, this method may reduce the risk of loss of motor control, paralysis, or paraplegia. Subjects at risk of loss of motor control, paralysis, or paraplegia may include those who have suffered traumatic injuries, as well as those who are about to undergo thoracic and / or abdominal aortic surgery. These methods may involve administering an effective amount of a GSSSG composition, as described herein, within minutes to hours after the traumatic injury occurs and / or before the scheduled thoracic and / or abdominal aortic surgical procedure, for example, several hours to several days prior.

[0021] An “effective dose” is an amount sufficient to produce a beneficial or desired result. For example, a therapeutic dose is an amount that achieves a desired therapeutic effect. This amount may be the same as or different from a prophylactic effective dose, which is the amount required to prevent the onset of a disease or disease symptom. The effective dose may be administered in one or more doses, applications, or dosages. The composition may be administered in one, once daily, or multiple times, or in multiple doses including once a week or once every other day. In some embodiments, GSSSG is administered daily for at least two, three, four, five, six, or seven days prior to a scheduled thoracic and / or abdominal aortic surgery procedure. Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or disorder in question, previous treatments, overall health status, and / or age, as well as other pre-existing conditions, may influence the dose and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutic effective dose of the therapeutic compounds described herein may include a single treatment or a series of treatments.

[0022] The dosage, toxicity, and therapeutic efficacy of therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as procedures for determining the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic factor, which can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a high therapeutic factor are preferred. While compounds exhibiting toxic side effects may be used, care should be taken to design a delivery system that targets the site of the affected tissue to reduce side effects by minimizing potential damage to non-infectious cells.

[0023] Data obtained from cell culture assays and animal studies can be used to determine a range of dosage for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes an ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the present invention, a therapeutically effective dosage can first be estimated from cell culture assays. The dosage may be determined to achieve a range of circulating plasma concentrations that includes the IC50 (i.e., the concentration of the test compound that achieves maximal half-maximal inhibition of symptoms) in an animal model as determined in cell culture. Such information can be used to more accurately determine useful dosages in humans. Plasma levels may be measured, for example, by high performance liquid chromatography.

[0024] Pharmaceutical Compositions and Methods of Administration The methods described herein include the use of a pharmaceutical composition comprising GSSSG as an active ingredient, which composition is prepared by dissolving this crystalline GSSSG in a buffer, such as physiological saline at pH 3 - 6, using a crystalline form of GSSSG as described in European Patent Application Publication No. 3560947. A method for preparing a crystalline form of glutathione trisulfide dihydrate may include precipitating crystals of glutathione trisulfide dihydrate in an aqueous solution in which glutathione trisulfide is dissolved, and collecting the precipitated crystals of glutathione trisulfide dihydrate.

[0025] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the expression "pharmaceutically acceptable carrier" includes physiological saline, solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, etc., which are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the composition.

[0026] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as, for example, intravenous.

[0027] Methods of formulating suitable pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY) series. For example, solutions or suspensions for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and a drug for tonicity adjustment such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0028] Suitable pharmaceutical compositions for injectable use include sterile aqueous solutions (water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be easily injected with a syringe. The composition must be stable under manufacturing and storage conditions and stored in a manner that prevents contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene (polyetheylene) glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of a surfactant. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols like mannitol and sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0029] Sterile injectable solutions can be prepared by mixing the required amount of the active compound with one or a combination of the components listed above in a suitable solvent, and subsequently sterilizing by filtration, if necessary. Generally, dispersions are prepared by mixing the active compound in a sterile vehicle containing a base dispersion medium and other required components from the components listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield powders of the active ingredient and any additional desired components from the sterile filtered solution.

[0030] The pharmaceutical composition can be placed in a container, box, or dispenser along with instructions for administration. For example, GSSSG can be supplied in crystalline form as a kit, along with a sterile buffer (e.g., physiological saline) with a pH of 3-6 used to dissolve the crystals and prepare the solution for injection. [Examples]

[0031] The present invention will be further described in the following examples, but these examples are not intended to limit the scope of the present invention as described in the claims.

[0032] [Example 1] The preventive effect of GSSG on neurological dysfunction after SCI. To elucidate the molecular mechanisms leading to delayed paraplegia, the inventors recently developed and thoroughly characterized a mouse model of SCI in which mice exhibit delayed paraplegia and have the lowest surgical mortality rate (Kakinohana et al., 2011). Briefly, SCI was induced by placing a first clip in the aortic arch between the left common carotid artery and the left subclavian artery, and a second clip on the origin of the left subclavian artery, under anesthesia and mechanical ventilation via endotracheal intubation. Perfect occlusion was confirmed by the immediate loss and maintenance of pulse pressure, which was detectable by tracking femoral artery pressure. After 5 minutes of ischemia, the clips were removed and the chest was closed by overlapping. Ten minutes after blood flow was restored, the arterial catheter was removed, the incision was closed, and the animals were allowed to recover from anesthesia. Throughout the surgical period, from anesthesia to recovery, the temperature of the erector spinae muscles was monitored and maintained at 37.5°C. In sham-operated mice, all surgical procedures were performed as described, but clipping was not performed. Motor function was continuously quantified using the Basso Mouse Scale (BMS) before spinal cord ischemia (SCI), and at 24, 48, and 72 hours after spinal cord ischemia (Basso et al., 2006; Kakinohana et al., 2011). The maximum deficit is indicated by a score of 0. A BMS score < 6 (0-5) indicates paraplegia, while a BMS score > 6 (6-9) indicates the ability to walk.

[0033] To investigate the prophylactic effect of GSSSG on neuronal dysfunction after SCI, mice were subjected to pre-conditioning with GSSSG treatment before induction of SCI. Briefly, GSSSG was ground using an agate mortar and dispersed in DMSO using an ultrasonic water bath, and administered as intracellular therapy (IP) at a dose of 200 mg / kg per day for 4 days. Control mice were administered DMSO alone. Mice were induced to undergo SCI 24 hours after the final dose of either GSSSG or DMSO alone.

[0034] The results showed that all mice treated with DMSO alone developed paraplegia after SCI, while all mice pre-conditioned with GSSSG prevented motor dysfunction and paraplegia (Figure 1A). Pre-conditioning with GSSSG did not affect the survival rate of mice after SCI (Figure 1B).

[0035] [Example 2] Protective effects of GSSG in neurodegeneration models The effect of GSSSG on 1-methyl-4-phenylpyridinium (MPP+)-induced neuronal cell (SH-SY5Y cell) death was evaluated. MPP+ toxin administration is an in vitro model of Parkinson's disease.

[0036] SH-SY5Y cells were incubated at 37°C for 24 hours in DMEM / F12 (20% FBS) with or without the drug, with or without MPP+ (2 mM). Cell viability was measured using the Crystal Violet assay.

[0037] The results shown in Figure 2 demonstrate that polysulfides, rather than Na2S, protected SH-SY5Y cells from MPP+-induced cell death.

[0038] References cited

[0039] [Table 1]

[0040] Other Embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to illustrate, not limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the following claims. The present invention includes the following embodiments. [1] A method for treating or reducing the risk of neurodegenerative disorders in a subject, comprising administering a therapeutically or prophylactically effective amount of a composition prepared using glutathione trisulfide (GSSSG) crystals to a subject in need thereof. [2] The method according to [1] above, further comprising preparing the composition containing GSSSG by dissolving the crystalline form of GSSSG in physiological saline at pH 3 to 6. [3] The method according to [1] above, wherein the disorder is post-ischemic neuronal cell death. [4] The method according to [1] above, wherein the disorder is a chronic neurodegenerative disease. [5] The method according to [1] above, wherein the chronic cerebral degenerative disease is multiple cerebral infarction dementia, Alzheimer's disease, Parkinson's disease, or Lewy body dementia. [6] The method according to [1] above, comprising administering an effective amount of a composition containing GSSSG within a few minutes to a few hours after a traumatic injury occurs. [7] The method according to [1] above, comprising administering an effective amount of a composition containing GSSSG prior to a scheduled thoracic and / or abdominal aortic surgical procedure. [8] The method according to [7] above, comprising administering an effective amount of a composition containing GSSSG several hours to several days prior to a scheduled thoracic and / or abdominal aortic surgical procedure. [9] The method according to [8] above, comprising administering an effective amount of a composition containing GSSSG 2 to 24 hours and / or 1, 2, 3, 4, 5, 6 and / or 7 days prior to the scheduled thoracic and / or abdominal aortic surgical procedure.

[10] A composition prepared using glutathione trisulfide (GSSSG) crystals for use in the treatment of or reduction of the risk of neurodegenerative disorders in a subject, wherein the method comprises administering a therapeutically or prophylactically effective amount to a subject in need thereof.

[11] The composition for the use described in

[10] above, wherein the composition is prepared by a method comprising dissolving the crystalline form of GSSSG in physiological saline at pH 3 to 6.

[12] The composition for the use described in

[10] above, wherein the impairment is post-ischemic neuronal cell death.

[13] The composition for the use described above in

[10] , wherein the disorder is a chronic neurodegenerative disease.

[14] The composition for the use described above

[10] , wherein the chronic cerebral degenerative disease is multiple cerebral infarction dementia, Alzheimer's disease, Parkinson's disease, or Lewy body dementia.

[15] The composition for use according to

[10] above, wherein the method comprises administering an effective amount of the composition containing GSSSG within a few minutes to several hours after a traumatic injury occurs.

[16] The composition for the use described in

[10] above, wherein the method comprises administering an effective amount of the composition comprising GSSSG prior to a planned thoracic and / or abdominal aortic surgical procedure.

[17] The composition for the use described above

[16] , wherein the method comprises administering an effective amount of the composition comprising GSSSG several hours to several days before a scheduled thoracic and / or abdominal aortic surgical procedure.

[18] The composition for use according to

[17] above, wherein the method comprises administering an effective amount of the composition containing GSSSG 2 to 24 hours and / or 1, 2, 3, 4, 5, 6 and / or 7 days prior to the scheduled thoracic and / or abdominal aortic surgical procedure.

Claims

1. A composition for the treatment of or reduction of the risk of neurodegenerative disorders in a subject, comprising glutathione trisulfide (GSSSG), wherein the disorder is post-ischemic neuronal cell death, and the composition is administered to the subject prior to a planned thoracic and / or abdominal aortic surgical procedure.

2. The composition according to claim 1, wherein the crystalline form of GSSSG is dissolved in physiological saline at a pH of 3 to 6.

3. The composition according to claim 1, which is administered several hours to several days before a scheduled thoracic and / or abdominal aortic surgical procedure.

4. The composition according to claim 3, which is administered 2 to 24 hours and / or 1, 2, 3, 4, 5, 6 and / or 7 days before the scheduled thoracic and / or abdominal aortic surgical procedure.

Images