Intranasal Administration of Polysulfides

Abstract

Methods and devices for the nasal administration of compositions comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS) or lipoic acid trisulfide (LA-SSS) for neuroprotection in neurodegenerative diseases and for reducing the risk of ischemic injury. The method can be used, for example, to reduce the risk of injury to the brain, spinal cord, and peripheral nerves from a state of ischemia or low blood flow that can be caused by, for example, surgery, trauma, and other conditions that reduce / impair blood flow and / or oxygen delivery to the nervous system.

Description

Technical Field

[0001] Priority Claim This application claims the benefit of U.S. Provisional Application No. 63 / 344,095, filed May 20, 2022. The entire content of the foregoing is incorporated herein by reference.

[0002] Methods and devices for the nasal administration of compositions comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS) or lipoic acid trisulfide (LA-SSS) for neuroprotection in neurodegenerative diseases and for reducing the risk of ischemic injury. The methods can be used to reduce the risk of injury to the brain, spinal cord, and peripheral nerves from ischemic or low blood flow conditions that can be caused, for example, by surgery, trauma, and other conditions that reduce / impair blood flow and / or oxygen delivery to the nervous system.

Background Art

[0003] Approximately 2-12% of patients undergoing thoracoabdominal aortic surgery experience devastating complications of paraplegia 1,2 . More than 80% of postoperative paraplegia is reported to be delayed and caused by secondary neuronal injury within the spinal cord 3,4 . Although the pathogenesis of secondary neuronal injury is incompletely understood, increased oxidative stress, mitochondrial dysfunction, inflammation, apoptosis, and glutamate-mediated excitotoxicity have been suggested to play important roles 5,6 . Since the onset of paraplegia is delayed in these patients, there is an excellent opportunity for potential preventive intervention. However, to date, there has been no pharmacological treatment shown to reduce delayed paraplegia after thoracoabdominal aortic surgery.

Summary of the Invention

[0004] As shown herein, when delivered nasally after reperfusion, glutathione trisulfide (GSSSG) and pantethine trisulfide (PTN-SSS) prevented delayed paraplegia after SCI. The neuroprotective effect of GSSSG was accompanied by an increase in local sulfenyl sulfur concentration in the lumbar spinal cord.

[0005] Accordingly, provided herein is a method of using nasal administration of GSSSG, PTN-SSS, or lipoic acid trisulfide (LA-SSS) to treat a disorder associated with neurodegeneration or reduce the risk of a disorder in a subject. In some embodiments, the method includes preparing a composition comprising GSSSG by dissolving a crystalline form of GSSSG in saline at pH 3-6, such as pH 4.8-5.0. Further provided is a composition for nasal administration, optionally a composition prepared by dissolving a crystalline form of GSSSG in saline at pH 3-6, such as pH 4.8-5.0, comprising GSSSG, PTN-SSS, or LA-SSS for use in a method of treating a disorder associated with neurodegeneration or reducing the risk of a disorder in a subject.

[0006] In some embodiments, the disorder is post-ischemic neuronal death.

[0007] In some embodiments, the disorder is a chronic neurodegenerative disease, such as multi-infarct dementia, Alzheimer's disease, Parkinson's disease, or Lewy body dementia.

[0008] In some embodiments, the method includes administering an effective amount of a composition comprising GSSSG, PTN-SSS, or LA-SSS within minutes to hours after the occurrence of a traumatic injury.

[0009] In some embodiments, the method includes administering an effective amount of a composition comprising GSSSG, PTN-SSS, or LA-SSS prior to a scheduled thoracic and / or abdominal aortic surgical procedure.

[0010] In some embodiments, the method includes administering an effective amount of a composition comprising GSSSG, PTN-SSS, or LA-SSS several hours to several days prior to a scheduled thoracic and / or abdominal aorta surgical procedure.

[0011] In some embodiments, the method includes administering an effective amount of a composition comprising GSSSG, PTN-SSS, or LA-SSS 2 to 24 hours and / or 1, 2, 3, 4, 5, 6, and / or 7 days prior to a scheduled thoracic and / or abdominal aorta surgical procedure.

[0012] Furthermore, provided herein is an apparatus for the nasal administration of GSSSG, PTN-SSS, or LA-SSS to a subject.

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The methods and materials used in the present invention are described herein. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0014] Other features and advantages of the present invention will become apparent from the following detailed description, the drawings, and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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Mode for Carrying Out the Invention

[0016] Hydrogen sulfide (H2S), a colorless gas with a characteristic rotten - egg odor, is an environmental hazardous substance produced by various natural and industrial sources. H2S is also considered a signaling molecule that plays diverse physiological roles. 7 . Many effects of H2S are attributed to sulfenyl sulfur molecular species, such as persulfides (RSSH) and polysulfides (RS n H). The cytoprotective effects of sulfenyl sulfur molecular species can be mediated by multiple mechanisms including antioxidant 8 and anti - inflammatory effects 9,10 , inhibition of lipid peroxidation and ferroptosis by scavenging of free radicals 10,11 , as well as post - translational modification of proteins 12 . Sulfenyl sulfur (S), a sulfur atom with six valence electrons but no charge, 13,14 . 0In the process known as persulfidation, sulfen sulfur molecular species are readily donated to receptor thiols in target proteins, resulting in post-translational modification in proteins and regulating the function of target proteins. 13,14 . In previous studies, we showed that inhaled H2S prevented delayed paraplegia in mice under transient spinal cord ischemia (SCI). The neuroprotective effect of H2S seemed to be associated with persulfidation of nuclear factor kappa B (NF-kB) p65. 15 .

[0017] The mechanism by which systemically administered H2S donor compounds regulate the concentration of reactive sulfur molecular species in target tissues is not fully defined, partly because of the short half-life of H2S in the blood. 16 . This knowledge gap regarding the in vivo pharmacokinetics of sulfides has hindered the application of sulfide-based therapies to patient care. To enable future use of polysulfides in the treatment of neurodegenerative diseases, it is essential to determine whether polysulfide administration regulates the local concentration of sulfen sulfur molecular species in the central nervous system (CNS).

[0018] Glutathione trisulfide (GSSSG) is an endogenous polysulfide (Figure 1A) and is in dynamic equilibrium with various reactive sulfur molecular species including glutathione (GSH), glutathione hydroperysulfide (GSSH), glutathione hydroepolysulfide, and other glutathione polysulfides. 9,17 GSSSG could be an important endogenous reservoir of sulfen sulfur molecular species. The observation that the levels of sulfen sulfur molecular species vary in patients according to disease type and severity suggests that sulfen sulfur molecular species may have a protective role in pathological conditions. 18,19 .

[0019] GSH, a natural tripeptide of glutamic acid, cysteine, and glycine, is ubiquitous and the most common thiol (RSH) in mammalian cells. GSH is a nucleophile and acts as the major intracellular antioxidant in mammalian cells. 20。GSH has been reported to have a neuroprotective effect against ischemia-reperfusion injury 21 。Glutathione disulfide (GSSG) is the oxidized form of GSH and is mainly produced by glutathione peroxidase-mediated catalysis or by the direct reaction of GSH with electrophilic compounds such as radical species 20 。Both GSH and GSSG can cause post-translational modification of proteins by "glutathionylation", which protects protein cysteines from irreversible oxidation and regulates the structure and function of diverse proteins 20,22~24 。

[0020] Pantethine (PTN), a precursor for the synthesis of coenzyme A, transfers an acetyl group from pyruvate to oxaloacetate, initiating the tricarboxylic acid cycle 25 。Preclinical studies have suggested the beneficial effects of PTN in mouse models of neurodegenerative diseases 26,27 。Pantethine trisulfide (PTN-SSS), a polysulfide, consists of one molecule of sulfenic sulfur and one molecule of PTN (see Figure 1B; refer to International Publication No. WO 2022 / 045052)

[0021] Lipoic acid trisulfide is a relatively small molecule (MW = 238.39) expected to cross the blood-brain barrier and be delivered into the CNS (see, for example, International Publication No. WO 2022 / 045212). Lipoic acid trisulfide, such as GSSSG, improved cell viability in a cellular model of Parkinson's disease, while lipoic acid did not. After delivery of its "sulfenic sulfur" into cells, the resulting lipoic acid and dihydrolipoic acid can exert their own biological properties, such as antioxidants, metal chelators, cofactors for mitochondrial enzymes. LA-SSS consists of one molecule of sulfenic sulfur and one molecule of LA. In some embodiments, LA-SSS is alpha lipoic acid, or LA-SSS-βCD (lipoic acid trisulfide-beta cyclodextrin), or LA-SSS-CE (choline ester); refer to International Publication No. WO 2022 / 045212

[0022] This study investigated the neuroprotective effect and pharmacokinetics of intranasal administration of polysulfides in an established mouse model of spinal cord ischemia. In this mouse model, neurodegeneration mainly occurs in the anterior horn of the lumbar spinal cord 24 - 48 hours after reperfusion. 6,28,29 This experiment determined whether intranasal administration of polysulfides preferentially increases the levels of polysulfides in the CNS. 30~32 It was hypothesized that intranasal administration of polysulfides targeting the CNS prevents neurodegeneration in the lumbar spinal cord by increasing the local concentration of sulfenyl sulfur molecular species and rescues mice from delayed paraplegia.

[0023] In this study, we showed that intranasal administration of GSSSG after reperfusion prevented the extensive loss of surviving neurons in the anterior horn of the lumbar spinal cord and rescued mice from delayed paraplegia after SCI, while GSH or GSSG did not. In primary cortical neurons, GSSSG improved cell viability after OGD / R, while GSH or GSSG did not. The beneficial effects of GSSSG were accompanied by inhibition of the increase in inflammatory cytokine levels and inhibition of microglial activation and caspase - 3 activation. Some 34 significant increases in S - labeled sulfenyl sulfur molecular species were observed in GS 34It was detected in the lumbar spinal cord immediately after intranasal administration of SSG. Furthermore, we observed that the protective effect of GSSSG was accompanied by an increase in sulfenyl sulfur levels in the lumbar spinal cord after intranasal administration of GSSSG and in primary cortical neurons after incubation with GSSSG. Furthermore, incubation of SH-SY5Y cells with PTN-SSS increased intracellular sulfenyl sulfur levels and improved cell viability after OGD / R, and intranasal administration of PTN-SSS after reperfusion rescued mice from delayed paraplegia after SCI but PTN did not. PTN-SSS increased sulfenyl sulfur levels in the central nervous system immediately after intranasal administration. These observations suggest that sulfenyl sulfur can be readily delivered to the central nervous system by intranasal administration of polysulfides and that this treatment prevents delayed neurodegeneration in the lumbar spinal cord and reduces delayed paraplegia. The results of this study highlight the important therapeutic potential of polysulfides in preventing spinal cord neurodegeneration.

[0024] Previously, we used a chemically induced cytotoxicity model using SH-SY5Y cells to show that the cytoprotective effects of H2S donor compounds correlate with their ability to increase intracellular sulfenyl sulfur levels. 45 We also reported that administration of sodium thiosulfate improved the survival and neurological function of mice subjected to global cerebral ischemia-reperfusion, and the cytoprotective effect was accompanied by a marked increase in thiosulfate (sulfenyl sulfur molecular species) in plasma and brain tissue. 46 These results suggest that an increase in the concentration of sulfenyl sulfur may be neuroprotective in pathological conditions. In this study, we showed that the neuroprotective effect of intranasal GSSSG in SCI-induced spinal cord injury was accompanied by an increase in sulfenyl sulfur levels in the lumbar spinal cord. This result supports the hypothesis that the neuroprotective effect of GSSSG is mediated by an increase in sulfenyl sulfur.

[0025] Previous preclinical studies have suggested that administration of GSH improves neuronal cell death after cerebral ischemia-reperfusion. 21,47 Since GSH is a metabolite of GSSSG 9,17It was possible that the increase in the concentration of GSH could explain the neuroprotective effect of GSSSG. However, in this study, we observed that intranasal administration of GSSSG, rather than GSH, prevented delayed paraplegia after SCI. The reason for the contradiction between this report and previous reports regarding the effect of GSH might have arisen from differences in the dose of GSH, the route of administration, and the animal model. In particular, the dose of GSH used in this study was one-tenth of the dose used in previous studies. The results of this study support the hypothesis that the mechanism of GSSSG-mediated neuroprotection is independent of the conversion of GSH.

[0026] Intracellular sulfenyl sulfur molecular species can react with GSH, resulting in the production of GSSH. 9 Compared with GSH, GSSH is more nucleophilic and a better intracellular antioxidant. Furthermore, GSSH can be directly generated from GSSSG. 9 Akaike and co-researchers reported that the concentration of endogenous GSSH in the mouse brain was 222 pmol / mg protein, which was significantly higher than the concentrations of other endogenous sulfenyl sulfur molecular species, including GSSSG (1 pmol / mg protein), CysSSH (2 pmol / mg protein), or CysSSSCys (not detected). 9,37 In this study, 34 30 minutes after intranasal administration of GSSG, 34 S-labeled GSSSG was detected in the lumbar spinal cord at 317 ± 111 pmol / mg protein. Furthermore, based on the previously reported levels of endogenous GSSH and CysSSH, 37 the levels of 34 S-labeled GSSH and 34 S-labeled CysSSH in the lumbar spinal cord after intranasal administration of GSSG should have been approximately 1,600 pmol / mg protein and 18 pmol / mg protein, respectively. 34 9,37These results indicate that intranasal administration of GSSSG can increase the levels of multiple sulfenyl sulfur molecular species by about 10- to 100-fold in the lumbar spinal cord, which is the center of neuronal death after SCI. Considering that GSSH is the most quantitatively prominent sulfenyl sulfur molecular species in the lumbar spinal cord after intranasal administration of GSSSG, most of the neuroprotective effect of GSSSG may be exerted via GSSH.

[0027] Previous studies have shown that molecules over 500 Da cannot pass through the blood-brain barrier and blood-spinal cord barrier after systemic administration. 31 Due to the large size of GSSSG (644.7 Da), we chose to administer this compound intranasally. After intranasal administration, large molecules can bypass the blood-brain barrier and blood-spinal cord barrier through the olfactory nerve pathway and trigeminal nerve pathway and rapidly reach the parenchyma of the central nervous system. 30,32 The peripheral olfactory system connects the nasal cavity to the olfactory bulb and rostral brain, and the peripheral trigeminal system connects the nasal cavity to the brainstem and spinal cord. 30 For example, a previous report compared the intravenous and intranasal routes of administration regarding the amount of methylprednisolone sodium succinate (497.5 Da) that reached the spinal cord. 48 A relatively large amount of methylprednisolone was detected in the spinal cord parenchyma after intranasal administration, whereas much less methylprednisolone was detected after intravenous administration. 48 Various other high-molecular-weight therapeutic agents (>500 Da) have been effectively delivered to the central nervous system by intranasal administration. 49 In the present study, 34 30 minutes after intranasal administration of GSSSG, 34 we observed that the concentration of S-labeled GSSSG (268 ± 140 pmol / mg protein) in the central nervous system was significantly higher than the concentration of endogenous GSSSG (1 pmol / mg protein) in the mouse brain. 37 In contrast, in plasma, 34The concentration of S-labeled GSSSG (25 ± 10 nM) was significantly lower than the endogenous GSSSG in the plasma of wild-type mice (125 nM, unpublished data by Akaike and coworkers). These results suggest that intranasally administered GSSSG readily and preferentially reaches the central nervous system, including the spinal cord, through the olfactory and trigeminal nerve pathways rather than through the bloodstream.

[0028] There are several limitations to this study. First, we did not determine the detailed mechanism responsible for the beneficial effects of polysulfides beyond the fact that the effects were accompanied by increased sulfen sulfur levels in target organs, inhibition of elevated levels of inflammatory cytokines, and inhibition of microglial activation and caspase-3 activation, and are unlikely to be due to transglutathionylation. Polysulfides act as antioxidants. 9,10 and anti-inflammatory drugs 10,11 Acts as a phospholipid inhibitor, inhibits lipid peroxidation and ferroptosis 12 , as well as improved post-translational modifications of proteins 13,14 Polysulfides appear to be cytoprotective through multiple mechanisms, including the suppression of sulfide-catabolizing effect and the induction of sulphuric acid synthesis. The exact mechanism responsible for the neuroprotective effect of polysulfides remains to be elucidated in future studies. Second, we did not investigate the mechanism by which GSSSG suppresses the mRNA level of SQOR, a protein that catabolizes sulphide to GSSH. The increase in the level of GSSH after intranasal administration of GSSSG may downregulate the expression of SQOR through negative feedback. Third, although GSSSG and PTN-SSS were administered intranasally under light sedation, some mice spit or swallowed the drugs. The cytoprotective effect conferred by polysulfides in this study seems to reflect the effect of a low dose administered effectively. As better drug formulations that are more suitable for clinical application are developed, we expect that the beneficial effects of polysulfides will be achieved using lower doses.

[0029] This study revealed that intranasal administration of GSSSG or PTN-SSS after reperfusion rescued mice under the influence of SCI from delayed paraplegia, while GSH, GSSG, or PTN did not. Intranasally administered GSSSG accumulated in the lumbar spinal cord, increased the local concentrations of persulfides, polysulfides, and sulfenyl sulfur, and reduced neuroinflammation, apoptosis, and neurodegeneration. The potent neuroprotective effect of intranasally administered GSSSG is similar to the effect of inhaled H2S, but the use of polysulfides, including GSSSG and PTN-SSS, is far more practical in clinical medicine than the administration of gaseous H2S. In particular, the excellent physical properties of PTN-SSS justify further evaluation for clinical development. This study opens up the possibility of a novel polysulfide-based therapy to prevent the development of delayed paraplegia and other neurodegenerative diseases of the spinal cord after thoracoabdominal aortic surgery.

[0030] We also observed the protective effect of trisulfide compounds in MPTP / MPP + induced cell injury. The small size of Na2S3 allowed for its intraperitoneal delivery based on the prediction that this molecule would cross the blood-brain barrier. Due to the large size of GSSSG, we delivered this compound intranasally in an animal model of PD, and the results demonstrated a protective effect and the usefulness of providing neuroprotection in neurodegenerative diseases.

[0031] Method of treatment The methods described herein include methods for treating or reducing the risk of a disorder associated with neurodegeneration in a subject, such as a mammalian subject, such as a human or non-human veterinary subject. In some embodiments, the disorder is, for example, post-ischemic neuronal death in the spinal cord. In some embodiments, the disorder is a chronic neurodegenerative disease (e.g., multi-infarct dementia, Alzheimer's disease, Parkinson's disease, or Lewy body dementia). Generally, the method includes transnasal administration of a therapeutically effective amount of a composition comprising a crystalline form of GSSSG, PTN-SSS, or LA-SSS described herein to a subject in need of such treatment or a subject determined to be in need of such treatment.

[0032] When used in this context, "treating" means ameliorating at least one symptom of a disorder associated with neurodegeneration. Conditions that can be treated using the methods described herein can involve loss of motor control, paralysis or paraplegia. Administration of a therapeutically effective amount of the compounds described herein can result in an improvement in motor control, a reduction in paralysis or paraplegia.

[0033] Furthermore, the methods can result in a reduction in the risk of development of loss of motor control, paralysis or paraplegia. Subjects at risk of developing loss of motor control, paralysis or paraplegia include those who have suffered a traumatic injury and those who are about to undergo thoracic and / or abdominal aorta surgery. Those methods can include administration, within minutes to hours after a traumatic injury has occurred and / or, for example, hours to days before a scheduled thoracic and / or abdominal aorta surgical procedure, of an effective amount of the GSSSG, PTN-SSS or LA-SSS composition described herein by nasal administration.

[0034] "Effective amount" is an amount sufficient to achieve a useful or desired result. For example, a therapeutic amount is an amount that achieves a desired therapeutic effect. This amount may or may not be the same as a prophylactically effective amount that is necessary to prevent the onset of a disease or disease symptom. An effective amount can be administered in one or more administrations, applications or dosages. The composition can be administered from once or more per day to once or more per week, including once every other day. In some embodiments, GSSSG, PTN-SSS or LA-SSS is administered daily for at least 2, 3, 4, 5, 6 or 7 days prior to a scheduled thoracic and / or abdominal aorta surgical procedure. One of ordinary skill in the art will understand that certain factors including, but not limited to, the degree of the disease or disorder, previous treatments, the general health and / or age of the subject, and current other diseases can affect the dosage and timing required to effectively treat the subject. Further, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.

[0035] The dosage, toxicity, and therapeutic efficacy of a therapeutic compound can be determined by standard pharmaceutical procedures in cell culture or animal models, for example, with regard to the determination of LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dosage ratio between the toxic effect and the therapeutic effect is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compounds showing a high therapeutic index are preferred. Although compounds showing toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of the diseased tissue in order to minimize the potential for damage to non-infected cells and thereby reduce side effects.

[0036] Data obtained from cell culture assays and animal tests can be used to formulate the dosage range for use in humans. The dosage of such a compound is preferably within the range of circulating concentrations that include the ED50 and have minimal or no toxicity. The dosage can vary within that range depending on the dosage form utilized and the route of administration. For any compound used in the methods of the present invention, a therapeutically effective dosage can first be evaluated from cell culture assays. The dosage can be formulated in an animal model to achieve a range of circulating plasma concentrations that includes the IC50 (i.e., the test compound concentration that achieves 50% inhibition of the symptoms) determined in cell culture. Such information can be used to more accurately determine useful dosages in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0037] Pharmaceutical Compositions and Methods of Administration The methods described herein include the use of pharmaceutical compositions comprising GSSSG, PTN-SSS or LA-SSS as active ingredients. In some embodiments, the composition uses a crystalline form of GSSSG and is prepared by dissolving crystalline GSSSG in a buffer, such as saline at pH 3-6, for example pH 4.8-5, using the method described in European Patent Application Publication No. 3560947. The pH can be adjusted using an acid or a base, such as hydrochloric acid or sodium hydroxide. Compositions comprising PTN-SSS or LA-SSS can be prepared by dissolving them in a buffer, such as saline or water at pH 4-9, for example 5-8.

[0038] An exemplary method for producing a crystalline form of glutathione trisulfide dihydrate can include the steps of precipitating crystals of glutathione trisulfide dihydrate in an aqueous solution in which glutathione trisulfide is dissolved, and recovering the precipitated crystals of glutathione trisulfide dihydrate. PTN-SSS or LA-SSS can be prepared as described in International Publication No. 2022 / 045212 (LA-SSS) and International Publication No. 2022 / 045052 (PTN-SSS).

[0039] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, and absorption delaying agents, etc., which are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the composition.

[0040] The pharmaceutical compositions used in the method are formulated to be compatible with nasal administration. Examples of the route of administration include parenteral administration, such as intravenous administration.

[0041] Methods of formulating suitable pharmaceutical compositions are known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and in the works of the Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY) series.

[0042] For mucosal therapeutic administration, an active compound (e.g., GSSSG, PTN-SSS or LA-SSS) may be incorporated with excipients or carriers suitable for administration by inhalation or absorption, e.g., via a nasal spray or nasal drops. For nasal administration, the formulation may be an aerosol in a sealed vial or other suitable container.

[0043] The pharmaceutical compositions and nasal formulations may further comprise one or more compounds that reduce the rate at which the active ingredient decomposes. Thus, the nasal formulations described herein may be processed into immediate-release or sustained-release formulations. An immediate-release formulation may release GSSSG, PTN-SSS or LA-SSS in a fairly short time, e.g., within minutes to within a few hours. A sustained-release formulation may release GSSSG, PTN-SSS or LA-SSS over a period of several hours, e.g., up to 24 hours or more if desired. In either case, delivery may be controlled so as to be at a substantially particular predetermined rate over the period of delivery.

[0044] Nasal delivery is considered an attractive route of systemic drug delivery without needles, especially when rapid absorption and effects are desired. Furthermore, nasal delivery can help address problems related to poor bioavailability, slow absorption, drug degradation, and adverse events (AEs) in the gastrointestinal tract, and avoids first-pass metabolism in the liver.

[0045] Liquid nasal formulations are mainly aqueous solutions, but suspensions and emulsions can also be delivered. In conventional spray pump systems, antibacterial preservatives are typically required to maintain the microbiological stability in the formulation.

[0046] Since its introduction, the metered spray pump has dominated the nasal drug delivery market. The pump typically delivers about 25 - 200 μL per spray and provides high reproducibility of the emitted dose and plume geometry. The particle size and plume geometry can vary within certain limits and depend on the characteristics of the pump, formulation, opening of the actuator, and the applied force. Conventional spray pumps require preservatives to prevent contamination as they replace the ejected liquid with air.

[0047] Alternative spray systems or devices that eliminate the need for preservatives can also be used. These systems use collapsible bags, movable pistons, or compressed gas to replenish the ejected liquid volume. Solutions using collapsible bags and movable pistons to replenish the ejected liquid volume offer the additional advantage of being able to eject in an inverted position without the risk of air being sucked into the dip tube and spoiling the next spray. This can be useful for some products when the patient is bedridden and for head down applications. Another way to use without preservatives is that the air replacing the ejected liquid is filtered through a sterile air filter. Additionally, some systems have a ball valve at the tip to prevent contamination of the liquid within the applicator tip.

[0048] For administration by inhalation, the GSSSG, PTN - SSS or LA - SSS compounds may be delivered in the form of a dry powder, or as an aerosol spray from a pressurized container or dispenser containing a suitable high - pressure gas such as carbon dioxide gas, or in the form of a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0049] Also provided herein are, for example, nasal administration devices as described herein that contain GSSSG, PTN-SSS, or LA-SSS.

[0050] Also described herein are kits that can contain a composition comprising GSSSG, PTN-SSS, or LA-SSS, for example, in a pre-prepared dry powder or in a ready-to-administer liquid nasal form, or kits that can contain a solid pharmaceutical composition comprising a composition comprising GSSSG, PTN-SSS, or LA-SSS that can be reconstituted with a solvent to provide a liquid nasal dosage form. When the kit contains a GSSSG composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (for example, for oral or nasal administration), the kit can optionally contain a reconstitution solvent having a pH of 3 to 6, for example, a pH of 4.8 to 5.0. When the kit contains a PTN-SSS or LA-SSS composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (for example, for oral or nasal administration), the kit can optionally contain a reconstitution solvent having a pH of 4 to 9, for example, a pH of 5 to 8. In that case, the solvent for constitution or reconstitution is combined with the active ingredient to provide a liquid oral dosage form of the active ingredient. Typically, the active ingredient is soluble in the solvent and forms a solution. The solvent can be, for example, water, a non-aqueous liquid, or a combination of a non-aqueous and an aqueous component. Suitable non-aqueous components include, but are not limited to, oils; alcohols such as ethanol; glycerin; and glycols such as polyethylene glycol and propylene glycol. In some embodiments, the solvent is phosphate buffered saline (PBS).

[0051] The pharmaceutical composition can be contained in a container, pack, or dispenser, together with instructions for administration. For example, GSSSG can be provided in the kit in crystalline form, together with a sterile buffer solution (for example, saline) having a pH of 3 to 6 for use in dissolving the crystals to prepare a solution for nasal administration.

Examples

[0052] The present invention will be further described in the following examples which are set forth in the claims and which do not limit the scope of the present invention.

[0053] Materials and Methods The following materials and methods were used in the following examples.

[0054] Materials The stable form of GSSSG dihydrate was synthesized and provided by Kyowa Hakko Bio Co., Ltd. (Tokyo, Japan). GSSSG was suspended in distilled water and dissolved by titrating with sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) to a pH between 4.8 and 5.0. A fresh GSSSG solution was prepared immediately before each experiment so that the compound would not decompose. GSH (Sigma-Aldrich, St. Louis, MO, USA) and GSSG hexahydrate (provided by Kyowa Hakko Bio Co., Ltd.) were dissolved in distilled water. In the GSSSG test, the vehicle alone was distilled water and the pH was adjusted between 4.8 and 5.0 with hydrochloric acid.

[0055] PTN-SSS was synthesized and provided by Kyowa Hakko Bio Co., Ltd. The purity of PTN-SSS was 96.3% and PTN-SSS was highly water-soluble (>50 g / L). PTN-SSS and PTN (Toronto Research Chemicals, Toronto, ON, Canada) were suspended in distilled water. PTN-SSS was stable at room temperature in a solution with a pH of 4.0 to 9.0 for at least 4 days. In the PTN-SSS test, distilled water was used as the vehicle alone.

[0056] In Vivo Tests Animals All animal procedures were conducted in accordance with protocols approved by the Massachusetts General Hospital Committee on Animal Research and the National Research Council's Guide for the Care and Use of Laboratory Animals. The description of the study design and experiments followed the ARRIVE guidelines. Adult C57BL / 6J mice (12–18 weeks old) of both sexes were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). In experiments investigating the neuroprotective effect of GSSSG on motor function after SCI, both male and female mice were used. Since the neuroprotective effect of GSSSG appeared to be independent of sex, subsequent in vivo experiments were performed in male mice only. All mice were housed in a temperature- and humidity-controlled room with a 12-h light–dark cycle at the animal facility and had free access to food and water. To allow access by mice recovering from surgery, additional food pellets were inserted into hydrated gel placed on top of the bedding. A randomized paired design was used to minimize variation between treatment groups. Mice were paired based on body weight, age, date of delivery, and, if possible, housing cage. After pairing, mice were randomly assigned to different treatments.

[0057] Surgery to induce spinal cord ischemia and administration of the test drug Surgical procedures to produce delayed paraplegia in mice were performed as previously described 6(FIG. 2A). The mice were anesthetized with 5% isoflurane in 100% O2 and intubated tracheally with a 20-gauge catheter (Angiocath; Becton Dickinson, Franklin Lakes, NJ, USA). The mice were mechanically ventilated at a tidal volume of 8 μl / g (MiniVent model 845; Harvard Apparatus, Holliston, MA, USA) and anesthesia was maintained with 2% isoflurane in 100% O2. Paraspinal muscle temperature was measured using a T-type implantable thermocouple probe (IT-18) and a T-type pod (ADInstruments, Colorado Springs, CO, USA). After making a skin incision on the back, an 18-gauge needle was used to place the tip of the probe at the level of L1-L3, and a warming pad and a DC temperature controller (FHC, Bowdoin, ME, USA) were used to maintain the temperature at 37.5 ± 0.5 °C. A median sternotomy was made from the tip of the manubrium to the second rib. The aortic arch was gently isolated between the left common carotid artery (LCCA) and the left subclavian artery (LSA), avoiding the vagus nerve and the left recurrent laryngeal nerve. A first clip (straight microclip, RS-5424; Roboz Surgical Instrument Company, Inc., Gaithersburg, MD, USA) was placed on the aortic arch between the LCCA and the LSA, and then a second clip (45° angled microclip, RS-5435; Roboz Surgical Instrument Company, Inc., Gaithersburg, MD, USA) was placed at the origin of the LSA within 15 seconds. After ischemia, the clips were removed in the reverse order. The incisions were closed in layers and mechanical ventilation was discontinued. The mice were extubated when stable spontaneous respiration was confirmed.

[0058] The previously described surgical procedure 6 was modified as follows. The respiratory rate during the procedure was increased to 230 breaths / min because hyperventilation promotes the development of delayed paraplegia resulting from spinal cord ischemia. 33 . To monitor femoral artery blood flow, a laser Doppler perfusion monitor (moorVMS-LDF1; Moor Instruments, Millwey, UK) was used. 28,29To confirm that aortic occlusion resulted in an immediate and sustained decrease (>90%) in femoral artery blood flow, a plastic fiber (POF500; Moor Instruments, Millwey, UK) was attached perpendicular to the left femoral artery. 28,29 To improve the accuracy of the surgical procedure, a microscope (Leica MZ95; Leica Microsystems, Buffalo Grove, IL, USA) was used to isolate the aortic arch and place the clips. Based on the results of preliminary tests, the ischemia time with the lowest mortality 72 hours after surgery, which induced late-onset paraplegia in all mice, was selected (3 minutes in male mice and 3.5 minutes in female mice) (Table 1).

[0059]

Table 1

[0060] The test drug was administered intranasally using a single-channel pipettor. Mice were anesthetized with 3% isoflurane using a non-rebreathing circuit with a mouse nose cone (VetEquip, Inc., Livermore, CA, USA). During drug administration, the nose cone was removed and the mice were allowed to breathe air. Approximately 6 μL of each drug was administered into the nostrils of the mice, and the mice inhaled the droplets during inspiration. This procedure was repeated at intervals until the total volume of the drug was administered, which took approximately 10 minutes. The interruption time of isoflurane administration was approximately 10 seconds per intranasal dose. The intranasal drug was administered 0 hours, 8 hours, 23 hours, and 32 hours after surgery (Figure 2B). In the sham procedure group, the above surgical procedure was performed, but the aorta was not cross-clamped.

[0061] For pain management, 0.1 mg / kg of buprenorphine was administered intraperitoneally before surgery and every 12 hours until 60 hours after surgery. Immediately after surgery, 0.5 mg / kg of 0.25% bupivacaine was also administered subcutaneously around the wound incision site.

[0062] Evaluation of motor function after spinal cord ischemia Hindlimb motor function was quantified using the Basso Mouse Scale (BMS) for locomotion 34 at 0, 8, 24, 48, and 72 h after surgery (Figure 2B). This score ranged from 0 for complete paralysis to 9 for normal motor function. A BMS score <6 (0 - 5) indicated paralysis or incomplete paralysis, and a BMS score ≥6 (6 - 9) indicated that the mouse was able to walk.

[0063] A study to investigate the neuroprotective effect of post - perfusion treatment with GSSSG on motor function after spinal cord ischemia Mice were randomly assigned to each treatment group, and the investigator performing the surgical procedure was blinded to the group assignment. Based on preliminary tests, 50 mg / kg of GSSSG, 45.2 mg / kg of GSH (since 1 molecule of GSSSG contains 2 molecules of GSH, 2 times the molar amount of GSSSG) (Figure 1A), or 53 mg / kg of GSSG (equimolar dose of GSSSG) was administered.

[0064] Histological tests The lumbar enlargement of the spinal cord was removed 48 h after surgery and sectioned into 5 - μm - thick slices using a cryostat (CM1850UV; Leica Biosystems, Heidelberg, Germany). Nissl staining was performed using a Nissl staining kit (VitroVivo Biotech, Rockville, MD, USA) according to the manufacturer's recommended protocol. Immunohistochemical staining for ionized calcium - binding adapter molecule 1 (Iba - 1) and cleaved caspase - 3 was performed as previously described 6,15 . The stained sections were examined using an epifluorescence microscope (Nikon Eclipse 80i; Nikon Instruments, Inc., Melville, NY, USA).

[0065] An investigator blinded to the nature of the samples counted the number of stained cells in 3 different sections of the spinal cord from each mouse at high magnification (200x) in one area (0.26 mm 2) were counted. The average number of stained cells per mouse was calculated. For the quantitative analysis of Iba-1 staining, the ImageJ image processing program (National Institutes of Health, Bethesda, MD, USA) was used to calculate the Iba-1 positive area per region (0.26 mm 2 ) at high magnification (200×) in three different ventral horn sections per mouse. The average area of Iba-1 staining in the spinal cord of mice in the sham procedure group was set to 1, and the relative amount of Iba-1 staining was determined for each experimental group of mice. Each group included 5 mice.

[0066] Measurement of gene expression Messenger RNA (mRNA) levels were measured as previously described using the lumbar spinal cord 48 hours after surgery. 15 . The mRNA levels of C-C motif chemokine 2 (CCL2), C-X-C motif chemokine ligand 1 (CXCL1), interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)α, B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL), cystathionine beta-synthase, cystathionine gamma-lyase, 3-mercaptopyruvate sulfurtransferase, sulfide quinone oxidoreductase (SQOR), ethylmalonic encephalopathy 1, thiosulfate sulfurtransferase, sulfite oxidase, cysteinyl-tRNA synthetase 1, and cysteinyl-tRNA synthetase 2 were standardized against the level of 18S ribosomal RNA using quantitative real-time polymerase chain reaction (7500 Fast Real-Time PCR System, Thermo Fisher Scientific, Waltham, MA, USA). The primer sequences are listed in Table 2.

[0067]

Table 2

[0068] Liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis GS, an isotope of endogenous GSSSG 34After intranasal administration of SSG, in the CNS and plasma 34 To measure the amount of S-labeled GSSSG( 32 S2, 34 S]GSSSG), LC-MS / MS analysis was used 9,35,36 . 34 S-labeled sulfenyl sulfur molecular species( 32 S, 34 S]GSSH, 32 S, 34 S]cysteine hydroperoxidesulfide (CysSSH), and 32 S2, 34 S]cysteine trisulfide (CysSSSCys)) to endogenous sulfenyl sulfur molecular species( 32 S2]GSSH, 32 S2]CysSSH, and 32 S3]CysSSSCys) ratio was also quantitatively evaluated. 30 minutes after intranasal administration of 50 mg / kg of GS 34 SSG, blood was obtained, and four different central nervous tissues, namely, olfactory bulb and forebrain; brainstem; cervical and thoracic spinal cords, and lumbar spinal cord were collected. A total of 30 mg of each tissue was homogenized in 300 μl of ice-cold methanol solution containing 5 mM β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM; Santa Cruz Biotechnology, Dallas, TX, USA). 25 μl of plasma was mixed with 75 μl of ice-cold methanol solution containing 5 mM HPE-IAM. The samples were incubated in the dark at 37 °C for 20 minutes. After centrifugation (tissue at 14,000×g, plasma at 1,870×g, 10 minutes, 4 °C), the supernatant was separated, diluted with 0.1% formic acid for LC-MS / MS analysis, and the pellet was sonicated to measure the protein concentration by BCA assay. 32 S2, 34 The amount of S]GSSSG was quantified by selected reaction monitoring (SRM) with a precursor ion (647.14 m / z), a product ion (389.1 m / z), and high-energy collision dissociation (21 v). 32 S, 34 S]GSSH vs 32 S2]GSSH, 32 S, 34S]CysSSH pair 32 S2]CysSSH, and 32 S2, 34 S]CysSSSCys pair 32 The ratio of S3]CysSSSCys was calculated from their peak areas measured by a Dionex UltiMate 3000 RS UPLC-Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the sample was exposed to a UPLC system using a Hypersil Gold C-18 (100×2.1 mm, 3.0 μm, Thermo Fisher Scientific, Waltham, MA, USA) column and then eluted at a flow rate of 0.2 ml / min at 40 °C with a linear gradient of methanol in the mobile phase (0 - 90%, 15 min) in the presence of 0.1% formic acid. The raw data was analyzed by Compound Discoverer 3.3 software (Thermo Fisher Scientific, Waltham, MA, USA). The molecular weights of those sulfenyl sulfur molecular species combined with HPE-IAM were determined based on previous reports 9,11,37 。

[0069] Measurement of relative sulfenyl sulfur levels in the lumbar spinal cord after spinal cord ischemia The lumbar spinal cord was harvested 48 hours after surgery and snap-frozen in liquid nitrogen. Subsequently, the spinal cord was homogenized in Hank's balanced salt solution (HBSS; Thermo Fisher Scientific, Waltham, MA, USA) containing SSip-1 (5 μM), incubated for 20 minutes in the dark at room temperature, and then centrifuged. SSip-1 was synthesized and provided by the Hanaoka laboratory 38 。The fluorescence intensity of the supernatant was measured at a wavelength of λex / λem = 491 nm / 525 nm using a microplate reader (SpectraMax M5; Molecular Devices, San Jose, CA, USA). The fluorescence intensity was normalized to the weight of the spinal cord

[0070] Measurement of relative sulfenyl sulfur levels in the olfactory bulb, forebrain, and entire spinal cord after intranasal administration of PTN-SSS Thirty minutes after intranasal administration of 50 mg / kg of PTN-SSS, the olfactory bulb, forebrain, and entire spinal cord were harvested. Tissues were homogenized in HBSS containing 10 μM of SSip-1. The fluorescence intensity of the supernatant was measured.

[0071] In vitro assays Mouse primary cortical neuron culture Primary cortical neurons were isolated from the cerebral cortex of embryonic day 15 C57BL / 6J mice of both sexes as previously described. 39 Primary cortical neurons were maintained in a humidified tissue culture chamber at 37 °C and 5% CO2 and used on day 11 after collection.

[0072] Measurement of relative sulfenyl sulfur levels in primary cortical neurons or SH-SY5Y cells after incubation with polysulfides Primary cortical neurons, or SH-SY5Y cells, a human neuroblastoma cell line (American Type Culture Collection, Manassas, VA, USA), were incubated with SSip-1 DA (5 μM) 38 for 20 minutes in the dark at 37 °C. Cells were washed with pre-warmed HBSS containing calcium and magnesium (Thermo Fisher Scientific, Waltham, MA, USA) and then incubated with GSSSG, sodium trisulfide (Na2S3; Dojindo Molecular Technologies, Inc., Rockville, MD, USA), PTN-SSS, or vehicle alone for 20 minutes in the dark at 37 °C. Fluorescence intensity was measured using a microplate reader.

[0073] Effect of polysulfides on cell viability after oxygen-glucose deprivation / reoxygenation (OGD / R) As previously described 39, Primary cortical neurons or SH-SY5Y cells were exposed to OGD for 2.5 hours or 15 hours in an incubation chamber (MIC-101; Billups-Rothenberg, Inc., San Diego, CA, USA), and then the cells were incubated with GSSSG, GSH, GSSG, PTN-SSS, or vehicle only, and then exposed to reoxygenation for 21 hours (primary cortical neurons) or 24 hours (SH-SY5Y cells). As previously described 39 , After reoxygenation, crystal violet assay and lactate dehydrogenase (LDH) assay were used to evaluate cell viability and cell injury.

[0074] Cytotoxic effect of GSSSG on cell viability Primary cortical neurons were incubated with 0 μM (vehicle alone), 10 μM, 30 μM, 60 μM, or 100 μM GSSSG for 21 hours in a humidified incubator at 37 °C with 95% air and 5% CO2. Cell viability was evaluated using the LDH assay.

[0075] Cytotoxic effect of PTN-SSS on cell viability SH-SY5Y cells were incubated with 0 μM (vehicle alone), 5 μM, 10 μM, 25 μM, 50 μM, or 100 μM PTN-SSS for 24 hours in a humidified incubator at 37 °C with 95% air and 5% CO2. Cell viability was evaluated using the crystal violet assay.

[0076] Statistical analysis Data were presented as mean and standard deviation (parametric data) or median and interquartile range (nonparametric data). Descriptive statistics were used to describe the study population. Shapiro–Wilk test and Q–Q plot were used to evaluate data normality. Parametric data were analyzed using independent t-test for comparison between two groups and one-way analysis of variance (ANOVA) with Tukey's multiple comparison test or Dunnett's multiple comparison test for comparison among three or more groups. Nonparametric data were analyzed using Mann–Whitney test for comparison between two groups and Kruskal–Wallis test with Dunn's multiple comparison test for comparison among three or more groups. Significance was considered at the level of P < 0.05. Statistical analysis was performed using GraphPad Prism 9.2.0 (GraphPad Software Inc., San Diego, CA, USA).

[0077] Sample size calculation for the experiment to compare the BMS scores 72 h after surgery among the GSSSG group, GSH group, and vehicle-alone group was performed using the F-test for fixed-effect one-way ANOVA (G * Power3.1; Heinrich-Heine University, Düsseldorf, Germany) 40 was used. Based on a pilot study for this experiment, it was estimated that 11 male and 8 female mice would be required per group (α = 0.05, β = 0.1 [power = 0.9], effect sizes were f = 0.7022756 for male mice and f = 0.8709832 for female mice, number of groups = 3). Sample size calculation for the experiment to compare the BMS scores 72 h after surgery among the PTN-SSS group, PTN group, and vehicle-alone group was performed using the F-test for fixed-effect one-way ANOVA. Based on a pilot study for this experiment, it was estimated that 9 mice would be required per group (α = 0.05, β = 0.1 [power = 0.9], effect size f = 0.7747206, number of groups = 3).

[0078] Intermittent H2S inhalation-sulfide preconditioning (SPC) We previously reported that 80 ppm inhaled H2S significantly increased the plasma and brain levels of H2S and its oxide, thiosulfate 50,51 . Male C57BL6 / J mice were exposed to air alone or air mixed with 80 ppm H2S (Airgas Inc., Radnor, PA) in custom-made plastic chambers for 4 h per day for 5 consecutive days. H2S exposure sessions were conducted from 8 AM to 12 PM from day 1 to day 5 as previously described 51 (Figure 1). A portable gas monitor (VENTIS MX4 multi-gas monitor, Industrial Scientific Corporation, Oakdale, PA) was used to continuously measure H2S concentration and FiO2

[0079] MPTP-induced Parkinson's disease model in mice As previously described 3 , male C57BL6 / J mice and CD-1 mice were intraperitoneally injected with MPTP (20 mg / kg) or saline 4 times at 2-h intervals for a total of 4 doses of MPTP (80 mg / kg total) or the corresponding volume of saline. To evaluate the effect of MPTP on dopaminergic neurons, all mice were sacrificed 7 days after MPTP administration. Then, tyrosine hydroxylase protein levels were detected by immunoblotting

[0080] Cell viability of neurons MPP +(5 mM for SH-SY5Y cells and 50 μM for primary cortical neurons), and vehicle (PBS), Na2S (Sigma-Aldrich), Na2S3 (Dojindo Molecular Technologies, Inc.), GSSSG (glutathione trisulfide, Kyowa Hakko Bio Co., Ltd., Tokyo, Japan), LA (α-lipoic acid, Sigma-Aldrich), or LASSS (α-lipoic acid trisulfide, Kyowa Hakko Bio Co., Ltd., Tokyo, Japan). Twenty-four hours after the addition, a cell viability assay was performed to examine the cell viability of SH-SY5Y cells or mouse primary cortical neurons. Cell viability was measured using a crystal violet assay.

[0081] Administration of Na2S3 to mice after treatment with MPTP To determine the effect of polysulfides on the loss of tyrosine hydroxylase in the substantia nigra and striatum of mice treated with MPTP, starting immediately after the first injection of MPTP on day 0, Na2S3 (20 mg / kg) or saline was administered intraperitoneally every 2 h. Subsequently, mice were injected intraperitoneally with Na2S3 (20 mg / kg) or saline twice every 12 h from day 1 to day 6. Seven days after MPTP administration, all mice were sacrificed. Then, the tyrosine hydroxylase protein level was detected by immunoblotting.

[0082] Administration of GSSSG to mice after treatment with MPTP To determine the effect of polysulfides on the loss of tyrosine hydroxylase in the substantia nigra and striatum of mice treated with MPTP, immediately after the first and third injections of MPTP on day 0, GSSSG (50 mg / kg) or saline was administered via the intranasal route. Subsequently, mice were injected intranasally with GSSSG (50 mg / kg) or saline twice every 12 h from day 1 to day 6. Seven days after MPTP administration, all mice were sacrificed. Then, the tyrosine hydroxylase protein level was detected by immunoblotting.

[0083] [Example 1] Intranasal administration of GSSSG rescued mice from delayed paraplegia after transient spinal cord ischemia To investigate whether polysulfides could prevent delayed paraplegia, mice were subjected to SCI and treated with GSSSG, GSH, or vehicle alone at 0, 8, 23, and 32 h after surgery. Thirty-six male mice were subjected to SCI, but three of those mice were excluded from further testing because a reduction of more than 90% in femoral artery blood flow was not achieved. The remaining 33 male mice received one of three treatments, namely, GSSSG (n = 11), GSH (n = 11), or vehicle alone (n = 11). Hindlimb motor function was quantified using the Basso Mouse Scale (BMS) for locomotion, which evaluates hindlimb movement, forelimb–hindlimb coordination, and trunk stability. As previously described by Kakinohana and co-workers, mice were considered to have delayed paraplegia if neurological deficit symptoms (BMS < 6) occurred after a period during which the mice were able to walk (BMS ≥ 6). 6 。

[0084] After approximately 36 h after surgery and later, the hindlimb motor function of mice treated with vehicle alone deteriorated gradually, and all mice treated with vehicle alone developed paraplegia by 48 h after surgery (Figure 3). In contrast, intranasal administration of GSSSG prevented the development of delayed paraplegia in 8 of 11 mice (73%) and improved the BMS score at 72 h after surgery compared with vehicle alone (GSSSG vs vehicle alone, BMS; 9 [4–9] vs 0 [0–0]; P < 0.0001 by Kruskal–Wallis test with Dunn's multiple comparison test, Figure 3). Intranasal administration of GSH also prevented the development of delayed paraplegia in 3 of 11 mice (27%), but did not improve the BMS score at 72 h after surgery (GSH vs vehicle alone, BMS; 1 [0–7] vs 0 [0–0]; P = 0.0914, Figure 3). These results indicate that in male mice, GSSSG prevents the development of delayed paraplegia after SCI, whereas GSH does not.

[0085] To investigate the gender effect on the ability of GSSSG to prevent delayed paraplegia after SCI, 28 female mice were subjected to SCI and treated with GSSSG, GSH, or vehicle alone at 0, 8, 23, and 32 hours after surgery. Four of the 28 female mice had labored breathing immediately after extubation and were euthanized. The remaining 24 female mice were treated with GSSSG (n = 8), GSH (n = 8), or vehicle alone (n = 8). Mice receiving intranasal GSSSG improved their BMS scores at 72 hours after surgery compared to mice treated with vehicle alone (GSSSG vs. vehicle alone, BMS; 6.5 [1.5 - 9.0] vs. 0.0 [0.0 - 0.0]; P = 0.0060 by Kruskal - Wallis test with Dunn's multiple comparison test, Figure 3). In contrast, intranasal administration of GSH did not improve the BMS scores at 72 hours after surgery (GSH vs. vehicle alone, BMS; 0.5 [0.0 - 6.7] vs. 0.0 [0.0 - 0.0]; P = 0.2887, Figure 3). These results indicate that in both genders, GSSSG can prevent delayed paraplegia after SCI, while GSH cannot.

[0086] GSSG can react with thiols in proteins by glutathionylation (PSH+GSSG→←PSSG+GSH) 23 。GSSSG can also potentially react with thiols in proteins by the same process 41。To investigate whether the beneficial effects of GSSSG are associated with glutathionylation, the effects of intranasal administration of GSSSG or GSSG on the outcome after SCI in male mice were compared. Intranasal administration of GSSSG rescued 5 out of 6 male mice (83%) from delayed paraplegia, whereas, in contrast, intranasal administration of GSSG rescued none of the male mice from delayed paraplegia (0%). The BMS score 72 hours after surgery in the GSSSG group was significantly higher than that in the GSSG group (GSSSG vs GSSG, BMS; 8.0 [5.2 - 9.0] vs 0.0 [0.0 - 0.0]; P = 0.0152 by Mann - Whitney test, Figure 11). GSSSG may potentially mediate glutathionylation of target proteins by the same process as GSSG, but since only GSSSG protected against delayed paraplegia, this result suggests that the neuroprotective effect of GSSSG is not a result of glutathionylation.

[0087] [Example 2] Intranasal administration of GSSSG decreased neurodegeneration, microglial activation, and caspase - 3 activation in the lumbar spinal cord after spinal cord ischemia Spinal cord ischemia is associated with degenerative changes in neurons in the anterior horn of the lumbar spinal cord, and the survival rate of the affected neurons can be evaluated using Nissl staining. 6 。Nissl staining detects Nissl bodies in the cytoplasm of neurons, and this purple cytoplasmic staining is an indicator of neuronal integrity. 6,42 。To investigate the effect of GSSSG on neurodegeneration, GSSSG or vehicle alone was administered at 0, 8, 23, and 32 hours after surgery. Forty - eight hours after surgery, the lumbar spinal cord was harvested, fixed, sectioned, and incubated with Nissl staining. Compared with mice that received sham surgical procedures, spinal cord ischemia was associated with a marked decrease in neurons that were positive for Nissl staining, suggesting that the majority of the cells were not viable (Figures 4A - 4B). Compared with mice treated with vehicle alone after SCI, intranasal administration of GSSSG was associated with a marked increase in the number of viable neurons (Figures 4A - 4B).

[0088] Previous studies have shown that enhanced microglial activation in the anterior horn of the lumbar spinal cord parallels the development of delayed paraplegia after SCI 6,29 and that inhibition of microglial activation attenuates neuronal damage and prevents the development of delayed paraplegia after SCI. 43 An increase in the expression of ionized calcium-binding adapter molecule 1 (Iba-1) is a sensitive marker of microglial activation. 44 To investigate the mechanism by which GSSSG prevents neurodegeneration, mice were subjected to SCI and treated with GSSSG or vehicle alone. Sections were prepared from the lumbar spinal cord and stained for Iba-1. Compared with the staining of the spinal cord obtained 48 hours after sham surgery, the staining of the spinal cord from SCI mice had a significantly increased relative area of Iba-1 positive staining (Figures 5A, 5B). These results indicate that SCI is associated with an increase in microglial activation. In contrast, mice treated with intranasal GSSSG after SCI had a marked decrease in Iba-1 staining, indicating a decrease in microglial activation (Figures 5A, 5B).

[0089] Previous researchers have shown that caspase-3 activation is a central factor in the neurodegeneration that occurs in the anterior horn of the lumbar spinal cord after SCI. 6 To investigate whether attenuation of caspase-3 activation could be the mechanism by which GSSSG prevents neurodegeneration after SCI, immunohistochemical staining for cleaved caspase-3 was used. Compared with the spinal cord from mice 48 hours after sham surgery, the lumbar spinal cord from SCI mice had an increase in the number of cleaved caspase-3 positive neurons in the anterior horn (Figures 5C, 5D). Intranasal administration of GSSSG prevented the SCI-induced increase in the number of cleaved caspase-3 positive neurons in the lumbar spinal cord (Figures 5C, 5D). These results suggest that intranasal administration of GSSSG prevents neurodegeneration in the anterior horn of the lumbar spinal cord and is associated with a decrease in microglial activation and attenuation of caspase-3 activation.

[0090] [Example 3] Intranasal administration of GSSSG attenuated the upregulation of inflammatory cytokines after spinal cord ischemia Previous studies have shown that a marked increase in inflammatory cytokines produced by activated microglia precedes the onset of delayed paraplegia after SCI 15,29 Inhibition of microglial activation suppressed the upregulation of inflammatory cytokines and prevented the development of delayed paraplegia after SCI 43 To further evaluate the effect of GSSSG on SCI-induced inflammatory cytokines, the levels of mRNA encoding CCL2, CXCL1, IL-1β, IL-6, and TNF-α were measured in the lumbar spinal cord from mice subjected to SCI and treated with GSSSG or vehicle alone. The lumbar spinal cord was harvested 48 hours after surgery. Compared with sham-operated mice, mice subjected to SCI had a marked increase in the levels of mRNA encoding cytokines related to inflammation. In contrast, GSSSG attenuated the upregulation of mRNA encoding inflammatory cytokines 48 hours after surgery (Figure 6). Intranasal administration of GSSSG also decreased the level of mRNA encoding SQOR, an enzyme that oxidizes sulfide to persulfide. GSSSG had no effect on the levels of mRNA encoding other enzymes involved in the synthesis or metabolism of sulfide or persulfide (Figure 12). There was no significant difference in the levels of mRNA of anti-apoptotic genes including Bcl-2 and Bcl-XL between the vehicle-alone treatment group and the GSSSG treatment group (Figure 6). These results suggest that the beneficial effect of GSSSG may be mediated by the inhibition of inflammatory cytokines

[0091] [Example 4] 34 S-labeled GSSSG reached the spinal cord immediately after intranasal administration and was metabolized into other sulfen sulfur molecular species To examine the pharmacokinetics of intranasally administered GSSSG 34 S-labeled GSSSG was used to investigate the distribution of GSSSG and its metabolites (GSSH, CysSSH, and CysSSSCys) in the CNS after intranasal administration. GS 34Thirty minutes after intranasal administration of SSG, using LC-MS / MS 32 S2, 34 the amount of S]GSSSG was measured, and the ratio of S-labeled sulfenyl sulfur molecular species to endogenous sulfenyl sulfur molecular species in the brain, spinal cord, and plasma was determined. 34 S- labeled sulfenyl sulfur molecular species to endogenous sulfenyl sulfur molecular species was determined. 32 S2, 34 S]GSSSG was detected in the olfactory bulb and forebrain (128 ± 67 pmol / mg protein), brainstem (226 ± 101 pmol / mg protein), cervical and thoracic spinal cords (414 ± 98 pmol / mg protein), and lumbar spinal cord (317 ± 111 pmol / mg protein) (Figure 7A). Furthermore, 32 S2, 34 S]GSSSG was detected in plasma at a concentration of 25 ± 10 nM. 32 S, 34 S]GSSH to 32 S2]GSSH, 32 S, 34 S]CysSSH to 32 S2]CysSSH, and 32 S2, 34 S]CysSSSCys to 32 S3]CysSSSCys ratios in the brain and spinal cord were 6.5 ± 2.8, 10.6 [9.8 - 12.5], and 31.8 ± 9.8, respectively (Figure 7B). These results indicate that GSSSG reaches different parts of the central nervous system, including the spinal cord, within 30 minutes after intranasal administration and is converted to other sulfenyl sulfur molecular species.

[0092] [Example 5] Intranasal administration of GSSSG increased sulfenyl sulfur levels in the lumbar spinal cord after spinal cord ischemia. GSSSG protects the lumbar spinal cord from neurodegeneration 48 hours after SCI. To investigate whether an increase in the level of sulfane sulfur in the lumbar spinal cord could contribute to the neuroprotective effect, the change in sulfane sulfur level 48 hours after SCI was measured. Compared with sham-operated mice, vehicle alone treatment following SCI did not change the sulfane sulfur level in the lumbar spinal cord (sham operation vs. vehicle alone following SCI; 1.00 ± 0.30 vs. 0.56 ± 0.13; one-way ANOVA using Dunnett's multiple comparison test, P = 0.0870, Figure 8A). In contrast, the level of sulfane sulfur in the lumbar spinal cord of mice that received intranasal administration of GSSSG after SCI was higher than that in mice that received vehicle alone after SCI (GSSSG following SCI vs. vehicle alone; 1.37 ± 0.50 vs. 0.56 ± 0.13; P = 0.0023, Figure 8A). These results suggest that the beneficial effect of GSSSG in preventing delayed paralysis is associated with an increase in the level of sulfane sulfur in the lumbar spinal cord.

[0093] [Example 7] Effect of GSSSG on primary cortical neurons To determine whether polysulfides increase the level of sulfane sulfur in neurons, relative sulfane sulfur levels in primary cortical neurons were measured in the presence and absence of polysulfides using SSip-1 DA. SSip-1 DA is a fluorescent probe that can be used to measure the concentration of intracellular sulfane sulfur 38 . Neurons incubated with GSSSG or Na2S3 had an increase in the level of intracellular sulfane sulfur compared to non-treated primary cortical neurons (Figure 8B).

[0094] In vivo studies have shown that GSSSG can prevent neurodegeneration after SCI. To determine whether GSSSG can protect neurons from similar damage in vitro, primary cells were incubated with GSSSG after oxygen-glucose deprivation / reoxygenation (OGD / R). As previously described 39, cell viability was evaluated using crystal violet assay and lactate dehydrogenase (LDH) assay. Crystal violet dye stains intracellular DNA and proteins, confirming that cells are alive and can maintain adhesion to tissue culture plates despite OGD / R. The LDH assay measures the level of lactate dehydrogenase in the tissue culture medium and is an indirect measurement of cell membrane damage. Treatment with OGD / R was associated with a significant decrease in the number of viable adherent cells (Figure 9A) and a significant increase in the concentration of LDH in the tissue culture medium (Figure 9B) compared to control cells. Compared to vehicle-only treated cells, 30 μM GSSSG increased cell viability as measured by crystal violet assay (Figure 9A), and 30 μM or 60 μM GSSSG decreased LDH release after OGD / R (Figure 9B). High-dose GSSSG (100 μM) itself increased the LDH level in the tissue culture medium (Figure 13) and could not improve cell survival after OGD / R. These results indicate that GSSSG protects neurons in a dose-dependent manner from the effects of OGD / R.

[0095] In vivo studies showed that GSSSG inhibits the development of delayed paralysis after SCI, while GSH or GSSG does not, and suggested that the neuroprotective effect of GSSSG is derived from persulfide sulfur. To determine whether the neuroprotective effect of GSSSG on primary cultured neurons is derived from persulfide sulfur, the ability of GSSSG, GSH, and GSSG to protect neurons from OGD / R was tested. Compared to vehicle-only treated cells, 30 μM GSSSG improved cell viability after OGD / R (Figure 9C). In contrast, 60 μM GSH and 30 μM GSSG did not improve cell viability after OGD / R (Figure 9C). These results suggest that the neuroprotective effect of GSSSG is derived from persulfide sulfur.

[0096] [Example 8] Effect of PTN-SSS on SH-SY5Y cells In vitro assays were used to show that GSSSG protects neurons after OGD / R and to suggest that the neuroprotective effect of GSSSG is derived from sulfenyl sulfur. To investigate whether other polysulfides are cytoprotective after OGD / R, the effect of the polysulfide PTN-SSS on the viability of SH-SY5Y cells after OGD / R was examined. Similar to primary cortical neurons, treatment with OGD / R decreased the viability of SH-SY5Y cells. Incubation with PTN-SSS at 10 μM, 25 μM, or 50 μM significantly improved SH-SY5Y viability after OGD / R in a dose-dependent manner compared to vehicle-only treated cells (Figure 10A). In contrast, PTN-SSS did not improve cell viability at 100 μM, due to the direct cytotoxic effect of high-dose PTN-SSS (Figure 14). These results show that PTN-SSS rescues SH-SY5Y cells in a dose-dependent manner after OGD / R and support that polysulfides are cytoprotective after OGD / R.

[0097] To determine whether the cytoprotective effect of PTN-SSS is related to the amount of intracellular sulfenyl sulfur, SSip-1 DA was used to measure the sulfenyl sulfur levels in SH-SY5Y cells after incubation with PTN-SSS. The levels of sulfenyl sulfur in SH-SY5Y cells increased with increasing concentrations of PTN-SSS (Figure 10B). The ability of SSip-1 DA (5 μM) to measure intracellular sulfenyl sulfur reached its maximum at a concentration of 100 μM of PTN-SSS (Figure 10B). Compared to vehicle-only treated cells, the levels of sulfenyl sulfur in SH-SY5Y cells increased significantly at doses of PTN-SSS (10 - 50 μM), which significantly improved cell viability after OGD / R. These results suggest that the cytoprotective effect of PTN-SSS is related to the amount of sulfenyl sulfur released from PTN-SSS.

[0098] [Example 10] PTN-SSS increased sulfenyl sulfur levels in the olfactory bulb, forebrain, and entire spinal cord immediately after intranasal administration To test whether PTN-SSS raises the sulfenyl sulfur level in the central nervous system immediately after intranasal administration, 30 minutes after intranasal administration of PTN-SSS, the sulfenyl sulfur levels in the olfactory bulb, forebrain, and entire spinal cord were measured using SSip-1. Compared with mice that received intranasal administration of vehicle alone, intranasal administration of PTN-SSS increased the sulfenyl sulfur levels in the olfactory bulb, forebrain, and entire spinal cord (Figure 10C). These results suggest that PTN-SSS raises the sulfenyl sulfur level in the central nervous system within 30 minutes after intranasal administration.

[0099] [Example 11] Intranasal administration of PTN-SSS rescued mice from delayed paraplegia after transient spinal cord ischemia In vitro tests showed that PTN-SSS was cytoprotective after OGD / R and that the cytoprotective effect of PTN-SSS was related to the amount of sulfenyl sulfur released from PTN-SSS. Since GSSSG inhibited the development of delayed contralateral paralysis after SCI and the neuroprotective effect of GSSSG was related to sulfenyl sulfur, we hypothesized that PTN-SSS could also rescue mice from delayed contralateral paralysis after SCI. Furthermore, similar to GSSSG, we tested whether sulfenyl sulfur in PTN-SSS was more neuroprotective than PTN alone. Male mice received intranasal administration of PTN-SSS, PTN, or vehicle alone at 0 h, 8 h, 23 h, and 32 h after surgery. Equimolar doses of PTN-SSS (50 mg / kg) or PTN (47.3 mg / kg) were administered. Intranasal administration of PTN-SSS rescued 6 out of 9 mice (66%) from delayed contralateral paralysis. In contrast, intranasal administration of PTN did not rescue any male mice from delayed contralateral paralysis. The BMS score at 72 h after surgery in the PTN-SSS group was significantly higher than that in the PTN or vehicle-alone groups (PTN-SSS vs. PTN, BMS; 7.0 [1.5 - 9.0] vs. 1.0 [0.0 - 2.0]; P = 0.0323. PTN-SSS vs. vehicle alone, BMS; 7.0 [1.5 - 9.0] vs. 1.0 [0.0 - 1.5]; P = 0.0258, Figure 10D). These results indicate that PTN-SSS inhibits the development of delayed contralateral paralysis after SCI in mice, while PTN does not, and that the neuroprotective effect of PTN-SSS is derived from sulfenyl sulfur rather than PTN.

[0100] [Example 12] Inhalation of H2S resulted in neuroprotection in a PD animal model Sulfide levels are tightly regulated in the brain. We recently reported a robust protective effect of sulfide metabolism in hypoxic / ischemic brain injury. See Marutani et al, Nature Communications 12, Article number: 3108; 25 May 2021. Since other forms of brain injury may result from changes in sulfide homeostasis, we examined the effect of sulfide metabolism in Parkinson's disease (PD), one of the most common neurodegenerative disorders. See also Kida, K., Ichinose, F. (2015). Hydrogen Sulfide and Neuroinflammation. In: Moore, P., Whiteman, M. (eds) Chemistry, Biochemistry and Pharmacology of Hydrogen Sulfide. Handbook of Experimental Pharmacology, vol 230. Springer, Cham; Kida et al., Antioxidants & Redox Signaling 2010;15:343-352; and Kida et al, Antioxidents and Redox Signaling 15(2): 343-52 (2011).

[0101] Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused the loss of dopaminergic neurons, as measured by the levels of tyrosine hydroxylase (TH) in the substantia nigra of mice and the level of motor impairment. Respiration with air mixed with 80 ppm H2S for 4 h daily for 5 days prior to MPTP administration (sulfide preconditioning) protected mice from MPTP-induced loss of dopaminergic neurons and motor impairment. The protective effect of sulfide preconditioning was associated with a marked increase in the level of sulfide quinone oxidoreductase (SQOR) in the substantia nigra and striatum. AAV-mediated SQOR expression in the substantia nigra-striatum region prevented MPTP-induced loss of dopaminergic neurons in mice.

[0102] This study revealed the neuroprotective effect of sulfide catabolism by SQOR in the MPTP-induced mouse model of PD. The protective effect of upregulation of SQOR may be mediated by elevated persulfide levels.

[0103] [Example 13] Inhalation of H2S provided neuroprotection in PD animal models Because SQOR catalyzes the conversion of sulfides to polysulfides, and because SPC- or AAV-mediated SQOR overexpression prevented the reduction of sulfene sulfur after MPTP administration, we hypothesized that the beneficial effects of SQOR upregulation after MPTP administration might be mediated by increasing the levels of polysulfides. + We examined the effect of polysulfides on the viability of SH-SY5Y cells incubated with MPP. + (5 mM) for 24 h and viability was assessed by crystal violet assay. 41 Compared to control cells, treatment with Na2S (at doses between 1 and 100 μM) reduced MPP + did not improve the viability of SH-SY5Y cells treated with 42 In contrast, treatment with Na2S3 or GSSSG (10 μM each) reduced the MPP + The results showed that IFN-γ increased the viability of treated SH-SY5Y cells (Figure 15A).

[0104] To further examine the cytoprotective effects of polysulfides in physiologically relevant situations, MPP + We examined the effects of GSSSG, glutathione (GSH, the parent molecule of GSSSG that does not contain sulfane sulfur), α-lipoic acid trisulfide (1,2,3-trithiane-4-pentanoic acid, LA-SSS), and α-lipoic acid (the parent molecule of LA-SSS that does not contain sulfane sulfur) on mouse primary cortical neurons incubated with MPP. + (50 μM) and each of the above compounds for 24 hours. The polysulfide compounds GSSSG and LA-SSS induce MPP in mouse primary cortical neurons. +Protected against the effects of the treatment, but did not protect the parent compounds GSH and LA (Figures 15B and 15C). These results suggest that the sulfenyl sulfur-releasing molecule protects neurons from MPP + -induced cytotoxicity.

[0105] [Example 14] Intranasal administration of GSSSG resulted in neuroprotection in a PD animal model The large molecular weight of GSSSG (Mw: 644.7 DA) makes it impossible to cross the blood-brain barrier after systemic administration. However, recent studies suggest that intranasal administration may allow for the effective passage of relatively large molecules into the central nervous system 43 . To examine the possibility that GSSSG may have a protective effect against MPTP-induced neurodegeneration when administered intranasally, GSSSG (50 mg / kg) or saline was administered intranasally immediately after the administration of MPTP or saline (control) on day 0. From day 1 to day 6, mice were administered 50 mg / kg of GSSSG or saline intranasally every 12 hours. Intranasal administration of GSSSG was observed to prevent the MPTP-induced decrease in tyrosine hydroxylase in the substantia nigra-striatal region (Figure 16). Collectively, these results suggest that polysulfides have a robust therapeutic effect in a mouse model of PD.

[0106] References

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[0113] Other embodiments Although the present invention has been described in connection with its detailed description, it should be understood that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A pharmaceutical composition for use in a method for treating neurodegenerative disorders in a subject or for reducing the risk of such disorders, wherein the pharmaceutical composition comprises glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS), and the pharmaceutical composition is for intranasal administration to the subject.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises GSSSG, and the GSSSG is prepared by dissolving the crystalline form of GSSSG in a buffered saline solution with a pH of 3 to 6.

3. The pharmaceutical composition according to claim 1, wherein the disorder is post-ischemic neuronal cell death.

4. The pharmaceutical composition according to claim 1, wherein the disorder is a chronic neurodegenerative disease.

5. The pharmaceutical composition according to claim 1, wherein the chronic cerebral degenerative disease is polyinfarct dementia, Alzheimer's disease, Parkinson's disease, or Lewy body dementia.

6. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is intended to be administered in an effective amount within a few minutes to several hours after the occurrence of traumatic injury.

7. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is intended to be administered in an effective amount prior to a planned thoracic and / or abdominal aortic surgical procedure.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is intended to be administered in an effective amount several hours to several days before a scheduled thoracic and / or abdominal aortic surgical procedure.

9. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is to be administered in an effective amount 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.

10. A nasal administration composition comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS) for use in a method comprising the step of administering a therapeutically effective or prophylactically effective amount intranasally to a subject in need for the treatment of neurodegenerative disorders or for reducing the risk of such disorders.

11. The composition for use according to claim 10, which is prepared by a method comprising the step of dissolving the crystalline form of GSSSG in a buffered saline solution with a pH of 3 to 6.

12. The composition for use according to claim 10, wherein the disorder is post-ischemic neuronal cell death.

13. The composition for use according to claim 10, wherein the said disorder is a chronic neurodegenerative disease.

14. The composition for use according to claim 10, wherein the chronic cerebral degenerative disease is polyinfarct dementia, Alzheimer's disease, Parkinson's disease, or Lewy body dementia.

15. The composition for use according to claim 10, wherein the method comprises the step of administering an effective amount of a composition comprising GSSSG, PTN-SSS, or LA-SSS within a few minutes to several hours after the occurrence of a traumatic injury.

16. The composition for use according to claim 10, wherein the method comprises the step of administering an effective amount of the composition comprising GSSSG, PTN-SSS, or LA-SSS prior to a planned thoracic and / or abdominal aortic surgical procedure.

17. The composition for use according to claim 16, wherein the method comprises administering an effective amount of the composition comprising GSSSG, PTN-SSS, or LA-SSS several hours to several days before a scheduled thoracic and / or abdominal aortic surgical procedure.

18. The composition for use according to claim 17, wherein the method comprises administering an effective amount of the composition comprising GSSSG, PTN-SSS, or LA-SSS 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.

19. A device for intranasal administration of compositions containing glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS).

20. The apparatus according to claim 19, which is a nebulizer, or a pressurized container or aerosol dispenser further containing a spray agent.

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