Intranasal administration of polysulfides

Abstract

Methods and devices for nasal administration of a composition comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS) in neuroprotection (e.g., in neurodegenerative disease) and in order to reduce the risk of ischemic injury. The methods may be used, for example, to reduce the risk of ischemia or low blood flow conditions causing injury to the brain, spinal cord, and peripheral nerves that may result from surgery, trauma, and other conditions that reduce / damage blood flow and / or oxygen delivery to the nervous system.

Description

[0001] Priority declaration

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 344,095, filed May 20, 2020. The entire contents of the foregoing application are incorporated herein by reference. Technical Field

[0003] Methods and devices for nasal administration of compositions comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS) for use in neuroprotection (e.g., in neurodegenerative diseases) and to reduce the risk of ischemic injury. The method can be used, for example, to reduce the risk of damage to the brain, spinal cord, and peripheral nerves from ischemic or low blood flow states that may be caused by surgery, trauma, and other conditions that reduce / impair blood flow and / or oxygen delivery to the nervous system. Background Art

[0004] Approximately 2 to 12% of patients undergoing thoracoabdominal aortic surgery experience devastating paraplegic complications 1,2 It has been reported that more than 80% of postoperative paraplegia is delayed and is caused by secondary nerve damage in the spinal cord. 3,4 Although the pathogenic mechanisms of secondary neuronal injury are not fully understood, increased oxidative stress, mitochondrial dysfunction, inflammation, apoptosis, and glutamate-mediated excitotoxicity have been suggested to play important roles. 5,6 Because the development of paraplegia is delayed in these patients, there is a window of opportunity for potential preventive intervention. However, to date no pharmacological treatment has been shown to alleviate delayed paraplegia following thoracoabdominal aortic surgery. Summary of the invention

[0005] As shown herein, glutathione trisulfide (GSSSG) and pantethine trisulfide (PTN-SSS) prevent delayed paraplegia after SCI when delivered nasally after reperfusion. The neuroprotective effects of GSSSG are associated with increased local sulfane sulfur concentrations in the lumbar spinal cord.

[0006] Therefore, provided herein is a method for treating a disease associated with neurodegeneration or reducing the risk of a disease associated with neurodegeneration in a subject using nasal administration of GSSSG, PTN-SSS, or lipoic acid trisulfide (SSS). In some embodiments, the method includes preparing a composition comprising GSSSG by dissolving the crystalline form of GSSSG in a saline of pH 3-6 (e.g., pH 4.8-5.0). Also provided is a composition comprising GSSSG, PTN-SSS, or LA-SSS for nasal administration in a method for treating a disease associated with neurodegeneration or reducing the risk of a disease associated with neurodegeneration in a subject, optionally by dissolving the crystalline form of GSSSG in a saline of pH 3-6 (e.g., pH 4.8-5.0).

[0007] In some embodiments, the disorder is post-ischemic neuronal death.

[0008] In some embodiments, the disorder is a chronic brain degenerative disease, for example, multi-infarct dementia, Alzheimer's disease, Parkinson's disease, or dementia with Lewy bodies.

[0009] In some embodiments, the method comprises administering an effective amount of a composition comprising GSSSG, PTN-SSS, or LA-SSS within minutes to hours after the traumatic injury.

[0010] In some embodiments, the method comprises 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.

[0011] In some embodiments, the method comprises 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 aortic surgery.

[0012] In some embodiments, the method comprises administering an effective amount of a composition comprising GSSSG, PTN-SSS, or LA-SSS 2-24 hours, and / or 1, 2, 3, 4, 5, 6, and / or 7 days prior to a scheduled thoracic and / or abdominal aortic surgery.

[0013] Also provided herein are devices for nasally administering GSSSG, PTN-SSS, or LA-SSS to a subject.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail.

[0015] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A -C. Chemical structures of polysulfides. A. Chemical structures of GSSSG and GSH. One molecule of GSSSG consists of one molecule of sulfane sulfur (arrow) and two molecules of GSH. B. Chemical structures of PTN-SSS and PTN. One molecule of PTN-SSS consists of one molecule of sulfane sulfur (arrow) and one molecule of PTN. C. Chemical structures of LA-SSS and LA. One molecule of LA-SSS consists of one molecule of sulfane sulfur (red) and one molecule of LA. Abbreviations: GSH, glutathione; GSSSG, glutathione trisulfide; PTN, pantethine; PTN-SSS, pantethine trisulfide; LA-SSS, lipoic acid trisulfide.

[0017] Figure 2A -B. Mouse model of spinal cord ischemia-reperfusion injury for exploring the neuroprotective effects of polysulfides. A. Schematic diagram of the time course of the surgical procedure to produce delayed paraplegia. SCI was induced by clamping the distal aortic arch and the left subclavian artery. B. Scheme of intranasal administration of study drug after surgery and recovery of perfusion. Intranasal administration of study drug was performed under 3% isoflurane at 0, 8, 23, and 32 hours after surgery. BMS was measured at 0, 8, 24, 48, and 72 hours after surgery to evaluate hindlimb motor function. Abbreviations: BMS, Basso mouse scale for locomotion; PEEP, positive end-expiratory pressure; RR, respiratory rate; SCI, spinal cord ischemia; TV, tidal volume.

[0018] Figure 3.Post-reperfusion treatment with GSSSG prevents paraplegia after spinal cord ischemia in male and female mice. Changes in BMS scores 72 hours after SCI in male and female mice subjected to sham surgery, or male and female mice subjected to SCI and treated with GSSSG, GSH, or vehicle alone. n=11 for each of GSSSG, GSH, and vehicle alone treatment in the male mouse group, and n=8 for each of GSSSG, GSH, and vehicle alone treatment in the female mouse group. n=5 mice in the sham surgery group. Data are expressed as mean and standard deviation. Abbreviations: BMS, Basso mouse motor scale; GSH, glutathione; GSSSG, glutathione trisulfide; SCI, spinal cord ischemia.

[0019] Figure 4A -B. Post-reperfusion treatment with GSSSG prevents the loss of motor neurons in the lumbar spinal cord 48 hours after spinal cord ischemia. A. Representative photomicrographs of Nissl-stained lumbar spinal cord cross sections from mice subjected to sham surgery, or mice subjected to SCI and treated with GSSSG or vehicle alone. Magnifications of the boxed areas are provided below each cross section. Scale bar = 200 μm in low-magnification images (40×), and scale bar = 100 μm in high-magnification images (200×). B. Each field of view (0.26 mm) at high magnification (200×) in the ventral corners of lumbar spinal cord sections from mice subjected to sham surgery, or SCI and treated with vehicle alone, or GSSSG. 2 ). Comparisons were made using one-way ANOVA with Tukey's multiple comparison test. n = 5 mice per group. Data are presented as mean and standard deviation. Abbreviations: GSSSG, glutathione trisulfide; LSC, lumbar spinal cord; SCI, spinal cord ischemia.

[0020] Figure 5A -D. Post-reperfusion treatment with GSSSG prevents microglial activation and caspase-3 activation in the lumbar spinal cord 48 hours after spinal cord ischemia. A. Lumbar spinal cords were obtained from mice subjected to sham surgery, or mice subjected to SCI and treated with GSSSG or vehicle alone. Representative photomicrographs of cross sections of lumbar spinal cord stained with anti-Iba-1 antiserum are shown. Magnifications of the boxed areas are provided below each cross section. Scale bar = 200 μm in low-magnification images (40×), and scale bar = 100 μm in high-magnification images (200×). B. Each field of view (0.26 mm) at high magnification (200×) in the ventral corners of lumbar spinal cord sections from mice subjected to sham surgery, or mice subjected to SCI and treated with GSSSG or vehicle alone.2 ). Comparisons were made using one-way ANOVA with Tukey's multiple comparison test. The mean of the Iba-1 positive area in mice in the sham-operated group was set to 1. n = 5 mice per group. Data are expressed as mean and standard deviation. C. Representative photomicrographs of the ventral horn in a cross-section of the lumbar spinal cord stained with an antibody that recognizes cleaved caspase-3. Scale bar = 100 μm. D. Each field of view (0.26 mm) at high magnification (200×) in the ventral horn of a lumbar spinal cord section. 2 ). Comparisons were made using the Kruskal-Wallis test with Dunn's multiple comparison test. n = 5 mice per group. Data are presented as median and interquartile range. Abbreviations: GSSSG, glutathione trisulfide; Iba-1, ionized calcium-binding adaptor molecule 1; LSC, lumbar spinal cord; SCI, spinal cord ischemia.

[0021] Figure 6 .Reperfusion posttreatment with GSSSG inhibits upregulation of inflammatory mediators in the lumbar spinal cord 48 hours after spinal cord ischemia. Mice were subjected to sham surgery, or SCI followed by intranasal administration of GSSSG or vehicle alone. The lumbar spinal cord was harvested, and the mRNA level of each gene was normalized to 18S ribosomal RNA. The mean value of lumbar spinal cord mRNA levels of mice in the sham group was set to 1. n = 4-5 mice per group. *P<0.05, **P<0.01 relative to sham surgery. Data are expressed as medians and interquartile ranges for CCL2, CXCL1, IL-1β, IL-6, and TNF-α, and as means and standard deviations for Bcl-2 and Bcl-XL. Abbreviations: Bcl-2, B-cell lymphoma 2; Bcl-XL, B-cell lymphoma extra large; CCL2, C-C motif chemokine 2; CXCL1, C-X-C motif chemokine ligand 1; GSSSG, glutathione trisulfide; IL, interleukin; mRNA, messenger RNA; SCI, spinal cord ischemia; TNF-α, tumor necrosis factor α.

[0022] Fig. 7A -B.GS 34 Intranasal administration of SSG increases CNS 34 The levels of S-labeled GSSSG and 34 The ratio of S-labeled sulfane sulfur species to endogenous sulfane sulfur species. A. At 50 mg / kg GS 34 Detection of OB+FB, BS, C+ThSC, and LSC 30 min after intranasal administration of SSG 34Amount of S-labeled GSSSG. n = 4 mice for OB+FB, BS, and C+ThSC. n = 3 mice for LSC. Data are presented as mean and standard deviation. B. At 50 mg / kg of GS 34 OB+FB, BS, C+ThSC, and LSC 30 minutes after intranasal administration of SSG 32 S, 34 S]GSSH and 32 S2]GSSH ratio, [ 32 S, 34 CysSSH and 32 S2] CysSSH ratio, and [ 32 S2, 34 S]CysSSSCys and [ 32 S3] CysSSSCys ratio. n = 4 mice per organ. Data for [ 32 S, 34 S]GSSH and 32 S2]GSSH ratio and [ 32 S2, 34 S]CysSSSCys and [ 32 S3]CysSSSCys ratios are presented as mean and standard deviation and for [ 32 S, 34 CysSSH and 32 [S2] The proportion of CysSSH is expressed as median and interquartile range. Abbreviations: BS, brainstem; CNS, central nervous system; C+ThSC, cervical and thoracic spinal cord; CysSSH, cysteine ​​hydropersulfide; CysSSSCys, cysteine ​​trisulfide; GSSH, glutathione hydropersulfide; GSSSG, glutathione trisulfide; LC-MS / MS, liquid chromatography-tandem mass spectrometry; LSC, lumbar spinal cord; OB+FB, olfactory bulb and forebrain.

[0023] Fig. 8A-B. GSSSG increases relative sulfane sulfur levels in lumbar spinal cord and primary cortical neurons. A. Relative sulfane sulfur levels in lumbar spinal cord 48 hours after surgery in mice subjected to sham surgery, or mice subjected to SCI and treated with GSSSG or vehicle alone. Relative sulfane sulfur levels in lumbar spinal cord were estimated using the fluorescent probe SSip-1. n = 6 mice per group. Data are expressed as mean and standard deviation. B. Relative sulfane sulfur levels in primary cortical neurons after incubation with 10 μM GSSSG, 10 μM Na2S3, or vehicle alone. Data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. n = 10 for GSSSG group and vehicle group. n = 5 for Na2S3 group. Data are expressed as mean and standard deviation. Abbreviations: GSSSG, glutathione trisulfide; Na2S3, disodium trisulfide; SCI, spinal cord ischemia.

[0024] Fig. 9A -C.GSSSSG (but not GSH or GSSG) improves cell viability after oxygen and glucose deprivation / reoxygenation. A. Cell viability assessed by crystal violet assay after OGD / R when primary cortical neurons were incubated with different doses of GSSSG. One-way ANOVA with Dunnett's multiple comparison test was used for comparison. n=10 per group. Data are expressed as mean and standard deviation. B. Cell viability assessed by LDH assay after OGD / R when primary cortical neurons were incubated with different doses of GSSSG. One-way ANOVA with Dunnett's multiple comparison test was used for comparison. n=10 per group. Data are expressed as mean and standard deviation. C. Cell viability after OGD / R was assessed by crystal violet assay when primary cortical neurons were incubated with 30 μM GSSSG, 60 μM GSH, 30 μM GSSG, or vehicle alone. Kruskal-Wallis test with Dunn's multiple comparison test was used for comparison. n = 10 per group. Data are expressed as median and interquartile range. Abbreviations: CV, crystal violet; GSH, glutathione; GSSG, glutathione disulfide; GSSSG, glutathione trisulfide; LDH, lactate dehydrogenase; OGD / R, oxygen and glucose deprivation / reoxygenation.

[0025] Fig. 10A-D.PTN-SSS as a post-reperfusion treatment improves cell viability after OGD / R and prevents delayed paraplegia after SCI. A. Cell viability was assessed using a crystal violet assay after OGD / R when SH-SY5Y cells were incubated with 5μM, 10μM, 25μM, 50μM, 100μM PTN-SSS or vehicle alone. One-way ANOVA with Dunnett's multiple comparison test was used for comparison. n=12 for control, vehicle alone, and 5μM, 10μM, and 25μM PTN-SSS. n=6 for 50μM and 100μM PTN-SSS. Data are expressed as mean and standard deviation. B. Relative sulfane sulfur levels in SH-SY5Y cells after incubation with 5μM, 10μM, 50μM, 100μM, 300μM PTN-SSS or vehicle alone. Comparisons were made using one-way ANOVA with Dunnett's multiple comparison test. n=6 per group. Data are expressed as mean and standard deviation. C. Relative sulfane sulfur levels in the olfactory bulb and forebrain and whole spinal cord 30 minutes after intranasal administration of PTN-SSS. Relative sulfane sulfur levels were estimated using the fluorescent probe SSip-1. Unpaired t-test was used for comparison. n=5 mice per group. Data are expressed as mean and standard deviation. D. Changes in BMS scores 72 hours after SCI in male mice subjected to SCI and treated with vehicle alone, PTN, or PTN-SSS. Comparisons were made using the Kruskal-Wallis test with Dunn's multiple comparison test. n=9 mice per group. Data are expressed as mean and standard deviation. Abbreviations: BMS, Basso mouse motor scale; CV, crystal violet; LDH, lactate dehydrogenase; OB+FB, olfactory bulb and forebrain; OGD / R, oxygen and glucose deprivation / reoxygenation; PTN, pantethine; PTN-SSS, pantethine trisulfide; SCI, spinal cord ischemia.

[0026] Fig.11 Comparison of the neuroprotective effects of GSSSG and GSSG on hindlimb motor function after SCI. Changes in BMS scores 72 hours after SCI in male mice subjected to SCI and treated with GSSSG or GSSG. n = 6 mice per group. Data are expressed as mean and standard deviation. Abbreviations: BMS, Basso mouse motor scale; GSSG, glutathione disulfide; GSSSG, glutathione trisulfide; SCI, spinal cord ischemia.

[0027] Fig.12.Relative mRNA levels of enzymes that synthesize or metabolize sulfide or persulfide in the lumbar spinal cord obtained 48 hours after surgery. Mice were subjected to sham surgery, or SCI followed by intranasal administration of GSSSG or a single carrier. The lumbar spinal cord was harvested, and the mRNA level of each gene was normalized to 18S ribosomal RNA. The mean value of the lumbar spinal cord mRNA level of mice in the sham group was set to 1. n = 4-5 mice per group. *P<0.05, ****P<0.0001 relative to sham surgery. Data are expressed as medians and interquartile ranges for CBS, TST, and SUOX, and as mean values ​​and standard deviations for CSE, 3-MST, SQOR, ETHE1, CARS1, and CARS2. Abbreviations: CARS, cysteinyl-tRNA synthetase; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; ETHE1, ethylmaronicencephalopathy 1; GSSSG, glutathione trisulfide; mRNA, messenger RNA; SCI, spinal cord ischemia; SQOR, sulfide:quinone oxidoreductase; SUOX, sulfite oxidase; 3-MST, 3-mercaptopyruvate sulfotransferase; TST, thiosulfate sulfotransferase.

[0028] Fig.13 .Cytotoxic effect of GSSSG on cell viability. Primary cortical neurons were incubated with 10 μM, 30 μM, 60 μM, 100 μM of GSSSG, or vehicle alone. Cell viability was assessed using the LDH assay. Incubation with 100 μM of GSSSG significantly increased LDH release compared to vehicle-only treated cells (vehicle alone vs. GSSSG (100 μM); 105 [85-110]% vs. 149 [133-159]%; P < 0.0001). Comparisons were made using the Kruskal-Wallis test with Dunn's multiple comparison test. n = 10 per group. Data are expressed as median and interquartile range. Abbreviations: LDH, lactate dehydrogenase; GSSSG, glutathione trisulfide.

[0029] Fig.14.Cytotoxic effect of PTN-SSS on cell viability. SH-SY5Y cells were incubated with 5 μM, 10 μM, 25 μM, 50 μM, 100 μM PTN-SSS, or vehicle alone. Cell viability was assessed using a crystal violet assay. Incubation with 100 μM PTN-SSS significantly reduced cell viability compared to vehicle-treated cells alone (vehicle alone vs. PTN-SSS (100 μM); 1.00 ± 0.25 vs. 0.59 ± 0.21; P = 0.0105). One-way ANOVA with Dunnett's multiple comparison test was used for comparison. n = 12 for vehicle alone and 5 μM, 10 μM, and 25 μM PTN-SSS. n = 6 for 50 μM and 100 μM PTN-SSS. Data are expressed as mean and standard deviation. Abbreviations: CV, crystal violet; PTN-SSS, pantethine trisulfide.

[0030] Figure 15A-C .Sulfide and polysulfide use MPP + Effects on cell viability of incubated SH-SY5Y cells and primary cortical neurons. (A) SH-SY5Y cells or (B and C) mouse primary cortical neurons were incubated with or without MPP in the presence or absence of trisulfide compounds at 37°C. + Incubate for 24 h. Cell viability was measured using the crystal violet assay (n=4 or 5 per group). *, **, ***p<0.05, 0.01, 0.001 relative to controls without trisulfide treatment.

[0031] Fig.16 Effect of intranasal administration of GSSSG on MPTP-induced neurodegeneration. Representative immunoblots show the levels of tyrosine hydroxylase in the nigrostriatal region of mice 7 days after administration of MPTP or saline with or without Na2S3 treatment. Quantification of relative levels of tyrosine hydroxylase was determined using densitometry, comparing the levels of tyrosine hydroxylase to focal adhesion proteins (n=5, 4, 5 mice, respectively). **p<0.01 relative to saline+saline. ##p<0.05 relative to MPTP+saline. DETAILED DESCRIPTION

[0032] Hydrogen sulfide (H2S), a colorless gas with a characteristic rotten egg odor, is an environmental hazard produced by a variety of natural and industrial sources. H2S is also considered a signaling molecule that plays a variety of physiological roles. 7 Many of the effects of H2S have been attributed to persulfides (RSSH) and polysulfides (RS n H) and other sulfane sulfur species 8The cytoprotective effects of sulfane sulfur compounds may be mediated by multiple mechanisms, including antioxidant effects. 9,10 and anti-inflammatory effects 10,11 , inhibit lipid peroxidation and ferroptosis by scavenging free radicals 12 , and post-translational modifications of proteins 13,14 Sulfane sulfur compounds produce post-translational modifications in proteins because sulfane sulfur (S 0 ) (a sulfur atom with 6 valence electrons but no charge) is readily donated to acceptor thiols in target proteins in a process called persulfidation, which modulates the function of the target protein 13,14 In previous studies, we showed that breathing H2S prevented delayed paraplegia in mice subjected to transient spinal cord ischemia (SCI). The neuroprotective effect of H2S appears to be associated with the hypersulfidation of nuclear factor kappa B (NF-kB) p65 15 .

[0033] The mechanism by which systemically administered H2S donor compounds modulate the concentration of reactive sulfur species in target tissues is not well defined, in part because of the short half-life of H2S in the blood. 16 This knowledge gap concerning the in vivo pharmacokinetics of sulfides has hampered the implementation of sulfide-based therapies into patient care. To allow for the future use of polysulfides to treat neurodegenerative diseases, it is essential to determine whether administration of polysulfides modulates local concentrations of sulfane sulfur species in the central nervous system (CNS).

[0034] Glutathione trisulfide (GSSSG) is an endogenous polysulfide ( Figure 1A ), and is in dynamic equilibrium with various reactive sulfur species including glutathione (GSH), glutathione hydropersulfide (GSSH), glutathione hydropolysulfide, and other glutathione polysulfides. 9,17 GSSSG may be an important endogenous reservoir of sulfane sulfur species. It has been observed that the levels of sulfane sulfur species vary in patients depending on the type and severity of the disease, suggesting that sulfane sulfur species may have a protective role in pathological conditions. 18,19 .

[0035] GSH, a natural tripeptide of glutamate, cysteine, and glycine, is ubiquitous in mammalian cells and is the most prevalent thiol (RSH) in mammalian cells. GSH is a nucleophile and functions as a major intracellular antioxidant in mammalian cells. 20 GSH has been reported to have a neuroprotective effect against ischemia-reperfusion injury 21Glutathione disulfide (GSSG) is the oxidized form of GSH and is primarily produced by GSH peroxidase-mediated catalysis or from the direct reaction of GSH with electrophilic compounds such as free radicals. 20 Both GSH and GSSG can produce post-translational modifications of proteins through "glutathionylation", which protects protein cysteines from irreversible oxidation and regulates the structure and function of a wide variety of proteins. 20,22-24 .

[0036] Pantethine (PTN), a precursor for the synthesis of coenzyme A, transfers the acetyl group from pyruvate to oxaloacetate, initiating the tricarboxylic acid cycle 25 Preclinical studies demonstrate beneficial effects of PTN in mouse models of neurodegenerative diseases 26,27 Pantethine trisulfide (PTN-SSS) is a polysulfide composed of one sulfane sulfur molecule and one PTN molecule ( Figure 1B ); see WO2022 / 045052.

[0037] Lipoic acid trisulfide is a relatively small molecule (MW = 238.39; see, e.g., WO2022 / 045212) that is expected to cross the blood-brain barrier and be delivered to the CNS. Lipoic acid trisulfide (rather than lipoic acid) improves cell viability in a cell model of Parkinson's disease, similar to GSSSG. After its "sulfane sulfur" is delivered into cells, the resulting lipoic acid and dihydrolipoic acid can exert their own biological properties, such as antioxidants, metal chelators, and cofactors for mitochondrial enzymes. LA-SSS consists of one molecule of sulfane sulfur and one molecule of LA. In some embodiments, LA-SSS is alpha lipoic acid, or LA-SSS-βCD (lipoic acid trisulfide-β cyclodextrin or LA-SSS-CE (choline ester); see WO2022 / 045212.

[0038] This study investigated the neuroprotective effects and pharmacokinetics of intranasal polysulfides in an established mouse model of spinal cord ischemia. In this mouse model, neurodegeneration occurs primarily in the ventral horn of the lumbar spinal cord 24-48 hours after reperfusion. 6,28,29 The experiments determined whether intranasal administration of polysulfides would preferentially increase polysulfide levels in the CNS 30-32 We hypothesized that CNS-targeted, intranasal administration of sulfide would prevent neurodegeneration in the lumbar spinal cord by increasing local concentrations of sulfane sulfur species and would rescue mice from delayed onset paraplegia.

[0039] In the present study, we show that post-reperfusion intranasal administration of GSSSG (but not GSH or GSSG) prevents the massive loss of viable neurons in the ventral horn of the lumbar spinal cord and rescues mice from delayed paraplegia after SCI. In primary cortical neurons, GSSSG (but not GSH or GSSG) improved cell viability after OGD / R. The beneficial effects of GSSSG were associated with the suppression of increased levels of inflammatory cytokines as well as the inhibition of microglial activation and caspase-3 activation. 34 The significant increase of S-labeled sulfane sulfur species in GS 34 SSG was detected in the lumbar spinal cord shortly after intranasal administration. In addition, we observed that the protective effect of GSSSG was associated with increased sulfane sulfur levels in the lumbar spinal cord after intranasal administration of GSSSG and increased sulfane sulfur levels in primary cortical neurons after incubation with GSSSG. In addition, incubation of SH-SY5Y cells with PTN-SSS increased intracellular sulfane sulfur levels and improved cell viability after OGD / R, while intranasal administration after reperfusion of PTN-SSS (rather than PTN) rescued mice from delayed paraplegia after SCI. PTN-SSS increased sulfane sulfur levels in the central nervous system shortly after intranasal administration. These observations indicate that sulfane sulfur can be easily delivered to the central nervous system with intranasal administration of polysulfides, and indicate that this treatment prevents delayed neurodegeneration in the lumbar spinal cord and alleviates delayed paraplegia. The results of this study emphasize the important therapeutic potential of polysulfides in preventing neurodegeneration of the spinal cord.

[0040] Previously, we used a chemically induced cytotoxicity model with SH-SY5Y cells to show that the cytoprotective effects of H2S-donating compounds correlated with their ability to increase intracellular sulfane sulfur levels. 45 We also report that administration of sodium thiosulfate improves survival and neurological function in mice subjected to global cerebral ischemia-reperfusion; the cytoprotective effect is associated with a significant increase in thiosulfate, a sulfane sulfur species, in plasma and brain tissue. 46 These results suggest that increased concentrations of sulfane sulfur can be neuroprotective in pathological conditions. In this study, we showed that the neuroprotective effect of intranasal GSSSG in SCI-induced spinal cord injury was associated with increased sulfane sulfur levels in the lumbar spinal cord. The results support the hypothesis that the neuroprotective effect of GSSSG is mediated through increased sulfane sulfur.

[0041] Previous preclinical studies have shown that administration of GSH attenuates neuronal cell death after cerebral ischemia-reperfusion 21,47 Because GSH is a metabolite of GSSSG 9,17, it is possible that the increased concentration of GSH can explain the neuroprotective effect of GSSSG. However, in the current study, we observed that intranasal administration of GSSSG (but not GSH) prevented delayed paraplegia after SCI. The reasons for the differences between the current reports and previous reports on the effects of GSH may be caused by differences in the dose of GSH, the route of administration, and the animal model. In particular, the dose of GSH used in the current study was one-tenth of the dose of GSH used in previous studies. The results of this study support the hypothesis that the mechanism of GSSSG-mediated neuroprotection is independent of the conversion to GSH.

[0042] Intracellular sulfane sulfur substances can react with GSH to produce GSSH 9 Compared with GSH, GSSH is more nucleophilic and a better intracellular antioxidant. In addition, GSSH can be directly generated from GSSSG 9 Akaike and colleagues reported that the concentration of endogenous GSSH in mouse brain was 222 pmol / mg protein, which was significantly greater than the concentrations of other endogenous sulfane sulfur species, including GSSSG (1 pmol / mg protein), CysSSH (2 pmol / mg protein), or CysSSSCys (not detected). 9,37 In the current study, 34 S-labeled GSSSG in GS 34 SSG was detected at 317 ± 111 pmol / mg protein in the lumbar spinal cord 30 min after intranasal administration. In addition, based on previously reported levels of endogenous GSSH and CysSSH 37 , in GS 34 Lumbar spinal cord after intranasal administration of SSG 34 S-labeled GSSH and 34 The levels of S-labeled CysSSH were approximately 1,600 pmol / mg protein and 18 pmol / mg protein, respectively. 9,37 These results show that intranasal administration of GSSSG can increase the levels of multiple sulfane sulfur species by about 10- to 100-fold in the lumbar spinal cord, the epicenter of neuronal death after SCI. Considering that GSSH is the most quantitatively predominant sulfane sulfur species in the lumbar spinal cord after intranasal administration of GSSSG, the majority of the neuroprotective effects of GSSSG may be conferred via GSSH.

[0043] Previous studies have shown that molecules larger than 500 Da are unable to cross the blood-brain barrier and the blood-spinal cord barrier after systemic administration. 31Due to the large size of GSSSG (644.7 Da), we chose to administer this compound intranasally. After intranasal administration, the macromolecule can bypass the blood-brain barrier and blood-spinal cord barrier via the olfactory and trigeminal nerve pathways and can rapidly reach the parenchyma of the central nervous system. 30,32 The peripheral olfactory system connects the nasal passages to the olfactory bulbs and rostral brain, and the peripheral trigeminal system connects the nasal passages to the brainstem and spinal cord. 30 For example, a previous report compared intravenous and intranasal routes of administration in terms of the amount of methylprednisolone sodium succinate (497.5 Da) that reached the spinal cord. 48 Although relatively large amounts of methylprednisolone were detected in the parenchyma of the spinal cord after intranasal administration, much smaller amounts of methylprednisolone were detected after intravenous administration. 48 Various other high molecular weight therapeutics (>500 Da) have been successfully delivered to the central nervous system via intranasal administration 49 In the current study, we observed that 34 The concentration of S-labeled GSSSG (268 ± 140 pmol / mg protein) was 34 30 minutes after intranasal administration of SSG, the concentration of endogenous GSSSG in the brain of mice was significantly higher than that in mice (1 pmol / mg protein). 37 In contrast, plasma 34 The 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 of Akaike and colleagues). These results indicate that intranasally administered GSSSG readily and preferentially reaches the central nervous system, including the spinal cord, via the olfactory and trigeminal pathways rather than the bloodstream.

[0044] There are several limitations to this study. First, we were unable to determine the detailed mechanisms responsible for the beneficial effects of polysulfides, other than the fact that these effects were associated with increased levels of sulfane sulfur in target organs, inhibition of increased levels of inflammatory cytokines, and inhibition of microglial activation and caspase-3 activation, and were unlikely to be attributed to transglutathionylation. Polysulfides appear to be cytoprotective via multiple mechanisms, including acting as antioxidants. 9,10 and anti-inflammatory agents 10,11 , inhibit lipid peroxidation and ferroptosis 12 , and enhance post-translational modification of proteins 13,14. The precise mechanisms responsible for the neuroprotective effects of polysulfides remain to be elucidated in future studies. Second, we did not explore the mechanism by which GSSSG inhibits the mRNA levels of SQOR, a protein that metabolizes sulfide to GSSH. Increased levels of GSSH following intranasal administration of GSSSG could downregulate the expression of SQOR via negative feedback. Third, although GSSSG and PTN-SSS were administered intranasally under mild sedation, some mice coughed up the drugs from their noses or swallowed them. The cytoprotective effects conferred by polysulfides in this study most likely reflect the effects of successfully administering lower doses. As better drug formulations more suitable for clinical application are developed, we expect that the beneficial effects of polysulfides will be achieved using lower doses.

[0045] The current study revealed that intranasal administration of GSSSG or PTN-SSS (rather than GSH, GSSG, or PTN) after reperfusion rescued mice subjected to SCI from delayed paraplegia. Intranasally administered GSSSG accumulates in the lumbar spinal cord, increases the local concentration of persulfide, polysulfide, and sulfane sulfur, and reduces neuroinflammation, apoptosis, and neurodegeneration. The potent neuroprotective effect of intranasally administered GSSSG is similar to that of inhaled H2S, but the use of polysulfides including GSSSG and PTN-SSS is more practical in clinical medicine than the administration of gaseous H2S. In particular, the excellent physical properties of PTN-SSS are worthy of further evaluation for clinical development. This study opens up the possibility of novel polysulfide-based therapies to prevent the occurrence of delayed paraplegia after thoracic and abdominal aortic surgery and other neurodegenerative diseases of the spinal cord.

[0046] We also observed that trisulfide compounds in MPTP / MPP + The small size of Na2S3 allowed us to deliver the molecule intraperitoneally, expecting it to cross the blood-brain barrier. Due to the large size of GSSSG, we delivered the compound intranasally in an animal model of PD and saw protective effects, demonstrating its usefulness in providing neuroprotection in neurodegenerative diseases.

[0047] Treatment

[0048] The methods described herein include methods for treating a disorder associated with neurodegeneration or reducing the risk of a disorder associated with neurodegeneration in a subject, e.g., a mammalian subject, e.g., a human subject or a non-human veterinary subject. In some embodiments, the disorder is post-ischemic neuronal death, e.g., post-ischemic neuronal death in the spinal cord. In some embodiments, the disorder is a chronic brain degenerative disease (e.g., multi-infarct dementia, Alzheimer's disease, Parkinson's disease, or Lewy body dementia). Typically, the method includes administering a therapeutically effective amount of a composition comprising a crystalline form of GSSSG, PTN-SSS, or LA-SSS to a subject in need of such treatment or who has been determined to be in need of such treatment.

[0049] As used herein, "treating" means alleviating at least one symptom of a disorder associated with neurodegeneration. Conditions that can be treated using the methods described herein can be associated with loss of motor control, paralysis, or paraplegia. Administration of a therapeutically effective amount of a compound described herein can result in improved motor control, reduced paralysis, or paraplegia.

[0050] In addition, the method can result in a reduction in the risk of loss of motor control, paralysis or paraplegia. Subjects at risk of loss of motor control, paralysis or paraplegia can include those subjects with traumatic injuries and those subjects about to undergo thoracic aorta and / or abdominal aorta surgery. These methods can include nasal administration of an effective amount of GSSSG, PTN-SSS or LA-SSS compositions as described herein within a few minutes to a few hours after the traumatic injury occurs and / or within a few hours to a few days before a scheduled thoracic aorta and / or abdominal aorta surgery, for example.

[0051] "Effective amount" is an amount sufficient to achieve a beneficial result or a desired result. For example, a therapeutic amount is an amount that achieves a desired therapeutic effect. This amount may be the same or different from a preventive effective amount, which is an amount necessary to prevent the onset of a disease or disease symptom. An effective amount may be administered in one or more administrations, applications or dosages. The composition may be administered from one or more times a day to one or more times a 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 before a scheduled thoracic aorta and / or abdominal aorta surgery. It will be appreciated by the technician that certain factors may affect the dosage and timing required for the effective treatment of a subject, including but not limited to the severity of the disease or condition, previous treatment, the general health and / or age of the subject, and other diseases present. In addition, treating a subject with a therapeutic compound described herein in a therapeutically effective amount may include a single treatment or a series of treatments.

[0052] The dosage, toxicity and therapeutic efficacy of therapeutic compounds can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, for determining LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indexes are preferred. Although compounds that exhibit toxic side effects can be used, it should be noted that the delivery system designed to target the site of such compounds to the affected tissues is minimized and side effects are thereby reduced to potential damage to uninfected cells.

[0053] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for humans. The dosage of such compounds is preferably within the range of circulating concentrations including ED50 with little toxicity or no toxicity. The dosage can vary within this range according to the dosage form used and the route of administration utilized. For any compound used in the method of the present invention, the therapeutically effective dose can be preliminarily estimated from the cell culture assay. The dosage can be formulated in an animal model to reach a circulating plasma concentration range including the IC50 (i.e., the concentration of the test compound that reaches half the maximum inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine a useful dosage in the human body. The level in plasma can be measured, for example, by high performance liquid chromatography.

[0054] Pharmaceutical compositions and methods of administration

[0055] The methods described herein include using pharmaceutical compositions comprising GSSSG, PTN-SSS, or LA-SSS as active ingredients. In some embodiments, the compositions use GSSSG in crystalline form, using the method described in EP 3560947, by dissolving crystalline GSSSG in a buffer (e.g., saline) at pH 3-6 (e.g., pH 4.8-5). The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Compositions comprising PTN-SSS or LA-SSS can be prepared by dissolving in a buffer (e.g., saline or water) at pH 4-9 (e.g., 5-8).

[0056] An exemplary method for producing glutathione trisulfide dehydrate in crystalline form 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. PTN-SSS or LA-SSS may be prepared as described in WO2022 / 045212 (LA-SSS) and WO2022 / 045052 (PTN-SSS).

[0057] Pharmaceutical compositions generally include a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with drug administration. Supplementary active compounds may also be incorporated into the composition.

[0058] The pharmaceutical compositions used in the present method are formulated to be compatible with nasal administration.Examples of routes of administration include parenteral administration, such as intravenous administration.

[0059] Methods of formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, 2005; and books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).

[0060] For the purpose of mucosal therapeutic administration, the active compound (e.g., GSSSG, PTN-SSS, or LA-SSS) can be combined with an excipient or carrier suitable for administration by inhalation or absorption, for example, via nasal spray or drops. For nasal administration, the formulation can be an aerosol in a sealed vial or other suitable container.

[0061] Pharmaceutical composition and nasal dosage form can further comprise one or more compounds that reduce the rate that active ingredient will decompose.Therefore, nasal dosage form described herein can be processed into quick-release dosage form or sustained-release dosage form.Quick-release dosage form can release GSSSG, PTN-SSS or LA-SSS in quite a short time, for example, in a few minutes to within a few hours.Sustained-release dosage form can release GSSSG, PTN-SSS or LA-SSS in a few hours, for example, up to 24 hours or longer if needed.In either case, delivery can be controlled to be substantially at a certain predetermined rate during delivery.

[0062] Nasal delivery is considered an attractive route for needle-free systemic drug delivery, especially when rapid absorption and action are desired. In addition, nasal delivery can help solve problems associated with poor bioavailability, slow absorption, drug degradation, and adverse events (AEs) in the gastrointestinal tract, and avoid first-pass metabolism in the liver.

[0063] Liquid nasal formulations are primarily aqueous solutions, but suspensions and emulsions can also be delivered. In conventional spray pump systems, antimicrobial preservatives are often required to maintain microbial stability in liquid formulations.

[0064] Metered spray pumps have dominated the nasal drug delivery market since their introduction. The pumps typically deliver about 25-200 μL / spray, and they provide high reproducibility of spray dose and plume geometry. Particle size and plume geometry can vary within certain limits and depend on the characteristics of the pump, formulation, actuator orifice, and applied force. Traditional spray pumps replace the sprayed liquid with air, and therefore require preservatives to prevent contamination.

[0065] Optional spray systems or devices that avoid the need for preservatives can also be used. These systems use a foldable bag, a movable piston, or compressed gas to compensate for the volume of liquid sprayed. The solution of a foldable bag and a movable piston that compensates for the volume of liquid sprayed provides an additional advantage, that is, they can be sprayed upside down without the risk of sucking air into the dip tube and damaging the subsequent spray. This can be useful for some products where the patient is bedridden and where it is recommended to apply with the head down. Another method for avoiding preservatives is to filter the air that replaces the liquid sprayed through a sterile air filter. In addition, some systems have a ball valve at the tip to prevent contamination of the liquid inside the applicator tip.

[0066] For administration by inhalation, the GSSSG, PTN-SSS, or LA-SSS compound can be delivered as a dry powder or aerosol spray from a pressure container or dispenser containing a suitable propellant (e.g., a gas such as carbon dioxide), or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[0067] Also provided herein are devices for nasal administration comprising GSSSG, PTN-SSS, or LA-SSS, e.g., as described herein.

[0068] Described herein is a kit, which may include a composition comprising GSSSG, PTN-SSS, or LA-SSS, such as a dry powder form or liquid nasal form prepared for administration, or alternatively, the kit may include a composition comprising GSSSG, PTN-SSS, or LA-SSS as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid nasal dosage form. When the kit includes a GSSSG composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (e.g., for oral administration or nasal administration), the kit may optionally include a reconstitution solvent of pH 3-6 (e.g., pH 4.8-5.0). When the kit includes 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 (e.g., for oral administration or nasal administration), the kit may optionally include a reconstitution solvent of pH 4-9 (e.g., pH 5-8). In this case, the composition or reconstitution solvent is combined with the active ingredient to provide a liquid oral dosage form of the active component. Typically, the active ingredient is soluble in a solvent and forms a solution. The solvent can be, for example, water, a non-aqueous liquid, or a combination of a non-aqueous component and an aqueous component. Suitable non-aqueous components include, but are not limited to, oils; alcohols, such as ethanol, glycerol; and glycols, such as polyethylene glycol and propylene glycol. In some embodiments, the solvent is phosphate buffered saline (PBS).

[0069] The pharmaceutical composition can be included in a container, package, or dispenser together with instructions for administration. For example, GSSSG can be provided in a kit in crystalline form with a sterile buffer (e.g., saline) at pH 3-6 for dissolving the crystals to prepare a solution for nasal administration.

[0070] Example

[0071] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0072] Materials and methods

[0073] The following materials and methods were used in the examples below.

[0074] Material

[0075] The GSSSG dihydrate in a stable form was synthesized and provided by Kyowa Hakko Bio Co., Ltd. (Tokyo, Japan). GSSSG was suspended in distilled water and dissolved by titrating the pH to between 4.8 and 5.0 with sodium bicarbonate (Sigma-Aldrich, Saint Louis, MO, USA). In order to avoid degradation of the compound, a fresh GSSSG solution was prepared immediately before each experiment. GSH (Sigma-Aldrich, Saint Louis, MO, USA) and GSSG hexahydrate (provided by Kyowa Hakko Bio Co., Ltd.) were dissolved in distilled water. In the GSSSG study, the single carrier was distilled water, and the pH was adjusted to between 4.8 and 5.0 with hydrochloric acid.

[0076] 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 in solutions of pH 4.0-9.0 at room temperature for at least 4 days. In the PTN-SSS study, distilled water was used as the sole carrier.

[0077] In vivo studies

[0078] animal

[0079] All animal procedures were performed in accordance with the protocol approved by the Animal Care and Use Committee of the Massachusetts General Hospital Institution and the National Research Council of the United States "Guide to the Care and Use of Laboratory Animals". The description of the research design and experiment follows the ARRIVE guidelines. Adult C57BL / 6J mice (12-18 weeks old) of both sexes were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Male mice and female mice were used in the experiment to explore the neuroprotective effect of GSSSG on motor function after SCI. Because the neuroprotective effect of GSSSG does not seem to depend on gender, subsequent in vivo experiments were performed only in male mice. All mice were kept in a temperature and humidity controlled room in an animal center with a 12-hour light / dark cycle, and food and water were freely provided. In order to allow mice recovering from surgery to approach, additional food pellets were inserted into the hydrated gel placed on the mat (bedding). Random pairing was designed to minimize the variability between each treatment group. Mice were paired based on body weight, age, delivery date, and the cage to be treated when possible. After pairing, mice were randomly assigned to different treatments.

[0080] Surgery to induce spinal cord ischemia and administration of investigational drugs

[0081] The surgical procedure to produce delayed paraplegia in mice was performed as previously described. 6 implement( Figure 2A). Mice were anesthetized with 5% isoflurane in 100% O2 and intubated with a 20-gauge catheter (Angiocath; Becton Dickinson, Franklin Lakes, NJ, USA). Mice were mechanically ventilated (MiniVent model 845; Harvard Apparatus, Holliston, MA, USA) and anesthesia was maintained with 2% isoflurane in 100% O2 with a tidal volume of 8 μl / g. Paraspinal muscle temperature was measured using a T-type implantable thermocouple probe (IT-18) and a T-pod (AD Instruments, Colorado Springs, CO, USA). A skin incision was made on the back, the tip of the probe was placed at the level of L1-L3 using an 18-gauge needle, and the temperature was maintained at 37.5±0.5°C using a heating pad and a DC temperature controller (FHC, Bowdoin, ME, USA). The median sternotomy extends from the top of the manubrium to the second rib. The aortic arch is 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. The first vascular clip (straight microvascular clip, RS-5424; Roboz Surgical Instrument Company, Inc., Gaithersburg, MD, USA) is placed on the aortic arch between the LCCA and the LSA, and then, within 15 seconds, the second vascular clip (45° angle microvascular clip, RS-5435; Roboz Surgical Instrument Company, Inc., Gaithersburg, MD, USA) is placed at the origin of the LSA. After ischemia, the vascular clips are removed in the opposite order. The incisions are sutured layer by layer and mechanical ventilation is stopped. After confirming stable spontaneous breathing, the mice are extubated.

[0082] Previously described surgical procedures 6 The respiratory rate during surgery was increased to 230 breaths / min because hyperventilation promotes delayed paraplegia caused by spinal cord ischemia. 33 A laser Doppler perfusion monitor (moorVMS-LDF1; Moor Instruments, Millwey, UK) was used to monitor femoral artery blood flow. 28,29 A plastic fiber (POF500; Moor Instruments, Millwey, UK) was fixed perpendicular to the left femoral artery to confirm that occlusion of the aorta resulted in an immediate and sustained reduction (>90%) in femoral artery blood flow. 28,29To improve surgical accuracy, the aortic arch was isolated and vascular clamps were placed using a microscope (Leica MZ95; Leica Microsystems, Buffalo Grove, IL, USA). Based on the results of the pilot study, we chose an ischemic time to induce delayed paraplegia in all mice (3 minutes in male mice and 3.5 minutes in female mice) with the lowest mortality rate at 72 hours after surgery (Table 1).

[0083] Table 1. Spinal cord ischemia time causing delayed paraplegia in each sex.

[0084]

[0085] Abbreviations: SCI, spinal cord ischemia.

[0086] The investigational drug was administered intranasally using a single-channel pipette. Mice were anesthetized with 3% isoflurane using a non-rebreathing circuit with a mouse nose cone (VetEquip, Inc., Livermore, CA, USA). When administering the drug, the nose cone was removed and the mouse was allowed to breathe air. Approximately 6 μL of each drug was administered into the nostrils of the mouse, and the mouse inhaled the droplets during inspiration. The procedure was repeated at intervals until the total volume of the drug was administered, which took about 10 minutes. The interruption time for each intranasal dose of isoflurane administration was about 10 seconds. Intranasal drugs were administered at 0, 8, 23, and 32 hours after surgery ( Figure 2B In the sham procedure group, the above surgical procedures were performed, but the aorta was not cross-clamped.

[0087] For pain control, 0.1 mg / kg of buprenorphine was administered intraperitoneally before surgery and every 12 hours after surgery until 60 hours. We also administered 0.5 mg / kg of 0.25% bupivacaine subcutaneously around the wound incision site immediately after surgery.

[0088] Assessment of motor function after spinal cord ischemia

[0089] Hindlimb motor function was assessed using the Basso Mouse Motor Scale (BMS) at 0, 8, 24, 48, and 72 hours after surgery. 34 To quantify ( Figure 2B ). The score ranges from 0 for complete paraplegia to 9 for normal motor function. A BMS score <6 (0-5) indicates paraplegia or mild paraplegia, while a BMS score ≥6 (6-9) indicates that the mouse is able to walk.

[0090] Study to investigate the neuroprotective effect of post-perfusion conditioning with GSSSG on motor function after spinal cord ischemia

[0091] Mice were randomly assigned to treatment groups, and the investigator performing the surgery was blinded to group assignment. Based on a pilot study, 50 mg / kg of GSSSG, 45.2 mg / kg of GSH (twice the molar amount of GSSSG, since one molecule of GSSSG contains two molecules of GSH) were administered ( Figure 1A ), or 53 mg / kg of GSSG (equimolar dose of GSSSG).

[0092] Histological studies

[0093] The lumbar bulge of the spinal cord was removed 48 hours after surgery and sectioned to 5 μm thickness using a cryostat (CM1850UV; Leica Biosystems, Heidelberger, Germany). Nissl staining was performed using a Nissl staining kit (VitroVivo Biotech, Rockville, MD, USA) according to the protocol recommended by the manufacturer. Immunohistochemical staining for ionized calcium binding adaptor 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).

[0094] The number of stained cells was determined by an investigator blinded to the identity of the samples in three different spinal cord sections from each mouse at high magnification (200×) in one field of view (0.26 mm 2 ) were counted. The average number of stained cells was calculated for each mouse. For quantitative analysis of Iba-1 staining, each field (0.26 mm) was analyzed at high magnification (200×) in three different ventral horn sections of each mouse. 2 ) were calculated using ImageJ image processing program (National Institutes of Health, Bethesda, MD, USA). The average area of ​​Iba-1 staining in the spinal cord of mice in the sham operation group was set to 1, and the relative amount of Iba-1 staining of mice in each experimental group was determined. Each group included 5 mice.

[0095] Measurement of gene expression

[0096] Messenger RNA (mRNA) levels were as described previously 15Lumbar spinal cord was used to measure 48 hours after surgery.CC motif chemokine 2 (CCL2), CXC motif chemokine ligand 1 (CXCL1), interleukin (IL) -1 β, IL-6, tumor necrosis factor (TNF) -α, B- cell lymphoma 2 (Bcl-2), B- cell lymphoma super large (Bcl-XL), cystathionine β synthetase, cystathionine γ lyase, 3-mercaptopyruvate sulfotransferase, sulfide: quinone oxidoreductase (SQOR), ethylmalonic encephalopathy protein 1, thiosulfate sulfotransferase, sulfite oxidase, cysteinyl-tRNA synthetase 1 and cysteinyl-tRNA synthetase 2 mRNA levels are standardized to the level of 18S ribosomal RNA using quantitative real-time polymerase chain reaction (7500 fast real-time PCR systems, ThermoFisher Scientific, Waltham, MA, USA).Primer sequences are listed in Table 2.

[0097] Table 2. List of primer sequences used for quantitative polymerase chain reaction.

[0098]

[0099] Abbreviations: Bcl-2, B-cell lymphoma 2; Bcl-XL, B-cell lymphoma extra large; CARS, cysteinyl-tRNA synthetase; CBS, cystathionine β synthetase; CCL2, C-C motif chemokine 2; CSE, cystathionine γ lyase; CXCL1, C-X-C motif chemokine ligand 1; ETHE1, ethylmalonic encephalopathy protein 1; IL, interleukin; SQOR, sulfide:quinone oxidoreductase; SUOX, sulfite oxidase; 3-MST, 3-mercaptopyruvate sulfotransferase; TNF-α, tumor necrosis factor α; TST, thiosulfate sulfotransferase.

[0100] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis

[0101] We measured GS using LC-MS / MS analysis 34 CNS and plasma levels of SSG (an isotope of endogenous GSSSG) after intranasal administration 34 S-labeled GSSSG ([ 32 S2, 34 S]GSSSG) 9,35,36 . Also 34 S-labeled sulfane sulfur species ([ 32 S, 34 S]GSSH, 32 S, 34 S]cysteine ​​hydropersulfide (CysSSH), and [ 32S2, 34 S]cysteine ​​trisulfide (CysSSSCys)) and endogenous sulfane sulfur species ([ 32 S2]GSSH, 32 S2]CysSSH, and 32 The ratio of S3]CysSSSCys) was quantitatively evaluated. 34 Thirty minutes after intranasal administration of SSG, blood was obtained and four different central nervous tissues were harvested: olfactory bulb and forebrain; brainstem; cervical and thoracic spinal cord and lumbar spinal cord. 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 at 37°C for 20 minutes in the dark. After centrifugation (14,000×g for tissues and 1,870×g for plasma, 10 minutes, 4°C), the supernatant was separated and diluted with 0.1% formic acid for LC-MS / MS analysis, and the pellet was sonicated to measure protein concentration by BCA assay. [ 32 S2, 34 The amount of S]GSSSG was quantified in selected reaction monitoring (SRM) using precursor ion (647.14 m / z), product ion (389.1 m / z), and higher energy collision dissociation (21 V). 32 S, 34 S]GSSH and 32 S2]GSSH ratio, 32 S, 34 CysSSH and 32 S2] CysSSH ratio, and [ 32 S2, 34 S]CysSSSCys and [ 32S3] The ratio of CysSSSCys was calculated from their peak areas measured by a Dionex UltiMate 3000RS UPLC-Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the samples were subjected to a UPLC system with a Hypersil Gold C-18 (100×2.1 mm, 3.0 μm, Thermo Fisher Scientific, Waltham, MA, USA) column and then eluted by a linear methanol gradient (0-90%, 15 min) of the mobile phase in the presence of 0.1% formic acid at a flow rate of 0.2 ml / min at 40°C. The raw data were analyzed by Compound Discoverer 3.3 software (Thermo Fisher Scientific, Waltham, MA, USA). The molecular weights of these sulfane sulfur species bound to HPE-IAM were based on previous reports. 9,11,37 Sure.

[0102] Measurement of relative sulfane sulfur levels in the lumbar spinal cord following spinal cord ischemia

[0103] Lumbar spinal cords were harvested 48 hours after surgery and snap-frozen in liquid nitrogen. The spinal cords were then homogenized in Hanks balanced salt solution (HBSS; Thermo Fisher Scientific, Waltham, MA, USA) containing SSip-1 (5 μM), incubated at room temperature in the dark for 20 minutes, and then subjected to centrifugation. SSip-1 was synthesized and provided by Hanaoka laboratory 38 The fluorescence intensity of the supernatant was measured using a microplate reader (SpectraMax M5; Molecular Devices, San Jose, CA, USA) at a wavelength of λex / λem=491 nm / 525 nm. The fluorescence intensity was normalized to the weight of the spinal cord.

[0104] Measurement of relative sulfane sulfur levels in the olfactory bulb and forebrain and whole spinal cord following intranasal administration of PTN-SSS

[0105] At 30 minutes after intranasal administration of 50 mg / kg of PTN-SSS, the olfactory bulb and forebrain as well as the whole spinal cord were harvested. The tissues were homogenized in HBSS containing 10 μM SSip-1. The fluorescence intensity of the supernatant was measured.

[0106] In vitro studies

[0107] Rat primary cortical neuron culture

[0108] Primary cortical neurons were isolated from the cerebral cortex of C57BL / 6J mice of both sexes at embryonic day 15 as described previously. 39 Primary cortical neurons were maintained at 37°C in a humidified tissue culture chamber with 5% CO2, and cells were used on day 11 after harvesting.

[0109] Measurement of relative sulfane sulfur levels in primary cortical neurons or SH-SY5Y cells after incubation with polysulfides

[0110] Primary cortical neurons or SH-SY5Y cells (human neuroblastoma cell line) (American Type Culture Collection, Manassas, VA, USA) were incubated with SSip-1 DA (5 μM) at 37°C in the dark. 38 Incubate for 20 minutes. The cells were washed with preheated 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.

[0111] Effects of polysulfides on cell viability after oxygen and glucose deprivation / reoxygenation (OGD / R)

[0112] After primary cortical neurons or SH-SY5Y cells were subjected to 2.5 or 15 h of OGD in an incubation chamber (MIC-101; Billups-Rothenberg, Inc., San Diego, CA, USA), cells were incubated with GSSSG, GSH, GSSG, PTN-SSS, or vehicle alone and then subjected to 21 (primary cortical neurons) h or 24 (SH-SY5Y cells) h of reoxygenation as previously described. 39 After reoxygenation, cell viability and cell damage were assessed using crystal violet and lactate dehydrogenase (LDH) assays as previously described. 39 .

[0113] Cytotoxic effect of GSSSG on cell viability

[0114] Primary cortical neurons were incubated with 0 μM (vehicle alone), 10 μM, 30 μM, 60 μM, or 100 μM GSSSG for 21 hours at 37° C. in a humidified incubator with 95% air and 5% CO 2 . Cell viability was assessed using the LDH assay.

[0115] Cytotoxic effect of PTN-SSS on cell viability

[0116] 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 h at 37° C. in a humidified incubator with 95% air and 5% CO 2 . Cell viability was assessed using the crystal violet assay.

[0117] Statistical analysis

[0118] Data are expressed as mean and standard deviation (parametric data) or median and interquartile range (nonparametric data).Descriptive statistics are used to describe the study population.We use Shapiro-Wilk test (Shapiro-Wilktest) and QQ figure to evaluate the normality of data.Use unpaired t test to analyze parametric data to compare two groups, and use one-way analysis of variance (ANOVA) with Tukey multiple comparison test or Dunnett multiple comparison test to analyze parametric data to compare three or more groups.Use Mann-Whitney test (Mann-Whitney test) to analyze nonparametric data to compare two groups, and use Kruskal-Wallis test with Dunn multiple comparison test to analyze nonparametric data to compare three or more groups.Significance is considered to be at the level of P<0.05.Statistical analysis is carried out using GraphPad Prism9.2.0 (GraphPad Software Inc., San Diego, CA, USA).

[0119] Sample size calculation for the experiment comparing BMS scores 72 hours after surgery between the GSSSG group, the GSH group, and the vehicle alone group was performed using a fixed-effects one-way ANOVA with an F-test (G*Power 3.1; Heinrich-Heine- Düsseldorf, Germany) 40. Based on our pilot study for this experiment, we speculated that 11 male mice and 8 female mice would be needed per group (α=0.05, β=0.1 [power=0.9], effect size f=0.7022756 for male mice and f=0.8709832 for female mice, number of groups=3). Sample size calculations for experiments comparing BMS scores 72 hours after surgery between the PTN-SSS group, the PTN group, and the vehicle alone group were performed using a fixed-effects one-way ANOVA F-test. Based on our pilot study for this experiment, we speculated that 9 mice would be needed per group (α=0.05, β=0.1 [power=0.9], effect size f=0.7747206, number of groups=3).

[0120] Intermittent H2S Inhalation - Sulfide Pretreatment (SPC)

[0121] We previously reported that breathing H2S at 80 ppm significantly increased plasma and brain levels of H2S and thiosulfate (an oxidation product of H2S). 50,51 Male C57BL6 / J mice breathed air alone and air mixed with 80 ppm H2S (Airgas Inc., Radnor, PA) for 4 h per day for 5 consecutive days in custom-made plastic chambers. 51 The H2S breathing session was performed from 8 am to 12 pm from day 1 to day 5 (Figure 1). H2S concentration and FiO2 were continuously measured using a portable gas monitor (VENTIS MX4 Multi-Gas Monitor, Industrial Scientific Corporation, Oakdale, PA).

[0122] MPTP-induced Parkinson's disease model in mice

[0123] Male C57BL6 / J and CD-1 mice received four intraperitoneal injections of MPTP (20 mg / kg) or saline every 2 h for a total of four doses of MPTP (total, 80 mg / kg) or an equal amount of saline as previously described. 3 To evaluate the effects of MPTP on dopaminergic neurons, all mice were harvested 7 days after MPTP administration. Then, tyrosine hydroxylase protein levels were detected by immunoblotting.

[0124] Cell viability of neuronal cells

[0125] Perform cell viability assay to add MPP +Cell viability of SH-SY5Y cells or mouse primary cortical neurons was examined 24 h after addition of 5 mM (5 mM for SH-SY5Y and 50 μM for primary cortical neurons) with 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). Cell viability was measured using a crystal violet assay.

[0126] Administration of Na2S3 to MPTP-treated mice

[0127] In order to determine the impact of polysulfide on the loss of tyrosine hydroxylase in the substantia nigra and striatum in the mice treated with MPTP, we used Na2S3 (20mg / kg) or saline intraperitoneally every 2h, starting immediately after the first injection of MPTP on the 0th day. Subsequently, mice received two intraperitoneal injections of Na2S3 (20mg / kg) or saline every 12h from the 1st day to the 6th day. All mice were harvested in the 7 days after MPTP was used. Then, tyrosine hydroxylase protein level was detected by immunoblotting.

[0128] GSSSG administration to MPTP-treated mice

[0129] In order to determine the impact of polysulfide on the loss of tyrosine hydroxylase in the substantia nigra and striatum in the mice treated with MPTP, we immediately applied GSSSG (50mg / kg) or saline via intranasal route after the first and third injections of MPTP on the 0th day. Subsequently, mice received two intranasal injections of GSSSG (50mg / kg) or saline every 12h from the 1st day to the 6th day. All mice were harvested in the 7 days after MPTP was used. Then, tyrosine hydroxylase protein level was detected by immunoblotting.

[0130] Example 1. Intranasal administration of GSSSG rescues mice from delayed paraplegia following transient spinal cord ischemia

[0131] To explore whether polysulfides can prevent delayed paraplegia, mice were subjected to SCI and treated with GSSSG, GSH, or vehicle alone at 0, 8, 23, and 32 hours after surgery. 36 male mice were subjected to SCI; however, three of these mice were excluded from further studies because they did not achieve a greater than 90% reduction in femoral artery blood flow. The remaining 33 male mice received one of the following three treatments: GSSSG (n=11), GSH (n=11), or vehicle alone (n=11). Hindlimb motor function was quantified using the Basso Mouse Motor Scale (BMS), which evaluates hindlimb movement, forelimb-hindlimb coordination, and trunk stability. As previously described by Kakinohana and colleagues, mice were considered to have delayed paraplegia if neurological deficits (BMS<6) occurred after a period of time in which the mouse was able to walk (BMS≥6). 6 .

[0132] Starting approximately 36 hours after surgery, hindlimb motor function of mice treated with vehicle alone gradually deteriorated, and all mice treated with vehicle alone developed paraplegia by 48 hours after surgery ( Figure 3 In contrast, intranasal administration of GSSSG prevented the development of delayed paraplegia in 8 of 11 mice (73%) and improved BMS scores 72 hours 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 delayed paraplegia in 3 of 11 mice (27%), but did not improve the BMS scores 72 hours after surgery (GSH vs. vehicle alone, BMS; 1 [0-7] vs. 0 [0-0]; P = 0.0914, Figure 3 ). The results showed that GSSSG, but not GSH, prevented the development of delayed paraplegia after SCI in male mice.

[0133] To explore the effect of gender 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. 4 of the 28 female mice experienced dyspnea 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). Compared with mice treated with vehicle alone, mice receiving intranasal GSSSG had improved BMS scores 72 hours after surgery (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 BMS scores 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 show that GSSSG, but not GSH, can prevent delayed paraplegia after SCI in both sexes.

[0134] GSSG can be glutathionylated Reacts with thiols in proteins 23 GSSSG could potentially also react with thiols in proteins via the same process. 41 . To explore whether the beneficial effects of GSSSG are related to glutathionylation, we compared the effects of intranasal administration of GSSSG or GSSG on outcomes after SCI in male mice. Intranasal administration of GSSSG rescued 5 of 6 male mice (83%) from delayed paraplegia; conversely, intranasal administration of GSSG did not rescue any male mice (0%) from delayed paraplegia. The BMS scores 72 hours after surgery in the GSSSG group were significantly higher than those 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, Fig.11 ). Because GSSSG could potentially mediate glutathionylation of target proteins through the same process as GSSG, but only GSSSG prevented delayed paraplegia, the results suggest that the neuroprotective effect of GSSSG is not a result of glutathionylation.

[0135] Example 2. Intranasal administration of GSSSG reduces neurodegeneration and microglial activation and caspase-3 activation following spinal cord ischemia in the lumbar spinal cord

[0136] Spinal cord ischemia is associated with degenerative changes in neurons in the ventral horn of the lumbar spinal cord, and the viability of affected neurons can be assessed using Nissl staining 6 Nissl staining detects Nissl bodies in the cytoplasm of neurons, and the presence of this purple cytoplasmic stain is an indicator of neuronal integrity. 6,42 To explore the effects of GSSSG on neurodegeneration, GSSSG or vehicle alone was administered at 0, 8, 23, and 32 hours after surgery. Lumbar spinal cords were harvested 48 hours after surgery, fixed, sectioned, and incubated with Nissl stain. Spinal cord ischemia was associated with a significant reduction in Nissl-positive neurons compared to mice subjected to sham surgery, indicating that most cells were not viable ( Figure 4A -B). Intranasal administration of GSSSG was associated with a significant increase in the number of viable neurons compared to mice subjected to SCI and treated with vehicle alone ( Figure 4A -B).

[0137] Previous studies have shown that increased microglial activation in the ventral horn of the lumbar spinal cord parallels the onset of delayed paraplegia after SCI. 6,29 , and inhibition of microglial activation attenuates neuronal damage and prevents the development of delayed paraplegia after SCI 43 Increased expression of ionized calcium binding adaptor molecule 1 (Iba-1) is a sensitive marker of microglial activation 44 To explore the mechanism by which GSSSG prevents neurodegeneration, mice were subjected to SCI and treated with GSSSG or vehicle alone. Sections were prepared from lumbar spinal cords and stained for Iba-1. Staining of spinal cords from mice subjected to SCI had a significantly increased relative area of ​​Iba-1 positive staining ( 1.44 × 10 cm) compared to staining of spinal cords obtained from mice 48 hours after sham surgery. Figure 5A , 5B ). The results showed that SCI was associated with increased microglial activation. Conversely, mice subjected to SCI and treated with intranasal GSSSG had a significant decrease in Iba-1 staining, indicating reduced microglial activation ( Figure 5A , 5B ).

[0138] Previous researchers have shown that activation of caspase-3 is a core component of neurodegeneration in the ventral horn of the lumbar spinal cord after SCI. 6Immunohistochemical staining for cleaved caspase-3 was used to explore whether attenuation of caspase-3 activation might be a mechanism by which GSSSG protects against neurodegeneration after SCI. Lumbar spinal cords from mice subjected to SCI had an increased number of cleaved caspase-3-positive neurons in the ventral horn compared with spinal cords from mice 48 hours after sham surgery ( Figure 5C , 5D Intranasal administration of GSSSG prevents SCI-induced increase in the number of cleaved caspase-3-positive neurons in the lumbar spinal cord ( Figure 5C , 5D ). These results indicate that intranasal administration of GSSSG prevents neurodegeneration in the ventral horn of the lumbar spinal cord and is associated with reduced microglial activation and attenuated caspase-3 activation.

[0139] Example 3. Intranasal administration of GSSSG attenuates upregulation of proinflammatory cytokines following spinal cord ischemia

[0140] Previous studies have shown that a significant increase in proinflammatory cytokines produced by activated microglia precedes the onset of delayed paraplegia after SCI 15,29 Inhibition of microglial activation blocks the upregulation of proinflammatory cytokines and prevents the development of delayed paraplegia after SCI 43 . To further evaluate the effect of GSSSG on proinflammatory cytokines induced by SCI, we measured the levels of mRNA encoding CCL2, CXCL1, IL-1β, IL-6, and TNF-α in the lumbar spinal cords of mice subjected to SCI and treated with GSSSG or vehicle alone. The lumbar spinal cords were harvested 48 hours after surgery. Compared with mice subjected to sham surgery, mice subjected to SCI had a significant increase in the levels of mRNA encoding cytokines associated with inflammation. In contrast, GSSSG attenuated the upregulation of mRNA encoding proinflammatory cytokines 48 hours after surgery ( Figure 6 Intranasal administration of GSSSG also reduced the levels of mRNA encoding SQOR, an enzyme that oxidizes sulfide to persulfide. GSSSG had no effect on the levels of mRNA encoding other enzymes that synthesize or metabolize sulfide or persulfide ( Fig.12 There were no significant differences in the mRNA levels of anti-apoptotic genes, including Bcl-2 and Bcl-XL, between the vehicle alone and GSSSG treatment groups ( Figure 6 ). These results suggest that the beneficial effects of GSSSG may be mediated through the suppression of proinflammatory cytokines.

[0141] Example 4. 34 S-labeled GSSSG reaches the spinal cord shortly after intranasal administration and is metabolized to other sulfane sulfur species.

[0142] To investigate the pharmacokinetics of intranasally administered GSSSG, we used 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. 34 Thirty minutes after intranasal administration of SSG, LC-MS / MS was used to measure [ 32 S2, 34 S]GSSSG and was used to determine the amount of 34 The ratio of S-labeled sulfane sulfur species to endogenous sulfane sulfur species. 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 cord (414 ± 98 pmol / mg protein), and lumbar spinal cord (317 ± 111 pmol / mg protein). Fig. 7A ).also,[ 32 S2, 34 S]GSSSG was detected in plasma at a concentration of 25 ± 10 nM. 32 S, 34 S]GSSH and 32 S2]GSSH ratio, 32 S, 34 CysSSH and 32 S2] CysSSH ratio, and [ 32 S2, 34 S]CysSSSCys and [ 32 The ratios of CysSSSCys were 6.5±2.8, 10.6[9.8-12.5], and 31.8±9.8( Figure 7B ). These results show that GSSSG reaches different parts of the central nervous system including the spinal cord within 30 minutes after intranasal administration and is converted into other sulfane sulfur species.

[0143] Example 5. Intranasal administration of GSSSG increases sulfane sulfur levels in the lumbar spinal cord following spinal cord ischemia

[0144] GSSSG protects the lumbar spinal cord from neurodegeneration 48 hours after SCI. To explore whether increased levels of sulfane sulfur in the lumbar spinal cord contribute to neuroprotection, we measured changes in sulfane sulfur levels 48 hours after SCI. SCI followed by treatment with vehicle alone did not alter sulfane sulfur levels in the lumbar spinal cord compared to sham-operated mice (sham vs. SCI followed by vehicle alone; 1.00 ± 0.30 vs. 0.56 ± 0.13; P = 0.0870 by one-way ANOVA with Dunnett's multiple comparison test, Fig. 8A In contrast, levels of sulfane sulfur in the lumbar spinal cord of mice that underwent SCI and received intranasal administration of GSSSG were greater than those in mice that underwent SCI and received vehicle alone (SCI followed by GSSSG vs. vehicle alone; 1.37 ± 0.50 vs. 0.56 ± 0.13; P = 0.0023, Fig. 8A ). These results suggest that the beneficial effects of GSSSG in preventing delayed onset paraplegia are associated with increased sulfane sulfur levels in the lumbar spinal cord.

[0145] Example 7. Effects of GSSSG on primary cortical neurons

[0146] To determine whether polysulfides increase sulfane sulfur levels within neurons, we measured relative sulfane sulfur levels in primary cortical neurons in the presence and absence of polysulfides using SSip-1 DA, a fluorescent probe that can be used to measure the concentration of sulfane sulfur inside cells. 38 Relative to untreated primary cortical neurons, neurons incubated with GSSSG or Na2S3 had increased levels of intracellular sulfane sulfur ( Figure 8B ).

[0147] In vivo studies have shown that GSSSG can prevent neurodegeneration after SCI. To determine whether GSSSG can protect neurons from similar injury in vitro, primary cells were incubated with GSSSG after oxygen and glucose deprivation / reoxygenation (OGD / R). Cell viability was assessed using crystal violet and lactate dehydrogenase (LDH) assays as previously described. 39 Crystal violet dye stains DNA and proteins in cells and confirms that cells are viable and able to maintain attachment to tissue culture plates despite OGD / R. The LDH assay measures the level of lactate dehydrogenase in tissue culture medium and is an indirect measure of plasma membrane damage. Treatment with OGD / R was associated with a significant decrease in the number of viable adherent cells compared to control cells ( Fig. 9A ) and was associated with a significant increase in the concentration of LDH in the tissue culture medium ( Fig. 9B). GSSSG at 30 μM increased cell viability as measured by crystal violet assay compared to vehicle-only treated cells ( Fig. 9A ), and 30 μM or 60 μM GSSSG reduced the release of LDH after OGD / R ( Fig. 9B Higher doses of GSSSG (100 μM) alone increased LDH levels in tissue culture medium ( Fig.13 ), and failed to improve cell survival after OGD / R. These results show that GSSSG dose-dependently protects neurons from the effects of OGD / R.

[0148] In vivo studies have shown that GSSSG, but not GSH or GSSG, prevents the development of delayed paraplegia after SCI and suggest that the neuroprotective effects of GSSSG are derived from sulfane sulfide. To determine whether the neuroprotective effects of GSSSG on primary cultured neurons are derived from sulfane sulfide, we tested the ability of GSSSG, GSH, and GSSG to protect neurons from OGD / R. GSSSG at 30 μM improved cell viability after OGD / R compared to cells treated with vehicle alone ( Fig. 9C In contrast, 60 μM GSH and 30 μM GSSG did not improve cell viability after OGD / R ( Fig. 9C ). These results suggest that the neuroprotective effect of GSSSG originates from sulfane sulfur.

[0149] Example 8. Effect of PTN-SSS on SH-SY5Y cells

[0150] We used in vitro studies to show that GSSSG protects neurons after OGD / R and indicated that the neuroprotective effect of GSSSG is derived from sulfane sulfur. To explore whether other polysulfides are cell protective after OGD / R, we examined the effect of another polysulfide, PTN-SSS, on the viability of SH-SY5Y cells after OGD / R. As with primary cortical neurons, treatment with OGD / R reduced the viability of SH-SY5Y cells. Incubation with 10 μM, 25 μM, or 50 μM PTN-SSS significantly improved SH-SY5Y viability after OGD / R in a dose-dependent manner compared to cells treated with vehicle alone ( Fig. 10A In contrast, PTN-SSS did not improve cell viability at 100 μM due to the direct cytotoxic effect of PTN-SSS at high doses ( Fig.14 ). These results show that PTN-SSS dose-dependently rescues SH-SY5Y cells after OGD / R and support that polysulfides are cytoprotective after OGD / R.

[0151] To determine whether the cytoprotective effect of PTN-SSS is related to the amount of intracellular sulfane sulfur, we used SSip-1 DA to measure the level of sulfane sulfur in SH-SY5Y cells after incubation with PTN-SSS. The level of sulfane sulfur in SH-SY5Y cells increased with increasing concentrations of PTN-SSS ( Fig. 10B The ability of SSip-1 DA (5 μM) to measure intracellular sulfane sulfur reached a maximum at a concentration of 100 μM PTN-SSS ( Fig. 10B ). The sulfane sulfur levels in SH-SY5Y cells were significantly increased at doses of PTN-SSS (10-50 μM) compared to cells treated with vehicle alone, which significantly improved cell viability after OGD / R. These results suggest that the cytoprotective effect of PTN-SSS is related to the amount of sulfane sulfur released from PTN-SSS.

[0152] Example 10. PTN-SSS increases sulfane sulfur levels in the olfactory bulb and forebrain, and throughout the spinal cord shortly after intranasal administration

[0153] To investigate whether PTN-SSS increases sulfane sulfur levels in the central nervous system shortly after intranasal administration, we measured sulfane sulfur levels in the olfactory bulb and forebrain and whole spinal cord 30 min after intranasal administration of PTN-SSS using SSip-1. Intranasal administration of PTN-SSS increased sulfane sulfur levels in the olfactory bulb and forebrain and whole spinal cord compared to mice receiving intranasal administration of vehicle alone ( Fig. 10C ). These results indicate that PTN-SSS increases sulfane sulfur levels in the central nervous system within 30 minutes after intranasal administration.

[0154] Example 11. Intranasal administration of PTN-SSS rescues mice from delayed paraplegia following transient spinal cord ischemia

[0155] In vitro studies have shown that PTN-SSS is cytoprotective after OGD / R, and the cytoprotective effect of PTN-SSS is related to the amount of sulfane sulfur released from PTN-SSS. Since GSSSG prevents the occurrence of delayed paraplegia after SCI, and the neuroprotective effect of GSSSG is related to sulfane sulfur, we assume that PTN-SSS can also rescue mice from delayed paraplegia after SCI. In addition, as with GSSSG, we tested whether the sulfane sulfur in PTN-SSS (rather than PTN alone) is neuroprotective. Male mice received intranasal administration of PTN-SSS, PTN, or a single vehicle at 0, 8, 23, and 32 hours 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 of 9 male mice (66%) from delayed paraplegia. In contrast, intranasal administration of PTN did not rescue any male mice from delayed paraplegia. The BMS scores at 72 hours after surgery in the PTN-SSS group were significantly higher than those in the PTN group or the vehicle alone group (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, Fig. 10D ). These results show that PTN-SSS, but not PTN, prevents the development of delayed paraplegia after SCI in mice, suggesting that the neuroprotective effect of PTN-SSS originates from sulfane sulfur, rather than PTN.

[0156] Example 12. Inhalation of H2S provides neuroprotection in PD animal models

[0157] Sulfide levels are strictly controlled in the brain. We recently reported a robust protective effect of sulfide catabolism in hypoxic / ischemic brain injury. See Marutani et al., Nature Communications 12, Article number: 3108; 25 May 2021. Because other forms of brain damage can be caused by altered sulfide homeostasis, we examined the impact of sulfide catabolism 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., Antioxidants and Redox Signaling 15(2): 343-52 (2011).

[0158] The administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes the loss of dopaminergic neurons and dyskinesia measured by the level of tyrosine hydroxylase (TH) in the substantia nigra in mice. Before the administration of MPTP, breathing air mixed with 80ppm H2S for 4h every day for 5 consecutive days (sulfide pretreatment) protects mice from the loss of dopaminergic neurons and dyskinesia induced by MPTP. The protective effect of sulfide pretreatment is related to the obvious increase of sulfide:quinone oxidoreductase (SQOR) level in the substantia nigra and striatum. SQOR expression in the substantia nigra striatum region mediated by AAV prevents the loss of dopaminergic neurons induced by MPTP in mice.

[0159] This study revealed a neuroprotective effect of sulfide catabolism via SQOR in an MPTP-induced PD mouse model. The protective effect of upregulation of SQOR could be mediated by increased persulfide levels.

[0160] Example 13. Inhalation of H2S provides neuroprotection in PD animal models

[0161] Because SQOR catalyzes the conversion of sulfide to polysulfide, and because SPC- or AAV-mediated overexpression of SQOR prevents the reduction of sulfane sulfur after MPTP administration, we hypothesized that the beneficial effects of upregulated SQOR after MPTP administration are mediated by increased levels of polysulfide. To consider this possibility, we examined the effects of polysulfide on the expression of MPP. + Effect of MPP on the viability of SH-SY5Y cells incubated with + (5 mM) for 24 h, and viability was assessed by crystal violet assay 41 Compared with control cells, treatment with Na2S (at doses between 1 and 100 μM) did not improve the + Viability of treated SH-SY5Y cells 42 In contrast, treatment with Na2S3 or GSSSG (10 μM each) increased MPP + Viability of treated SH-SY5Y cells ( FIG. 15A ).

[0162] To further examine the cytoprotective effects of polysulfides in a physiologically relevant context, we examined the effects of GSSSG, glutathione (GSH, the parent molecule of GSSSG without the 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 without the sulfane sulfur) on the cell proliferation and survival of patients with MPP. + Effects of incubation of primary rat cortical neurons. Cells were treated with MPP + (50 μM) and incubated with the above compounds for 24 h. The polysulfide compounds GSSSG and LA-SSS (but not the parent compounds GSH and LA) protected primary mouse cortical neurons from MPP + These results suggest that sulfane sulfur-releasing molecules protect neurons from MPP. + Induced cytotoxicity.

[0163] Example 14. Nasal administration of GSSG provides neuroprotection in PD animal models

[0164] The large molecular weight of GSSSG (Mw: 644.7 DA) prevents it from crossing the blood-brain barrier after systemic administration. However, recent studies have shown that intranasal administration can allow relatively large molecules to successfully pass into the central nervous system. 43To consider the possibility that GSSSG could have a protective effect against MPTP-induced neurodegeneration when administered intranasally, GSSSG (50 mg / kg) or saline was administered IN immediately after administration of MPTP or saline (control) on day 0. Between days 1 and 6, mice received 50 mg / kg of GSSSG or saline IN every 12 hours. We observed that IN administration of GSSSG prevented the MPTP-induced decrease in tyrosine hydroxylase in the nigrostriatal region ( Fig.16 ). Taken together, these results suggest that polysulfides have robust therapeutic effects in mouse models of PD.

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[0217] Other embodiments

[0218] It should be understood that although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A method for treating or reducing the risk of a disorder associated with neurodegeneration in a subject, the method comprising nasally administering to a subject in need thereof a therapeutically or prophylactically effective amount of a composition comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS).

2. The method according to claim 1, further wherein the composition comprising GSSSG is prepared by dissolving GSSSG in a crystalline form in buffered saline at pH 3-6.

3. The method of claim 1, wherein the condition is post-ischemic neuronal death. The method according to claim 1 , wherein the disorder is a chronic brain degenerative disease.

5. The method of claim 1, wherein the chronic brain degenerative disease is multi-infarct dementia, Alzheimer's disease, Parkinson's disease, or Lewy body dementia.

6. The method of claim 1, comprising administering an effective amount of the composition comprising GSSSG, PTN-SSS, or LA-SSS within a few minutes to a few hours after the traumatic injury occurs.

7. The method of claim 1, comprising administering an effective amount of the composition comprising GSSSG, PTN-SSS, or LA-SSS prior to a scheduled thoracic and / or abdominal aortic surgery.

8. The method of claim 7, comprising administering an effective amount of the composition comprising GSSSG, PTN-SSS, or LA-SSS several hours to several days prior to a scheduled thoracic and / or abdominal aortic surgery.

9. The method of claim 8, comprising administering an effective amount of the composition comprising GSSSG, PTN-SSS, or LA-SSS 2-24 hours and / or 1, 2, 3, 4, 5, 6, and / or 7 days prior to the scheduled thoracic and / or abdominal aortic surgery.

10. A composition for nasal administration comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS), for use in treating a disorder associated with neurodegeneration or reducing the risk of a disorder associated with neurodegeneration in a subject, the method comprising nasally administering a therapeutically or prophylactically effective amount to a subject in need thereof.

11. The composition for use according to claim 10, wherein the composition is prepared by a process comprising dissolving a crystalline form of GSSSG in a buffered saline at pH 3-6.

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

13. The composition for use according to claim 10, wherein the disease is a chronic brain degenerative disease.

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

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

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

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

18. The composition for use according to claim 17, wherein the method comprises administering an effective amount of a composition comprising GSSSG, PTN-SSS, or LA-SSS 2-24 hours and / or 1, 2, 3, 4, 5, 6, and / or 7 days prior to the scheduled thoracic and / or abdominal aortic surgery.

19. A device for nasal administration of a composition comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS).

20. The device according to claim 19, which is a nebulizer or a pressurized container or an aerosol dispenser further comprising a propellant.

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