Intranasal dantrolene nanoparticles, alone or in combination with lithium, for treating depression and amyotrophic lateral sclerosis (ALS)

Abstract

The present invention relates to methods for treating, preventing or delaying onset, and alleviating the symptoms of amyotrophic lateral sclerosis (ALS) through nasally or intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. The present invention further relates to methods for treating depression comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. The present invention additionally relates to methods for treating depression in an Alzheimer's Disease subject comprising administering a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

Description

P-654697-PCINTRANASAL DANTROLENE NANOPARTICLES, ALONE OR IN COMBINATION WITH LITHIUM, FOR TREATING DEPRESSION AND AMYOTROPHIC LATERAL SCLEROSIS (ALS)STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under AG061447 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0002] The present invention relates to methods for treating, preventing or delaying onset, and alleviating the symptoms of amyotrophic lateral sclerosis (ALS) through nasally or intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. The present invention further relates to methods for treating depression comprising administering to a subject in need thereof a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. The present invention additionally relates to methods for treating depression in an Alzheimer’s Disease subject comprising administering a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. The depression also may be in an Alzheimer’s Disease and ALS subject. The present invention also relates to inhibiting molecular and cellular mechanisms underlying amyotrophic lateral sclerosis (ALS) pathologies through nasally or intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. The present invention furthermore relates to methods for inhibiting a pathological intracellular Ca2+and / or Fe2+dysregulation, oxidative stress and inflammatory pyroptosis activation, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting astrogliosis and microgliosis in a subject having depression, comprising administering a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. The invention also relates to methods for increasing cytoprotective IL-10 and inhibiting a PSD-95 synapse protein loss in a subject having depression, comprising administering a pharmaceutical composition comprising dantrolene or a combinationP-654697-PCof dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. The aforementioned methods increasing cytoprotective IL-10 and inhibiting a PSD-95 synapse protein loss in a subject having depression may be utilized for an Alzheimer’s Disease subject having depression.BACKGROUND OF THE INVENTION

[0003] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the degeneration of selective motor neurons, leading to progressive muscle weakness and eventual death due to respiratory failure. ALS has a prevalence of 6-9 per 100,000 persons and a lifetime risk of approximately 1 in 350. ALS has a poor prognosis, with an average survival of 3-5 years following diagnosis. ALS is categorized into the familial (fALS, generic) and sporadic (sALS) types, with the former accounting for about 10% of cases and the latter the remaining 90% with unknown etiology. Common ALS-related genes include superoxide dismutase 1 (SOD1), chromosome 9 open reading frame 72 (C9orf72), fused in sarcoma (FUS), and TAR DNA-binding protein 43 (TDP-43 / TARDBP), which are involved in a variety of cellular functions, including RNA metabolism, protein folding, autophagy, and inflammation. Pathologically, key features of ALS include the loss of upper and lower motor neuron cell bodies, as well as the degeneration of the corticospinal tracts and lower motor neuron axons, with denervation changes occurring within muscles. The proposed pathophysiology for ALS includes glutamate excitotoxicity, mitochondrial dysfunction, endoplasmic reticulum (ER) stress and proteinopathy, oxidative stresses, neuroinflammation, dysregulated nucleocytoplasmic or vesicle transport, impaired DNA damage repair, aberrant RNA metabolism, axonopathy, etc. Although there is no cure for ALS yet, there are six FDA-approved drugs to manage symptoms and improve the quality and length of life.

[0004] Physiological activation of RyRs located on the membrane of the endoplasmic reticulum (ER) maintains cytosolic Ca2+levels required for normal cell functions. However, overactivation of RyRs and excessive Ca2+release from intracellular Ca2+stores is detrimental, having been implicated in many human neurodegenerative diseases, including ALS. Glutamate excitotoxicity in ALS, primarily via overactivation of N-methyl-D-Aspartic acid receptors (NMDARs) and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), results in excessive Ca2+influx and abnormally increased Ca2+concentration in the cytosol ([Ca2+]c). This in turn over-activates RyRs via the Ca2+induced Ca2+release (CICR) mechanism. In fact, up to 78% of NMDAR activation-mediated elevation of [Ca2+]c originates from Ca2+release from the ER via RyRs. This Ca2+then transfers into mitochondria, leading to ER Ca2+depletion and stress andP-654697-PCmitochondria Ca2+overloading and dysfunction. This Ca2+dysregulation is linked to various neurodegenerative diseases, including ALS. Peripheral inflammation resulting from LPS generated by gut microbiota dysbiosis can enter the CNS and activate toll-like receptors (TLR) in neurons. This increases endogenous agonist of RyRs, cADPR, leading to overactivation of RyRs, ER stress and mitochondrial dysfunction.

[0005] Three hundred million people worldwide suffer from major depressive disorder (MDD). MDD is a chronic and recurrent disease affecting about 20% of the population and is the leading cause of suicide. The prevalence of MDD in patients of Alzheimer’s disease is particularly high, impacting up to 40%. MDD has been considered a prodrome of dementia, and depression and dementia are two pathologies making a vicious cycle leading to symptoms in AD. There are several classes of drugs to treat MDD, but with limited effectiveness and considerable side effects. No disease-modifying drugs are available to treat both depression and dementia in AD.

[0006] Although the molecular mechanisms for MDD pathology are unclear, increasing evidence suggests that disruption of intracellular Ca2+homeostasis and associated downstream multiple pathologies, including pathological inflammation, mitochondrial dysfunction, oxidative stress, pyroptosis and synapse dysfunction play a critical role in MDD pathologies. The abnormally increased glutamate and associated excitotoxicity via overactivation of N-methyl-D-aspartate (NMDA) receptors (NMDAR) result in downstream elevation of cytosolic and mitochondria Ca2+concentrations. Extracellular Ca2+influx via the NMDAR can be potentiated by the ApoE4 gene, a primary risk factor for sporadic AD (SAD, 95% AD patients). On the other hand, the overactivation of ryanodine (RyR) and InsP3 (InsP3R) receptors and associated excessive Ca2+release from endoplasmic reticulum (ER) results in depletion of ER and pathological elevation of cytosol and mitochondrial Ca2+concentrations, detrimental to cell survival. The type 2 RyR (RyR-2) is pathologically increased in brains of AD patients. The number and activity of RyR pathologically increase in different cell and animal models of AD. Similarly, the number and activity of InsP3R also is pathologically increased in AD. The numbers and activity of InsP3R also pathologically increase in AD. The related upstream Ca2+dysregulation results in downstream mitochondrial dysfunction, oxidative stresses, initiation of the pyroptosis pathway and pathological inflammation, eventually leading to synapse and cell / neuron damage, and depression and cognitive dysfunction.P-654697-PC

[0007] Numerous studies have reported that oxidative stress and neuroinflammation play an important role in the pathogenesis of MDD. Animal studies have revealed that depressed rats have increased levels of oxidative markers (MDA and SOD activity) in the brain. Increasing clinical studies have demonstrated significantly upregulated levels of IL-10, IL-6 and TNF-a in the serum of MDD patients. It has been reported that microglial NLRP3 inflammasome activation is crucial to generate IL-1β and associated pathological inflammation leading to depression. Similarly, the excessive release of the ER Ca2+causes mitochondrial Ca2+overloading, which result in excessive production of reactive oxygen species (ROS), NLRP3 inflammasome activation and initiation of pyroptosis pathway. Pyroptosis is a form of programmed Gasdermins (GSDMs)-mediated inflammatory cell death which demonstrates both necrosis and apoptosis-like morphology. Studies have reported that the NLRP3 inflammasome initiated pyroptosis pathway plays an important role in synapse and neuronal dysfunction or destruction in both depression and cognitive dysfunction, especially in AD. The NLRP3 inflammasome-forming complex activates associated proteins that regulate pyroptosis (caspase-1 and GSDMD pore formation (cleaved GSDMD). This results in releases of inflammatory cytokines (IL- 10 and IL- 18) and pathological inflammation mediated cell death by pyroptosis. Considering the critical roles of synapse dysfunction and cell or neurons damage in both depression and cognitive dysfunction, inhibition of upper stream Ca2+dysregulation and downstream pathological inflammation and pyroptosis is expected to treat both depression and cognitive dysfunction, especially in AD.

[0008] Accordingly, there remains a need for developing improved drug compositions to correct / impede upstream critical Ca2+dysregulation by inhibition of both NMDAR and RyR, as well as for methods to treat ALS.

[0009] Accordingly, there remains a need for developing improved drug compositions to correct / impede upstream critical Ca2+dysregulation by inhibition of both NMDAR and RyR, as well as for methods to treat depression, including depression in subjects having Alzheimer’s Disease.SUMMARY OF THE INVENTION

[0010] In one aspect, provided herein is a method for treating and / or alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.P-654697-PC

[0011] In another aspect, provided herein is a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier.

[0012] In another aspect, provided herein is a method for treating depression comprising administering to a subject in need thereof a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier.

[0013] In another aspect, provided herein is a method for treating depression in an Alzheimer’s Disease subject comprising administering to the subject in need thereof a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier.

[0014] In another aspect, provided herein is a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL-10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

[0015] In one aspect, provided herein is a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, synapse destruction, and impairment of neurogenesis in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

[0016] In one aspect, provided herein is a method for restoring motor neuron function and / or muscle strength, and / or reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.P-654697-PC

[0017] In one aspect, provided herein is a method for ameliorating muscle weakness, muscle atrophy, and motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

[0018] In another aspect, provided herein is a method for inhibiting a pathological increase of intracellular Ca2+and Fe2+dysregulation, oxidative stress and pyroptosis activation, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting astrogliosis and microgliosis in an Alzheimer’s Disease subject having depression, the method comprising administering to the subject a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

[0019] In a further aspect, provided herein is a method for increasing cytoprotective IL-10 and inhibiting a PSD-95 synapse protein loss in a subject having depression, the method comprising administering to the subject a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

[0020] In another aspect, provided herein is a method for increasing cytoprotective IL- 10 and inhibiting a PSD-95 synapse protein loss to an Alzheimer’s Disease subject having depression, the method comprising administering to the subject a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

[0021] In one aspect, provided herein is a pharmaceutical composition comprising of dantrolene, a lithium salt, and a pharmaceutically acceptable carrier.

[0022] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.P-654697-PCBRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0024] Figure 1 shows the proposed mechanisms of dantrolene in treatment of amyotrophic lateral sclerosis (ALS). Pathological activation of N-methyl-D-aspartate receptors (NMDAR) or a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) by glutamate excitotoxicity leads to excessive Ca2+influx into the cytosol, pathologically increasing cytosolic Ca2+concentration ([Ca2+]c) and leading to Ca2+induced Ca2+release (CICR) from endoplasmic reticula (ER) via ryanodine receptors (RyRs). Gut dysbiosis mediated release of lipopolysaccharides (LPS), an inflammation inducer, increases endogenous agonists of RyRs, the cyclic ADP -ribose (cADPR), via the activation of toll-like receptor 4 (TLR4), causing excessive Ca2+release from the ER into the cytosol. Glutamate and LPS mediated Ca2+dysregulation results in mitochondrial dysfunction and activation of Ca2+-dependent calpain protease and ER stress, which results in downstream mutated TDP-43 or SOD1 aggregation, oxidative stress, inflammatory pyroptosis in motor neurons, and synapse destruction. The above pathologies eventually cause motor neuron destruction in the brain and spinal cord and lead to ALS.

[0025] Figures 2A-2C show dantrolene-mediated inhibition of LPS-induced cell damage in an ALS cell model. QBL293 stable cell lines expressing cytoplasmic TDP-43 (iGFP-NLSm) and its corresponding wild-type TDP-43 control (iGFP-WT) were treated with different concentrations of dantrolene for 24 hours (Fig.2A) and LPS for 4 hours. (Fig.2B) Dose-response cell viability was then determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. (Fig. 2C) Both types of cells were pretreated with dantrolene (0.25pM) for 24 hours, then treated with vehicle (DMSO), followed by LPS at the toxic dose 50% (TD50, 5pg / mL) for 4 hours. Mean±SD from 10 repeated experiments (N=10). Two-way (A, B, C) ANOVA followed by Tukey post-hoc tests. *, ****, P<0.05, P<0.0001 compared to No Tx in iGFP-WT cells; ###, ####, P<0.001, P<0.0001 compared to No Tx in iGFP-NLSm (A). **, ****, P<0.01, P<0.0001 compared to No Tx in iGFP-WT cells; ###, ####, P<0.001, P<0.0001 compared to No Tx in iGFP-NLSm (B). ** P<0.01, **** P<0.0001 (C).P-654697-PC

[0026] Figures 3A-3D show dantrolene-mediated inhibition of LPS-induced mitochondrial and cell damage. QBI-293 stable cell lines expressing cytoplasmic TDP-43 (iGFP-NLSm) and its corresponding wild-type TDP-43 control (iGFP-WT) cells were pretreated with dantrolene (Dan, 0.25pM) for 24 hours, then challenged with LPS (5pg / mL). Typical tracings of mitochondrial respiration OCR measurements in iGFP-WT (Fig. 3A) and iGFP-NLSm (Fig. 3B). Basal OCR (Fig. 3C) and proton leak as an indicator of mitochondria cell damage (Fig. 3D). Mean±SD from 6 repeats of three experiments, N=3. Two-way ANOVA followed by Tukey post-hoc test. *P<0.05, **P<0.01.

[0027] Figure 4 shows dantrolene-mediated inhibition of LPS-induced pathological elevation of reactive oxygen species (ROS) in an ALS cell model. GFP-NLSm and GFP-WT expressing cells were pretreated with dantrolene (DAN, 0.25pM) for 24 hours, then treated with LPS (5pg / mL) for 4 hours. ROS levels were measured with DCFH-DA fluorescence assay. Mean±SD from 10 repeated experiments. Two-way ANOVA followed by Tukey post-hoc test. **** P<0.0001.

[0028] Figures 5A-5B show dantrolene-mediated inhibition of LPS-induced elevation of cytokines IL-18 and IL-ip in an ALS cell model. QBI-293 stable cell lines transfected with the TDP-43 mutation at the nuclear localization signal (iGFP-NLSm) and its corresponding wild-type TDP-43 control (iGFP-WT) cells were pretreated with dantrolene (DAN, 0.25pM) for 24 hours, then treated with LPS (5 g / mL) for 4 hours. Thereafter, the secretion of inflammatory cytokines related to pyroptosis, IL-ip (Fig. 5A), and IL-18 (Fig. 5B), in the cell culture medium were analyzed by ELISA kit. Means±SD from 9 repeated experiments (N=9). Two-way ANOVA followed by Tukey post-hoc test. *P<0.05, ***P<0.001, ***P<0.0001.

[0029] Figure 6 shows intranasal dantrolene nanoparticles but not vehicle significantly improves the overall motor phenotype neurological scores in SOD1-G93A mice. The higher the neurological score, the worse the ALS symptoms and signs. N=12, 4 and 12 for Control and 12, 11 and 16 for SOD1G93A mice for groups: No Tx, Vehicle and Dantrolene, respectively. Means ± 95%CI, two-way ANOVA, followed with the Tukey multiple comparison test (MCT). P<0.05 is considered statistically significant.Figures 7A-7H Intranasal dantrolene nanoparticles robustly and significantly inhibit impairments in motor coordination and movement balance in SOD1-G93A ALS mice. Overall motor coordination and balance were evaluated at the end of a 30-day treatment period (No treatment (NO TX), intranasal vehicle (IN-VEH) or intranasal dantrolene nanoparticles (IN-DAN) at 120P-654697-PCdays of age, using the beam balance test at two beam widths: 12 mm (Fig. 7A and Fig. 7B) and 6 mm (Fig. 7C and Fig. 7D). Longer beam crossing time (Fig. 7A and Fig. 7C) and increased number of foot slips (Fig.7B and Fig. 7D) indicate greater impairment of motor coordination and movement balance. N = 12 (NO TX), 10 (IN-VEH), and 12 (IN-DAN) for the n on-transgenic control mice and 12 (NO TX), 10 (IN-VEH), and 16 (IN-DAN) for the transgenic SOD1-G93A ALS mice. Data are presented as means ± 95% CI and were analyzed using two-way ANOVA, followed by Tukey’s multiple comparison test (MCT). P<0.05 was statistically significant. Motor coordination and balance were further evaluated weekly over a four-week treatment period using the rotarod test (Fig. 7E- Fig. 7H), following the initiation of intranasal dantrolene particle treatment (IN-DAN), vehicle control (INVEH), or no treatment (NO TX) between the ages of 90 and 118 days. A longer latency to fall from the rotarod indicates improved motor coordination and balance. At treatment initiation, N = 12 (NO TX), 10 (IN-VEH) and 12 (IN-DAN) for the non-transgenic control mice. N = 18 (No TX), 15 (IN-VEH) and 18 (IN-DAN) for the SOD1-G93A transgenic ALS mice. Data are presented as means ± 95%CI and were analyzed using two-way ANOVA, followed by Tukey’s multiple comparison test (MCT)Figures 8A-8B show intranasal dantrolene nanoparticles significantly inhibit body weight loss and muscle weakness in SOD1-G93A transgenic ALS mice. Fig 8A shows intranasal dantrolene significantly inhibited the muscle weakness in SOD1G93A mice. Kondziela’s inverted screen test was performed to evaluate the muscle strength (grips) of mice. The longer time falls from the inverted screen, the better muscle strength. N=12, 4 and 12 for Control and 12, 11 and 16 for SOD1G93A mice for groups: No Tx, Vehicle and Dantrolene, respectively. Means ± 95%CI, Two-way ANOVA, followed with the Tukey multiple comparison test (MCT). P<0.05 is statistically significant. Fig 8B shows dynamic changes of body weight for 4 weeks treatment. Body weight was measured weekly from 1 week (1W) up to 4 weeks (W) after initiation of treatment. Means ± 95%CI, Data were analyzed by repeated measures two-way ANOVA, followed with the Tukey multiple comparison test.Figure 9 Intranasal dantrolene nanoparticles significantly inhibit spinal cord degeneration in SOD1-G93A ALS mice. Spinal cord degeneration was evaluated following 30 days of treatment with no treatment (NO TX), intranasal vehicle control (IN-VEH) or intranasal dantrolene nanoparticles (IN-DAN), at 135 days of age, by measuring levels of the neuronal skeletal protein neurofilament light chain (NFL) and spinal cord weight. (Fig. 9A) Representative ImmunoblotP-654697-PCillustrating changes in NFL protein level in the lumbar and sacral spinal cord (~L1 to S5). (Fig.9B) Quantification and statistical analysis of NFL protein expression in the same region. N = 8 per group in both control and SOD1-G93A mice. (Fig. 9C) Fresh weight of lumbar and sacral spinal cord tissue collected for Western blot (~L1 to S5). N=10 per group in control mice, N=5 (NO TX), 9 (IN-VEH) and 7 (IN-DAN) in SOD1-G93A transgenic mice. Fig. 9D. Representative images of 4% paraformaldehyde-fixed cervical and thoracic spinal cords (~C1 to T12). Fig. 9E. Weight measurements of the cervical and spinal cord segments (~C1 to T12). N = 5 per experimental group. Data are presented as means ± 95% Cl and were analyzed using two-way ANOVA, followed by Tukey’s multiple comparison test (MCT). (Fig. 9B, Fig. 9C, Fig. 9E). P<0.05 was considered statistically significant.Figure 10 Intranasal dantrolene nanoparticle treatment robustly prolongs survival probability in SOD1-G93A transgenic ALS mice. The probability of survival in SOD1-G93A transgenic ALS mice was monitored daily over a 30-day treatment period with intranasal dantrolene nanoparticle (IN-DAN), vehicle control (IN-VEH), or no treatment (NO TX), beginning at 90 days of age and ending at 120 days. At treatment onset, N = 18 (NO TX), 15 (IN-VEH), and 18 (IN-DAN) for SOD1-G93A mice. Kaplan-Meier survival analysis was performed to evaluate survival probability. At the end of the treatment period, the survival rate for all non-transgenic control mice was 100% (data not shown). In contrast, among SOD1-G93A mice: No TX: 67% survival (12 / 18), IN-VEH: 67% survival (10 / 15), IN-DAN: 89% survival (16 / 18).

[0030] Figure 11 shows the weekly survival rate for 4 weeks treatment, rate was recorded weekly from 1 to 4 weeks after initiation of treatment. N=12, 4 and 12 for Control mice and 12, 11 and 16 for SOD1G93A mice for groups: No Tx, Vehicle and Dantrolene respectively at the beginning of treatment. Kaplan-Meier Survival analysis. Survival rate at the end of fourth week treatment: Control mice: all 100%, SOD1G93A mice: No TX (25%), IN-DAN (87.5%), IN- Veh (80%).

[0031] Figure 12 shows the experimental design in transgenic mice with TDP43 mutation ALS animal model. M: F: Male: Female, WT: Wild type, IN-DNRF: Intranasal dantrolene in Ryanodex formulation; Veh: vehicle, RyRs: Ryanodine receptors, ELISA: Enzyme-linked immunosorbent assay; TNF: Tumor necrosis factor; NFL: Neurofilament light chain; ALT: Alanine transaminase; IHC: Immunohistochemistry, RyRs: ryanodine receptors, NMDARs: N-Methyl D-Aspartate (NMDA) receptors, AMPARs: alpha-amino-3-hydroxy-5-methyl-4-isooxazole-propionic acid receptors, TDP43: Tar DNA-binding protein 43, MDA; Malondialdehyde, 4-HNE: 4-P-654697-PCHydroxynonenal, NLRP3: nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3; GSDMD-NT: N-terminal Gasdermin D. PSD-95: Postsynaptic density 95.

[0032] Figure 13 shows the experimental design in SOD1G93A transgenic mice ALS animal model. M: F: Male: Female, WT: Wild type, IN-DNRF: Intranasal dantrolene in Ryanodex formulation; Veh: vehicle, RyRs: Ryanodine receptors, ELISA: Enzyme-linked immunosorbent assay; TNF: Tumor necrosis factor; NFL: Neurofilament light chain; ALT: Alanine transaminase; IHC: Immunohistochemistry, RyRs: ryanodine receptors, NMDARs: N-Methyl D-Aspartate (NMDA) receptors, AMPARs: alpha-amino-3-hydroxy-5-methyl-4-isooxazole-propionic acid receptors, TDP43: Tar DNA-binding protein 43, MDA; Malondialdehyde, 4-HNE: 4-Hydroxynonenal, NLRP3: nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3; GSDMD-NT: N-terminal Gasdermin D. PSD-95: Postsynaptic density 95.

[0033] Figures 14A-14B shows the mechanism of dantrolene treatment for major depressive disorder (MDD) and dementia in AD and the experimental designs for early treatment group (ETG) and late treatment group (LTG) according to embodiments of the invention. Fig. 14A shows excessive and pathological activation of N-methyl-D-aspartate receptors (NMDAR) by glutamate in MDD leads to excessive Ca2+influx into the cytosol via the NMDARs. This can be aggravated by the major sporadic AD (SAD, 95% of AD patients) risk factor, ApoE4. Lipopolysaccharides (LPS) pathologically increase endogenous agonists of the ryanodine receptor (RyR), cyclic ADP-ribose (cADPR), via the activation of toll like receptor 4 (TLR4), leading to excessive Ca2+release from the endoplasmic reticulum (ER) into the cytosol. Overall, the cytosolic ([Ca2+]c) and mitochondrial Ca2+([Ca2+]m) are pathologically elevated in MDD, resulting in excess production of reactive oxygen species (ROS), which in turn activates NLRP3 and consequently activating caspase- 1 which then cleave full Gasdermin D into N terminal Gasdermin D (N-GSDMD). Eventually, this pathology leads to cell or neuron death via pyroptosis pathway, and synaptic destruction or dysfunction, depression, and dementia. Dantrolene, through inhibition of NMDAR and RyR, ameliorates upstream Ca2+dysregulation followed by downstream oxidative stresses, inflammation, pyroptosis and synapse destruction, effectively treating MDD and dementia, particularly in AD.

[0034] Figures 15A-15B show that dantrolene abolished depression behavior in 5xFAD mice. Wild type (WT) or 5xFAD mice were treated with intranasal (IN) dantrolene (DAN 5mg / kg) nanoparticles, daily, Monday to Friday for 12 consecutive weeks. Depression behavior wasP-654697-PCassessed with the tail suspension test (TST), which was performed at the age of 6 (Fig. 15A) or 12 (Fig. 15B) months old after completion of treatments. The more immobile time, the more severe the depression behavior. Means±SD. The data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (Fig. 15B) and the 2-way ANOVA followed by Dunnett’s multiple comparison test (MCT) (2A). **, P<0.01 (Figs. 15A, 15B).

[0035] Figures 16A-16D show that dantrolene inhibits memory loss in 5XFAD mice. Wild type (WT) or 5xFAD mice were treated with intranasal (IN) dantrolene (DAN 5mg / kg) nanoparticles, daily, Monday to Friday for 12 consecutive weeks. Both Contextual fear conditioning (CFC) and Cued fear conditioning were performed at the age of 6 (Fig. 16A, hippocampus-dependent; Fig.16B, hippocampus-independent) or 12 (Fig. 16C, hippocampus-dependent; Fig. 16D, hippocampus-independent) months old after completion of treatments. The less freezing time, the more severe the memory loss. Data represents Means±SD from 4-6 mice, and were analyzed using the one-way ANOVA (C, D) followed by Tukey’s multiple comparison test and the 2-way ANOVA (A, B) followed by Dunnett’s multiple comparison test (MCT). *, **, ***, ****, P<0.05, P<0.01, P<0.001, P<0.0001, respectively.

[0036] Figures 17A-17B show dantrolene inhibits the pathological increase of type 2 ryanodine receptor (RyR-2) and type 1 InsPiR (InsPsR-l) proteins in 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9 months old were treated with intranasal (IN) dantrolene (DAN, 5 mg / kg) nanoparticles, daily, Monday to Friday for 12 consecutive weeks. Mice brains were harvested at 13 months old after behavioral tests. Immunoblot was used to determine the protein levels of RyR-2 (Fig. 17A) and InsPaR-l (Fig. 17B). GAPDH was used as the housekeeping protein. Data are Means±SD from 4 separate mice in each treatment group (N=4) and were analyzed using the oneway ANOVA followed by Tukey’s multiple comparison test. * P<0.05, **P<0.01, **** PO. OOOl.

[0037] Figures 18A-18B show dantrolene inhibits oxidative stress in 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9 months old were treated with intranasal (IN) dantrolene (DAN, 5 mg / kg) nanoparticles, daily, Monday to Friday for 12 consecutive weeks. Mice brains were harvested at 13 months old after behavior tests. Fig. 18A shows a representative Western blot that was used to measure malondialdehyde (MDA, lipid peroxidation byproduct) modified proteins marked by *. Fig. 18B shows statistical analysis of changes of MDA-modified proteins. Data represented as mean ± standard deviations (Means±SD) from 4 separate mice brain proteins (N=4)P-654697-PCand were analyzed using the one-way ANOVA followed by Tukey’s multiple comparison test. *** P<0.001, **** P<0.0001.

[0038] Figures 19A-19D show dantrolene inhibits pathological activation of pyroptosis pathway in 5xFAD mice. Wild type (WT) or 5xFAD mice at 9 months old were treated with intranasal (IN) dantrolene (DAN, 5 mg / kg) nanoparticles, daily, Monday to Friday for 12 consecutive weeks. Mice brains were harvested at 13 months old after behavior tests. Representative Western blots and associated statistical analysis were used to determine changes of critical regulatory proteins of pyroptosis pathway (Fig. 19A, NLR family pyrin domain containing 3 (NLRP3); Fig. 19B, Caspase-1; Fig. 19C, N terminal Gasdermin D (N-GSDMD) and Fig. 19D, Gasdermin D full length (GSDMD-FL). GAPDH was used as the loading control. Data are mean ± standard deviation (Means±SD) from 4 separate mice brains (N=4) and were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. ***, **** P<0.001, P<0.0001.

[0039] Figures 20A-20E show dantrolene inhibits pathological increase of neurotoxic cytokines but increase neuroprotective cytokines in 5xFAD mice. Wild type (WT) or 5xFAD mice at 9 months old were treated with intranasal (IN) dantrolene (DAN, 5 mg / kg) nanoparticles, daily, Monday to Friday for 12 consecutive weeks. Mice brains were then harvested at the age of 13 months old after all behavior tests. Representative Western blot and associated statistical analysis of neurotoxic cytokines proteins IL-ip (Fig.20A), IL-18 (Fig.20B), IL-6 (Fig.20C), TNF-a (Fig.20D) and neuroprotective cytokine IL- 10 (Fig. 20E) were determined and analyzed. GAPDH as loading control. Data are mean ± standard deviation (Means±SD) from 4 separate mice brains (N=4) and were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. **, ***, ****, P<0.01, P<0.001, P<0.0001.

[0040] Figures 34A-34B shows dantrolene inhibits synapse loss in aged 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9 months old were treated with intranasal (IN) dantrolene (DAN, 5 mg / kg) nanoparticles, daily, Monday to Friday for 12 consecutive weeks. Mice brains were then harvested at the age of 13 months old after all behavior tests. Western blot determined the PSD-95 (Fig. 34A) and Synapsin-1 (Fig. 34B) synapse protein levels. GAPDH was used as the housekeeping protein. Data are mean ± standard deviation (Means±SD) from 4 separate mice brains (N=4) and were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. **, ***, ****, P<0.01, P<0.001, P<0.0001, respectively.P-654697-PC

[0041] Figures 22A-22F show chronic intranasal dantrolene nanoparticles administration did not affect thyroid, kidney or liver function. Wild type (WT) or 5xFAD mice at 2 or 5 months old were treated with intranasal (IN) dantrolene (DAN, 5 mg / kg) nanoparticles, daily, Monday to Friday for 12 consecutive weeks. Mice bloods were then harvested at the age of 6 months old (Figs. 22A-9C) and 13 months old (Fig. 22D-22F) after all behavioral tests. Blood biomarkers for thyroid (TSH: Thyroid Stimulating Hormone), kidney (creatinine), liver (ALT: Alanine Aminotransferase) function was determined at indicated ages using ELISA kits. Data are mean ± standard deviation (Means±SD) from 4-6 separate mice and were analyzed using one-way ANOVA.Figures 23A-23B show the effects of dantrolene on cognitive function determined by the Y-maze test. Wild type (WT) or 5xFAD mice at 2 or 5 months old were treated with intranasal (IN) dantrolene (DAN, 5 mg / kg) nanoparticles, daily, Monday to Friday for 12 consecutive weeks. The Y-maze test was then performed at 6 (Fig. 23A) and 12 (Fig. 23B) months old to determine cognitive function. Data are mean ± standard deviation (Means±SD) from 4-5 separate mice and were analyzed using one-way ANOVA.

[0042] Figures 24A-24C show the effects of chronic intranasal dantrolene nanoparticles on motor and smell functions and body weight. Wild type (WT) or 5xFAD mice at 2 or 5 months old were treated with intranasal (IN) dantrolene (DAN, 5 mg / kg) nanoparticles daily, Monday to Friday for 12 consecutive weeks. The motor (Fig. 24A) and smell (Fig. 24B) functions were determined by rotarod, and food buried tests at 6 months of age. Body weight was determined monthly from initiation of IN dantrolene treatment at 9 months old till the end of all behavioral tests at 12 months old (Fig. 24C). Data are mean ± standard deviation (Means±SD) from 4-6 mice (N=3) and were analyzed using two-way ANOVA.

[0043] Figures25A-25C show dantrolene differentially affected ryanodine receptor (RyR) subtype mRNA expression in a dose-, ApoE4-, and cell type-dependent manner. Induced pluripotent stem cells (iPSCs) were treated with 0.5 pM or 2.5 pM dantrolene for seven days. mRNA were collected and measured. Control received no treatment. mRNA levels are shown as folds change and were compared between the four cell types and two dantrolene concentrations for (A) ryanodine receptor subtype 1 (RyR-1), (B) subtype 2 (RyR-2) and (C) subtype 3 (RyR-3). N=l.

[0044] Figures 26A-26B show dantrolene inhibits lipopolysaccharide (LPS)-induced elevation of cytosolic Ca2+concentration ([Ca2+]c). iPSCs from a SAD patient with parental ApoE4 / E4 (ApoE4 / E4) or its isogenic gene-edited ApoE3 / E3 (ApoE3 / E3) were pretreated with dantroleneP-654697-PC(DAN, 10 pM), then challenged by LPS (50 pg / ml). [Ca2+]cwas measured by the Fura-2 Ca2+indicator and expressed as 340 / 380 ratio. (Fig.26A) The response curve from an average of 40-50 iPSCs. Cell response rate is 100%. (Fig. 26B) Overall Ca2+increase defined by the Area under curve (AUC). The results are from one experiment (N=l).

[0045] Figures 27A-27D show dantrolene inhibits the glutamate-induced pathological elevation of mitochondrial Ca2+concentrations ([Ca2+]m) in neurons from Alzheimer’s disease (AD) patients. iPSC derived neurons (healthy control, sporadic (SAD), and familial (FAD) AD) were exposed to 20 mM glutamate (GLU) with or without 20pM dantrolene (DAN) pretreatment for 1 hour. [Ca2+]mwas measured using the jellyfish photoprotein aequorin-based probe. Typical curves of [Ca2+]mchanges without (Fig. 27A) and with dantrolene (Fig. 27B). Mean ± SD. Two-way ANOVA followed by Tukey test. **P<0.01 (Figs.27C, 27D).

[0046] Figures 28A-28F show that dantrolene inhibits the NMDA mediated pathological elevation of basal mitochondrial oxygen consumption rate (OCR) and proton leak (apoptosis) in ApoE4 iPSCs. iPSCs with parental ApoE4 / E4 (ApoE4 / E4) from a SAD patient or its isogenic gene-edited ApoE3 / E3 (ApoE3 / E3) were pretreated with dantrolene (dan, 0.25 pM), then challenged by vehicle (DMSO), N-methyl-D-aspartate (NMDA, IpM), or 5 uM RSL3. Typical tracings of OCR measurements in ApoE3 (A) and ApoE4 (B) iPSCs. Basal OCR (C, E). Proton leak as an indicator of apoptosis (D, F). Means±SD, N=3 (C, D) and N=2 (E, F). Two-way ANOVA followed by Tukey post-hoc test. *P<0.05,**P<0.01, ***P<0.001.

[0047] Figures 29A-29E show dantrolene inhibits NMDA mediated pathological elevation of ROS and oxidative stress in ApoE4 iPSCs. iPSC with parental ApoE4 / E4 (ApoeE4 / E4) from a SAD patient or its isogenic gene-edited ApoE3 / E3 (ApoE3 / E3) were pretreated with dantrolene (Dan, 0.25pM) for 1 hour, then co-treated without (Figs. 29A, 29C) or with (Figs. 29B, 29D) N-metheyl-D-aspartate (NMDA, IpM) for 24 hours. Means±SE, N=3, Two-way ANOVA followed by Tukey’s test. *P<0.05, ****P<0.0001 (Figs. 29A, 29B, 29C, 29D). ROS: Reactive oxygen species; MDA: Malondialdehyde (lipid peroxide byproduct). Fig. 29E shows expression of oxidized multiple proteins and protective effects of dantrolene.

[0048] Figures 30A-30C show dantrolene inhibit NMDA-induced mitochondria and cell damage in ApoE4 iPSCs. iPSCs with parental ApoE4 / E4 (ApoE4 / E4) from a SAD patient or its isogenic gene-edited ApoE3 / E3 (ApoE3 / E3) were treated with dantrolene (Dan, 0.25 or 10 pM) (30A) or intracellular Ca2+chelator (B APTA-AM) (Fig.30B) for 24 hours. (Fig.30C). Cells were pretreatedP-654697-PCwith Dan (0.25 pM) for one hour, then co-treated with the N-methyl-D-aspartate (NMDA 1 pM) for 24 hours. Means±SD, N=3 separate experiments (Fig. 30A, 30B, 30C). Two-way ANOVA followed by Tukey’s test. **, ***, ****, P<0.01, P<0.001, P<0.0001 compared between the groups indicated in the figures (Figs. 30A, 30C). (Fig. 30B). Nonparametric test followed by Wilcoxon matched-pairs signed rank text. * ** *** **** p<0.05, P<0.01, P<0.001, P<0.0001, compared to control in same types of iPSCs. # P<0.05, ## P<0.01, ### P<0.001 compared to ApoE3 / E3 iPSCs.

[0049] Figures 31A-31C show dantrolene inhibits lipopolysaccharide (LPS)-induced elevation of ROS, mitochondria and cell damage in ReNcell Human Cortical Neural Progenitor Cells. Dantrolene (Dan, 0.25 pM) inhibits LPS (30 pg / mL) mediated pathological elevation of ROS (10 repeats from two experiments) (Fig. 31A). LPS decreased cell viability in a dose-dependent manner (Fig. 31B). Dantrolene inhibits LPS mediated mitochondria and cell damage determined by MTT reduction assay (Fig. 31C). Cells were pretreated with dantrolene 0.25 pM for 2 hours, then co-treated with LPS (30pg / mL) for 24 hours. (16 repeats from 2 experiments) (31C).Mean±SE, two-way ANOVA followed by Tukey’s test. ****P<0.0001 (Figs. 31A, 31C).

[0050] Figures 32A-32C show that dantrolene inhibits antidepressant sensitive behavior (depression) and memory loss in 5xFAD mice. Mice were treated with intranasal dantrolene nanoparticles only (Dan, 5mg / kg), Monday to Friday each week for 3 consecutive months. Memory was determined using the fear conditioning test at the age of 6 months (Fig.32A).Depression behavior using the tail suspension test was determined at the age of 6 (Fig. 32B) and 13 (Fig. 32C) months. The more immobility time, the more depression behavior was observed. Mean±SD. Two-way (Figs. 32A, 32B) and one-way (Fig. 32C) ANOVA followed by post-hoc Tukey tests. *P<0.05, **P<0.01, ***P<0.001.

[0051] Figures 33A-33E show that dantrolene inhibits pathological inflammation in 5XFAD mice brains. 10 month old 5xFAD Mice were treated with dantrolene (Dan 5mg / kg), Monday to Friday for 3 months. Western blot analysis of neurotoxic cytokines of IL-10 (Fig.33A), IL-18 (Fig.33B), IL-6 (Fig. 33C), TNF-a (Fig. 33D) and neuroprotective cytokine IL- 10 (Fig. 33E) protein expression in both WT and 5xFAD mice. GAPDH as loading control. N=2 (Figs.33A, 33B, 33E) and N=3 (Figs.33C, 33D).

[0052] Figures 34A-34D show that dantrolene inhibits pathological pyroptosis pathway in 5XFAD mice brain. 10 month old 5xFAD mice were treated with intranasal dantrolene (Dan, 5mg / kg), weekly (Monday to Friday) for 3 consecutive months. Representative Western blots andP-654697-PCanalyzed data was used to detect the therapeutic effect of dantrolene on the expression of NLPR3 (Fig. 34A), cleaved-caspase-1 (Fig. 34B), GSDMD (Fig. 34C), and N-term GSDMD (Fig. 34D) proteins. Western blot determined the protein levels. GAPDH was the loading control.

[0053] Figure 35 shows intranasal dantrolene nanoparticles inhibits synapse loss in 5xFAD mice.10 month old 5xFAD mice were treated with dantrolene (Dan, 5 mg / kg), weekly (Monday to Friday) for 3 months. Western blots were used to determine PSD-95 synapse protein levels. GAPDH was used as the housekeeping protein. N=3. Two-way ANOVA followed by Tukey ’s test. *, ** meanP<0.05, and P<0.01, respectively.

[0054] Figures 36A-36F show dantrolene did not cause side effects with chronic treatment.Mice were treated with dantrolene (Dan, 5mg / kg), weekly (Monday to Friday) for 3 consecutive months. Olfactory and motor function was determined by the food buried (Fig. 36A) and rotarod (Fig. 36B) tests. Body weight (Fig 36C), blood biomarkers for thyroid (Fig 36D. TSH: thyroid stimulating hormone), kidney (Fig 36E, creatinine) and liver (Fig 36F, ALT: Alanine Aminotransferase) function were determined using ELISA kits at the indicated ages. Mean±SD. Two-way ANOVA followed by Tukey tests. *P<0.05. This is also shown in Figs. 9 and 11 supra.

[0055] Figures 37A-37B show intranasal dantrolene (DAN) nanoparticles inhibit lipopolysaccharide (LPS)-induced depression-like behavior in mice. Wild type mice at 4 months of age were treated with intranasal dantrolene nanoparticles (5 mg / kg), Monday to Friday for 4 continuous weeks. Then, mice received a single LPS (5 mg / kg) intraperitoneal injection. Tail suspension (Fig.37A) and forced swimming tests (Fig.37B) were performed at 24 hours after the LPS injection. Control mice (CON) received no treatments. Mean±SD, N=6-8. One-way ANOVA followed by Tukey post-hoc test. *P<0.05, **P<0.01, ***P<0.001.

[0056] Figure 38 shows a detailed animal study experimental design. IN: intranasal, IP: intraperitoneal, nano: nanoparticles, ETG: early treatment group, LTG: late treatment group, LPS: lipopolysaccharide, IHC: immunohistochemistry, ELISA: enzyme linked immunosorbent assay, NRLP3: nucleotide-binding domain, leucine-rich-containing family, pyrin domain-con taining-3, GSDMD: Gasdermin D, N-GSDMD: N-terminal GSDMD, PSD95: postsynaptic density protin-95.

[0057] Figure 39 shows an experimental design for using lithium salts to inhibit inflammatory pyroptosis and abolish memory loss and depression behavior in 5xFAD mice. LiCl: LithiumP-654697-PCChloride, RFV: Ryanodex Formulation Vehicle, ELISA: Enzyme linked immunosorbent assay, TSH; Thyroid Stimulating Hormone.

[0058] Figures 40A-40C show that intranasal lithium chloride in a Ryanodex Formulation Vehicle (RFV) increased its duration in the brain but significantly decreased blood lithium concentrations than oral administration, with increased brain / blood lithium concentration ratio. Wild type mice at 2 months old were given LiCl (3 mM / kg) dissolved in Ryanodex formulation vehicle (RFV) either intranasally (RFV, red), in water (green) or oral administration of LiCl in RFV (Black). Brain (Fig.40A) vs. serum (Fig. 40B) lithium concentrations (cone) were measured and brain / blood concentration ratio were calculated (Fig. 40C), at 30, 60 and 120 minutes after drugs administration. Means±SD, N=6, Two-way ANOVA followed by Tukey post hoc tests. * ** **** P<0.05, P<0.01, P<0.0001, compared to oral administration (black).

[0059] Figures 41A-41D show intranasal LiCl in Ryanodex formulation vehicle (RFV) inhibited memory loss and depressive behavior in 5xF D mice. Wild type (WT) or 5xFAD mice at 2 or 9 months old were treated with intranasal LiCl in RFV (INLI 3 mM / Kg), 5days / week, for 3 consecutive months. Cognitive function was determined by fear conditioning at 6 (Fig. 41A hippocampus-dependent) or 12 (Fig. 41B, hippocampus-independent) months old. Depressive behavior was determined by tail suspension test at 6 months (Fig. 41C) or 12 (Fig. 41D) months old. Data represents Means±SD from 4-6 mice and were analyzed using the 2-way ANOVA followed by Dunnett’s post-hoc test (A, C) or one-way ANOVA (Figs. 41B, 41D) followed by Tukey’s post-hoc test. *, **, ***, ****, P<0.05, P<0.01, P<0.001, PO. OOOl, respectively.

[0060] Figure 42 shows intranasal LiCl in RFV inhibited the pathological increase of type 1 InsPsR (InsPsR-l) proteins in aged 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9-month-old were treated with intranasal LiCl in RFV (INLI, 3 mM / kg), 5 days / week for 3 consecutive months. Mice brains harvested at 13 months old. Data are Means±SD from 4 separate mice in each treatment group (N=4) and were analyzed using the one-way ANOVA followed by Tukey’s multiple comparison test. ****, P<0.0001.

[0061] Figures 43A-43C show intranasal LiCl in Ryanodex formulation vehicle (RFV) inhibited oxidative stress in aged 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9-month-old were treated with intranasal LiCl in RFV (INLI, 3 mM / kg), 5 days / week for 3 consecutive months. Mice brains harvested at 13 months old. Immunoblot determined the lipid peroxidation product 4HNE (Figs. 43A, 43C) or MDA (Figs. 43B, 43C) modified proteins. Data are Means±SD from 4P-654697-PCseparate mice in each treatment group (N=4) and were analyzed using the two-way ANOVA followed by Tukey’s post hoc test. ****P<0.0001.

[0062] Figures 44A-44C show intranasal LiCl in Ryanodex formulation vehicle (RFV) inhibited pathological activation of pyroptosis in 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9-month-old were treated with intranasal LiCl in RFV (INLI, 3 mM / kg), 5 days / week for 3 consecutive months. Mice brains harvested at 13 months old. NLRP3: NLR family pyrin domain containing 3, GSDMD-NT N terminal Gasdermin D, GSDMD-FL: full length GSDMD Gasdermin D. Fig. 44A. represents Western Blot, Fig. 44B shows statistical analysis. Data are Means±SD from 4 separate mice brains (N=4) and were analyzed using one-way ANOVA for each protein followed by Tukey’s post hoc test. **, ***, **** P<0.01, P<0.001, P<0.0001, respectively. Fig.44C, left panel shows western blot and the right panel shows the statistical analysis of full length GSDMD Gasdermin D (GSDMD-FL).

[0063] Figures 45A-45D show intranasal LiCl in Ryanodex formulation vehicle (RFV) inhibited astrocytosis / microgliosis and the pathological increase of neurotoxic cytokines and increase neuroprotective cytokine in aged 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9-month-old were treated with intranasal LiCl in RFV (INLI, 3 mM / kg), 5 days / week for 3 consecutive months. Mice brains harvested at 13 months old. Immunoblot was used to determine protein biomarkers of astrocytosis (GFAP) and microgliosis (IBA-1) (Figs. 45A, 45B), neurotoxic (TNF-a, IL-6) or neuroprotective (IL- 10) cytokines (Figs. 45 C, 45D). Data are Means±SD from 4 separate mice brains (N=4) and analyzed using one-way ANOVA followed by Tukey’s post-hoc test. **, **** P<0.01, P<0.0001, respectively (Figs.45B, 45D)

[0064] Figures 46A-46B shows intranasal LiCl in Ryanodex formulation vehicle (RFV) inhibited synapse protein loss in aged 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9-month-old were treated with intranasal LiCl in RFV (INLI, 3 mM / kg), 5 days / week for 3 consecutive months. Mice brains harvested at 13 months old. Immunoblot was used to determine levels of PSD-95 (Fig.46A) and Synapsin-1 (Fig. 46B). Data are Means±SD from 4 separate mice brains (N=4) and analyzed using one-way ANOVA followed by Tukey’s post-hoc ****, P<0.0001.

[0065] Figures 47A-47D show intranasal LiCl in RFV protected kidney dysfunction in 5XFAD mice. Wild type (WT) or 5xFAD mice at 2 or 9 months old were treated with intranasal LiCl in RFV (INLI 3 mM / Kg), 5 days / week, for 3 consecutive months. Mice bloods were harvested at the age of 6 (Figs. 47A, 47C) and 13 (Figs. 47B, 47D) months old. Blood biomarkers for function ofP-654697-PCthyroid (Figs. 34A, 34B) (TSH: thyroid stimulating hormone), kidney (Figs. 47C, 47D) (Creatinine) was determined at indicated ages. Data are Means±SD from blood of 4-6 separate mice (N=4-6) and analyzed using one-way ANOVA followed by Tukey’s post-hoc test. *, **** represents P<0.05, PO. OOOl, respectively.

[0066] Figures 48A-48B show the effect of intranasal LiCl in Ryanodex Formulation Vehicle (RFV) on cognitive function in 5xFAD mice. Wild type (WT) or 5xFAD mice were treated with intranasal (IN) lithium chloride (IN LI, 3 mM / kg) dissolved in Ryanodex Formulation Vehicle (RFV), daily, Monday to Friday for 12 consecutive weeks. Y-maze was performed at the age of 6 months (Fig. 48A) and 12 months (Fig. 48B) after completion of treatments. Data represents Means±SD from 4-6 mice (N=-6) and was analyzed using the one-way ANOVA (Fig. 48B) followed by Tukey’s multiple comparison test or the 2-way ANOVA (Fig. 48A) followed by Dunnett’s multiple comparison test (MCT).

[0067] Figures 49A-49D show the effects of chronic intranasal LiCl in Ryanodex Formulation Vehicle (RFV) on motor and smell functions and body weight. Wild type (WT) or 5xFAD mice were treated with intranasal (IN) LiCl in RFV (IN LI, 3 mM / kg) nanoparticles, daily, Monday to Friday for 12 consecutive weeks from 2 to 5 months old. Motor (Fig. 49A) and olfactory (Fig.49B) functions were determined by rotarod, and food buried tests respectively at 6 months old. Body weight was measured monthly from initiation of IN LiCl in RFV at 2 months old (Fig.49C) until the end of all behavioral tests at 12 months old (Fig.49D). Data is Means±SD from 4-6 mice.

[0068] Figure 50 shows the mechanism of lithium to inhibit calcium dysregulation mediated mitochondria dysfunction, oxidative stress, inflammatory pyroptosis, synapse loss, memory loss and depressive behavior in Alzheimer’s disease. NMDAR: N-methyl-D-aspartate (NMDA) receptor, ADPS: Alzheimer’s Presenilin, ROS: reactive oxygen species (ROS), NLRP3: (NLR family pyrin domain containing 3), GSDMD-N: N terminal Gasdermin D, ER: endoplasmic reticulum, MCU: Mitochondria calcium uniporter.

[0069] Figures 51A-51B show differentiation of iPSCs into neuroprogenitors (NPCs) and ReNcell NPC into mixed cortical neurons and astrocytes. Fig.51A shows ApoE4 / E4 vs. ApoE3 / E3 iPSCs. DAPI stain nuclei. Nanog, Sox2, OCT4 and Tra-1-81 are biomarkers of iPSCs. Nestin is biomarker for neuroprogenitors (NPCs). Fig. 51B shows immunostaining and immunoblotting to confirm differentiation of ReNcell NPC into mature neuron with biomarker of TuJ 1 (class III betatubulin) and astrocytes with biomarker of GFAP after 15 days ReNcell NPC differentiation.P-654697-PC

[0070] Figures 52A-52D show lithium inhibit ApoE4 / NMDA mediated cell damage. Fig. 52A shows ApoE4 / E4 or ApoE3 / E3 were treated with LiCl (Li, 0.25 mM) for 24 hours. Cell viability was determined by MTT reduction assay. Human ReNcell cortical neurons / astrocytes were treated with NMDA at different concentration for 24h. MTT (Fig. 52B) and LDH (Fig. 52C) assay determined cell viability. Lithium inhibits NMDA (30 pM. TD50%) mediated ReNcell cortical neurons / astrocyte damage (Fig. 52D. MTT) and death (Fig. 39E LDH). Means±SD from at least four separate experiments. One-way (Figs.52D, 52E) or Two-way (Figs.52A, 52B, 52C) ANOVA followed by Tukey post hoc tests. **** P<0.0001 (Figs. 52A, 52D, 52E), or compared to no NMDA (0 pM) treatment (Figs. 52B, 52C).

[0071] Figure 53 shows abnormal increase of cytosolic Ca2+concentration contributed to ApoE4 / E4 iPSCs damage. ApoE4 / E4 or ApoE3 / E3 iPSCs treated with cytosol Ca2+chelator (BAPTA-AM), for 24 hrs. Means±SD from at least four separate experiments with each experiments include three repeats. Two-way ANOVA followed by Tukey post hoc tests. *, **, ***, P<0.05, P<0.01, P<0.001 compared same concentration of BAPTA-AM treatment in ApoE4 / E4 iPSCs. #, ##, ####, P<0.05, P<0.01, P<0.0001 compared to no BATPA-AM (0 pM) treatment in the same ApoE4 / E4 iPSCs.

[0072] Figures 54A-54B show lithium inhibits ApoE4 / NMDA mediated increase of type 1 InsPsR (InsPsR-l) Ca2+channel proteins. Fig. 54A shows ApoE4 / E4 or ApoE3 / E3 iPSCs treated with LiCl (Li, 0.25 mM) or vehicle for 24 hours. Fig.54B shows human RenCell CX neurons / astrocytes 15 days after NPC differentiation were pretreated with LiCl (0.25, 1.5mM LiCl) for 3 days, then treated with N-methyl-D-aspartate (NMDA) at TD50 (30pM) for 24 hrs. (Figs. 54A and 54B).Means±SD from 3 independent experiments, One-way ANOVA followed by Tukey ’s post hoc test ****p<0.0001.

[0073] Figures 55A-55F show lithium inhibits ApoE4 / NMDA mediated pathological elevation of basal mitochondria oxygen consumption rate (OCR) and pyroptosis (proton leak). ApoE4 / E4 or ApoE3 / E3 iPSCs treated with LiCl (Li, 0.25 mM). Typical tracing of mitochondria oxygen consumption rate (OCR) measurement in ApoE3 / E3 (Fig. 55A) and ApoE4 / E4 (Fig. 55B) iPSCs and the effects of lithium treatments on Basal respiration (Fig.55C) and proton leak (Fig.55D) in ApoE3 / E3 vs. ApoE4 / E4. RenCell CX neuron / astrocytes were pretreated with LiCl (0.25, 1.5mM LiCl) for 3 days, then treated with NMDA at TD50 (30pM). Basal respiration (Fig. 55E), and proton leak (Fig. 55F) were measured. Data represent Means±SD of three separate experiments,P-654697-PC(N=3) and were analyzed by one-way (Figs. 55E, 55F) and two-way (Figs. 55C, 55D) ANOVA followed by Turkey post-hoc test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0074] Figures 56A-56D show lithium inhibits ApoE4 / NMDA mediated pathological elevation of ROS and associated lipid peroxidation. ApoE4 / E4 or ApoE3 / E3 iPSCs treated with LiCl (Li, 0.25 mM) for 24 hours. (Fig. 56A) Intracellular reactive oxygen species (ROS). (Fig. 56B) intracellular lipid peroxidation by-product malondialdehyde (MDA). RenCell CX neuron / astrocyte mixture were pre-treated with LiCl (Li, 0.25mM, 1,5mM) for 3 days, then treated with 30pM NMDA for 24 hours. Lipid peroxidation byproducts 4-HNE (Fig.56C) and MDA (Fig.56D) modified proteins were marked by *. Means±SD, from 3 separate experiments (N=3). Means±SD, Two-way ANOVA followed by Tukey post hoc test. **P<0.01, ****P<0.0001 (Figs.56A, 56B)

[0075] Figures 57A-57D show lithium inhibited NMDA-mediated activation of the pyroptosis pathway. RenCell CX mixed human neuron / astrocytes mixture were pretreated with LiCl (0.25, 1.5mM LiCl) for 72 hours, then treated with NMDA at TD50 (30pM) for 24 hrs. Fig. 57A represents a Western blot for primary pyroptosis activation regulatory proteins expression of NLRP3, Cleaved caspase-1, N terminal Gasdermin D (GSDMD-NT). Fig. 57B shows statistical analysis of pyroptosis regulatory protein changes. Figs. 57C, 57D show the secretion of inflammatory cytokines (IL-1β, and IL-18) in the cell culture. Data are Means±SD from 3 (Figs.57B) and 8 (Figs. 57C, 57D) independent experiments, analyzed using one-way (Figs. 57C, 57D) and two-way (Fig. 57B) ANOVA followed by Tukey’s post hoc test. **, ***, **** P<0.01, P<0.001, P<0.0001, respectively.

[0076] Figures 58A-58C show intranasal lithium chloride in Ryanodex formulation vehicle (RFV) increased its duration in the brain but significantly decreased blood lithium concentrations than oral administration, with increased brain / blood lithium concentration ratio. (Also shown in Figs. 40A-40C). Wild type mice at 2 months old were given LiCl (3 mM / kg) dissolved in Ryanodex formulation vehicle (RFV) either intranasally (RFV, red), in water (green) or oral administration of LiCl in RFV (Black). Brain (Fig. 58A) vs. serum (Fig. 58B) lithium concentrations (cone) were measured and brain / blood concentration ratio were calculated (Fig.58C), at 30, 60 and 120 minutes after drug administration. Means±SD, N=6, Two-way ANOVA followed by Tukey post hoc tests. *P<0.05, **P<0.01, ****P<0.0001, compared to oral administration (black).P-654697-PC

[0077] Figure 59 shows intranasal LiCl in RFV inhibited the pathological increase of type 1 InsPsR (InsPaR-l ) proteins in aged 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9-month-old were treated with intranasal LiCl in RFV (INLI, 3 mM / kg), 5 days / week for 3 consecutive months. Mice brains harvested at 13 months old. Data are Means±SD from 4 separate mice in each treatment group (N=4) and were analyzed using the one-way ANOVA followed by Tukey’s multiple comparison test. ****, P<0.0001.

[0078] Figures 60A-60C show intranasal LiCl in Ryanodex formulation vehicle (RFV) inhibited oxidative stress in aged 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9-month-old were treated with intranasal LiCl in RFV (INLI, 3 mM / kg), 5 days / week for 3 consecutive months. Mice brains harvested at 13 months old. Immunoblot determined the lipid peroxidation product 4HNE (Figs. 60A, 60C) or MDA (Figs. 60B, 60C) modified proteins. Data are Means±SD from 4 separate mice in each treatment group (N=4) and were analyzed using the two-way ANOVA followed by Tukey’s post hoc test. ****P<0.0001.

[0079] Figures 61A-61B show intranasal LiCl in Ryanodex formulation vehicle (RFV) inhibited pathological activation of pyroptosis in 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9-month-old were treated with intranasal LiCl in RFV (INLI, 3 mM / kg), 5 days / week for 3 consecutive months. Mice brains harvested at 13 months old. NLRP3: NLR family pyrin domain containing 3, GSDMD-NT N terminal Gasdermin D, GSDMD-FL: full length GSDMD Gasdermin D. Fig. 61A represents a Western Blot. Fig. 61B shows statistical analysis. Data are Means±SD from 4 separate mice brains (N=4) and were analyzed using one-way ANOVA for each protein followed by Tukey’s post hoc test. **, ***, **** P<0.01, P<0.001, P<0.0001, respectively.

[0080] Figures 62A-62D show intranasal LiCl in Ryanodex formulation vehicle (RFV) inhibited astrocytosis / microgliosis and the pathological increase of neurotoxic cytokines and increase neuroprotective cytokine in aged 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9-month-old were treated with intranasal LiCl in RFV (INLI, 3 mM / kg), 5 days / week for 3 consecutive months. Mice brains harvested at 13 months old. Immunoblot was used to determine protein biomarkers of astrocytosis (GFAP) and microgliosis (IBA-1) (Figs. 62A, 62B), neurotoxic (TNF-a, IL-6) or neuroprotective (IL-10) cytokines (Figs. 62C, 62D). Data are Means±SD from 4 separate mice brains (N=4) and analyzed using one-way ANOVA followed by Tukey’s post-hoc test. **, **** P<0.01, P<0.0001, respectively (Figs. 62B, 62D)P-654697-PC

[0081] Figure 63 shows intranasal LiCl in Ryanodex formulation vehicle (RFV) inhibited synapse protein loss in aged 5xFAD mice brains. Wild type (WT) or 5xFAD mice at 9-month-old were treated with intranasal LiCl in RFV (INLI, 3 mM / kg), 5 days / week for 3 consecutive months. Mice brains harvested at 13 months old. Immunoblot was used to determine. Data are Means±SD from 4 separate mice brains (N=4) and analyzed using one-way ANOVA followed by Tukey’s post-hoc ****, P<0.0001.

[0082] Figures 64A-64D show intranasal LiCl in Ryanodex formulation vehicle (RFV) inhibited memory loss and depressive behavior in 5xFAD mice. Wild type (WT) or 5xFAD mice at 2 or 9 months old were treated with intranasal LiCl in RFV (INLI 3 mM / Kg), 5days / week, for 3 consecutive months. Cognitive function was determined by fear conditioning at 6 (Fig. 64A hippocampus-dependent) or 12 (Fig. 64B, hippocampus-independent) months old. Depressive behavior was determined by tail suspension test at 6 (Fig. 64C) or 12 (Fig. 64D) months old. Data represents Means±SD from 4-6 mice and were analyzed using the 2-way ANOVA followed by Dunnett’s post-hoc test (Figs. 64A, 64C) or one-way ANOVA (Figs. 64B, 64D) followed by Tukey’s post-hoc test. *, **, ***, ****, P<0.05, P<0.01, P<0.001, P<0.0001, respectively.

[0083] Figures 65A-52D show intranasal LiCl in RFV protected kidney dysfunction in 5XFAD mice. Wild type (WT) or 5xFAD mice at 2 or 9 months old were treated with intranasal LiCl in RFV (INLI 3 mM / Kg), 5 days / week, for 3 consecutive months. Mice bloods were harvested at the age of 6 (Fig. 65A, 52C) and 13 (Figs. 65B, 65D) months old. Blood biomarkers for function of thyroid (Figs. 65A, 65B) (TSH: thyroid stimulating hormone), kidney (Fig. 65C, 65D) (Creatinine) was determined at indicated ages. Data are Means±SD from blood of 4-6 separate mice (N=4-6) and analyzed using one-way ANOVA followed by Tukey’s post-hoc test. *, **** represents P<0.05, PO. OOOl, respectively.

[0084] Figures 66 shows the experimental design for an animal study. AD mice include wild type (WT), 5XFAD, E4FAD and E3FAD. ETG: Early Treatment Group, LTG: Late Treatment Group, Lithium has three forms (LiCl, LiCOa, lithium orotate) in RFV or water. IN: Intranasal; NMDAR: N-methyl-D-aspartate receptor; NFL: neurofdament light chain; TSH: thyroid stimulating hormone; IHC: Immunohistochemistry. NLRP3: NLR family pyrin domain containing 3, GSDMD-NT: N terminal Adermin D.

[0085] Figures 67A-67D show the experimental design for treating depression with dantrolene. Wild type (WT) or 5XFAD mouse groups at 4 or 9 months old with an intranasalP-654697-PCadministration of dantrolene nanoparticles (Fig. 67C, 5mg / kg) or vehicle (Fig. 67D) for 4 weeks, followed by a one-time treatment with an intraparietal (IP) injection of lipopolysaccharide (LPS, 5mg / kg) at 5 or 10 months old, respectively, (Figs. 67B, 67C and 67D). Depression and behavior tests were done 24 hour after IP LPS injection. The control group received no treatment (Fig.67A).

[0086] Figures 68A-68F show intranasal dantrolene nanoparticles abolished lipopolysaccharide (LPS)-induced depression behavior in 5 or 10 month old mice. Wild type and 5xFAD mice at 4 (Figs. 68A, 68B, 68C) or 9 months (Figs. 68D, 68E, 68F) old were treated with intranasal dantrolene (DAN) nanoparticles (5 mg / kg) or vehicle (VEH) daily, Monday to Friday, for 4 continuous weeks, followed by intraparietal (IP) injection of LPS (5 mg / kg) to mice at 5 or 10 months old, respectively. Forced swimming test (FST, Figs.68A, 68D), Tail Suspension test (TST, Figs. 68B, 68E) and Open Field (OPF, Figs. 68C, 68F) were performed at 24 hours after a onetime IP LPS injection. The more immobility time, the more depression behavior for all three tests. Mice in the control group received no treatments. Means±SD, N=9-10 mice. Two-Way ANOVA followed by Tukey post hoc test.. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The test results demonstrated that administration of intranasal dantrolene nanoparticles abolished lipopolysaccharide (LPS)-induced depression behavior in 5 and 10 month old mice.

[0087] Figures 69A-69B show intranasal dantrolene nanoparticles inhibited lipopolysaccharide (LPS)-induced anxiety behavior in 5 or 10 month old mice. Wild type and 5xFAD mice at 4 (Fig. 69A) or 9 months (Fig. 69B) old were treated with intranasal dantrolene (DAN) nanoparticles (5 mg / kg) or vehicle (VEH) daily, Monday to Friday, for 4 continuous weeks, followed by treatment with intraparietal (IP) injection of LPS (5 mg / kg). Elevated plus maze (EPM) was performed at 24 hours after one-time IP LPS injection. Mice in control (CON) group received no treatments. The more time in the closed arm, the greater anxiety the mice had. Means±SD, N=9-10 mice. Two-Way ANOVA followed by Tukey post hoc test.. *P<0.05, **P<0.01. The test results showed that intranasal dantrolene nanoparticles inhibited lipopolysaccharide (LPS)-induced anxiety behavior in 5 and 10 month old mice.

[0088] Figures 70A-70B show treatment with intranasal dantrolene inhibited lipopolysaccharide (LPS)-induced pathological elevation of IL- 18 and IL- 10 cytokines in blood in mice. Wild type mice at 4 months of age (Figs. 70A, 70B) were treated with intranasal dantrolene (DAN) nanoparticles (5 mg / kg) or vehicle (VEH) daily, Monday to Friday, for 4 continuous weeks, followed by treatment with intraparietal (IP) injection LPS (5 mg / kg). Mice in controlP-654697-PC(CON) group received no treatments. IL10 (Fig.70A) and IL18 (Fig.70B) in blood were measured using an ELISA assay kit. Means±SD, N=7-8 mice. One-Way ANOVA followed by Tukey’s multiple comparison test (Figs. 70A, 70B),*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001The test results showed that intranasal dantrolene inhibited lipopolysaccharide (LPS)-induced pathological elevation of IL- 18 and IL- 10 cytokines in blood in mice.

[0089] Figures 71A-71F show the effects of intranasal dantrolene nanoparticles on lipopolysaccharide (LPS)-induced depression behavior (Figs.71A-71D) and on cognitive function in 5 or 10 month old mice (Figs. 71E-71F). Wild type and 5xFAD mice at 4 (Figs. 71A, 71B) or 9 months (Figs. 71C, 71D) of age were treated with intranasal dantrolene (DAN) nanoparticles (5 mg / kg) or vehicle (VEH) daily, Monday to Friday, for 4 continuous weeks, followed by treatment with intraparietal (IP) injection of LPS (5 mg / kg) for one time. Open field tests (OPF) were performed at 24 hours after one-time IP LPS injection. Mice in control group received no treatments. The less speed (Figs. 71A, 71C) or zone percentage (Figs. 71B, 71D), the greater the depression behavior. Means±SD, N=9-10 mice. Two-Way ANOVA followed by Tukey post hoc test.. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Although the IN dantrolene nanoparticle significantly inhibited the LPS-induced increase of immobility time (depression) in the open field test, as shown in Figures 71C, 71F. IN dantrolene did not show positive data to inhibit LPS-induced depression in two other variables in open field test (Figs. 71A-71D). Figs.71E-72F show the effects of intranasal dantrolene nanoparticles on cognitive function in 10 month old 5xFAD mice. Wild type and 5xFAD mice at 9 months old were treated with intranasal dantrolene (DAN) nanoparticles (5 mg / kg) or vehicle (VEH) daily, Monday to Friday, for continuous 4 weeks. Mice were then treated with intraparietal (IP) injection of LPS (5 mg / kg), Fear-conditioning dependent (Fig. 71E) and Fear-conditioning independent (Fig. 71F) tests were performed at 24 hours after one-time IP LPS inj ection. Mice in control (CON) group received no treatments. The more freezing time, the greater memory loss the mice had. Means±SD, N=9-10 mice. Two-Way ANOVA followed by Tukey post hoc test. *P<0.05, **P<0.01

[0090] Figure 72 shows the mechanisms of dantrolene and lithium neuroprotection in Alzheimer’s Disease (AD).

[0091] Figures 73A-73D show dantrolene (DAN) nanoparticles and lithium inhibit ApoE4 / NMDA mediated pathological elevation of ROS and associated lipid peroxidation. iPSCs with parental ApoE4 / E4 (ApoE4 / E4) from a SAD patient or its isogenic gene-edited (ApoE3 / E3)P-654697-PCwere pretreated with dantrolene (DAN, 0.25 pM), lithium chloride (Li, 0.25 mM) for one hour, then treated with or without vehicle, (DMSO) or the N-methyl-D-aspartate (Fig. 73C, NMDA, 1 pM) for 24 hours. Tx: treatment. Lipid peroxidation by-product malondialdehyde (MDA) was measured using MDA assay kit (Figs.73B, 73C). Western blot to measure MDA modified proteins marked by * as another biomarker of lipid peroxidation (Fig.73D). Means±SD, Two-way ANOVA followed by Tukey test* P < 0.001. ****P < 0.0001 (Figs. 73A, 73B, 73C).

[0092] Figures 74A-74C show dantrolene and lithium inhibit ApoE4 / NMDA mediated mitochondrial and cell damage. iPSCs with ApoE4 / E4 (ApoE4 / E4) from a SAD patient or its isogenic gene-edited (ApoE3 / E3) were treated with dantrolene (DAN, 0.25 pM or 10 pM), lithium chloride (Li, 0.25 mM) (Fig. 74A) or intracellular Ca2+plus chelator (BAPTAAM) (Fig. 74B) for 24 hours. Fig. 74C shows cells were pretreated with DAN (0.25 pM), Li 0.25 mM, or both for one hour, then co-treated with NMDA (1 pM) for 24 hours. Means±SD, Two-way ANOVA followed by Tukey Multiple tests. *, **, **** P < 0.05, P < 0.01, P < 0.0001 (Figs. 74A, 74C). *, **, *** P < 0.05, P < 0.01, P < 0.001 compared to control in ApoE3 cells; #, ##, ###, P < 0.05, P < 0.01, P < 0.0001 compared to ApoE3 (Fig. 74B).

[0093] Figures 75A-75C show dantrolene and lithium inhibit the increase of type one InsP3R (Insp3Rl) and ameliorate the decrease of PSD 5 or Synapsin-1 synapse proteins in 5XFAD mice. Wild type (WT) or 5XFAD mice were treated with dantrolene (DAN 5mg / kg) and lithium (li, 3mM / Kg) or their combination (DAN +Li), Monday to Friday for 3 consecutive months. Western Blot to determine protein level. GAPDH as housekeeping protein.

[0094] Figures 76A-76C show dantrolene and lithium inhibit memory loss and depression in 5XFAD mice. Hippocampus dependent- (Fig. 76A) and independent- (Fig. 76B) memory was determined using a fear conditioning test at the indicated ages. The more freezing time, the better the memory. Depression was determined by a tail suspension test determined at 6 months (Fig.76C) or 13 months of age (Fig. 76D). The more immobility time, the more depression. Mice were treated with dantrolene (Dan 5mg / kg) and lithium (Li,3mM / kg) or their combination (DAN+Li), Monday to Friday, once a day, 5 times a week for 3 consecutive months. Means±SD, Two-way ANOVA (Fig. 76A, 76C) and one-way ANOVA (Fig. 76B, 76D) followed by Tukey tests. *P < 0.05, **P< 0.01, ***P < 0.001.

[0095] Figures 77A-77F show dantrolene and lithium do not cause side effects / toxicity with chronic treatments. Smell and motor function were determined by buried food (Fig. 77A) andP-654697-PCrotarod (Fig.77B) tests. Body weight (Fig.77C), blood markers for function of thyroid (Fig. 77D. TSH: thyroid stimulating hormone), kidney (Fig. 77E) and liver (Fig. 77F, ALT: Alanine Aminotransferase) were determined at the indicated ages using ELISA kits. Mice were treated with dantrolene (DAN 5mg / kg) and lithium (Li, 3mM / kg) or their combination (Dan+Li) one time per day, 5 times per week per week, Monday to Friday, for three consecutive months. Means±SD, Two-way ANOVA followed by Tukey tests. P < 0.05.

[0096] Figures 78A-78D. Intranasal dantrolene nanoparticles significantly reduced LPS-induced acute helplessness and anxiety-related behaviors. Adult B6SJLF1 / J mice were treated with intranasal dantrolene nanoparticles (IN-DAN) or vehicle (VEH) for 28 days, followed by a single i.p. injection of lipopolysaccharide (LPS) (IN-DAN+LPS). Control mice either had no treatment (No Tx), only LPS (LPS), or VEH+LPS. The forced swim test (FST, Fig. 78A), tail suspension test (TST, Fig. 78B), elevated plus maze (EPMT, Fig. 78C) and open field test (OFT, Fig. 78D) were performed 24 hours after the LPS injection. Increased mobility time indicates helplessness or anxiety-related behaviors (A, B, D). Increased time in the closed arm indicates anxiety (C). N=18-19 mice, Mean±95%CI, One-Way ANOVA followed by Tukey post hoc test. *P<0.05, **P<0.01, ****P<0.0001.

[0097] Figures 79A-79D Intranasal dantrolene nanoparticles reduced LPS-induced acute helplessness and anxiety-related behaviors more in female than male mice. The sex of the adult B6SJLF1 / J mice, pretreated with intranasal dantrolene nanoparticles (IN-DAN) or vehicle (VEH) for 28 days, followed by a single i.p. injection of LPS (IN-DAN+LPS), was examined. No treatment controls (No Tx), LPS only (LPS), or VEH+LPS. The forced swimming test (FST, Fig.79A), tail suspension test (TST, Fig. 79B), elevated plus maze (EPMT, Fig. 79C), and open field test (OFT, Fig. 79D) were done 24 hours after the LPS injection. N=8-10 mice, Mean±95%CI, Two-Way ANOVA followed by Tukey post hoc test. *P<0.05, **P<0.01, ****P<0.0001.

[0098] Figures 80A-80F. Intranasal Dantrolene Significantly Inhibited lipopolysaccharide-induced pathological elevation of IL-18 and IL-ip cytokines in the blood and brains. B6SJLF1 / J adult mice were pretreated with intranasal dantrolene nanoparticles DAN, 5mg / kg) or vehicle (Veh) control daily, Monday to Friday, for continuous 4 weeks. Mice were then treated with onetime IP injection of lipopolysaccharide (LPS, 5 mg / kg). Mice in control (No Tx) group received no treatments. ILip in blood (Fig. 80A) and brain (Fig. 80C, Fig. 80E) and IL18 in blood (Fig.80B) and brains (Fig. 80D, Fig. 80F) were measured using ELISA assay kit (Fig. 80A, Fig. SOB)P-654697-PCor immunoblotting (Fig. 80C- Fig. 80F)., N=18-20 mice for blood measurement (Fig. 80A, Fig.80B) and N=4 brains for immunoblotting (Fig. 80C- Fig. 80F), Means±95% CI, One-Way ANOVA followed by Tukey post hoc test. **P<0.01, ****P<0.0001 respectively.

[0099] Figures 81A-81F. Effects of intranasal dantrolene nanoparticles on lipopolysaccharide-induced programed cell death by pyroptosis in the brains. B6SJLF1 / J adult mice (5-10 months old) were pretreated with intranasal dantrolene nanoparticles (Dan, 5mg / kg) or vehicle (Veh) daily, Monday to Friday, for continuous 4 weeks. Mice were then treated with one-time IP injection of lipopolysaccharide (LPS, 5 mg / kg). Mice in control (No Tx) group received no treatments. Representative western blot (Fig.81A, Fig.81C, Fig. 81E) and statistical analysis (Fig. 81B, Fig.81D, Fig.81F) determined the protein levels of critical regulatory proteins of pyroptosis pathway (Fig. 81A, Fig.81B, NLRP3; Fig. 81C, Fig. 81D, Active caspase-1 (P-20), Fig. 81E, Fig.81F, N terminal GSDMD (GSDMD-NT) in brains. N=4 different mice brain in each group. GAPDH as loading control. Means±95%CI, One-Way ANOVA followed by Tukey post hoc test. * P<0.05.

[0100] Figures 82A-82D. Intranasal dantrolene nanoparticles significantly inhibited lipopolysaccharide-induced synapse protein loss in brains. B6SJLF1 / J adult mice were pretreated with intranasal dantrolene nanoparticles (Dan, 5mg / kg) or vehicle (Veh) daily, Monday to Friday, for continuous 4 weeks. Mice were then treated with one-time IP injections of lipopolysaccharide (LPS, 5 mg / kg). Mice in control (No Tx) group received no treatment. Western blot measured changes of synapse proteins of PSD-95 (Fig.82A, Fig.82C) and Synapsin-1 (Fig.82B, Fig.82D) in the brains. GAPDH as loading control. N=4 different mice in each group, Means±95%CI, One-Way ANOVA followed by Tukey post hoc test. *P<0.05, **P<0.01, ***P<0.001, P<0.0001 respectively.

[0101] Figure 83. Adult B6SJLF1 / J mice were pretreated with intranasal dantrolene nanoparticles in Ryanodex formulation (DNRF, 5mg / kg) or vehicle (Veh) for 4 weeks. Control group received no treatment. Mice were then treated with intraperitoneal (IP) injection of lipopolysaccharide (LPS, 5mg / kg) for one time. Depression and anxiety behavior tests were performed 24 hours after one-time IP LPS injection. PreTX: Pretreatment, Tx: Treatment, IN: Intranasal, Dan: Dantrolene, OFT: Open Field Test, EPMT: Elevated Plus Maze Test, TST: Tail Suspension Test, FST: Forced Swimming Test

[0102] Figures 84A-84B. Effects of intranasal dantrolene nanoparticles on lipopolysaccharide-induced anxiety behavior in adult mice. B6SJLF1 / J adult mice (5-10 monthsP-654697-PCold) were pretreated with intranasal dantrolene nanoparticles (Dan, 5mg / kg) or vehicle (Veh) control daily, Monday to Friday, for continuous 4 weeks. Mice were then treated with one-time IP injection of lipopolysaccharide (LPS, 5 mg / kg). Mice in control (No Tx) group received no treatment. Open field test (OFT) was done 24 hours after one-time IP LPS injection. The less central zone distance (Fig. 84A) or less mean speed (Fig. 84B), the more anxiety behavior for OFT. N= 18-20 mice. Means±95%CI, One-Way ANOVA followed by Tukey post hoc test. ***p<0.001.

[0103] Figures 85A-85P. Dantrolene inhibits pathological activation of the pyroptosis pathway and synapse loss in 5XFAD mice brains. Wild-type (WT) or 5XFAD mice at 9 months old were treated with intranasal dantrolene nanoparticles (IN-DAN, 5 mg kgl), daily, Monday to Friday for 12 consecutive weeks. Mice brains were harvested at 13 months old. Representative immunoblots and associated statistical analysis were used to determine changes of critical regulatory proteins of pyroptosis activation pathway. (Fig. 85A and Fig. 85B): NLR family pyrin domain containing 3 (NLRP3); (Fig. 85C and Fig.85D): cleaved caspase-1 (p-20); (Fig.85E and Fig.85F): gasdermin D full-length (GSDMD-FL); (Fig. 85G and Fig. 85H): gasdermin D N-terminal (GSDMD-NT);(Fig. 85I and Fig. 85J): IL-1β; (Fig. 85K and Fig. 85L): IL-18; (Fig. 85M and Fig. 85N): PSD-95; (Fig. 85O and Fig. 85P): synapsin-1 (SYN-1). GAPDH was used as the loading control. Data are mean (SD) from four separate mice brains (N=4) in each experimental group and were analysed using two-way ANOVA followed by Tukey’s multiple comparison test. P<0.05 is considered statistically significant.

[0104] Figure 86. Comparison of physicochemical features between dantrolene in Ryanodex formulation prepared in WEI Lab and Eagle Pharmaceutical Company. RT: Room Temperature.

[0105] Figure 87 Intranasal dantrolene nanoparticles significantly inhibited pathological elevation of neurodegeneration biomarker neurofilament light chain (NFL) in SOD1-G93A mice blood. Mouse blood was collected at 18 weeks of age after completion of drug treatment and behavioral testing. NFL levels were measured using ELISA. Higher NFL levels indicate more severe neurodegeneration. Data presented as mean ± SD from blood samples of 6-7 mice per group (N=6-7) and analyzed using two-way ANOVA followed by Tukey’s multiple comparison test. **** represents P<0.0001.

[0106] Figure 88A-88C. Intranasal dantrolene nanoparticles significantly inhibited motor neuron degeneration in the spinal cords of SOD1-G93A mice. Motor neuron degeneration in the left (LVH)P-654697-PCand right (RVH) ventral horn of the spinal cord at the C1-T12 level was detected by Fluoro-Jade C (FJC) staining. Fig. 88A. Representative staining from the wide type control, SOD-G93A, or SOD1G93A mice with intranasal dantrolene nanoparticle treatment at 5 mg / kg from the age of 90 to 120 days in the preliminary study 14. Red arrows point to positive FJC staining. The level of motor neuron degeneration was expressed by the FJC+ gray value (Fig. 88B). The area of cross section of spinal cord was measured and compared (Fig.88C). Data presented as mean ± SD, with N = 5 mice in each group (Fig.88B, Fig.88C). Data were analyzed by two-way ANOVA followed with Tukey’s multiple comparison test (Fig. 88B) or one-way ANOVA (Fig. 88C).

[0107] Figure 89A-89C. Intranasal dantrolene nanoparticles significantly inhibited motor neuron degeneration in the spinal cords of SOD 1-G93 Amice. Motor neuron degeneration in the left (LVH) and right (RVH) ventral horn of the spinal cord at the Cl -T 12 level was detected by cresyl violet staining. Fig. 89A. Representative staining from the wide type control, SOD-G93A, or SOD1G93A mice with intranasal dantrolene nanoparticle treatment at 5 mg / kg from the age of 90 to 120 days in the preliminary study. Red arrows point to stained motor neurons. The level of motor neuron degeneration was expressed by the spinal cord cell density (Fig. 89B). The area of cross section of spinal cord was measured and compared (Fig. 89C). The Data presented as mean ± SD, with N=5 mice in each group (Fig. 89B, Fig. 89C). Data were analyzed by two-way ANOVA, followed with Tukey’s multiple comparison test (Fig. 89B) or one-way ANOVA (Fig.89C).

[0108] Figures 90A-90B. Intranasal dantrolene nanoparticles significantly inhibited pathological elevation of caspase-1 in the brain. Caspase-1 levels in the brain cortex were measured by Western blot (Fig. 90A, Fig. 90B). Data presented as mean ± SD, with N = 3 (SOD1G93A) mice per experimental group (SOD1G93A or wild type). Data were analyzed by two-way ANOVA, followed with the Tukey multiple comparison test. *** represents P<0.001 (Fig. 90B).

[0109] Figures 91A-91C Intranasal dantrolene nanoparticles significantly inhibited pathological elevation of IL-18 in both blood and brain in SOD1G93A mice. IL-18 protein levels in the brain cortex were determined by Western blot (Fig. 91A, Fig. 91B). ELISA (C) measured IL- 18 levels in blood. Data presented as mean ± SD, with N = 6 (SOD1G93A) or N = 7 (wild type) mice for ELISA and N = 3 mice for Western blot analysis. Data were analyzed using two-way ANOVA, followed by Tukey’s multiple-comparison test. **,*** represents P<0.01, P<0.001 respectively (Fig. 91B, Fig. 91C)

[0110] Figure 92 Working hypothesis.P-654697-PC

[0111] Figure 93 Detailed Experimental Design of the study. DNRF: Dantrolene nanoparticles in Ryanodex formulation.

[0112] Figure 94. Working hypothesis. NMDA: N-methyl-D-aspartate; NMDAR: NMDA receptors; LPS: lipopolysaccharide; TLR4: Toll-like receptor 4; MCU: Mitochondria Calcium Uniporter; ROS: CICR: Calcium Induced Calcium Release; ER: Endoplasmic Reticulum; ROS: Reactive Oxygen Species; TDP-43: Transactive Response DNA-Binding Protein 43.

[0113] Figure 95. Experimental design in transgenic mice with TDP-43 pathology ALS. M: F, Male: Female; WT, Wild Type; IN-DNRF, intranasal dantrolene in Ryanodex formulation; VEH, vehicle; RyR, ryanodine receptors; MRI, magnetic resonance imaging; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor; NFL, neurofilament light chain; ALT, alanine transaminase; IHC, immunohistochemistry; NMDARs: N-methyl-D-aspartate (NMDA) receptors; AMPARs, alpha-amino-3-hydroxy-5-methyl-4-isooxazole-propionic acid receptors; TDP43, TAR DNA-binding protein 43; MDA: malondialdehyde; 4-HNE, 4-Hydroxynonenal; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3; GSDMD-NT, N-terminal Gasdermin D; PSD-95, postsynaptic density 95DETAILED DESCRIPTION OF THE INVENTION

[0114] The present subject matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.

[0115] Amyotrophic lateral sclerosis (ALS) is a devastating degenerative disease characterized by progressive loss of motor neurons in the motor cortex, brainstem, and spinal cord. This rapidly progressing fatal disease leads to weakness of limb, respiratory, and bulbar muscles. Patients progressively lose control of voluntary muscles, leading to loss of limb function and the ability to chew, swallow, speak and eventually breathe. Clinical manifestations of ALS include, but not limited to, bulbar palsy or pseudobulbar affect (PBA), muscle weakness and hypotrophy, weight loss, decline in pulmonary function, decline in in the ability score for the lower extremities, fasciculations and cramps, spastic hypertonus, and hyperreflexia. Some patients also display difficulty speaking (dysarthria), difficulty swallowing (dysphagia) and difficulty breathingP-654697-PC(dyspnea). Non-motor symptoms include behavioral disturbances, dysexecutive impairment, and frontotemporal dementia.

[0116] The neuropathological features of ALS include muscle atrophy, degeneration and loss of motor neurons, loss of anterior horn cells, loss of motor neuron function, neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, synapse destruction, and sclerosis of the spinal cord lateral columns. Gliosis, defined as activation of astrocytes and microglia, is also a hallmark of ALS.

[0117] Increasing evidence indicates that inflammation plays a critical role in ALS pathology, implying it must be reduced to achieve effective therapeutic effects. Core inflammasome (NLRP3) and pyroptosis-related cytokine levels (IL-1 (3 and IL-18) are significantly increased in ALS; so is activated program cell death by pyroptosis.

[0118] A significant gap exists in understanding the mechanisms of ALS pathology, making it difficult to identify an effective disease-modifying drug to treat ALS. The mutation in the transactive response DNA binding protein of 43 kDa (TDP-43) and associated aggregation is present in all ALS patients, while the mutation and aggregation of superoxide dismutase type 1 (SOD1) is considered one of the primary pathologies in familiar ALS (fALS). Upstream Ca2+dysregulation, especially the excessive Ca2+release from the endoplasmic reticulum (ER) via overactivation of ryanodine receptors (RyRs) is critical in causing aggregation of mutated TDP-43 and SOD1 proteins and their associated pathologies, leading to cellular oxidative stress and inflammatory pyroptosis.

[0119] TDP-43 is an RNA-binding protein encoded by TARDBP and is localized in the nucleus. Nuclear depletion and cytoplasmic aggregation of TDP-43 are pathological features found in more than 97% of all ALS patients and nearly 50% of FTD cases (known as FTLD-TDP). At the molecular level, the TDP-43 mutation has been linked to upstream Ca2+dysregulation, mitochondrial dysfunction, ER stress and proteinopathy, oxidative stress, inflammation, and programmed cell death via pyroptosis. SOD1 is an enzyme that metabolizes superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals which may damage cells. SOD1 helps break down potentially harmful oxygen molecules in cells, inhibiting oxidative stress and associated pathologies, including inflammation, and protects cells from destruction. SOD1 mutation is commonly seen in fALS patients and SOD1G93A transgenic mice are frequently used to evaluate drug efficacy treating ALS.P-654697-PC

[0120] As shown in Fig. 1, Ca‘ dysregulation induced by over-activation of NMDAR / AMPAR and RyRs may be an upstream trigger of mitochondrial Ca2+overloading and dysfunction, resulting in a pathological increase of mitochondrial reactive oxygen species (mROS) and cytosolic ROS. On the other hand, pathological elevation of ROS deteriorates the RyRs and induces over activation, forming a vicious cycle of oxidative stress, over-activation of RyRs, and resultant Ca2+release from the ER. The pathological elevation of [Ca2+]cactivates the Ca2+-dependent calpain protease, which subsequently cleaves TDP-43 into toxic cleaved TDP-43. Furthermore, excessive Ca2+release from the ER via RyRs results in depletion of ER Ca2+and ER stress, leading to the misfolding of proteins (unfolded protein response, UPR) and aggregation of proteins, such as mutated TDP-43 or SOD1 in ALS. These pathological effects of mutated TDP-43 or SOD1 aggregation collectively led to increased oxidative stress, activation of the inflammatory pyroptosis pathway, and release of cytotoxic cytokines (IL-ip and IL-18), eventually resulting in programmed motor neuronal death via pyroptosis. Accordingly, drugs ameliorating upstream Ca2+dysregulation, and downstream oxidative stress and inflammation-mediated pyroptosis are herein proposed to be new and effective treatments of ALS.

[0121] Incidence of MDD imposes a tremendous psychological burden, as well as significant social repercussions and contributions to other disabilities. The expected direct and indirect costs of MDD are up to $6 trillion in the USA alone. A significant gap exists in the lack of an effective MDD therapy with minimal toxicity, as this is a chronic disease with high risk of relapse and resistance (up to 30% of patients are unresponsive to the first treatment) and relapse (up to 8%). Furthermore, MDD is highly prevalent in Alzheimer’s disease (AD), affecting up to 40% of patients. Calcium dysregulation in MDD may cause pathological inflammation and pyroptosis, leading to synapse destruction and MDD, and also plays an important role in AD pathogenesis.

[0122] Dantrolene, is a -methyl-D-Aspartate (NMDAR) and ryanodine receptor RyR inhibitor, is the only FDA-approved drug to treat malignant hyperthermia, a disease caused by over-activation of type 1 RyRs (RyRs-1). Furthermore, dantrolene has been proposed to treat other human diseases, such as cardiac arrhythemia, cardiac hypertrophy, heart failure, asthma, diabetes, etc., through inhibiting over-activation of different RyR subtypes. Dantrolene has demonstrated neuroprotection in different neurodegenerative diseases, such as stroke, Huntington’s Disease, and Alzheimer’s Disease (AD) through the inhibition of intracellular calcium dysregulation. Specifically, Dantrolene blocks both Ca2+influx from extracellular space and Ca2+release fromP-654697-PCintracellular stores, and is neuroprotective in multiple AD models, but has relatively low penetration into the central nervous system (“CNS”) in vivo. Dantrolene is also an FDA approved treatment for malignant hyperthermia. The use of intranasal dantrolene has been pioneered to reverse the pathological effects of elevated cytosolic and mitochondrial Ca2+. Although studies suggest that dantrolene inhibits NMDARs, on top of RyR, these findings must be further verified and confirmed. Nonetheless, dantrolene inhibits up to 78% of NMDA-mediated abnormal elevation of [Ca2+]cin cortical neurons and could be a strong candidate for ALS treatment due to its potent inhibition of glutamate- and LPS-induced upstream Ca2+dysregulation, downstream mitochondrial dysfunction, oxidative stress, activation of NLRP3 inflammasome and pathological inflammation, and resultant inflammatory pyroptosis (Fig. 1). Furthermore, dantrolene inhibited glutamate excitotoxicity in motor neurons. However, the previous attempts to use orally delivered dantrolene in ALS treatments were unsuccessful (Staats et al., Neuroscience (2012) 220:26-31) and, in fact, led to the conclusion that dantrolene is strongly contraindicated in amyotrophic lateral sclerosis (ALS) (Rivera et al., JAMA (1975) 233(8):863-864).

[0123] The use of intranasal dantrolene has been pioneered to reverse the pathological effects of elevated cytosolic and mitochondrial Ca2+. It has been shown that intranasal delivery of dantrolene nanoparticles increases its brain / blood concentration ratio and achieves high levels of dantrolene in the CNS without systemic toxicity. Intranasal dantrolene nanoparticles abolished memory loss as a disease-modifying drug, with no side effects or muscle or liver toxicity after up to 10 months of treatment. However, the therapeutic effects and potential mechanism of dantrolene to correct pathologies in ALS has not been investigated.

[0124] Clearly it is important to develop new drugs for the effective treatment of MDD, with no or minimal side effects. Calcium dysregulation, oxidative stress, inflammation and associated programmed cell death by pyroptosis have been considered important primary pathological etiologies of MDD. Inflammation and pyroptosis result in synapse damage and dysfunction, eventually leading to MDD. (Fig. 14A) Drugs affecting ameliorating upstream Ca2+dysregulation, and downstream oxidative stress and inflammation-mediated pyroptosis are herein proposed to be new and effective treatments of MDD.

[0125] Lithium, a first-line drug for depression and mania bipolar disorder, is also neuroprotective against many neurodegenerative diseases, including ALS and AD. Although multiple mechanisms have been proposed for lithium treatment for depression behaviors and cognitive dysfunctionP-654697-PC(including in AD), the ability of lithium to ameliorate upstream Ca2+dysregulation by inhibiting NMDAR and suppressing InsPaR, and the downstream multiple pathologies, including oxidative stresses, inflammation and pyroptosis and synapse and cells / neurons destruction is considered the primary mechanism for lithium to treat both cognitive dysfunction and depression behaviors in AD and other neurodegenerative diseases. However, a major concern or limitation of lithium in bipolar disorder patients is its narrow therapeutic window, proneness to side effects / toxicity, and intolerance in patients with chronic use. As lithium neuroprotection is typically dose-dependent, the effective dose for neuroprotection in the CNS will likely cause intolerable side effects / toxicity. Thus, a new strategy to promote its CNS therapeutic effects, while minimizing its peripheral side effects and organ toxicity is needed to promote lithium use for treatment of both dementia and depressive disorder, especially in AD or other neurodegenerative diseases.

[0126] Accordingly, in one aspect, the present invention provides methods for administration of dantrolene nanoparticles, alone or in combination with lithium salts in order to inhibit the pathological pathways underlying ALS in an animal model (SOD 1G93 A transgenic mice), thereby reducing the associated inflammation, pyroptosis, and synaptic destruction in this ALS animal model, with no or minimal side effects or organ toxicity after chronic use of the dantrolene nanoparticles, alone or combined with lithium salts.Definitions

[0127] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0128] In practicing the present technology, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. These techniques are well-known and are explained in, e.g., Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A PracticalP-654697-PCGuide to Molecular Cloning; the series, Meth. Enzymol., (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, N Y, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu & Grossman, and Wu, Eds., respectively.

[0129] As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings.

[0130] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a compound” is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment incudes from the one particular and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0131] As used herein, the terms “component,” “composition,” “composition of compounds,” “compound,” “drug,” “pharmacologically active agent,” “active agent,” “therapeutic,” “therapy,” “treatment,” or “medicament” are used interchangeably herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and / or physiologic effect by local and / or systemic action. As used herein, the terms “treatment” or “therapy” (as well as different forms thereof) include preventative (e.g., prophylactic), curative or palliative treatment. As used herein, the term “treating” includes alleviating or reducing at least one adverse or negative effect or symptom of a condition, disease or disorder.

[0132] The terms “subject,” “individual,” and “patient” are used interchangeably herein, and refer to an animal, for example a human, to whom treatment, including prophylactic treatment, with the pharmaceutical composition according to the present invention, is provided. The term “subject” as used herein refers to human and non-human animals. The terms “non-human animals” and “nonhuman mammals” are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent, (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses and non-mammals such as reptiles, amphibians, chickens, and turkeys.P-654697-PC

[0133] As used herein, “treatment” or “treating” is an approach: for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (eg., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delay disease progression, increase the quality of life, or prolong survival.

[0134] In the context of the present invention, the term “treatment,” includes the therapy, prophylaxis, retardation or reduction of pathological consequence of amyotrophic lateral sclerosis (ALS), depression (including in the context of AD) and / or related disorder. This includes, in particular, control of progression of ALS, depression (including in the context of AD) and / or related disorder, reduction, prevention, retardation, or delayed onset of associated symptoms, reduction of pain, as well as increase in survival. The methods of the invention contemplate any one or more of these aspects of treatment.

[0135] The term “ALS related disorders,” as used herein, refers to the spectrum of neurodegenerative syndromes known under the names of Classical (Charcot's) ALS, Lou Gehrig's disease, motor neuron disease (MND), progressive bulbar palsy (PRP), progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), bulbar onset ALS, spinal onset ALS, frontotemporal dementia (FTD), as well as or ALS with multi-system involvement.

[0136] The term “combination” or “combinatorial treating / therapy,” as used herein, designates a treatment wherein at least two or more drugs are co-administered to a subject to cause a biological effect. In a combined therapy according to this invention, the at least two drugs may be administered together or separately, at the same time or sequentially. Also, the at least two drugs may be administered through different routes and protocols. As a result, although they may be formulated together, the drugs of a combination may also be formulated separately.

[0137] The term “effective amount” used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and / or delay one or more of its symptoms. In reference to amyotrophic lateral sclerosis, an effective amount comprises an amount sufficient to prevent or delay other unwantedP-654697-PCsymptoms associated with amyotrophic lateral sclerosis. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations.

[0138] The term “prodrug” as used herein refers to any functional derivatives (or precursors) of a compound of the present invention, which, when administered to a biological system (e.g. a human organism), generates said compound as a result of e.g., spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), and / or metabolic chemical reaction(s). Prodrugs typically have the structure X-drug, wherein X is an inert carrier moiety, and drug is the active compound. Prodrugs are usually inactive or less active than the resulting drug and can be used, for example, to improve the physicochemical properties of the drug, to target the drug to a specific tissue, to improve the pharmacokinetic and pharmacodynamic properties of the drug and / or to reduce undesirable side effects. Some of the common functional groups that are amenable to prodrug design include, but are not limited to, carboxylic, hydroxyl, amine, phosphate / phosphonate and carbonyl groups. Prodrugs typically produced via the modification of these groups include, but are not limited to, esters, carbonates, carbamates, amides and phosphates. Specific technical guidance for the selection of suitable prodrugs is general common knowledge. Furthermore, the preparation of prodrugs may be performed by conventional methods known by those skilled in the art. Methods which can be used to synthesize other prodrugs are described in numerous reviews on the subject. Exemplary dantrolene prodrugs are described in WO2019 / 079721, the entirety of which is incorporated by reference herein.

[0139] The term “derivative” of a compound includes any molecule that is functionally and / or structurally related to said compound, such as an acid, amide, ester, ether, acetylated variant, hydroxylated variant, or an alkylated (Ci-Ce) variant of such a compound. The term derivative also includes structurally related compound having lost one or more substituent as listed above. Preferred derivatives of a compound are molecules having a substantial degree of similarity to said compound, as determined by known methods. Similar compounds along with their index of similarity to a parent molecule can be found in numerous databases such as PubChem (pubchem.ncbi.nlm.nih.gov / search) or DrugBank (www.drugbank.ca). In a more preferred embodiment, derivatives should have a Tanimoto similarity index greater than 0.4, preferably greater than 0.5, more preferably greater than 0.6, even more preferably greater than 0.7 with aP-654697-PCparent drug. The Tanimoto similarity index is widely used to measure the degree of structural similarity between two molecules. Tanimoto similarity index can be computed by software such as the Small Molecule Subgraph Detector available online (www.ebi.ac.uk / thomton- srv / software / SMSD / ) Preferred derivatives should be both structurally and functionally related to a parent compound, i.e., they should also retain at least part of the activity of the parent drug.

[0140] The term “derivative” also includes metabolites of a drug, e.g., a molecule which results from the (biochemical) modification) s) or processing of said drug after administration to an organism, usually through specialized enzymatic systems, and which displays or retains a biological activity of the drug Metabolites have been disclosed as being responsible for much of the therapeutic action of the parent drug. In a specific embodiment, a “metabolite” as used herein designates a modified or processed drug that retains at least part of the activity of the parent drug, more preferably they should show a protective activity against glutamate toxicity for the motor units (as exemplified in the experimental part). Examples of a dantrolene metabolite include 5- hydroxy dantrolene.

[0141] The term “salt” refers to a pharmaceutically acceptable and relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention. Pharmaceutical salt formation consists in pairing an acidic, basic or zwitterionic drug molecule with a counterion to create a salt version of the drug A wide variety of chemical species can be used in neutralization reactions Pharmaceutically acceptable salts of the invention thus include those obtained by reacting the main compound, functioning as a base, with an inorganic or organic acid to form a salt, for example, salts of acetic acid, nitric acid, tartric acid, hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonic acid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid or citric acid Pharmaceutically acceptable salts of the invention also include those in which the main compound functions as an acid and is reacted with an appropriate base to form, e.g., sodium, potassium, calcium, magnesium, ammonium, or choline salts Though most of salts of a given active principle are bioequivalents, some may have, among others, increased solubility or bioavailability properties. Salt selection is now a common standard operation in the process of drug development

[0142] Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the hydrochloride, the hydrobromide, the nitrate, the perchlorate, the phosphate, the sulphate, the formate, the acetate, the aconate, the ascorbate, the benzenesulphonate, the benzoate, the cinnamate, the citrate, the embonate, theP-654697-PCenantate, the fumarate,!’ the glutamate, the glycolate, the lactate, the maleate, the malonate, the mandelate, the methane sulphonate, the naphthalene-2-sulphonate, the phthalate, the salicylate, the sorbate, the stearate, the succinate, the tartrate, the toluene-p-sulphonate, and the like. Such salts may be formed by procedures well known and described in the art

[0143] As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered The carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate: a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate: and a glidant, such as colloidal silicon dioxide.

[0144] As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to an individual without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the FDA.

[0145] Dantrolene, a RyR antagonist, is a US Food and Drug Administration approved drug for treatment of malignant hyperthermia, muscle spasm and neuroleptic syndrome, with tolerable side effects and occasional liver toxicity at high dose. Dantrolene, with its ability to inhibit the common upstream critical Ca2+dysregulation, has been shown to be neuroprotective against many neurodegenerative diseases, including cerebral ischemia, Huntington’s disease, spinocerebellar ataxia, amyotrophic lateral sclerosis and seizures. It has been demonstrated that dantrolene abolishes or ameliorates the cognitive dysfunction in multiple AD animal models. Thus, a drug that corrects upstream critical Ca2+dysregulation by inhibition of both NMDAR and RyR, such as dantrolene, may be effective to treat both depression and cognitive dysfunction, especially in AD.Methods of the present invention

[0146] As discussed above, the invention relates to drugs compositions and methods for treating ALS or a related disorder in a subject in need thereof. In an embodiment of the invention, the ALS is sporadic ALS. In an embodiment of the invention, the ALS is familial ALS (FALS). In an embodiment of the invention, the ALS is Western Pacific ALS. In an embodiment of the invention,P-654697-PCthe ALS is Hirayama Disease. In an embodiment of the invention, the ALS is juvenile ALS (JALS). In an embodiment of the invention, the ALS is a trauma induced ALS. In some embodiments the ALS is not FALS. In some embodiments the ALS is not juvenile ALS (JALS).

[0147] In an embodiment of the invention, the subject carries a mutant version of a gene that causes or contributes to ALS pathogenesis. In some embodiments the mutant version of the gene is selected from the group of genes consisting of the superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP) encoding TDP-43, fused in sarcoma (FUS), p62 (SQSTM1), ubiliquin-2 (UBQLN2). TANK-binding kinase 1 (TBK 1), profilin 1 (PFN1), VCP or p97 (VCP), angiogenin (ANG), optineurin (OPTN), C9orf72, Sigma-1 Receptor (SIR), Tubulin alpha-4A (TUBA4A), Dynactin (DCTN1), hnRNPAl (HNRNPA1), Matrin 3 (MATR3), Coiled-coil-helix-coiled-coil -helix domain containing 10 (CHCHD10) genes and any combination thereof.

[0148] More particularly, the invention relates to a composition for use in the treatment of ALS or a related disorder, comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

[0149] The inventors have surprisingly found that these compounds, and their combination(s), show a protective activity against a pathological increase of intracellular Ca2+dysregulation, oxidative stress, pyroptosis activation, and cytotoxic and / or neurotoxic cytokines which cause neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS).

[0150] Accordingly, in one embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In a particular embodiment, the pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0151] In another embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceuticalP-654697-PCcomposition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0152] In another embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0153] In another embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0154] In another embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0155] In another embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0156] In one embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0157] In another embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene inP-654697-PCnanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0158] In another embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0159] In another embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0160] In another embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0161] In another embodiment, the invention provides a method for treating amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0162] In one embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulateP-654697-PCform. In a particular embodiment, the pharmaceutical composition comprising of dantrolene and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0163] In another embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0164] In another embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0165] In another embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0166] In another embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0167] In another embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0168] In one embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising aP-654697-PCcombination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In specific embodiments, the pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0169] In another embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0170] In another embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0171] In another embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0172] In another embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceuticallyP-654697-PCacceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0173] In another embodiment, the invention provides a method for alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0174] In one embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In a particular embodiment, the pharmaceutical composition comprising of dantrolene and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0175] In another embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0176] In another embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0177] In another embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical compositionP-654697-PCcomprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0178] In another embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0179] In another embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0180] In one embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0181] In another embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0182] In another embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptableP-654697-PCcarrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0183] In another embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0184] In another embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0185] In another embodiment, the invention provides a method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0186] In one embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL-10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In a particular embodiment, the pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0187] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation,P-654697-PCinhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0188] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0189] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0190] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.P-654697-PC

[0191] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0192] In one embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0193] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0194] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation,P-654697-PCinhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0195] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0196] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0197] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising aP-654697-PCcombination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0198] In one embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In a particular embodiment, the pharmaceutical composition comprising of dantrolene and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0199] In another embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0200] In another embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0201] In another embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.P-654697-PC

[0202] In another embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0203] In another embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0204] In one embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0205] In another embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0206] In another embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in anP-654697-PCamyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0207] In another embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0208] In another embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0209] In another embodiment, the invention provides a method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0210] In one embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In a particular embodiment, the pharmaceutical compositionP-654697-PCcomprising of dantrolene and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0211] In another embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0212] In another embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0213] In another embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0214] In another embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0215] In another embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, comprising: intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0216] In one embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, comprising: administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising aP-654697-PCcombination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0217] In another embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0218] In another embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0219] In another embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0220] In another embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt,P-654697-PCand a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0221] In another embodiment, the invention provides a method for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0222] In one embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In a particular embodiment, the pharmaceutical composition comprising of dantrolene and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0223] In another embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0224] In another embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0225] In another embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical compositionP-654697-PCcomprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0226] In another embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0227] In another embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0228] In one embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In some embodiments, the pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0229] In another embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0230] In another embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptableP-654697-PCcarrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0231] In another embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0232] In another embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0233] In another embodiment, the invention provides a method for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0234] In one embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In a particular embodiment, the pharmaceutical composition comprising of dantrolene and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0235] In another embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In a particularP-654697-PCembodiment, the pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0236] In another embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0237] In another embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0238] In another embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0239] In another embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0240] In one embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.P-654697-PC

[0241] In another embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0242] In another embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0243] In another embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0244] In another embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0245] In another embodiment, the invention provides a method for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and lithium salt, and aP-654697-PCpharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0246] In one embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In a particular embodiment, the pharmaceutical composition comprising of dantrolene and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0247] In another embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0248] In another embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0249] In another embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0250] In another embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.P-654697-PC

[0251] In another embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0252] In one embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0253] In another embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In some embodiments, the pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0254] In another embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0255] In another embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprisingP-654697-PCintrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0256] In another embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0257] In another embodiment, the invention provides a method for ameliorating muscle weakness and / or muscle atrophy in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0258] In one embodiment, the invention provides a method for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In a particular embodiment, the pharmaceutical composition comprising of dantrolene and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0259] In another embodiment, the invention provides a method for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.P-654697-PC

[0260] In another embodiment, the invention provides a method for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0261] In another embodiment, the invention provides a method for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier. In a particular embodiment, the dantrolene is in nanoparticulate form.

[0262] In another embodiment, provided herein is a method for ameliorating motor neuron dysfunction in an amyotrophic lateral sclerosis (ALS) subject, comprising: intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier. In another embodiment, provided herein is a method for ameliorating motor neuron dysfunction in an amyotrophic lateral sclerosis (ALS) subject, comprising: intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene in nanoparticulate form and a pharmaceutically acceptable carrier.

[0263] In one embodiment, the invention provides a method for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In particular embodiments, the pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0264] In another embodiment, the invention provides a method for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical compositionP-654697-PCcomprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate. In some embodiments, the pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally, or intravenously.

[0265] In another embodiment, the invention provides a method for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0266] In another embodiment, the invention provides a method for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0267] In another embodiment, the invention provides a method for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intranasally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.

[0268] In another embodiment, the invention provides a method for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising intrathecally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a combination of dantrolene in nanoparticulate form and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, or lithium orotate.P-654697-PC

[0269] In another embodiment, the present invention provides methods for administration of dantrolene nanoparticles, alone or in combination with lithium salts, that eliminate both antidepressant sensitive behaviors (depression) and memory loss in an animal model (5XFAD mice) and reduce associated inflammation, pyroptosis, and synaptic destruction in this AD animal model, with no or minimal side effects or organ toxicity after chronic use of the dantrolene nanoparticles, alone or combined with lithium salts.

[0270] In another embodiment, the invention provides a method for treating depression comprising administering to a subject in need thereof a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In particular embodiments, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

[0271] In another embodiment, the invention provides a method for treating depression in an Alzheimer’s Disease subject comprising administering to the subject in need thereof a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the dantrolene is in nanoparticulate form. In an embodiment, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In various embodiments, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

[0272] In another embodiment, the invention provides a method for inhibiting a pathological intracellular Ca2+and / or Fe2+dysregulation, oxidative stress and inflammatory' pyroptosis activation, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting astrogliosis and microgliosis in a subject having depression, the method comprising administering to the subject a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the dantrolene is in nanoparticulate form. In certain embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In an embodiment, the pharmaceuticalP-654697-PCcomposition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

[0273] In another embodiment, the invention provides a method for inhibiting a pathological increase of intracellular Ca2+and Fe2+dysregulation, oxidative stress and pyroptosis activation, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting astrogliosis and microgliosis in an Alzheimer’s Disease subject having depression, the method comprising administering a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In certain embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In some embodiments, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

[0274] In another embodiment, the invention provides a method for increasing cytoprotective IL-10 and inhibiting a PSD-95 synapse protein loss in a subject having depression, the method comprising administering to the subject a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In certain embodiments, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

[0275] In another embodiment, the invention provides a method for increasing cytoprotective IL-10 and inhibiting a PSD-95 synapse protein loss to an Alzheimer’s Disease subject having depression, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier. In some embodiments, the dantrolene is in nanoparticulate form. In an embodiment, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In some embodiments, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.P-654697-PC

[0276] In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene, lithium salt, and a pharmaceutically acceptable carrier. In some embodiments of the pharmaceutical composition, the dantrolene is in nanoparticulate form. In certain embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In an embodiment, the pharmaceutical composition comprising dantrolene, a lithium salt, and a pharmaceutically acceptable carrier if formulated for intranasal, oral or intravenous administration.

[0277] Recent studies pioneered a novel approach of administrating intranasal dantrolene nanoparticles to increase its brain / blood concentration ratio, to promoted dantrolene CNS therapeutic effects while minimizing its peripheral side effects / organ toxicity. Intranasal dantrolene nanoparticles abolished memory loss as a disease-modifying drug, with no side effects or muscle or liver toxicity after up to 10 months of treatment. However, the therapeutic effects and potential mechanism of dantrolene to correct pathologies in MDD has not been investigated, particularly in AD.

[0278] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for use in the treatment of amyotrophic lateral sclerosis (ALS). In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for the manufacture of a medicament for treating amyotrophic lateral sclerosis (ALS).

[0279] In one embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier for use in the treatment of amyotrophic lateral sclerosis (ALS). In another embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier for the manufacture of a medicament for treating amyotrophic lateral sclerosis (ALS).

[0280] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for use in alleviating symptoms of amyotrophic lateral sclerosis (ALS). In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for the manufacture of a medicament for alleviating symptoms of amyotrophic lateral sclerosis (ALS).

[0281] In one embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier for use inP-654697-PCalleviating symptoms of amyotrophic lateral sclerosis (ALS). In another embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier for the manufacture of a medicament for alleviating symptoms of amyotrophic lateral sclerosis (ALS).

[0282] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for use in preventing or delaying the onset of amyotrophic lateral sclerosis (ALS). In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for the manufacture of a medicament for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS).

[0283] In one embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for use in preventing or delaying the onset of amyotrophic lateral sclerosis (ALS). In another embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier for the manufacture of a medicament for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS).

[0284] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for use in inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL-10 in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for the manufacture of a medicament for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL-10 in an amyotrophic lateral sclerosis (ALS) subject.

[0285] In one embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for use in inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / orP-654697-PCneurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL-10 in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for the manufacture of a medicament for inhibiting a pathological increase of intracellular Ca2+dysregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, and / or increasing cytoprotective IL-10 in an amyotrophic lateral sclerosis (ALS) subject.

[0286] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutical acceptable carrier for use in inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for the manufacture of a medicament for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject.

[0287] In one embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier for use in inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier for the manufacture of a medicament for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject.

[0288] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for use in restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for the manufacture of a medicament for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject.P-654697-PC

[0289] In one embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for use in restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier for the manufacture of a medicament for restoring motor neuron function in an amyotrophic lateral sclerosis (ALS) subject.

[0290] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for use in restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for the manufacture of a medicament to restore muscle strength in an amyotrophic lateral sclerosis (ALS) subject.

[0291] In one embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and lithium salt, and a pharmaceutically acceptable carrier for use in restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for the manufacture of a medicament for restoring muscle strength in an amyotrophic lateral sclerosis (ALS) subject.

[0292] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for use in reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for the manufacture of a medicament for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject.

[0293] In one embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for use in reversing weight loss in amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for the manufacture of a medicament for reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject.P-654697-PC

[0294] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for use in ameliorating muscle weakness in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for the manufacture of a medicament for ameliorating muscle weakness in an amyotrophic lateral sclerosis (ALS) subject.

[0295] In one embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for use in ameliorating muscle weakness in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for the manufacture of a medicament for ameliorating muscle weakness in an amyotrophic lateral sclerosis (ALS) subject.

[0296] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for use in ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene and a pharmaceutically acceptable carrier for the manufacture of a medicament for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject.

[0297] In one embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for use in ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject. In another embodiment, the invention provides a pharmaceutical composition comprising a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier for the manufacture of a medicament for ameliorating motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject.

[0298] In one embodiment, the invention provides a pharmaceutical composition comprising dantrolene, a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments of the pharmaceutical composition, the dantrolene is in nanoparticulate form. In certain embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In an embodiment, the pharmaceutical composition comprising dantrolene, a lithium salt, and a pharmaceutically acceptable carrier if formulated for intranasal, intrathecal, oral, or intravenous administration.P-654697-PC

[0299] In another embodiment, the present invention provides a pharmaceutical composition comprising dantrolene nanoparticles, alone or in combination with lithium salts, for use in treatment of both antidepressant sensitive behaviors (depression) and memory loss in an animal model (5XFAD mice) and reduce associated inflammation, pyroptosis, and synaptic destruction in this AD animal model, with no or minimal side effects or organ toxicity after chronic use of the dantrolene nanoparticles, alone or combined with lithium salts.

[0300] In another embodiment, the present invention provides a pharmaceutical composition comprising dantrolene, alone or in combination with lithium salts, and a pharmaceutically acceptable carrier for use in treatment of depression in a subj ect in need thereof. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In particular embodiments, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

[0301] In another embodiment, the present invention provides a pharmaceutical composition comprising dantrolene, alone or in combination with lithium salts, and a pharmaceutically acceptable carrier for use in treatment of depression in an Alzheimer’s Disease subject. In some embodiments, the dantrolene is in nanoparticulate form. In an embodiment, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In various embodiments, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

[0302] In another embodiment, the present invention provides a pharmaceutical composition comprising dantrolene, alone or in combination with lithium salts, and a pharmaceutically acceptable carrier for use in inhibiting a pathological intracellular Ca2+and / or Fe2+dysregulation, oxidative stress and inflammatory pyroptosis activation, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting astrogliosis and microgliosis in a subject having depression, in a the subject in need thereof. In some embodiments, the dantrolene is in nanoparticulate form. In certain embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In an embodiment, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.P-654697-PC

[0303] In another embodiment, the present invention provides a pharmaceutical composition comprising dantrolene, alone or in combination with lithium salts, and a pharmaceutically acceptable carrier for use in inhibiting a pathological increase of intracellular Ca2+and Fe2+dysregulation, oxidative stress and pyroptosis activation, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting astrogliosis and microgliosis in an Alzheimer’s Disease subject having depression. In an embodiment, the dantrolene is in nanoparticulate form. In certain embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In some embodiments, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

[0304] In another embodiment, the present invention provides a pharmaceutical composition comprising dantrolene, alone or in combination with lithium salts, and a pharmaceutically acceptable carrier for use in increasing cytoprotective IL-10 and inhibiting a PSD-95 synapse protein loss in a subject having depression. In an embodiment, the dantrolene is in nanoparticulate form. In some embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In certain embodiments, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

[0305] In another embodiment, the present invention provides a pharmaceutical composition comprising dantrolene, alone or in combination with lithium salts, and a pharmaceutically acceptable carrier for use in increasing cytoprotective IL-10 and inhibiting a PSD-95 synapse protein loss to an Alzheimer’s Disease subject having depression. In some embodiments, the dantrolene is in nanoparticulate form. In an embodiment, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In some embodiments, the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

[0306] In another embodiment, the invention provides a pharmaceutical composition comprising dantrolene, a lithium salt, and a pharmaceutically acceptable carrier. In some embodiments of the pharmaceutical composition, the dantrolene is in nanoparticulate form. In certain embodiments, the lithium salt is lithium chloride, lithium carbonate or lithium orotate. In an embodiment, the pharmaceutical composition comprising dantrolene, a lithium salt, and a pharmaceutically acceptable carrier if formulated for intranasal, oral or intravenous administration.P-654697-PC

[0307] In some embodiments, the pharmaceutical compositions of the present invention comprise dantrolene, or salt(s) or prodrug(s) or derivative(s) of any purity or sustained release formulations thereof for use in the treatment of ALS or a related disorder. In some embodiments, the pharmaceutical compositions of the present invention comprise a combination of dantrolene or salt(s) or prodrug(s) or derivative(s) of any purity or sustained release formulations thereof and a lithium salt.

[0308] The methods described herein may also be useful for any one or more of the following: 1) preventing loss of skeletal muscle associated with amyotrophic lateral sclerosis; 2) strengthening skeletal muscle in an individual having amyotrophic lateral sclerosis; 3) treatment of muscle wasting associated with amyotrophic lateral sclerosis; 4) treating dyspnea associated with muscle changes in amyotrophic lateral sclerosis; 5) treating dysarthria associated with muscle changes in amyotrophic lateral sclerosis; 6) treating dysphagia associated with muscle changes in amyotrophic lateral sclerosis; and 7) improving fatigue resistance of muscle in amyotrophic lateral sclerosis. Skeletal muscle includes, but is not limited to, gastrocnemius muscle, tibialis muscle, soleus muscle, and extensor digitorum longus (EDL) muscle, quadriceps, hamstrings, postural muscles, hand muscles, triceps, biceps, masseter and other jaw muscles, and intercostal and other respiratory muscles. The present application encompasses any of these methods.

[0309] In some embodiments, the individual has been diagnosed with or is suspected of having amyotrophic lateral sclerosis. In some embodiments, the individual exhibits one or more symptoms associated with amyotrophic lateral sclerosis. In some embodiments, the individual has been diagnosed with or is suspected of having depression. In some embodiments, the individual has AD and has been diagnosed with or is suspected of having depression. In some embodiments, the individual is a human. In some embodiments, the individual is at least about any of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years old. In some embodiments, the individual is a male. In some embodiments, the individual is a female. In some embodiments, the individual has been previously treated for amyotrophic lateral sclerosis. In some embodiments, the individual has not previously been treated for amyotrophic lateral sclerosis.

[0310] Other additional therapies that can be used in conjunction with drug combination(s) according to the present invention, may comprise one or more drug(s) that ameliorate(s) symptoms of ALS, one or more drug(s) that could be used for palliative treatment of ALS or one or more drug(s) currently evaluated in the frame of clinical trials for treating of ALS. Preferably, said oneP-654697-PCor more drug(s) is / are selected from AEOL 10150, arimoclomol, AVP-923, botulinum toxin type B (Myobloc), ceftriaxone, celastrol, celecoxib, cistanche total glycosides, coenzyme Q10, copaxone, creatine, creatinine, dronabinol, erythropoietin, escitalopram (Lexapro), glatiramer acetate, granulocyte-colony stimulating factor (G-CSF), growth hormone (Somatropin), GSK1223249, indinavir, insulin-like growth factor-1 (IGF-I), IGF-l-AAV, KNS-760704, leteprinim, leuprolide, levetiracetam, MCI- 186, mecobalamin, minocycline, modafinil, Naaladase inhibitor, N-Acetylcysteine, NBQX, nimesulide, nimodipine, olanzapine, olesoxime (TRO 19622), ONO-2506, oxepa, pioglitazone, R(+) pramipexole dihydrochloride monohydrate, olesoxime, oxandrolone, quinidine, phenyl butyrate, SB-509, Scriptaid, sNN0029, somatropine, talampanel, tamoxifen, tauroursodeoxycholic acid, TCH346, testosterone, thalidomide, trehalose, tretinoin, vitamin E, YAM80 or from 17-beta-estradiol, 2-MPPA (2-(3-mercaptopropyl)pentanedioic acid), 3,4-diaminopyridine, 5 -hydroxytry ptophan, 7-nitroindazole, alpha-lipoic acid, AM1241, aminophylline, angiogenin, anti-human SOD1 antibody, antisense peptide nucleic acid directed against p75(NTR), AP7, apocynin, BAPTA-AM, BDNF, BN82451, cannabinol, cardiotrophin-1, CD4 antibodies, CNTF, colivelin, dietary copper, corticotrophin, cyclophosphamide, Delta(9)-tetrahydrocannabinol, DHEA, diazepam, dietary zinc, diltiazem, DMPO, DP- 109, DP-460, edaravone, EGCG, epigallocatechin gallate, etidronate, FeTCPP, fluvoxamine, folic acid, gabapentin, galectin-1, GDNF, ginseng, GPI-1046, guanidine, HGF, humanin, IFN-alpha, interleukin-3, ivermectin, L-745,870, L-carnitine, L-DOPA, lecithinized SOD, lenalidomide, leupeptin, LIF, L-NAME, lysine acetylsalicylate, melatonin, mepivacaine, methamphetamine, methylcobalamin, MK-801, MnTBAP, modafinil, morphine, Neu2000, NGF, nordihydroguaiaretic acid, nortriptyline, NT3, olmesartan, penicillamine, pentoxifylline, pimozide, polyamine-modified catalase, pramipexole, prednisone, progesterone, promethazine, putrescine-modified catalase, pyruvate, rasagiline, RK35, Ro 28-2653, rofecoxib, RPR 119990, RX77368, SB203580, selegiline, semapimod, sertraline, SS-31, SSR180575, stabilized siRNA against human Cu, Zn-superoxide dismutase (SOD1), tacrolimus, tamsulosin hydrochloride, TAT-modified Bcl-X(L), TGF-beta2, tianeptine, trientine, TRO 19622, U-74389F, VEGF, vincristine, WHI-P131, WIN55, 212-2, WX-340, xaliproden, ZK 187638 and zVAD-fmk.

[0311] Other additional therapies that can be used in conjunction with drug combination(s) according to the present invention, may comprise one or more drug(s) that ameliorate(s) symptoms of depression (including in the context of AD). Preferably, said one or more drug(s) is / are selectedP-654697-PCfrom mono-amine oxidase inhibitors (MAOI), tricyclic antidepressants (TCA), serotonin specific reuptake inhibitors (SSRI), serotonin noradrenergic reuptake inhibitors (SNRI), noradrenaline reuptake inhibitor (NRI), “natural products” (such as Kava-Kava, St. John's Wort), dietary supplement (such as s-adenosylmethionine) and others. More specifically, drugs used in the treatment of depression include, but are not limited to imipramine, amitriptyline, desipramine, nortriptyline, doxepin, protriptyline, trimipramine, maprotiline, amoxapine, trazodone, bupropion, chlomipramine, fluoxetine, citalopram, sertraline, paroxetine, tianeptine, nefazadone, venlafaxine, desvenlafaxine, duloxetine, reboxetine, mirtazapine, phenelzine, tranylcypromine, and / or moclobemide.

[0312] The invention also relates to a method of treating ALS, depression (including in the context of AD) or a related disorder, the method comprising simultaneously, separately or sequentially administering to a subject in need thereof a drug combination as disclosed above.

[0313] The drugs or compositions of the invention may be administered repeatedly to the subject.

[0314] The compositions of the invention typically comprise one or several pharmaceutically acceptable carriers or excipients.

[0315] Therapy according to the invention may be provided at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital, so that the doctor can observe the therapy's effects closely and make any adjustments that are needed.

[0316] The duration of the therapy depends on the stage of the disease, the age and condition of the patient, and how the patient responds to the treatment.

[0317] Additionally, a person having a greater risk of developing an additional neuropathic disorder (e.g., a person who is genetically predisposed to or have, for example, diabetes, or is being under treatment for an oncological condition, etc.) may receive prophylactic treatment to alleviate or to delay eventual neuropathic response.

[0318] The dosage, frequency and mode of administration of each drug can be controlled independently. Combination therapy may be given in on-and-off cycles that include rest periods so that the patient's body has a chance to recover from any as yet unforeseen side-effects. The drugs may also be formulated together such that one administration delivers both drugs.P-654697-PCFormulation of Pharmaceutical Compositions

[0319] The administration of each drug of the combination may be by any suitable means that results in a concentration of the drug that, combined with the other component, is able to ameliorate the patient’s condition.

[0320] While it is possible for the active ingredients of the combination to be administered as the pure chemical it is preferable to present them as a pharmaceutical composition, also referred to in this context as pharmaceutical formulation. Possible compositions include those suitable for intranasal, intrathecal, inhalant, oral, rectal, topical (including transdermal, buccal and sublingual), or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.

[0321] More commonly these pharmaceutical formulations are prescribed to the patient in “patient packs” containing a number of dosing units or other means for administration of metered unit doses for use during a distinct treatment period in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in traditional prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions. Thus, the invention further includes a pharmaceutical formulation, as herein before described, in combination with packaging material suitable for said formulations. In such a patient pack the intended use of a formulation for the combination treatment can be inferred by instructions, facilities, provisions, adaptations and / or other means to help using the formulation most suitable for the treatment. Such measures make a patient pack specifically suitable for and adapted for use for treatment with the combination of the present invention.

[0322] The drug may be contained in any appropriate amount in any suitable carrier substance and may be present in an amount of 1-99% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for the intranasal, intrathecal, oral, parenteral (e.g., intravenously, intramuscularly), rectal, cutaneous, vaginal, inhalant, skin (patch), or ocular administration route. Thus, the composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols.P-654697-PC

[0323] The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Gennaro and the Encyclopedia of Pharmaceutical Technology).

[0324] Pharmaceutical compositions according to the invention may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or time period after administration.

[0325] The controlled release formulations include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain drug action during a predetermined time period by maintaining a relatively, constant, effective drug level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active drug substance; (iv) formulations that localize drug action by, e.g., spatial placement of a controlled release composition adjacent to or in the diseased tissue or organ; and (v) formulations that target drug action by using carriers or chemical derivatives to deliver the drug to a particular target cell type.

[0326] Drug administration in the form of a controlled release formulation is especially preferred in cases in which the drug in combination, has (i) a narrow therapeutic index (i.e., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; in general, the therapeutic index, TI, is defined as the ratio of median lethal dose (LD50) to median effective dose (ED₅₀)); (ii) a narrow absorption window in the gastro-intestinal tract; or (iii) a very short biological half-life so that frequent dosing during a day is required in order to sustain the plasma level at a therapeutic level.

[0327] A number of strategies can be pursued to obtain controlled release in which the release rate outweighs the metabolism rate of the drug in question. Controlled release may be obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the drug is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the drug in a controlled manner (single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes).P-654697-PCAdministration Routes

[0328] The dosage of the compositions described herein administered to an individual (such as a human) may vary with the particular composition, the method of administration, and the particular stage of amyotrophic lateral sclerosis. The amount should be sufficient to produce a desirable response, such as a therapeutic or prophylactic response against amyotrophic lateral sclerosis. In some embodiments, the amount of the composition is a therapeutically effective amount. In some embodiments, that amount of the composition is a prophylactically effective amount.

[0329] The compositions described herein can be administered to an individual (such as human) via various routes, including, for example, inhalation (e.g., intranasal), intravenous, intra-arterial, intraperitoneal, intraportal, intrapulmonary, oral, intravesicular, intramuscular, intra-tracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, sustained continuous release formulation of the composition may be used.Compositions for Intranasal Administration

[0330] In a preferred embodiment, administration route for drugs and drug combinations as disclosed above is the intranasal route. In a particular embodiment, the drug is absorbed across the nasal mucosa.

[0331] The intranasal delivery compositions can appear in conventional forms, for example, aerosols, solutions, suspensions, or topical applications, or in lyophilized form. Typical compositions include drugs and drug combinations as disclosed above and a pharmaceutically acceptable excipient which can be a carrier or a diluent. For example, the active agent may be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier. When the active agent is mixed with a carrier, or when the carrier serves as a diluent, it can be solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for the active agent. Some examples of suitable carriers are water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, the carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.P-654697-PC

[0332] Intranasal delivery compositions can further contain a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

[0333] The formulations can be mixed with auxiliary agents which do not deleteriously react with the active agent. Such additives can include wetting agents, emulsifying and suspending agents, salt for influencing osmotic pressure, buffers and / or coloring substances preserving agents, sweetening agents or flavoring agents. The compositions can also be sterilized if desired.

[0334] If a liquid carrier is used, the preparation can be in the form of a liquid such as an aqueous liquid suspension or solution. Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution.

[0335] The drugs and drug combinations as disclosed above may be provided as a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, frozen dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. The composition can optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these. A unit dosage form can be in individual containers or in multi-dose containers.

[0336] Polymeric nanoparticles serve as carriers for a broad variety of ingredients. The active components may be either dissolved in the polymetric matrix or entrapped or adsorbed onto the particle surface. Polymers suitable for the preparation of organic nanoparticles include cellulose derivatives and polyesters such as poly(lactic acid), poly(glycolic acid) and their copolymer. Due to their small size, their large surface area / volume ratio and the possibility of functionalization of the interface, polymeric nanoparticles are ideal carrier and release systems. If the particle size is below 50 nm, they are no longer recognized as particles by many biological and synthetic barrier layers but act like molecularly disperse systems.

[0337] In one embodiment, the preparation can contain a drugs and drug combinations as disclosed above, dissolved or suspended in a liquid carrier, such as an aqueous carrier, for aerosol application. The carrier can contain additives such as solubilizing agents, e.g., propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabens

[0338] Changes in osmolarity of a formulation can cause cells to expand or shrink, enhancing intracellular or extracellular transport mechanisms along olfactory and trigeminal nerves to theP-654697-PCCNS. In addition to cell shrinking, it is possible that the hypertonic solution caused epithelial changes, such as increased mucus secretion, which hindered transport into the brain.

[0339] The pH of the nasal formulation and ionization state of the drug can affect the efficiency of intranasal delivery to the CNS. For example, positively charged drugs may form electrostatic interactions with the negatively charged nasal epithelial cells, effectively hindering transport beyond the nasal mucosa and into the CNS.

[0340] Mucociliary clearance mechanisms rapidly remove drugs from the delivery site, reducing contact with the nasal epithelium and delivery into the CNS after intranasal administration. Several approaches, including use of mucoadhesive agents, e.g., sodium hyaluronate, chitosan, acrylic acid derivatives, lectin, and low methylated pectin, surface-engineered nanoparticles, efflux transporter inhibitors, and vasoconstrictors, have been utilized to reduce clearance, to prolong the residence time of the formulation at the delivery site, and to increase transport along direct pathways to the CNS. Increasing the residence time at the delivery site potentially enhances delivery into the CNS along olfactory and trigeminal nerves, the vasculature, or CSF and lymphatic channels. Mucoadhesives used in combination with microemulsion formulations show the greatest potential in terms of enhancing brain uptake and drug targeting to the CNS.

[0341] Surface engineering of nanoparticles with ligands that bind to specific cell surfaces is a promising approach to reduce clearance and enhance targeted delivery to the CNS. For example, the lectin, ulex europeus agglutinin I (UEA I), binds to receptors located predominantly in the olfactory epithelium, while WGA recognizes sugar molecules and binds to receptors expressed throughout the olfactory and respiratory epithelia. UEA I nanoparticles could enhance delivery to the CNS along olfactory pathways, whereas WGA nanoparticles could enhance delivery to the CNS along multiple pathways, including neural and vascular pathways. Intranasal studies using UEA I or WGA conjugated PEG-PLA nanoparticles loaded with a fluorescent marker resulted in increased delivery to different brain areas, including the olfactory bulbs, olfactory tract, cerebrum, and cerebellum, compared to unmodified nanoparticles, without resulting in nasal ciliotoxicity.

[0342] Reducing clearance from the nasal cavity due to efflux from transport proteins or due to absorption into the nasal vasculature are additional strategies that have been explored to increase the residence time at the delivery site and to enhance the efficiency of intranasal delivery to the CNS. Intranasal pretreatment with an inhibitor (rifampin) of the P-gp efflux transport protein prior to intranasal administration of a P-gp substrate (verapamil) resulted in significantly greater brainP-654697-PCuptake as a result of reduced clearance from P-gp-mediated efflux. Reducing clearance into the blood from the site of delivery by using a vasoconstrictor could allow more of the drug to be available for direct transport into the CNS. The reduced clearance from the nasal epithelium into the blood leads to increased deposition in the olfactory epithelium and increased delivery along olfactory nerve pathways to the olfactory bulbs.

[0343] Optimal delivery to the CNS along neural pathways is associated with delivery of the agent to the upper third of the nasal cavity. Although a supine position may be used, another position for targeting the olfactory region is the “praying to Mecca” position, with the head down-and-forward. A supine position with the head angle at 70° or 90° may be suitable for efficient delivery to the CSF using a tube inserted into the nostrils to deliver the drug via intranasal administration.

[0344] For intranasal drug administration nose drops may be administered over a period of 10-20 minutes alternating nostrils every 1-2 minutes to allow the solution to be absorbed into the nasal epithelium. This non-invasive method does not involve inserting the device into the nostril. Instead, drops are placed at the opening of the nostril, allowing the individual to sniff the drop into the nasal cavity. Other administration methods in anesthetized individual involve sealing the esophagus and inserting a breathing tube into the trachea to prevent the nasal formulation from being swallowed and to eliminate issues related to respiratory distress. Flexible tubing can be inserted into the nostrils for localized delivery of a small volume of the drug solution to the respiratory or olfactory epithelia, depending on the length of the tubing

[0345] Intranasal delivery devices are known in the art. Thus, any device suitable for delivery of drug to nasal mucosa may be used. Non-limiting examples of devices useful for the administration of liquid compositions include vapor devices (e.g., vapor inhalers), drop devices (e.g., catheters, single-dose droppers, multi-dose droppers, and unit-dose pipettes), mechanical spray pump devices (e.g., squeeze bottles, multi-dose metered-dose spray pumps, and single / duo-dose spray pumps), bi-directional spray pumps (e.g., breath-actuated nasal delivery devices), gas-driven spray systems / atomizers (e.g., single- or multi-dose HFA or nitrogen propellant-driven metered-dose inhalers, including traditional and circumferential velocity inhalers), and electrically powered nebulizers / atomizers (e.g., pulsation membrane nebulizers, vibrating mechanical nebulizers, and hand-held mechanical nebulizers). Non-limiting examples of devices useful for the administration of powder compositions (e.g., lyophilized or otherwise dried pooled compositions) include mechanical powder sprayers (e.g., hand-actuated capsule-based powder spray devices and hand-P-654697-PCactuated powder spray devices, hand actuated gel delivery devices), breath-actuated inhalers (e.g., single- or multi-dose nasal inhalers and capsule-based single- or multi-dose nasal inhalers), and insufflators (e.g., breath-actuated nasal delivery devices).

[0346] Use of metered sprays for intranasal delivery can also be accomplished by including the active ingredient in a solution or dispersion in a suitable medium which can be administered as a spray. Representative devices of this type are disclosed in the following patents, patent applications, and publications: WO03 / 026559, WO02 / 011800, WO00 / 51672, WO02 / 068029, WO02 / 068030, WO02 / 068031, WO02 / 068032, WO03 / 000310, WO03 / 020350, WO03 / 082393, WO03 / 084591, WO03 / 090812, WO00 / 41755.

[0347] In addition to the foregoing, the compounds can also be administered intranasally in the form of irrigations and douches, as is known in the art. Nasal irrigation involves regularly flooding the nasal cavity with solution, which includes the drug. Nasal douches are typically used by filling a nasal douche with a solution including the drug, inserting the nozzle from the douche into one nostril, opening one's mouth to breathe, and causing the solution to flow into one nostril, rinse round the septum, and discharge from the other nostril. Means to deliver drug to the upper portion of the nasal cavity, such as the cribriform, are of particular interest herein.Compositions for Intrathecal Administration

[0348] In a preferred embodiment, administration route for drugs and drug combinations as disclosed above is the intrathecal route. In some embodiments, the pharmaceutical compositions of the present invention are preferably specially formulated for intrathecal administration by, for example, intrathecal injection as, for example, a sterile solution.

[0349] Pharmaceutical compositions of this invention suitable for intrathecal administration may comprise one or more compounds described herein in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain a preservative, sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the cerebrospinal fluid of the intended recipient or suspending or thickening agents.

[0350] Exemplary suitable aqueous and nonaqueous carriers which may be employed in the intrathecal compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils,P-654697-PCsuch as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0351] These intrathecal compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0352] In some embodiments, the solution is delivered to the CNS by administering into the cerebrospinal fluid (CSF) of a subject in need of treatment. In some embodiments, intrathecal administration is used to deliver a solution into the CSF. As used herein, intrathecal administration (also referred to as intrathecal injection) refers to an injection into the spinal canal (intrathecal space surrounding the spinal cord). Various techniques may be used including, without limitation, lateral cerebroventricular injection through a burrhole or cisternal or lumbar puncture or the like, via, for example, a catheter. Exemplary methods are described in Lazorthes et al., Advances in Drug Delivery Systems and Applications in Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1: 169-179, the contents of which are incorporated herein by reference.

[0353] According to the present invention, the solution may be injected at any region surrounding the spinal canal. In some embodiments, the solution is injected into the lumbar area or the cistema magna or intraventricularly into a cerebral ventricle space. As used herein, the term “lumbar region” or “lumbar area” refers to the area between the third and fourth lumbar (lower back) vertebrae and, more inclusively, the L2-S1 region of the spine. Typically, intrathecal injection via the lumbar region or lumber area is also referred to as “lumbar IT delivery” or “lumbar IT administration.”

[0354] In some embodiments, “intrathecal administration” or “intrathecal delivery” according to the present invention refers to lumbar intrathecal administration or delivery, for example, delivered between the third and fourth lumbar (lower back) vertebrae and, more inclusively, the L2-S 1 region of the spine. It is contemplated that lumbar intrathecal administration or delivery distinguishesP-654697-PCover cistema magna delivery in that lumbar intrathecal administration or delivery provides better and more effective delivery to the distal spinal canal, while cisterna magna delivery, among other things, typically does not deliver well to the distal spinal canal.

[0355] In some embodiments, the solution is injected into the thoracic or cervical region of the spinal column of said patient. In another embodiment, the solution is intrathecally injected into the thoracic or cervical region of the spinal column of said patient, preferably the cervical region. The term “cistema magna” refers to the space around and below the cerebellum via the opening between the skull and the top of the spine. Typically, intrathecal injection via cisterna magna is also referred to as “cistema magna delivery.” The term “cerebral ventricle” refers to the cavities in the brain that are continuous with the central canal of the spinal cord. Typically, injections via the cerebral ventricle cavities are referred to as intravetricular Cerebral (ICV) delivery. Thus, in some embodiments the solution is injected between the Cl and C7 vertebrae, between the Cl and C5 vertebrae, between the Cl and C3 vertebrae, or at the Cl vertebrate (i.e. immediately above or below the Cl vertebrae) when intrathecally administered.

[0356] Alternatively, the intrathecal administration occurs in the brain, via intracranial intrathecal administration. The catheter can be positioned in any of the temporal lobes, with the hippocampus the preferred anatomical target.Solid Dosage Forms for Oral Use

[0357] In some embodiments, administration route for drugs and drug combinations as disclosed above is the oral route. Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert excipients or fillers (e.g., sucrose, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., stearic acid, silicas, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.P-654697-PC

[0358] The drugs may be mixed in the tablet or may be partitioned. For example, a first drug is contained on the inside of the tablet, and a second drug is on the outside, such that a substantial portion of the second drug is released prior to the release of the first drug.

[0359] Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid excipient (e.g., potato starch, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, liquid paraffin, or olive oil. Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner.Liquids for Oral Administration

[0360] Powders, dispersible powders, or granules suitable for preparation of an aqueous suspension by addition of water are convenient dosage forms for oral administration. Formulation as a suspension provides the active ingredient in a mixture with a dispersing or wetting agent, suspending agent, and one or more preservatives. Suitable suspending agents are, for example, sodium carboxymethylcellulose, methylcellulose, sodium alginate, and the like.Parenteral Compositions

[0361] Although less preferred, the pharmaceutical composition may also be administered parenterally by injection, infusion or implantation (intravenous, intramuscular, subcutaneous, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. Formulating and preparing such compositions are known to those skilled in the art of pharmaceutical formulation.Alternative Routes

[0362] Although less preferred and less convenient, other administration routes, and therefore other formulation, may be contemplated. In this regard, for rectal application, suitable dosage forms for a composition include suppositories (emulsion or suspension type), and rectal gelatin capsules (solutions or suspensions). In a typical suppository formulation, the active drug(s) are combined with an appropriate pharmaceutically acceptable suppository base such as cocoa butter, esterified fatty acids, glycerinated gelatin, and various water-soluble or dispersible bases like polyethylene glycols. Various additives, enhancers, or surfactants may be incorporated.P-654697-PC

[0363] The pharmaceutical compositions may also be administered topically on the skin for percutaneous absorption in dosage forms or formulations containing conventionally non-toxic pharmaceutical acceptable carriers and excipients including microspheres and liposomes. The formulations include creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters, and other kinds of transdermal drug delivery systems. The pharmaceutically acceptable carriers or excipients may include emulsifying agents, antioxidants, buffering agents, preservatives, humectants, penetration enhancers, chelating agents, gel-forming agents, ointment bases, perfumes, and skin protective agents.

[0364] Emulsifying agents may be naturally occurring gums (e.g., gum acacia or gum tragacanth).

[0365] The preservatives, humectants, penetration enhancers may be parabens, such as methyl or propyl p-hydroxybenzoate, and benzalkonium chloride, glycerin, propylene glycol, urea, etc.

[0366] The pharmaceutical compositions described above for topical administration on the skin may also be used in connection with topical administration onto or close to the part of the body that is to be treated. The compositions may be adapted for direct application or for application by means of special drug delivery devices such as dressings or alternatively plasters, pads, sponges, strips, or other forms of suitable flexible material.Dosages and Duration of the Treatment

[0367] It will be appreciated that the drugs of the combination may be administered concomitantly, either in the same or different pharmaceutical formulation or sequentially. If there is sequential administration, the delay in administering one of the active ingredients should not be such as to lose the benefit of the efficacious effect of the combination of the active ingredients. A minimum requirement for a combination according to this description is that the combination should be intended for combined use with the benefit of the efficacious effect of the combination of the active ingredients. The intended use of a combination can be inferred by facilities, provisions, adaptations and / or other means to help using the combination according to the invention.

[0368] It will be understood that the amount of the drug actually administered will be determined by a physician, in the light of the relevant circumstances including the condition or conditions to be treated, the exact composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic,P-654697-PCpharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage used.

[0369] Although the active drugs of the present invention may be administered in divided doses, for example two or three times daily, a single daily dose of each drug in the combination is preferred, with a single daily dose of all drugs in a single pharmaceutical composition (unit dosage form) being most preferred. The term “unit dosage form” refers to physically discrete units (such as capsules, tablets, or loaded syringe cylinders) suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material or materials calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.

[0370] Administration can be one to several times daily for several days to several years and may even be for the life of the patient. Chronic or at least periodically repeated long-term administration will be indicated in most cases.

[0371] The drugs may be administered simultaneously, i.e., approximately at the same time, although not necessarily exactly at the same time or through the same formulation.

[0372] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.EXAMPLES

[0373] In the present studies, the therapeutic effectiveness versus side effects were investigated, as were the potential mechanisms of intranasal dantrolene nanoparticles to ameliorate Example 1Determining the effects and mechanisms of dantrolene in inhibiting upstream Ca2+dysregulation and downstream inflammatory pyroptosis in in vitro ALS cell models.

[0374] Materials and Methods

[0375] Dantrolene formulations. Dantrolene is lipophilic and 20 mg of dantrolene requires 60 mL of solution to dissolve for MH treatment. A new FDA-approved formulation of dantrolene, Ryanodex, can be reconstituted rapidly with 250 mg of dantrolene in 5 ml water. Ryanodex does not increase solubility but instead delivers dantrolene in the form of crystal nanoparticles. Dantrolene can be dissolved in the Ryanodex formulation vehicle. Physicochemical features of dantrolene in Ryanodex formulation (DNRF) prepared in the Wei lab have been determined usingP-654697-PChigh-performance liquid chromatography (HPLC) (Fig. 86) by the Eagle Pharmaceutical Company (Woodcliff Lake, NJ, USA). The particle size of DNRF prepared in the Wei lab, is 706 nm at -20°C and 713 nm at room temperature, as measured by dynamic light scattering (DLS). The Z-average diameter of Ryanodex is 518 nm at -20°C and 508 nm at room temperature. Preliminary data suggest that DNRF prepared in the Wei lab forms nanoparticles like the reconstituted Ryanodex produced by Eagle Pharmaceutical.

[0376] Selection of the ALS in vitro cell models and tissue cultures. The TDP-43 Dox-inducible QB 1-293 stable cell lines (iGFP-WT and iGFP-NLSm) were selected for the following reasons: 1) They possess the human TDP-43 mutation, which exists in almost all ALS patients. Thus, a drug capable of inhibiting the effect of TDP-43 mislocalization is likely translational and would be expected to work in ALS patients; 2) The mutated TDP-43 (iGFP-NLSm) demonstrated LPS-induced mitochondrial dysfunction, pathological elevation of intracellular ROS, and cell death, all of which could be inhibited with a clinically available concentration of dantrolene. To strengthen the scientific rigor use primary cortical neurons cultured from fetal brains at gestational day 16 from either SOD1G93A transgenic mice or wild type control will also be used for following rationale: 1) These cell cultures use the same ALS transgenic mice as in the in vivo animal study in Example 2) Primary cortical neuronal cultures are more translational to study drug effectiveness in the ALS. The cultures and maintenance of QBL293 cell lines is the same as previously described (Porta, S. et al. Neuropathol. Appl. Neurobiol. 47, 1033-1049, (2021)). The primary cortical neuronal cultures and maintenance will be same as described previously (Wei, H. et al. Brain Res 1037, 139-147, (2005); Zhao, Y. et al. J. Pharmacol. Exp. Ther. 333, 14-22, (2010)).

[0377] Determining cell viability or death and dose- and time-response. Both the MTT reduction assay and the LDH release assay to determine cell viability (MTT) and cell death (LDH) were used. Dose- and time-response of dantrolene protection and TD50 of inflammation (LPS) were assessed. In addition, glutamate excitotoxicity (glutamate / NMDA / AMPA) will be measured to determine the optimum dose of dantrolene to provide cytoprotection and TD50 of LPS / glutamate / NMDA / AMPA, as in Fig. 2 and previous studies (Gao, X. et al. Curr Alzheimer Res 17, 1311-1319, (2020); Qiao, H. et al. Anesthesiology 127, 490-501, (2017)).

[0378] Dantrolene and Vehicle Control. Dantrolene will be dissolved in pure DMSO, as performed in the cell culture studies in previous publications (Qiao, H. et al. Anesthesiology 127, 490-501, (2017); Wang, Y. et al. Anesthesiology 132, 1062-1079, (2020); Yang, M. et al. JP-654697-PCAlzheimers Dis 67, 137-147, (2019)). The lowest concentration of DMSO that is not toxic to cells by itself will be determined in preliminary experiments.

[0379] Determining the mutated TDP-43 or SOD1 protein aggregation. The translocation of TDP-43 from the nucleus into the cytosol and aggregation is one of the hallmarks of ALS TDP-43 pathology. Immunohistochemical (IHC) immunofluorescence (IF) assays using previously described methods (Porta, S. et al. Neuropathol. Appl. Neurobiol. 47, 1033-1049, (2021); Wang, Y. et al. Anesthesiology 132, 1062-1079, (2020)) will be utilized to compare the amount of aggregated cytosolic TDP-43 between the two cell lines and the effects of dantrolene to inhibit such proteins’ translocation and aggregation. Similarly, the aggregation of SOD1 G93A will be determined, and its enzyme activity will be measured and compared between WT and SOD1G93A mutation in cortical neurons, using a method described (Wang, Q. etal. Toxicol. Appl. Pharmacol.250, 291-298, (2011)) in detail previously.

[0380] LPS / glutamateNMDA / AMPA-mediated changes in Ca2+channel proteins. It is hypothesized that the TDP-43 or SOD1 mutation in ALS cell models will upregulate Ca2+channels that contribute to pathological Ca2+dysregulation and elevation (RyRs-2 / InsP3R- 1 / NMDARs / AMPARs). Western blot and IHC will be used to determine changes in various Ca2+channel proteins, using detailed methods described in previous publications (Wei, H. et al. Brain Res 1037, 139-147, (2005); Wang, Y. etal. Anesthesiology 132, 1062-1079, (2020)).

[0381] Determining LPS / glutamate / NMDA / AMPA-mediated changes of [Ca2+]c, [Ca2+]mand [Ca2+]er. Ca2+imaging will be used to measure [Ca2+]c / [Ca2+]m / [Ca2+]er, using methods described in previous publications (Wang, Y. et al. Anesthesiology 132, 1062-1079, (2020); Yang, H. et al. Anesthesiology 109, 243-250, (2008)). In order to increase scientific rigor, a second method will be used to measure [Ca2+]c / [Ca2+]m / [Ca2+]c, utilizing jellyfish-specific photoprotein aequorinbased probes in previously described setup (Gao, X. et al. Curr Alzheimer Res 17, 1311-1319, (2020)). Furthermore, advanced methods will be used to measure Ca2+concentrations in cell compartments as described previously (see, e.g, Joseph, J. D. et al. Anesthesiology 121, 528-537, (2014)).

[0382] Determining dantrolene ’s effect on mitochondrial Ca2+overloading-mediated mitochondrial dysfunction and cell damage. Basal oxygen consumption rate and ATP calculation were measured using Seahorse with the method described previously (Leung, D. T. H. & Chu, S. Methods Mol Biol 1710, 285-293, (2018)). Cells grown in Seahorse XFp miniplates were analyzedP-654697-PCwith cell energy phenotype tests and Seahorse XFp Analyzer software. Cytosolic ATP levels and luminescence are measured with the ATPLite Luminescence ATP Detection Assay System (Perkin-Elmer) and a BioTech Synergy H1 plate reader, as previously described (Ren, G. etal. Sci Rep 7, 12378, (2017)). This will be compared to the ATP levels calculated based on the oxygen consumption rate in the Seahorse setup. Mitochondrial proton leak indicates mitochondrial and cell damage by apoptosis. It will be measured based on OCR using Seahorse, following manufacturer’s instructions (Jang, D. H., et al. Clin Toxicol (Phila) 54, 303-307, (2016).

[0383] Dantrolene ’s effect on oxidative stress. Cellular ROS levels were measured using fluorescent dye DCFH-DA, described previously (Wei, H. et al. J Neurochem 75, 81-90, (2000); Liang, G. et al. Anesth Analg 106, 492-500, table of contents, (2008)). Protein oxidation will also be measured by determining the lipid byproduct, malondialdehyde (MDA) with a commercially available MDA assay kit (Abeam, abl 18970), following the manufacturer’s protocol (Phan, K. et al. Brain Commun 5, fcac340, (2023); Phan, K. et al. Sci Rep 10, 3640, (2020)). The MDA-modified protein and 4-HNE act as indicators of lipid peroxidation and have been determined using western blot, in accordance with the previously described methods (Wang, Y. etal. Anesthesiology 132, 1062-1079, (2020); Ren, G. etal. Sci Rep 7, 12378, (2017)).

[0384] Dantrolene ’s effect on activation of the Ca2+dependent calpain protease. The Calpain Activity Assay Kit (Abeam, ab65308) will be used to measure the enzyme activity of calpain, following company instructions and using the method described (Hoyle, C., e / al., Immunology 165, 460-480, doi:10.1111 / imm.13454 (2022)). Briefly, the cell lysate is collected in extraction buffer prior to centrifugation at 18000 g at 4°C for 5 min, after which the supernatant is incubated with reaction buffer and fluorescent substrate (Ac-LLY-AFC) for 1 h at 37°C, prior to measuring fluorescence (400 / 505 nm). Furthermore, calpain protein levels will be determined using both western blot and IHC / IF, with previously described methods (Wang, Y. et al. Anesthesiology 132, 1062-1079, (2020)).

[0385] Determination activation of the pyroptosis pathway. The changes in the primary pyroptosis pathway activation proteins (N RLP3, caspase- 1 and NT-GSDMD, Fig. 1), will be determined using Western Blot and IHC with detailed methods described previously. (Ren, G. et al. Sci Rep 7, 12378, (2017); Piplu Bhuiyan, W. Z., Ge Liang, Bailin Jiang, Robert Vera, Rebecca Chae, Kyulee Kim, Lauren St. Louis, Ying Wang, Jia Liu, Huafeng Wei. (bioRxiv, 2024); Wang, Y. et al. Anesthesiology 132, 1062-1079, (2020)) Mitochondrial damage and cell death were confirmedP-654697-PCwith the MTT reduction and LDH release assays using the methods detailed above (Wei, H. et al. Brain Res 1037, 139-147, (2005); Qiao, H. etal. Anesthesiology 127, 490-501, (2017)).

[0386] Determination of pathological inflammation. Primary inflammation biomarkers for pyroptosis (IL-ip and IL-18, Figure 1) were measured in the cell culture medium using ELISA with detailed methods that have been described previously (Piplu Bhuiyan, W. Z., Ge Liang, Bailin Jiang, Robert Vera, Rebecca Chae, Kyulee Kim, Lauren St. Louis, Ying Wang, Jia Liu, Huafeng Wei. (bioRxiv, 2024)). Other neurotoxic cytokines (IL-6 and TNF- a) and neuroprotective cytokines (IL- 10) are measured in the cell culture medium using ELISA in a similar fashion. In addition, the protein levels in the cells are measured using western blots, in accordance with previously described methods (Ren, G. et al. Sci Rep 7, 12378, (2017); Joseph, D. J., et al. PLoS One 14, e0223509, (2019); Wang, Y. etal. Anesthesiology 132, 1062-1079, (2020)).

[0387] Results

[0388] Preliminary studies demonstrated that intranasal DNRF (IN-DNRF), daily, 5 mg / kg, from 90 days of age (after full onset of disease pathology and symptoms) to 120 days, provided robust neuroprotection against overall disease severity, as determined by a comprehensive neurological scoring system, in SOD1-G93A ALS mice. Treatment improved motor coordination and movement balance (as determined using a beam balance test) and significantly increased survival probability. Although dantrolene is traditionally considered a muscle relaxant, IN-DNRF significantly inhibited overall decline in muscle strength, as determined using the grip strength test. It was hypothesized that the beneficial effects are due to dantrolene’s inhibition of motor neuron degeneration, the root cause of muscle weakness. Consistent with this G93A mechanism, intranasal DNRF significantly inhibited body and spinal cord weight loss. In addition, recent preliminary data in in SOD1G93A mice demonstrated that dantrolene significantly inhibited pathological elevation of plasma NFL, a sensitive biomarker of neurodegeneration and therapeutic efficacy (Fig. 87). Furthermore, IN-DNRF significantly inhibited motor neuron neurodegeneration, determined by cresyl violet both Furo-Jade C (FJC) specific staining to neurodegeneration (Fig. 88) and cresyl violet staining to detect cell density (Fig. 89) in the spinal cord of SOD1-G93A mice. Mechanistically, intranasal DNRF significantly reduced activation of caspase-1 (Fig. 90) and interleukin- 18 (IL-18) in the brain and blood in SOD1G93A mice (Fig.91). These data indicate dantrolene may inhibit motor neuron degeneration by suppressing inflammatory pyroptotic pathways, as found in AD mice. The preliminary study and additionalP-654697-PCpreliminary data (Figs. 88-91) strongly suggest intranasal DNRF could be effective in ALS patients with the SOD1 mutation.

[0389] As SOD1 mutations represent only 20% of fALS cases, it is therefore urgent to investigate whether dantrolene is also effective in TDP-43 ALS under the neurofilament heavy chain promoter (NEFH). The effects of dantrolene on cell viability, mitochondrial function, and reactive oxygen species (ROS), with and without the treatment of an inflammation inducer (LPS), were tested in TDP-43 Dox -inducible QBL293 stable cell lines transfected with the TDP-43 mutation at the nuclear localization signal (NLS) (iGFP-NLSm) and its corresponding wild type TDP-43 control (iGFP-WT). Compared with iGFP-WT cells, iGFP-NLSm cells possessed significantly lower baseline cell viability, which could be increased by dantrolene treatment in a bell shape dosedependent response. The optimum dose of dantrolene for protection was 0.25 pM, a clinically used blood concentration (Figure 2A). LPS decreased cell viability dose-dependently, with a toxic dose 50% (TD50) at 5pg / ml in iGFP-NLSm cells (Figure 2B). Dantrolene at 0.25 pM significantly inhibited LPS at TDso-induced reductions of cell viability (Figure 2C). Pretreatment with dantrolene for 24 hours significantly inhibited LPS-induced pathological elevation of the baseline oxygen consumption rate (OCR) and mitochondrial proton leak (an indicator of mitochondria and cell damage), as determined using the Seahorse system in iGFP-NLSm cells (Figure 3). Also, dantrolene significantly inhibited LPS-induced pathological elevation of ROS in iGFP-NLSm cells (Figure 4), and significantly inhibited LPS-induced elevation of pyroptosis related cytotoxic cytokines (IL-1β, IL-18) (Figure 5).

[0390] Overall, dantrolene demonstrated robust protection against LPS-induced mitochondrial and cell damage via its inhibition of mitochondrial dysfunction, oxidative stress, and elevation of pyroptosis-related cytotoxic cytokines in a cell model of TDP-43 mislocalization (Figures 2-5).These preliminary data strongly support the hypothesis that dantrolene could be neuroprotective against motor neurodegeneration in ALS (Figure 1) and needs further studies.

[0391] Further Experiments

[0392] Overall Experimental Design. Two types of cells for the ALS in vitro model are elected, the cultured immortal cells used in the experiments above (Figures 2-5) and primary cultures of cortical neurons from the fetal brains of WT or SOD1G93A mutation transgenic mice. Following the hypothesis in Fig. 1, dantrolene dose- and time-response experiments will be performed first to determine the maximum dose of dantrolene that is not toxic to cells by itself and the optimumP-654697-PCdose to maximize dantrolene protection and duration (e.g., 0.25 mM dantrolene in Figure 2). A dose- and time-response experiments will be performed using inflammation (LPS) and glutamate excitotoxicity (glutamate / NMDA / AMPA) inducers to determine the TD50 of each cell toxicity inducer for the establishment of ALS cell models (Figure 2). For the molecular mechanism studies, the following parameters will be measured: the changes in Ca2+channels protein (RyRs / NMDARs / AMPARs) that contribute to ALS pathology, Ca2+concentrations in the cytosol, mitochondria and ER, mitochondrial function and damage (Figure 4), oxidation of brain proteins (Figure 5), aggregation of muted TDP-43 or SOD1G93A proteins, Ca2+-dependent calpain enzyme activity, levels of primary proteins for activation of inflammatory pyroptosis (NLRP3, Caspase- 1 and N-GSDMD), and synapse proteins, under the challenge of LPS / glutamate / NMDA / AMPA at their TD50, and in the presence or absence of dantrolene pretreatment / treatment / posttreatments at its optimum neuroprotection dose or the selective antagonists of each receptors as positive controls.

[0393] Data Analyses. To assure adequate power for statistical analysis, the designed experiments will be performed using at least four repeats in each experiment and average the data from at least four separate experiments. Increasing the N in each experiment will be considered if the standard deviation is higher than compared with previous studies. Intracellular Ca2+concentration changes will be compared with multiple variables as in previous studies, including peak, area under control (AUC), and percentage of responding cells. Data will be analyzed using student t-tests for two comparison variables, and one-way or two-way ANOVA for multiple comparison by Prism version 10 software. Finally, the post-hoc multiple comparison tests in ANOVA will be used.Example 2Determining therapeutic effectiveness and side effects of intranasal dantrolene nanoparticle treatment to ameliorate motor neuron dysfunction and muscle weakness by inhibiting inflammatory pyroptosis and synapse loss in the brain and spinal cord of ALS mice.

[0394] Materials and Methods

[0395] Election of ALS animal models for drugs testing. Although there are no well -recognized ALS animal models to mimic the pathologies and symptoms in sporadic ALS patients, the rNLS8 mouse model of ALS is considered a relatively adequate ALS animal model for testing new drug efficacy. SOD1G93A transgenic mice were chosen as a initial ALS animal model with followingP-654697-PCrational: 1) SOD1G93A mutation is commonly seen in fALS patients and the SOD1G93A mouse model is dependable to demonstrate motor neuron damage and dysfunction, and muscle weakness.2) SOD1G93A mice have been commonly utilized to evaluate drug therapeutic efficacy. 3) The primary cortical neuronal cultures in will be established from fetal brains from SOD1G93A transgenic and wild type mice. In subsequent experiments ALS transgenic mice with doxycycline (Dox)-suppressible expression of mutant human TDP-43 (hTDP-43), which induces a defective nuclear localization signal (ANLS), as well as a control group of non-transgenic mice (nTg) will be used. Expression of hTDP-43 ANLS in these ‘regulatable NLS’ (rN LS8) mice occurs when the mice are no longer fed with doxycycline, a gene expression suppression agent. This results in the accumulation of insoluble phosphorylated cytoplasmic TDP-43 in the brain and spinal cord and cause detrimental effects, such as loss of endogenous nuclear mouse TDP-43 (mTDP-43), brain atrophy, muscle denervation and weakness, dramatic motor neuron loss, and progressive motor impairments leading to death. These symptoms mimic features of aggressive neurodegeneration, motor dysfunction, and muscle atrophy in ALS patients. ALS rNLS8 mice will be used to evaluate the neuroprotective efficacy of intranasal DNRF with the following rationales: 1) TDP-43 mutation exists in almost all ALS patients, rendering this animal model more translational to evaluate drug therapeutic efficacy. 2) It is an aggressive ALS animal model that resembles disease progression in almost all ALS patients and will thus be translational to patients while evaluating the efficacy of drug therapeutics; 3) It has the same TDP-43 gene mutation as the TDP-43 Dox-inducible QBI-293 stable cell lines (iGFP-NLSm) used the experiments described herein, with preliminary data demonstrating dantrolene neuroprotection against the TDP-43 mutation and LPS-induced mitochondrial dysfunction (Fig. 3), oxidative stress (Fig. 4), pathological elevation of pyroptosis-related cytotoxic cytokines (Il-ip, IL- 18, Fig. 5), and cell death (Fig. 2). Both male and female mice were used with an adequate number in each group based on the power analysis using standard deviation from previous studies.

[0396] Dantrolene nanoparticles and vehicle preparation. Dantrolene was dissolved in the RYANODEX® formulation vehicle (VEH) to generate crystal nanoparticles as described in previous studies (Shi, Y. et al. J Alzheimers Dis 76, 1375-1389, (2020); Vera, R. etal. J Alzheimers Dis, 98(2):549-562 (2024); Wang, J. et al. PLoS One 15, e0229156, (2020); Ryanodex-A New Dantrolene Formulation for Malignant Hyperthermia. Med Lett Drugs Ther 57, 100 (2015)). The nanoparticles’ size, distribution, and stability have been confirmed using dynamic light scatteringP-654697-PC(DLS). The size of the newly prepared dantrolene crystal nanoparticles has been measured at room temperature possess a Z average of d= 1.182 pM, and d= 1.183 pM after three hours under the same conditions. The drugs and vehicle were thawed and used for experiments within 3 hours of preparation, which is the duration during which nanoparticle size and distribution remain stable.

[0397] Monitoring of general health and neurological scoring. Body weight, general health and activity, and mortality were monitored starting from the first week of treatment. A neurological score representing disease progression was evaluated each week using the neurological scoring system described previously (Tungtur, S. K. etal. SciRep 11, 11051, (2021); Hatzipetros, T. el al. J Vis Exp, 6:(104):53257 (2015)). with the following criteria: (0) hind limb stretch similar to wild type mice when suspended by its tail, (1) tail hang + leg spray test, where the tail hang causes a collapse, partial collapse, or trembling of the hind limbs, (2) toes curl under at least twice when walking for 12 inches, (3) rigid paralysis of hind limbs with the foot unable to perform forward motion, and (4) the mouse cannot right itself within 30s from either side. These behaviors were video recorded and analyzed. Total neurological scores were summed from all experiments and compared. Mice were monitored for tremor and hindlimb clasping phenotype each week as described (Walker, A. K. etal. Acta Neuropathol 130, 643-660, (2015)). To evaluate for tremors, mice were held on their backs in the palm of the observer’s hand while being gripped gently between thumb and index finger. Forelimb and hindlimb movements were observed for ~30s. Ultimately, the presence of either a fast / fine tremor during this observation period or a resting tremor when in the home cage denotes a positive response. To evaluate hindlimb clasping, mice were suspended by the tail -30 cm above the cage and slowly lowered. If both hindlimbs are held together within 5 s of being raised and maintained for ~30s, a positive response is recorded.

[0398] Motor neuron function coordination tests. Beam balance test was used to evaluate motor function and movement coordination of the mice. This test was carried out according to a previously described protocol (Luong, T. N., et al., J Vis Exp, 10:(49):2376 (2011)). Briefly, the balance beam test was performed by allowing the mice to first travel across either a 12 mm wide or a 6 mm-wide balance beam. Time to travel across the beam and the number of times the mouse’s hind foot slips off the beam is recorded. Additionally, rotarod test will be used according to previously described protocols (Shi, Y. et al. J Alzheimers Dis 76, 1375-1389, (2020)).

[0399] Muscle strength measurements. Inverted screen test (Tungtur, S. K. et al. Sci Rep 11, 11051, (2021); Deacon, R. M. J Vis Exp, 2:(76):2610 (2013)) was used to measure the muscleP-654697-PCstrength (grip power). Briefly, the mouse was placed in the center of a stainless-steel wire mesh attached to a frame. Then, the screen is slowly inverted and held ~40 cm above a soft surface. The time for the mouse to fall from the wire is measured. Tests will be performed by personnel blinded to the treatment groups.

[0400] Blood / tissue collection and preparation. Blood was collected as previously described (Vera, R. et al. J Alzheimers Dis, 98(2):549-562 (2024)) during euthanasia, transcardiac perfusion, and exsanguination. The serum was separated from the heparinized blood samples, and the tissues (brain, spinal cord, liver, kidney, nose) were harvested. Tissue from one half of the brain or whole spinal cord was stored at -80°C for immunoblotting biochemical assays (Peng, J. et cd. Neurosci Lett 516, 274-279, (2012); Yang, B. etal. PLoS One 9, e99171, (2014); Peng, J. etal. AnesthAnalg 119, 939-946, (2014). The other half of the brain, muscles of hind legs, liver and nose were paraffin-embedded (Shi, Y. et al. J Alzheimers Dis 76, 1375-1389, (2020); Peng, J. etal. Neurosci Lett 516, 274-279, (2012); Wu, Z. et al. Alzheimer Dis Assoc Disord 29, 184-191, (2015)) and H& E stained (Jiang, B. et al. Eur Rev Med Pharmacol Set 26, 198-203, (2022); Shi, Y. et al. J Alzheimers Dis 76, 1375-1389, (2020)) for ALS pathology (brain / muscle) or structural examination to evaluate potential dantrolene organ toxicity (liver, nose).

[0401] Aggregation of TDP-43 or SOD1G93A proteins in the brain and spinal cord. The level of aggregation of TDP-43 will be determined by using fluorescence microscopy and specific antibodies to examine muted TDP-43 or SOD1G93A proteins, in the cytosol of various brain tissue sections according to previously described methods (Wang, Y. et al. Anesthesiology 132, 1062- 1079, (2020); Porta, S. etal. Neuropathol. Appl. Neurobiol. 47, 1033-1049, (2021)).

[0402] Measurements of blood pathological biomarkers Serum calpain activity will be measured using a fluorometric method (Abeam Inc., Cambridge, UK) on a fluorescence spectrophotometer (Loinjak, D. et al. Biomedicines 11(7): 1847, (2023)). The fluorometric assay is based on the detection of cleavage of calpain 1 substrate Ac-LLY-AFC that emits blue light (λmax= 400 nm). Upon cleavage of the substrate by calpain 1, free AFC emits yellow-green fluorescence ( max = 505 nm). The fluorometric signal of AFC is measured on a fluorescence plate reader. The activity is expressed as a change of Relative Fluorescent Units (RFU) per microliter in one hour. Using ELISA techniques, assays were conducted with serum samples to determine changes in neurodegeneration marker NFL (Meyer, T. et al. Eur. J. Neurol., 31(9) el6379, (2024)) using methods previously described in detail (Huang, W., Wang, X., Xie, F., Zhang, H. & Liu, D.P-654697-PCCytokine 149, 155725, (2022); Suzuki, S. et al. Respir Investig 60, 750-761, (2022)). Additional assays for caspase-1, pyroptosis-related cytotoxic cytokines (IL-ip and IL-18) (Sharma, B. et al. Role of NLRP3 Inflammation 46, 56-87, (2023); Xu, C. Z. et al. Heliyon 10, e28553, (2024)), inflammatory cytotoxic cytokines (IL-6, TNF-a), serum NLRP3, and N-GSDMD, will be carried out using ELISA assays with previously described antibodies (Peng, J. et al. Anesth Analg 119, 939-946, (2014); Wang, Y. S. et al. Chin. Med. J. (Engl.) 126, 3931-3935 (2013)).

[0403] Determining inflammation-mediated oxidative stress, pathological inflammation and pyroptosis in brains and spinal cords. Both Western blot with frozen brain tissue and immunohistochemistry (IHC) on 4% paraformaldehyde-fixed and paraffin embedded brain tissue sections will be used to measure the levels of pyroptosis pathway activation proteins (NLRP3, caspase-1, and N-GSDMD), malondialdehyde (MDA) oxidized protein levels (an oxidative stress marker), and cytotoxic (IL-1β, IL- 18, IL-6, TNF-a) / cytoprotective (IL- 10) cytokines will be measured in similar way using previously described methods (Shi, Y. et al. J Alzheimers Dis 76, 1375-1389, (2020); Jia Liu, Y. L., et al., bioRxiv, doi:0.1 iO 1 / 2024;09.06. 1461 yl. (2024); Peng, J. et al. Alzheimers Dement 7, e67 (2011)).

[0404] Measuring synaptic proteins. Immunoblotting and immunohistochemistry will be used in accordance with detailed methods described previously to measure synaptic proteins (PSD-95, synapsin-1) in the brain and spinal cord.

[0405] Results

[0406] WT and SOD1G93A mice at 3 months old were treated with intranasal dantrolene nanoparticles (IN-DAN) at 5 mg / kg, daily, 7 days per week, or its vehicle control (IN-VEH) for 4 weeks continuously until 4 months old. This treatment represented initiation of drug therapy after full onset of ALS pathology in the brain and spinal cord and was continued until the disease endstage. Control WT or SOD1G93A mice received no treatments (No TX). The integrated neurological score was determined using a protocol previously described (Tungtur, S. K. etal. Sci Rep 11, 11051, (2021). Motor neuron function and movement coordination was determined using rotarod and balance beam tests. Muscle strength was determined using inverted screen (grip force) test. All detailed methods are described above. Body weight, general health and rotarod tests were determined weekly, while other behavioral tests were determined at the end of 4 weeks of intranasal DNRF treatments. Blood and brain tissue and spinal cords were harvested after the lastP-654697-PCbehavioral test. Samples were examined for the neurodegeneration biomarker, neurofdament light chain (NFL) in brain cortex. Tissue lysates were measured using Western Blot. As illustrated in Figure 6, compared to wild type controls, the SODG93A mice suffered significantly impaired neurological function demonstrated by the remarkably increased integrated neurological score, which could be robustly inhibited by 4 weeks of intranasal DNRF treatment. Consistently, the SOD1G93S mice demonstrated significantly impaired motor neuron function and movement coordination by requiring significantly more time to cross both the 12 mm and 6 mm beams, with 10.78-fold (116.9 vs. 9.92 seconds, P<0.0001) and 5.27-fold (112.2 vs. 17.90 seconds, P<0.0001) increases, respectively (Figure. 7 A, C). IN-DAN treatment significantly reduced crossing times for the SOD1- G93A mice on both beams, by 78% (116.9 vs. 25.66 seconds, P<0.0001) on the 12 mm beam and 79% (112.2 vs 33.19 seconds, P<0.0001) on the 6 mm beam. While intranasal vehicle (IH-VEH) treatment also significantly reduced crossing time on the 6 mm beam (112.2 vs.50.90 seconds, P=0.0034), it did not significantly affect performance on the 12 mm beam. Similarly, compared to controls, SOD1-G93A mice exhibited a dramatic increase in the number of foot slips while crossing the 12 mm and 6 mm beams, 33.7-fold (0.250 vs. 8.67, P<0.0001) and 10.75-fold (0.83 vs. 9.750, P<0.0001), respectively. This impairment was significantly and robustly reduced by IN-DAN treatment, which decreased foot slips by 84% (8.67 vs. 1.38, P<0.0001) on the 12-mm balance beam and 73% (9.75 vs. 2.63, P<0.0001) on the 6-mm balance beam (Fig. 7B, and Fig. 7D). Although IN-VEH treatment resulted in a reduction in foot slips on both beams, the effect was not statistically significant (Fig. 7B, and Fig.7D). Supplemental Video 1 website shows representative trials on the 12 mm beam balance test across experimental groups.

[0407] Furthermore, a second method was employed, the rotarod test, to assess motor coordination and balance in mice. This test was conducted weekly over the 30-day period of treatment (Fig.7E-Fig. 7H). Longer latency to fall from the accelerating rod indicates better motor coordination and balance. Compared to control wild-type (WT) mice, SOD1- G93A transgenic mice exhibited significantly reduced latency to fall across all four weeks of testing (Fig. 7E- Fig.7H). The degree of statistical significance increased progressively from week one to week four, indicating a gradual decline in motor function over time in the ALS transgenic mice. While intranasal dantrolene nanoparticle treatment (IN-DAN) showed a trend towards increased latency on the rotarod, this effect did not reach statistical significance at any time point during the 30-day treatment period.P-654697-PC

[0408] To assess muscle strength, Kondziela’s inverted screen test was employed, which measures the duration a mouse can hang from an inverted metal screen. Longer latency indicates greater grip force, or stronger muscle strength mice showed a dramatic 4.52-fold reduction in hang time (108.2 vs. 19.61 seconds, P<0.0001), indicating development of significant muscle weakness. IN-DAN treatment significantly improved grip strength, increasing hang time by 2.13-fold (19.61 vs. 61.37, P=0.0026). While IH-VEH treatment showed a trend toward improvement, the change was not statistically significant (P=0.18). (Figure 8 A).

[0409] The SOD1G93A mice demonstrated progressive loss of body weight during the four week treatment period, while intranasal DNRF treatment reversed the progressive body weight loss with a progressive gain of body weight. Body weight was monitored weekly throughout the treatment period. As shown in Figure 8B, non-transgenic control mice displayed a consistent increase in body weight over the four-week treatment period. In contrast, SOD1-G93A transgenic ALS mice, whether untreated or treated with intranasal vehicle (IN-VEH), exhibited significant body weight loss. Intranasal dantrolene nanoparticle treatment (IN-DAN) completely reversed this weight loss in SOD1-G93A mice, however, resulting in a body weight trajectory comparable to that of non-transgenic controls. Weight loss in SOD1-G93A transgenic mice is likely attributable to skeletal muscle, atrophy, a common symptom in human ALS patients.

[0410] NFL levels in the lumbar and sacral spinal cord were assessed (approx. L1-S5). In SOD1-G93A mice, NFL levels were significantly decreased by 3.9 fold (1.47 vs. 0.30, P<0.0001) compared to control mice (Fig. 9A, and Fig. 9B), implying neurodegeneration consistent with other studies. IN-DAN treatment tended to increase spinal NFL protein levels, but not to a statistically significant degree (Fig. 9C). The weight of 4% paraformaldehyde-fixed cervical and thoracic spinal cord (approx. C1-T12) was significantly reduced by 43% in SOD1-G93A mice (73.10 vs. 41.62 mg, P=0.0008), compared to the controls. This loss was significantly ameliorated by IN-DAN treatment, with spinal cord weight increasing by 53% (41.62 vs 63.60 mg, P<0.019), but not by the IN-VEH treatment (Fig.9D, and Fig.9E). Similarly, the weight of fresh lumbar and sacral spinal cord (approx. L1-S5) was reduced by 71% (30.99 vs 8.90 mg, (approx. L1-S5) was reduced by 71% (30.99 vs 8.90 mg, IN-DAN treatment significantly restored spinal cord weight, increasing it by 1.43-fold (8.90 vs. 21.66 mg, P=0.034), while IH-VEH had no significant effect.

[0411] The NFL in blood was significantly increased in SOD1G93A mice in comparison with wild type mice, which could be abolished by intranasal DNRF treatment (Figure 10).P-654697-PC

[0412] Neither intranasal dantrolene nanoparticle (IN-DAN) nor vehicle (TH- VEH) treatments affected survival in non -transgenic control mice, which maintained 100% survival throughout the 30-day treatment period (data not shown). In contrast, in at the treatment onset to 67% by the end of the 30-day period, in both untreated and IH-VEH treated groups. Strikingly, however, IN-DAN treatment significantly improved survival, with 89% of SOD1-G93A mice surviving through the full treatment course (Figure 11), suggesting a robust neuroprotective effect of intranasal dantrolene nanoparticles.

[0413] Overall, intranasal dantrolene nanoparticles treatment does not affect motor neuron’s function, coordination and muscle strength in WT mice, but robustly and significantly inhibited impaired neurological function (Figure 6), the progression of motor neuron dysfunction and movement impairment (Figure 7), muscle weakness (Figure 8A), pathological increased neurodegeneration biomarker in blood (Figure 10), body weight loss (Figure 8B) and prolonged SODG93A mice survival rate (Figure 11).

[0414] Further Experiments

[0415] Overall Experimental Design. The experimental design to test the hypothesis whether IN-DNRF will be an effective ALS treatment for two ALS animal models, is shown in Figures 12 and 13. As in Example 1, ALS transgenic mice either with mutated TDP-43 or mutated SOD1G93A will be used. Initially dantrolene dose response studies will be carried out, and the lowest dose that is still neuroprotective will be used to treat the animal models for the mechanistic studies. Intranasal DNRF administration will be examined both before or after the beginning of ALS pathology to determine drug efficacy as a preventive or as a disease-modifying drug. After all behavioral tests, mice will be euthanized. Blood, brain, and spinal cord will be harvested to examine the various types of Ca2+channel proteins, oxidative stress, ER stress, neurodegeneration by pyroptosis programed cell death.

[0416] Experimental groups and treatments. Figure 12 shows the detailed experimental design for the first ALS animal model in transgenic mice with the TDP43 mutation. Two cohorts with a total of 20 experimental groups will be used. Each group will include 10 male and 10 female mice. Cohort 1 will investigate the preventative neuroprotection of dantrolene against ALS pathology and motor neurons degeneration and dysfunction. In 10 experimental groups, intranasal DNRF or vehicle control treatment will be initiated at 4 weeks of age and last for total of 14 weeks until 18 weeks of age. Doxycycline will be withdrawn to initiate expression of hTDP-43 rNLS in the brainP-654697-PCand spinal cord at 8 weeks of age. Tn contrast, cohort 2 will study dantrolene treatment administered after the full onset of ALS pathology and symptoms / signs in CNS. As with cohort 1, doxycycline will be withdrawn to start expression of rNLS in the brain and spinal cord at 8 weeks of age and then allow the ALS pathology and symptoms to progress for 6 weeks until 14 weeks of age. Intranasal DNRF or vehicle control at different doses will be initiated at 14 weeks of age, 7 days per week, continuously for 4 weeks until 18 weeks of age. The rationale for this experimental design is based on the known progression of full ALS pathology and following the signs of motor dysfunction and muscle atrophy in rNLS8 mice from the initiation of doxycycline withdrawal to 8 weeks laterlOl. For the second animal model with the SOD1G93A transgenic mice, two cohorts (prevention versus treatment) with three different doses (5, 2.5, 1 mg / kg) for a total of 20 experimental groups will be designed (Figure 13), similar to the ALS transgenic mice with the TDP43 mutation (Figure 12). In cohort 1 (prevention), with a total of 10 experimental groups, treatments will be performed continuously, 7 days per week, for 8 weeks, from 8 to 16 weeks of age. In cohort 2 (treatment)), with 10 experimental groups, as in our preliminary study (Fig. 6-13), mice will be treated with intranasal DNRF or vehicle, 7 days / week, for 4 weeks, from 12 to 16 weeks of age. For the dose-response study in all mice, intranasal DNRF will start at 5mg / kg or vehicle control, a dose effective to treat dementia and depression in 5XFAD mice without side effects or toxicity and a dose to inhibit motor neurodegeneration and dysfunction and muscle weakness in SOD1G93A mice (Figures 6-8). The Effectiveness of intranasal DNRF will then be examined at lower doses (2.5 and 1 mg / kg). In both ALS animal models, behavioral tests for neurological scores, motor function coordination, and muscles strength will be performed after the last day treatment and will last for two weeks (Figures 6-8). Then, mice will be euthanized and blood, brain and spinal cord will be harvested for mechanisms studies by examining changes in various biomarkers and pathologies (Figures 10-11).

[0417] Discussion

[0418] This study has demonstrated that intranasal dantrolene nanoparticles provide robust and significant therapeutic effects against motor neuron dysfunction, motor discoordination, balance impairments, muscle weakness, and reductions in body and spinal cord weight, while prolonging survival in an ALS mouse model carrying the SOD1-G93A mutation. These beneficial effects were observed even when treatment was initiated after the full onset of disease pathology and symptoms.P-654697-PCat 3 months of age, suggesting dantrolene might function as a potential disease-modifying therapy of ALS.

[0419] Two primary symptom-relief drugs currently used to treat ALS patients are riluzole and edaravone. Riluzule acts as an NMDAR antagonist and edaravone functions as a scavenger of reactive oxygen species. However, there remains an urgent need to develop effective diseasemodifying therapies. Repurposing clinically available, FDA-approved drugs with known safety profiles and minimal organ toxicity, such as dantrolene, offers a promising strategy to accelerate clinical translation. This new formulation of dantrolene, Ryanodex, consists of crystalline nanoparticles. While Ryanodex is FDA-approved for the treatment of malignant hyperthermia, its current application is intravenous, not intranasal. A prior study failed to demonstrate efficacy of dantrolene in the same SOD1-G93A model, most likely due to its limited ability to cross the bloodbrain barrier (BBB). Intranasal drug delivery, particularly in nanoparticle forms, has been well established to enhance BBB penetration, increasing drug concentration in the CNS and its retention time, thereby improving therapeutic efficacy while minimizing peripheral side effects and organ toxicity. This strategy is especially advantageous for treatment of long-term conditions, such as those involving chronic neurological diseases. In fact, intranasal administration of riluzole nanoemulsions has been shown to produce higher brain concentrations than standard oral administration, enhancing its therapeutic potential in ALS. Consistently, our previous studies demonstrated that intranasal administration of dantrolene nanoparticles in the Ryanodex formulation significantly increased brain concentration, prolonged drug duration, and enhanced the brain-to-blood concentration ratio compared to oral or subcutaneous administrations. Intranasal dantrolene nanoparticles administered at the same dose (5mg / kg), daily, 5 days per week for up to 12 months, significantly inhibited memory loss in the 5XFAD Alzheimer’s disease mouse model, without evidence of side effects or nasal / liver toxicity. Given the chronic nature of ALS, the low systemic toxicity and high CNS penetration of intranasal dantrolene nanoparticle treatment further favor its utility for long-term treatment. In comparison with prior studies, the robust and significant neuroprotective and muscle-preserving effects observed in SOD1-G93A mice suggest enhanced drug delivery to the brain and spinal cord, with promoted efficacy in the CNS. Moreover, because ALS patients frequently develop difficult swallowing, intranasal drug delivery may be a more convenient and patient-friendly route of administration. A significant proportion of ALS patients develop cognitive impairment and depression-related psychiatric symptoms. Accordingly,P-654697-PCas intranasal dantrolene nanoparticles have demonstrated efficacy in treating memory dysfunction and depression and anxiety-like behaviors, this approach may offer additional clinical benefit beyond motor function preservation in the treatment of ALS. Similar to our previous findings in 5XFAD mice, the intranasal administration of vehicle alone (only the Ryanodex formulation) demonstrated predominantly minor, non-significant trends towards improvement in motor function, balance, and muscle strength, but to a clearly less effective degree than dantrolene-loaded nanoparticles. This suggests that dantrolene is the primary active agent providing neuroprotection and preserving muscle strength, although the potential additive role of the vehicle alone warrants further investigation.

[0420] Besides the general side effects of dantrolene such as nausea and vomiting and sleepiness, some specific side effects or organ toxicity with chronic use of dantrolene may be concerned when it is proposed to treat ALS patients. For example, chronic administration of dantrolene has been demonstrated to cause pleural effusion in some rear cases. A sever ortan toxiciy after chronic use of dantrolene at high dose for long duration can cause liver toxicity, which typically happen in female adult older than 35 years old, so the liver function need to be examined periodically if dantrolene is used to treat ALS patients in the future. Intranasal Dantrolene nanoparticles for up to 10 months dis not cause liver structure and function changes in 5XFAD mice. Although there has been concern to use dantrolene in ALS patients with muscle weakness as dantrolene is considered a muscle relaxant, it is an FDA approved drug to treat muscle spasm due to upper motor dysfunction, spinal cord injury, multiple sclerosis, cerebral palsy and stroke etc. We speculate that the early use of dantrolene increase muscle strength by inhibiting the root cause (degeneration of motor neuron) as demonstrated in this study, while the late use of dantrolene for ALS patients with existing respiratory failure may need particular caution for potential deterioration of respiratory failure by dantrolene due to its effects to relax respiratory muscle, which need future clinical studies. Although dantrolene is typically considered a muscle relaxant, intranasal dantrolene nanoparticles for up to 10 months treatment did not impair muscle strength in 5XFAD mice. In this ALS model, it significantly reversed muscle weakness and improved muscle strength, likely due to its inhibition of motor neuron degeneration, and potentially the direct protection against muscle damage. Overall, this preclinical study strongly supports the need for urgent clinical trials to evaluate the therapeutic potential of intranasal dantrolene nanoparticles in ALS patients.P-654697-PC

[0421] The robust inhibition of reduction in spinal cord weight observed in this study suggest that intranasal dantrolene nanoparticles inhibit degeneration of spinal cord, although the specific neuronal populations affected remain to be clarified. Although our data strongly suggest protection in the spinal cord, the exact neuroprotective mechanisms warrant further investigation. Dantrolene has previously been shown to inhibit neuronal death through multiple pathways, including apoptosis, pyroptosis, and ferroptosis. At the molecular level, its ability to inhibit upstream intracellular Ca2+dysregulation, triggered by pathological activation of NMDAR

[0033] , RyR and InsP3R, may contribute significantly to its broad neuroprotective effects. However, further studies are needed to investigate these mechanisms in ALS pathology.

[0422] Intranasal dantrolene nanoparticles prolonged survival in SOD-G93A mice, even with a limited 30-day treatment period (from postnatal day 90 to 120). Since most ALS patients succumb to respiratory failure due to respiratory muscle weakness, the observed inhibition of spinal cord degeneration and muscle atrophy by dantrolene likely contributed to the increased survival probability. We speculate that continued treatment beyond 120 days would further extend survival in this ALS animal model.

[0423] In conclusion, intranasal dantrolene nanoparticles provided robust and significant improvements in overall disease severity, including motor discoordination, imbalance, muscle weakness, body and spinal cord weight loss, and survival in SOD-G93 A ALS mice. These findings support further preclinical and clinical studies to evaluate intranasal dantrolene nanoparticles as a future effective disease-modifying treatment for ALS patients.Example 3Intranasal dantrolene nanoparticles abolish depression behavior and memory loss as a disease-modifying drug in 5xFAD mice

[0424] In this study, the therapeutic effectiveness versus side effects were investigated, as were the potential mechanisms of intranasal dantrolene nanoparticles to ameliorate both depression behavior and memory loss in both wild type and 5XFAD transgenic mice. The results demonstrated that administration of intranasal dantrolene nanoparticles for 12 consecutive weeks abolished both depression behavior and memory loss, as a disease-modifying drug, in both young adult and aged 5XFAD mice, with no side effects or toxicity on liver, muscle and smell function. Furthermore, administration of intranasal dantrolene nanoparticles inhibited oxidative stresses, NLRP3 inflammasome activation mediated pyroptosis, pathological inflammation, and synapse damage inP-654697-PC5xFAD mice. This study provides proof of concept that intranasal administration of dantrolene nanoparticles is an effective treatment of both depression and memory loss in AD with no side effects or organ toxicity; the therapeutic effect is thought to achieved by inhibiting pathological inflammation and pyroptosis.Materials and methodsAnimals

[0425] All the procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania. Four pairs of 5XFAD mice (B6SJL-Tg (APPSwFlU on, PSEN1*M146U*L286V) 6799Vas / Mmjax) and wild type mice (B6SJLF1 / J) were purchased from the Jackson Uaboratory (Bar Harbor, ME) and bred. These 5XFAD transgenic mice over express mutant human APP with the Swedish (K670N, M671L), Florida (I716V), and London (V7171) Familial Alzheimer’s Disease (FAD) mutations along with human PS 1 harboring two FAD mutations, M146L and L286V. Food and water were available in the cage. All mice were weaned no later than one month of age and genetically identified by polymerase chain reaction (PCR) analysis before weaning. At this time, mice were divided into different cages according to age and gender, with no more than five mice per cage. Both male and female mice were used in this study.Dantrolene administration

[0426] Dantrolene (Sigma, St Louis, MO) was dissolved in the Ryanodex Formulation Vehicle (RFV: 125 mg mannitol, 25 mg polysorbate 80, 4mg povidone K12 in 5mL of sterile water and pH adjusted to 10.3), similarly as described in Shi Y, Zhang L, Gao X, et al. Intranasal Dantrolene as a Disease-Modifying Drug in Alzheimer 5XFAD Mice. J Alzheimer s Dis 2020; 76(4): 1375-89 and Wang J, Shi Y, Yu S, et al. Intranasal administration of dantrolene increased brain concentration and duration. PLoS One 2020 15(3): e0229156, each of which is hereby incorporated by reference in its entirety. For intranasal administration, the final concentration of dantrolene was 5 mg / mL as described previously, the mice were held and fixed by the scruff of their necks with one hand and with the other hand given a total of IpL / gram of body weight of dantrolene nanoparticle solution or RFV. A mouse weighing 20 g would be given 20µL solution. The solution was slowly delivered directly into the mouse’s nose. Care was taken to make sure that mice were minimally stressed, and that the solution stayed in the nasal cavity and did not enter the stomach or lungs.P-654697-PCExperimental treatment groups

[0427] As demonstrated in Figure 14, age-matched male and female mice were randomly divided into experimental groups when they were genotyped around 1 month of age. The Early Treatment Groups (ETG), including intranasal dantrolene (In Dan) and no treatment, were treated once a day, 5* / week (Monday to Friday) beginning at 2 months of age (before the onset of intracellular amyloid pathology and any cognitive dysfunction). The Late Treatment Groups (LTG) include intranasal dantrolene (In Dan) began the same treatment at 9 months of age, after the onset of extracellular amyloid plaques accumulation and cognitive dysfunction. The RFV was made fresh and contained all inactive ingredients in Ryanodex, as described by Shi Y, Zhang L, Gao X, et al. supra. Fresh dantrolene nanoparticle were made every time before administration. Intranasal dantrolene nanoparticle stock solution was made 5 mg / ml. All LTG mice continued to receive treatment until they were euthanized at 13 months of age after 1 months of behavior test started at the completion of drug treatment.Food buried test

[0428] Olfaction was assessed in ETG at 6 months of age as previously described by as described by Shi Y, Zhang L, Gao X, et al. and Wang J, Shi Y, Yu S, et al. supra with modification. On the first day, cookies (Galletas La Modema, S. A. de C. V.; one cookie for every two mice) were buried in the bedding of the home cages for 24 h, and then the number of cookies consumed was recorded. The mice were fasted starting on the second day at 4 pm and ending on the third day at 9 am. Water was freely available during this time. The buried food test was conducted on the third day at 9-11 am. They were acclimated to the testing room for at least 1 h before the test. Mice were individually placed into a clean cage containing clean bedding with one cookie buried beneath the bedding in a comer. The latency for the animal to find the cookie (identified as catching the cookie with its front paws) was recorded manually. If the animal failed to find the cookie within 15 min, it would be placed back into its home cage. A clean cage and bedding were used for each animal and investigators were blinded to the experimental conditions.Rotarod test

[0429] Motor function was examined for muscle weakness, a common side effect of dantrolene, as was previously described, e.g. by Shi Y, Zhang L, Gao X, et al. supra. The amount of time spent on the accelerating rotarod (IITC Series8, Life Sciences, Woodland Hills, CA) was assessed for mice in the ETG at 6 months of age, as previously described. Briefly, animals were acclimated toP-654697-PCthe testing room at least 1 h before the test. Two 60 s training trials at a constant speed (9 rpm) were performed with a 30 min interval. Then, three 120 s test trials were conducted at a gradually increasing speed (4-40 rpm) with a 60 min interval between trials. The latency to fall from the rotarod was recorded automatically and analyzed.Fear conditioning test

[0430] Memory was assessed at 6 of age for the ETGs, and at 13 months of age for LTGs. Both hippocampal-dependent and-independent memory were assessed using the fear conditioning test, using a protocol described previously by Shi Y, Zhang L, Gao X, et al. supra. Each day, animals were acclimated to the testing room at least 1 h before the test. On the first day, each mouse was placed in the test chamber and went through three condition stimulation parings with a 60 s interval between each cycle. A 30 s tone of 2000 Hz and 85 dB was used as the tone stimulation and a 2-s electrical foot shock of 0.7mA was used as the shock stimulation. The mice were removed from the chamber 30 s after the last stimulation. On the second day, the contextual fear conditioning test was first performed to measure the hippocampal-dependent memory. The mouse was placed in the same chamber for 4 min with no tone or shock and then removed from the chamber. Two hours later, the cued fear conditioning test was performed to measure hippocampal-independent memory. The mouse was placed in another chamber that was different in size and smell using different cleaning solutions. There was no tone or shock during the first 1 min. Later the mouse went through three cycles of the same tone with a 60 s interval between each cycle with the freezing time recorded. Animals were then removed from the chamber 60 s after the last tone. The ANY-maze controlled Fear Conditioning System consisted of a sound-attenuating chamber (Model: 46000-...

Claims

1. P-654697-PCWHAT IS CLAIMED IS:

1. A method for treating and / or alleviating symptoms of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

2. A method for preventing or delaying the onset of amyotrophic lateral sclerosis (ALS), the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

3. A method for inhibiting a pathological increase of intracellular Ca2+dy sregulation, inhibiting oxidative stress and pyroptosis activation, inhibiting mitochondrial disfunction, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting PSD-95 synapse protein loss, inhibiting impairment of neurogenesis, and / or increasing cytoprotective IL- 10 in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

4. A method for inhibiting neuroinflammation, neurodegeneration, neuropathy, axonopathy, excitotoxicity, and synapse destruction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

5. A method for restoring motor neuron function and / or muscle strength, and / or reversing weight loss in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

6. A method for ameliorating muscle weakness, muscle atrophy, and motor neuron disfunction in an amyotrophic lateral sclerosis (ALS) subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of aP-654697-PCpharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

7. The method according to any one of the preceding claims, wherein the dantrolene is in nanoparticulate form.

8. The method according to any one of the preceding claims, wherein the lithium salt is lithium chloride, lithium carbonate or lithium orotate.

9. The method according to any one of the preceding claims, wherein the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intranasally, intrathecally, orally or intravenously.

10. The method according to claim 9, wherein the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intranasally.

11. The method according to claim 9, wherein the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intrathecally.

12. The method according to any one of claims 1-11, wherein the amyotrophic lateral sclerosis (ALS) is familial amyotrophic lateral sclerosis (fALS).

13. The method according to any one of claims 1-11, wherein the amyotrophic lateral sclerosis (ALS) is sporadic amyotrophic lateral sclerosis (sALS).

14. The method according to any one of claims 1-11, wherein the amyotrophic lateral sclerosis (ALS) is Western Pacific amyotrophic lateral sclerosis.

15. The method according to any one of claims 1-11, wherein the amyotrophic lateral sclerosis (ALS) is juvenile amyotrophic lateral sclerosis.

16. The method according to any one of claims 1-11, wherein the amyotrophic lateral sclerosis (ALS) is Hiramaya Disease.

17. The method according to any one of claims 1-11, wherein the amyotrophic lateral sclerosis (ALS) is trauma induced ALSP-654697-PC18. The method according to any one of claims 1-11, wherein the amyotrophic lateral sclerosis (ALS) is progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), or ALS with multi-system involvement.

19. The method according to any one of the preceding claims, wherein the subject is human.

20. A method for treating depression comprising administering to a subject in need thereof a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

21. The method of claim 20, wherein the dantrolene is in nanoparticulate form.

22. The method of claim 20, wherein the lithium salt is lithium chloride, lithium carbonate or lithium orotate.

23. The method of claim 20, wherein the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

24. The method of claim 20, wherein the subject is female.

25. A method for treating depression in an Alzheimer’s Disease subject comprising administering to the subject in need thereof a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

26. The method of claim 25, wherein the dantrolene is in nanoparticulate form.

27. The method of claim 25, wherein the lithium salt is lithium chloride, lithium carbonate or lithium orotate.

28. The method of claim 25, wherein the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

29. The method of claim 25, wherein the subject is female.

30. A method for inhibiting a pathological intracellular Ca2+and / or Fe“+dysregulation, oxidative stress and inflammatory pyroptosis activation, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting astrogliosis and microgliosis in a subject having depression, the method comprising administering to the subject a therapeutically effectP-654697-PCamount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

31. The method of claim 30, wherein the dantrolene is in nanoparticulate form.

32. The method of claim 30, wherein the lithium salt is lithium chloride, lithium carbonate or lithium orotate.

33. The method of claim 30, wherein the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

34. The method of claim 30, wherein the subject is female.

35. A method for inhibiting a pathological increase of intracellular Ca2+and Fe2’ dysregulation, oxidative stress and pyroptosis activation, inhibiting cytotoxic and / or neurotoxic cytokines, inhibiting astrogliosis and microgliosis in an Alzheimer’s Disease subject having depression, the method comprising administering to the subject a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

36. The method of claim 35, wherein the dantrolene is in nanoparticulate form.

37. The method of claim 35, wherein the lithium salt is lithium chloride, lithium carbonate or lithium orotate.

38. The method of claim 35, wherein the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

39. The method of claim 35, wherein the subject is female.

40. A method for increasing cytoprotective IL-10 and inhibiting a PSD-95 synapse protein loss in a subject having depression, the method comprising administering to the subject a therapeutically effect amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

41. The method of claim 40, wherein the dantrolene is in nanoparticulate form.

42. The method of claim 40, wherein the lithium salt is lithium chloride, lithium carbonate or lithium orotate.P-654697-PC43. The method of claim 40, wherein the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

44. The method of claim 40, wherein the subject is female.

45. A method for increasing cytoprotective IL-10 and inhibiting a PSD-95 synapse protein loss to an Alzheimer’s Disease subject having depression, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier.

46. The method of claim 45, wherein the dantrolene is in nanoparticulate form.

47. The method of claim 45, wherein the lithium salt is lithium chloride, lithium carbonate or lithium orotate.

48. The method of claim 45, wherein the pharmaceutical composition comprising dantrolene or a combination of dantrolene and a lithium salt, and a pharmaceutically acceptable carrier is administered intranasally, orally or intravenously.

49. The method of claim 45, wherein the subject is female.

50. A pharmaceutical composition comprising dantrolene, a lithium salt, and a pharmaceutically acceptable carrier.

51. The pharmaceutical composition of claim 50, wherein the dantrolene is in nanoparticulate form.

52. The method of claim 50, wherein the lithium salt is lithium chloride, lithium carbonate or lithium orotate.

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