FIELD OF THE INVENTION

The present invention relates to dapansutrile, or its pharmaceutically acceptable solvates, for treating spinal cord injury.

BACKGROUND

The nervous system is divided into two parts: the central nervous system (CNS), which includes the brain and the spinal cord, and the peripheral nervous system, which includes nerves and ganglions outside of the brain and the spinal cord. While the peripheral nervous system is capable of repair and regeneration, the central nervous system is unable to self-repair and regenerate.

In the United States, traumatic injuries to the CNS such as traumatic brain injury and spinal cord injury (SCI) affect over 90,000 people each year. These traumatic insults to the CNS cause axonal loss, disrupt neuronal connections, and ultimately result in permanent blindness, paralysis, and other losses in cognitive, motor, and sensory functions.

SCI leads to the loss of motor, sensory, and autonomic functions below the lesion site. Despite SCI being one of the main causes of death and disability, there is currently no effective treatment. Neurological deficits after SCI are due to the immediate neural damage produced by the injury itself but also to a secondary phase of tissue degeneration that occurs over several weeks after initial trauma. Various events are involved in triggering secondary damage following SCI, such as ischemia, excitotoxicity, necrosis, apoptosis, and inflammation, among others. The inflammatory response elicited after neurotrauma is thought to be one of the main contributors to secondary tissue damage [1] Therefore, suppressing inflammation could be a clinically beneficial approach to minimize functional impairments after acute SCI.

Today, there is a wide variety of drugs including biologies with anti-inflammatory properties. However, the use of anti-inflammatory reagents can be associated with unwanted side effects, including the risk of opportunistic infections [2] For instance, reactivation of Mycobacterium tuberculosis in patients receiving anti-TNF therapies can be 25 times higher than in untreated individuals [3] Indeed, methylprednisolone for acute SCI, which was a standard of care for years following the publication of NASCIS II, was discontinued due to the lack of efficacy in neurological recovery and the adverse side effects, including the increased risk of opportunistic infections [4, 5] Due to the nature of opportunistic infections, they become a major concern in many anti-inflammatory therapies. What distinguishes IL-1- based therapies from other agents is the lack of opportunistic infections. Although IL1 blocking biologies increase the risk of routine bacterial infection, these easily treated with antibiotics; opportunistic infections are rare with IL1 blockers [6]

There is a need to develop a new method for effectively treating SCI. The method should be effective with minimal side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1B. Levels of IL-Ib (1A) and IL-18 (IB) in the spinal cord at different time points after injury. Spinal cords without injury were used as naive condition. Dashed line marks the detection limit of each cytokine based on Luminex assay (1.49 pg/mg protein for IL-Ib and 9.88 pg/mg protein for IL-18). Data are represented as mean ± SEM. N = 4 per time point. One way ANOVA with Dunnetf s post hoc correction was used to analyze significant differences. * p < 0.05, ** pO.Ol, *** pO.001 vs Naive.

FIGs. 2A-2D. Effects of dapansutrile in the functional recovery and tissue preservation after SCI. (2A) BMS score showing the locomotor progression of mice receiving 60 mg/kg twice a day (middle curve) or 200 mg/kg once a day (top curve) of Dapansutrile (OLT1177 ^® ) for 7 days after SCI. Control mice were treated with saline (bottom curve). (2B) Functional distribution of different groups at 28dpi. Darker colors represent more skilled functional performances. BMS values of each type of movement are indicated in parenthesis. (2C) Myelin sparing in the lesion area of mice treated with 60 mg/kg twice a day (middle curve) or 200 mg/kg once a day (top curve) of dapansutrile for 7 days after SCI. Control mice were treated with saline (bottom curve). (2D) Representative micrographs of each group showing the myelinated are at the injury epicenter. Data are represented as mean ± SEM. N = 15 for Saline,

N = 12 for 60 mg/kg dose, and N = 8 for 200 mg/kg dose of dapansutrile. Two-way RM- ANOVA with Bonferroni’s post hoc correction was used to analyze differences between groups in (2A). Chi-square test to compare proportions in (2B). Multiple t-test comparisons with Holm- Sidak’s post hoc were used to analyze significant differences between groups in (2C). & p <

0.05 for dapansutrile 60 mg/kg (x2) vs Saline; * p < 0.05, ** p < 0.01, and *** p < 0.001 for dapansutrile 200 mg/kg vs Saline.

FIGs. 3A-3B. Assessment of neurons and astrocytes after treatment with dapansutrile.

(3 A) Quantification of NeuN-positive cells in the ventral horns at various rostral and caudal distances from the injury epicenter in mice treated with saline (bottom curve) or dapansutrile at 200 mg/kg (top curve). (3B) Rostro-caudal quantification of GFAP immunoreactivity in the spinal cord of mice treated with saline (bottom curve) of OLT177 at 200 mg/kg (top curve).

Data are represented as mean ± SEM. N = 6 for Saline and N = 7 for dapansutrile (200 mg/kg). Multiple t-test comparisons with Holm-Sidak’s post hoc were used to analyze significant differences between groups. * p < 0.05 vs Saline.

FIGs. 4A-4C. Effects of dapansutrile (OLT1177 ^® ) on NLRP3 activation in the spinal cord at 24 h post-injury. (4A) Representative immunoblotting showing NLRP3, pro-IL-Ib, ASC, andcaspase-1. Molecular weight (KDa) of each protein is marked on the left side b-acting was used as loading control. (4B) Graphs showing the quantification of NLRP3, pro-IL-Ib, ASC, and caspase-1 in mice treated with saline or dapansutrile (200 mg/kg) after SCI. (c) ELISA levels of active IL-Ib in the injured spinal cord after treatment with dapansutrile or vehicle. Data are represented as mean ± SEM. N = 5 for each group. Multiple t-test comparisons with Holm- Sidak’s post hoc were used to analyze significant differences between groups. * p < 0,01 and ***p < 0,0001 vs Saline.

FIGs. 5A-5E. Effects of dapansutrile (OLT1177 ^® ) in myeloid cell accumulation in the spinal cord after contusion injury. (5A-5C) Number of macrophages, microglia, and neutrophils in the spinal cord of mice treated with daily administration of dapansutrile (200mg/kg) or saline at 1 (A), 3 (B), and 5 (C) days after lesion. (5D, 5E) Representative density plots of flow cytometry analysis showing microglia, macrophages, and neutrophils in the spinal cord at 3 days (D) and 5 days (E) days post-injury. Cell numbers are included in parenthesis. Note the marked reduction in the number of neutrophils and macrophages in the dapansutrile group in comparison with Saline. Data are represented as mean ± SEM. N = 4 for each group. Multiple t- test comparisons with Holm- Sidak’s post hoc was to analyze significant differences between groups. * p<0.05 vs Saline.

DETAILED DESCRIPTION OF THE INVENTION

Spinal cord injury (SCI) leads to irreversible functional deficits due to the disruption of axons and the death of neurons and glial cells. The inflammatory response that occurs in the injured spinal cord results in tissue degeneration. The present invention targets inflammation after acute SCI, and thus ameliorates histopathological evidence indicative of damage, and consequently reduces functional disabilities induces functional protection and histological improvement after spinal cord injury.

The present invention is directed to a method of treating SCI. The method comprises administering to a subject suffering from SCI an effective amount of a compound of 3- methanesulfonylpropionitrile (dapansutrile), or a pharmaceutically acceptable solvate thereof, to treat SCI.

Dapansutrile is a b-sulfonyl nitrile synthetic compound of 140 KDa that selectively inhibits NLRP3 inflammasome [7] Dapansutrile is safe for use in humans.

3 -methanesulfonylpropionitrile

“Solvates,” as used herein, are addition complexes in which the compound is combined with an acceptable co-solvent in some fixed proportion. Co-solvents include, but are not limited to, water, acetic acid, ethanol, and other appropriate organic solvents.

“An effective amount,” as used herein, is an amount effective to treat SCI by ameliorating the symptoms of SCI.

Spinal cord injury may result in death or impairment of cells, e.g., neurons and associated loss of function. Death or impairment of cells can negatively impact recovery. For example, neurodegeneration may reduce the potential for recovery of neuronal functions following spinal cord injury. Cells impacted by spinal cord injury may undergo immediate death or impairment or, alternatively, delayed death or impairment.

Outcomes of spinal cord injury include the impairment or death of cells, e.g. neurons (neurodegeneration). Aspects of neurodegeneration may include decreased neuronal survival, decreased axon sparing, and/or decreased axon growth. Neurodegeneration may limit functional recovery following spinal cord injury or inhibit other post-injury neuronal activities, such as neuronal (e.g., axonal) growth, neuronal (e.g. axonal) regeneration (e.g. axonal sprouting), or neuronal repair.

The present invention targets the NLRP3 inflammasome with dapansutrile to arrest neuroinflammation and reduce functional impairments after acute SCI in humans. The present method treats SCI by improving or ameliorating one or more post SCI symptoms as described above.

In one embodiment, the present method decreases cellular impairment or cell death (e.g., neurodegeneration), improves functional recovery, and/or improves post-injury neuronal activities. In one embodiment, the present method promotes nerve function following injury to a CNS neuron.

In one embodiment, the present method provides a method for preserving neuron viability and/or promoting axon regeneration and nerve function in a subject affected with SCI.

In one embodiment, the present method protects against neurological deficits, myelin degeneration, and myelin loss.

In one embodiment, the present method ameliorates inflammation after SCI and leads to beneficial effects on functional and histopathological outcomes after SCI.

In one embodiment, the present method results in one or more responses selected from the group consisting of improved neuronal survival, improved neuronal regeneration, improvement/recovery of motor function, improvement/recovery of fine motor coordination, improvement/recovery from muscle spasticity, improvement/recovery from paresis or paralysis of one or both sides, reduction in severity and/or number of seizure disorders, improvement/recovery of balance, improvement/recovery of gait, improvement/recovery of sensory function, ameliorate impairment of sensation, and ameliorate impairment of motor function.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers and an active compound of dapansutrile, or a pharmaceutically acceptable solvate thereof. The active compound or its pharmaceutically acceptable solvate in the pharmaceutical compositions in general is in an amount of about 0.01- 20%, or 0.05-20%, or 0.1-20%, or 0.2-15%, or 0.5-10%, or 1-5% (w/w), for a topical formulation; about 0.1-5% for an injectable formulation, 0.1-5% for a patch formulation, about 1-90% for a tablet formulation, and 1-100% for a capsule formulation. The active compound used in the pharmaceutical composition in general is at least 90%, preferably 95%, or 98%, or 99% (w/w) pure.

In one embodiment, the pharmaceutical composition is in a dosage form such as tablets, capsules, granules, fine granules, powders, syrups, suppositories, injectable solutions, patches, or the like.

In one embodiment, the pharmaceutical composition is in the form of an aerosol suspension of respirable particles comprising the active compound, which the subject inhales. The respirable particles can be liquid or solid, with a particle size sufficiently small to pass through the mouth and larynx upon inhalation. In general, particles having a size of about 1 to 10 microns, preferably 1-5 microns, are considered respirable. The respirable particles including dapansutrile can be prepared into dry powder using well-known art of super critical fluid technology. In such a case, the compound is admixed with appropriate excipients and milled into a homogenous mass using suitable solvents or adjuvants. Following this, this mass is subjected to mixing using super critical fluid technology and suitable particle size distribution achieved. The particles in the formulation need to be within a desired particle size range such that the particles can be directly inhaled into the lungs using a suitable inhalation technique or introduced into the lungs via a mechanical ventilator.

In one embodiment, the active compound is incorporated into an acceptable carrier, including creams, gels, lotions or other types of suspensions that can stabilize the active compound and deliver it to the affected area by topical applications. The above pharmaceutical composition can be prepared by conventional methods.

Pharmaceutically acceptable carriers, which are inactive ingredients, can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous based solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments. The pharmaceutically acceptable carriers may also contain ingredients that include, but are not limited to, saline and aqueous electrolyte solutions; ionic and nonionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusters and buffers such as salts of hydroxide, phosphate, citrate, acetate, borate; and trolamine; antioxidants such as salts, acids and/or bases of bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetyl cysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxy toluene, tocopherols, and ascorbyl palmitate; surfactants such as lecithin, phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine and phosphatidyl inositiol; poloxamers and poloxamines, polysorbates such as polysorbate 80, polysorbate 60, and polysorbate 20, polyethers such as polyethylene glycols and polypropylene glycols; polyvinyls such as polyvinyl alcohol and povidone; cellulose derivatives such as methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose and hydroxypropyl methylcellulose and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di-, and triglycerides; polymers of acrylic acid such as carboxypolymethylene gel, and hydrophobically modified cross-linked acrylate copolymer; polysaccharides such as dextrans and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be preserved against bacterial contamination using well-known preservatives, these include, but are not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as a non-preserved formulation for either single or multiple use.

For example, a tablet formulation or a capsule formulation of the active compound may contain other excipients that have no bioactivity and no reaction with the active compound. Excipients of a tablet may include fillers, binders, lubricants and glidants, disintegrators, wetting agents, and release rate modifiers. Binders promote the adhesion of particles of the formulation and are important for a tablet formulation. Examples of binders include, but not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, starch, and tragacanth gum, poly(acrylic acid), and polyvinylpyrrolidone.

For example, a patch formulation of the active compound may comprise some inactive ingredients such as 1,3-butylene glycol, dihydroxyaluminum aminoacetate, disodium edetate, D- sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone (polyvinylpyrrolidone), propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. A patch formulation may also contain skin permeability enhancer such as lactate esters (e.g., lauryl lactate) or di ethylene glycol monoethyl ether.

Topical formulations including the active compound can be in a form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution, and suspension. The inactive ingredients in the topical formulations for example include, but not limited to, lauryl lactate (emollient/permeation enhancer), diethylene glycol monoethyl ether (emollient/permeation enhancer), DMSO (solubility enhancer), silicone elastomer (rheology/texture modifier), caprylic/capric triglyceride, (emollient), octisalate, (emollient/ETV filter), silicone fluid (emollient/diluent), squalene (emollient), sunflower oil (emollient), and silicone dioxide (thickening agent). Method of Treatment

The present invention is directed to a method of treating SCI. The method comprises the steps of first identifying a subject suffering from SCI, and administering to the subject the active compound dapansutrile, in an amount effective to treat SCI.

Dapansutrile is small and penetrates the CNS rapidly for reducing the local inflammation. The inventors have shown that NLRP3 is activated after spinal cord injury and that dapansutrile can inhibit NLRP3 activation in the contused spinal cord in an animal model. Dapansutrile ameliorates inflammation after SCI and leads to beneficial effects on functional and histopathological outcomes after SCI.

The inventors have shown that dapansutrile is able to promote significant protections against myelin loss at more distant regions from the injury epicenter. Dapansutrile leads to significant enhancement in myelin preservation at the injury epicenter and at some rostral and caudal regions.

The inventors have shown that dapansutrile protects against neurological deficits, myelin degeneration, and neuronal loss in a lower extent after SCI in mice.

The inventors have shown that dapansutrile reduces the infiltration of neutrophils and monocytes. This mechanism of dapansutrile’ s efficacy is highly relevant to SCI because early infiltration of neutrophils and monocytes into the lesioned area of the spinal cord significantly contributes to secondary damage and locomotor impairments. Dapansutrile is small and penetrates the CNS rapidly for reducing the local inflammation. There is no blood brain barrier that limits its penetration into the CNS.

The pharmaceutical composition of the present invention can be applied by systemic administration and local administration. Systemic administration includes oral, parenteral (such as intravenous, intramuscular, subcutaneous or rectal), and other systemic routes of administration. In systemic administration, the active compound first reaches plasma and then distributes into target tissues. Local administration includes topical administration.

Dosing of the composition can vary based on the extent of the disease and each patient’s individual response. For systemic administration, plasma concentrations of the active compound delivered can vary; but are generally 0.1-1000 pg/mL or 1-100 pg/mL.

In one embodiment, the pharmaceutical composition is administrated orally to the subject. The dosage for oral administration is generally at least 1 mg/kg/day and less than 100 mg/kg/day. For example, the dosage for oral administration is 1-100, or 5-50, or 10-50 mg/kg/day, for a human subject. For example, the dosage for oral administration is 100- 10,000 mg/day, and preferably 200-1000, 200-2000, 500-2000, 500-4000, 500-4000, 1000- 5000, 2000-5000, 2000-6000, or 2000-8000 mg/day for a human subject. The drug can be orally taken once, twice, three times, or four times a day. The patient is treated daily for 7- 14 days, up to 1 month, 2 months, or 3 months, or for lifespan.

In one embodiment, the pharmaceutical composition is administrated intravenously to the subject. The dosage for intravenous bolus injection or intravenous infusion is generally 0.03 to 20 or 0.03 to 10 mg/kg/day.

In one embodiment, the pharmaceutical composition is administrated subcutaneously to the subject. The dosage for subcutaneous administration is generally 0.3-20 or 0.3-3 mg/kg/day.

Those of skill in the art will recognize that a wide variety of delivery mechanisms are also suitable for the present invention.

The present invention may be used in combination with one or more other treatments that treat SCI.

The present invention is useful in treating a mammal subject or a mammal patient.

The present invention is particularly useful in treating humans. A “subject” and a “patient” are used interchangeably in the application.

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.

EXAMPLES

Example 1. Materials and Methods Spinal cord injury surgical procedure

Spinal cord injury procedure was performed according to described protocols [28] Adult (8 - 10 weeks old) female mice were anesthetized by intramuscular injection with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg). After skin and muscle incision, laminectomy at 11 th thoracic vertebrae was performed and the exposed spinal cord was contused using the Infinite Horizon Impactor device (Precision Scientific Instrumentation). A force of 60 kdynes and a tissue displacement of 450 - 550 pm were applied.

Dapansutrile treatment

Dapansutrile (OLT1177 ^® ; Olatec Therapeutics LLC) diluted in sterile saline solution was injected intraperitoneally once a day, starting 1 hour after lesion, for seven days. Two different administration protocols were used: (i) 60 mg/kg twice a day, and (ii) 200 mg/kg once a day. Control mice were treated with sterile saline solution using the same administration protocol.

Protein detection

Mice received an intraperitoneal injection of sodium pentobarbital (Dolethal). Blood was removed by perfusion with 60 mL of 0.9% NaCl in distilled water and a 6 mm piece of spinal cord, centered into the injury site, was taken from each mouse. Samples were frozen in liquid nitrogen and homogenized in RIPA buffer (Thermo Fisher) supplemented with protease inhibitor (Roche) using a TissueRuptor (Qiagen) and an Ultrasonic Homogenizer (Biologies Inc.). Protein concentration was quantified using BCA Protein Assay Kit (Thermo Fisher) according to manufacturer’s instructions.

To measure levels of IL-Ib and IL-18 in the naive condition and from 1 hour to 28 days after injury, samples were quantified by Luminex (bead-based multiplex assay).

Samples were diluted to 2 pg/pl and processed using a ProcartaPlex Panel (ThermoFisher) on MAGPIX Luminex reader (Millipore) as previously described [28] Final values were normalized by the protein concentration of each sample. 4 mice per time-point were used.

For immunoblotting studies, 60 pg of protein per sample were resolved on a Mini- PROTEAN TGX 4-20% gel (Bio-Rad) and transferred to a 0.1 pM nitrocellulose membrane (GE Healthcare). Membranes were blocked in 5% dried milk in PBS-T 0.5% for 1 hour at room temperature. Samples were incubated with primary antibody for mouse NLRP3 (1:1000, AdipoGen), caspase-1 (1:200, Santa Cruz Biotechnology), and IL-Ib (1:500, Santa Cruz Biotechnology). Peroxidase-conjugated secondary antibodies and chemiluminescence were used to detect the protein concentration. Conjugated antibody against b-actin (1:1000, Santa Cruz Biotechnology) was used to normalize protein concentrations. 5 mice per group were used.

Functional Assessment

Locomotor recovery after SCI was evaluated at 1, 3, 5, 7, 10, 14, 21, and 28 days post-injury (dpi) in an open-field using the nine-point Basso Mouse Scale (BMS) [29] BMS evaluation was performed by two researchers who were blinded to the experimental groups. Consensus score between two researchers was taken. 15 mice were used for the saline group. 12 and 8 mice were used for the groups treated with the 60mg/kg and 200mg/kg doses of dapansutrile, respectively.

Histology

At 28 dpi, mice were perfused with 4% paraformaldehyde (Sigma- Aldrich) in 0.1 M phosphate buffer. A piece of 6 mm of spinal cord, centered into the lesion site, was removed from each mouse and cryoprotected with 30% sucrose in 0.1 M phosphate buffer saline (PBS) at 4°C. Samples with 15 pm thick were cut in the cryostat and picked up with a glass slide. Samples were arranged following a serial distribution. Adjacent sections in the same slide were 150 pm apart.

For myelin quantification, sections were placed in a 1 mg/ml Luxol Fast Blue (LFB) (Sigma-Aldrich) solution in 95% ethanol and 0.05% acetic acid and left overnight at 37°C. Sections were then washed in 95% ethanol and distilled water before clearing with 0.5 mg/ml of LriCCh in distilled water for 1.5 min. After several washes, sections were dehydrated and mounted in DPX mounting media (Sigma-Aldrich). After fixation, the epicenter of the contusion was localized by determining the tissue section with the lowest LFB stained area. NIH ImageJ software was used to perform the quantifications. 15 mice were used for the saline group. 12 and 8 mice were used for the groups treated with the 60 mg/kg and 200 mg/kg doses of dapansutrile, respectively.

For neuron and astrocyte assessment, sections were blocked with 5% Normal Donkey Serum (Millipore) for 1 hour at room temperature. Samples for neuron quantification were also treated with Endogenous Biotin Blocking Kit (Invitrogen) following manufacture’s protocol. Sections were then incubated with antibodies against mouse NeuN (1 :200, biotinylated, Sigma-Aldrich) and GFAP (1:200, rabbit, Millipore) overnight at 4°C. Streptavidin conjugated to Alexa fluor 594 (1:200, Invitrogen) and donkey anti-rabbit secondary antibody conjugated to Alexa fluor 488 (1:200, Invitrogen) were applied for 1 hour at room temperature. Finally, slides were covered by coverslips in Mowiol mounting media (Sigma-Aldrich). Neuronal survival was assessed by manually counting the number of NeuN+ cells in the ventral horns. Astrocyte immunoreactivity was calculated at the whole section by measuring the integrated density using NIH ImageJ software. 6 and 7 mice were used for the saline and dapansutrile (200mg/kg) groups, respectively. Flow cytometry

Flow cytometry from spinal cord was performed according to described protocol [28] At 1, 3, and 5 days after injury, animals received an intraperitoneal injection of sodium pentobarbital (Dolethal). Blood was removed by perfusion with 60 mL of 0.9% NaCl and a piece of 6 mm of the spinal cord, centered into the injury site, was taken from each mouse. Cell suspensions of the spinal cord were obtained by enzymatic disaggregation using a mixture of collagenase (Sigma- Aldrich) and DNase (Roche) and mechanic disaggregation followed by passage through 70-pm cell strainers (Fisher). Cells were labeled with the following antibodies: CD45- PerCP (eBioscience), CDllb-PE-Cy7 (eBioscience), and F4/80- PE (eBioscience). 1:200 dilutions were used for each antibody. Proper isotypes for each antibody were used.

After cell staining, cells were fixed with 1% paraformaldehyde (Sigma- Aldrich) in 0.1 M of phosphate buffer and analyzed using the FACSCanto flow cytometer (BD Bioscience). Cell gating and quantification were performed using FlowJo ^® software. Microglia cells were defined as CD451ow and CD1 lb+, whereas macrophages were defined as CD45high, CDllb+, and F4/80+. Granulocytes, mainly neutrophils, were defined as CD45high, CDllb+, and F4/80- [28,30] 4 mice per group were used.

Statistics

All analyses were conducted through GraphPad Prism v8. Cytokine levels obtained from Luminex were analyzed using one-way ANOVA with Dunnett’s post hoc correction. Functional follow-ups for BMS score were analyzed using two-way ANOVA with repeated measures (RM) with Bonferroni’s post hoc correction. Functional proportions from BMS were compared by chi-square test. Myelin, neuron, and astrocyte sparing were analyzed using multiple t-test comparisons with Holm-Sidak’s post hoc correction. Protein levels obtained by western blot and ELISA were analyzed using t-test. Numbers of myeloid cells from flow cytometry were analyzed using t-test. Results from all the experiments were expressed as mean ± SEM and differences were considered significant at p<0.05.

Example 2. Protein levels of IL-Ib and IL-18 increase after SCI

Since damage to the spinal cord leads to the immediate increase in the production of pro-inflammatory cytokines, we first evaluated the dynamics of IL-Ib and IL-18 at protein level in the injury environment from lh to 28 days after contusion surgery using Luminex technology.

The data revealed that the protein levels of IL-Ib and IL-18 were very low in the spinal cord at physiological conditions, being in some samples below the detection limit (1.49 pg/mg protein and 9.88 pg/mg protein, respectively). However, levels of both cytokines changed significantly after contusion injury. Levels of IL-Ib in the spinal cord parenchyma peaked at 12 hours post-lesion and remained at significantly high levels up to 24 hours post injury (FIG. 1A). Expression of IL-18 in the injured spinal cord was increased at later time points after SCI. Indeed, levels of IL-18 did not show any increase until day 3 post-lesion, reaching maximal levels at day 7, and remained at significant greater levels up to day 21 (FIG. IB). Since activation of the NLRP3 inflammasome is required for the processing and release of IL-Ib and IL-18, these findings indicate that NLRP3 is activated after spinal cord injury.

The results show that levels of IL-Ib and IL18 were increased in the spinal cord parenchyma after SCI, but with different expression profiles. The levels of IL-Ib were rapidly increased, reaching a peak at 12 hours after the injury; whereas levels of IL-18 did not peak until day 7 after the injury. At a dose of 200mg/kg, dapansutrile reduced the levels of active IL-Ib in the spinal cord at early time points after contusion injury and diminished the accumulation of neutrophils and macrophages. These changes protected against neurological deficits and histological evidence of damage.

Example 3. Dapansutrile improves locomotor recovery and tissue preservation after SCI

In this example, we tested the effect of dapansutrile, on functional outcomes after

SCI.

In FIG. 2A, BMS scores show the locomotor progression of mice receiving 60 mg/kg twice a day (middle curve) or 200mg/kg once a day (top curve) of dapansutrile (OLT1177 ^® ) for 7 days after SCI. Control mice were treated with saline (bottom curve). The results show that mice treated with 60 mg/kg of dapansutrile twice a day for 7 days had an improved functional recovery, although statistical differences against saline group were not observed (FIG. 2A). Mice treated with 200 mg/kg of dapansutrile once a day for 7 days showed a significant enhancement in the functional performance in comparison with mice treated with saline. Differences in the BMS score between dapansutrile (200 mg/kg) and saline groups were statistically significant from day 10 to day 28 after SCI. FIG. 2B shows functional distribution of different groups at 28dpi. At the end of the follow-up, the 73% of the mice treated with saline showed hindlimb plantar placement, meaning that they were able to place the full feet on the ground, but in which only the 6% showed occasional stepping (FIG. 2B). In the case of the animals treated with the 60 mg/kg dose of dapansutrile, all of them showed hindlimb plantar placement and 50% of them also showed hindlimb plantar stepping, being occasional (42%) or frequent (8%). Finally, all mice treated with 200 mg/kg of dapansutrile showed hindlimb plantar placement and 75% of them were able to perform occasional (37.5%) or frequent (37.5%) hindlimb plantar stepping (FIG. 2B). The results are summarized in Table 1. Chi-square test verified that functional proportions were significantly determined by the group.

Table 1. Per cent of mice after saline treatment or dapansutrile treatment

FIG. 2C shows myelin sparing in the lesion area of mice treated with 60mg/kg twice a day (green) or 200mg/kg once a day (top curve) of dapansutrile for 7 days after SCI. Control mice were treated with saline (bottom curve). FIG. 2D shows the representative micrograph of each group; the myelinated are at the injury epicenter. The results show that spinal cord cross sections stained for LFB revealed that These findings indicate that NLRP3 is activated after spinal cord injury. Differences were evident from 300 pm rostral to 150 pm caudal sections from the lesion core (FIGs. 2C and 2D). However, the 200 mg/kg dose, but not 60 mg/kg dose of dapansutrile, was also able to promote significant protections against myelin loss at more distant regions from the injury epicenter (FIGs. 2C and 2D).

Since the 200 mg/kg dose of dapansutrile resulted in greater efficacy than the 60 mg/kg dose, we then evaluated whether the high dose of dapansutrile minimize neuronal loss and astrocytes reactivity.

FIGs. 3A-3B show the assessment of neurons and astrocytes after treatment with dapansutrile. We observed that dapansutrile increased number of NeuN+ cells at ventral horns at rostral and caudal regions from the injury epicenter. Significant differences against saline-treated mice were only found at sections located at 750 pm rostral (R) and 600 pm caudal (C) to the lesion core (FIG. 3A). No significant differences in the GFAP signal were found after dapansutrile treatment, however, this drug tended to increase astrocyte immunoreactivity at the lesion epicenter and adjacent sections (FIG. 3B).

These results therefore demonstrate that dapansutrile protects against neurological deficits, myelin degeneration, and neuronal loss in a lower extent after SCI in mice.

Example 4. dapansutrile reduces the inflammatory response after SCI

After demonstrating that dapansutrile has therapeutic effects after SCI, we next assessed the effects of this compound on the neuroinflammatory response.

First, we evaluated whether dapansutrile has some effect on the levels of IL-Ib. For this purpose, we measured the levels of the proteins implicated in the processing of IL-Ib at 24 hours after SCI. We observed that the levels of NLRP3 were maintained in all the samples (FIGs. 4A/4B). However, levels of cleaved caspase-1 were significantly reduced in those animals treated with dapansutrile (200mg/kg). This reduction in the levels of cleaved caspase-1 by dapansutrile correlated with a reduction in the levels of the active form of IL-Ib (FIG. 4C) while the levels of the inactive form of this cytokine remained unaltered (FIGs. 4A/4B). These data demonstrate the ability of dapansutrile to inhibit NLRP3 activation in the contused spinal cord.

We also assessed whether dapansutrile affected the number of microglia, macrophages, and neutrophils in the spinal cord at 1, 3, and 5 days after injury. We observed that dapansutrile did not reduce to the number of any of these myeloid cell populations at day 1 after SCI, after one single injection of 200 mg/kg (FIG. 5 A). However, the number of neutrophils in the spinal cord were reduced -50% in mice treated with dapansutrile at day 3 after injury (FIGs. 5B/5D). Finally, at day 5 after injury, the number of macrophages were reduced -50% in mice treated with dapansutrile further demonstrating the effects of this compound on minimizing inflammation after SCI (FIGs. 5C/5E). Dapansutrile also appeared to reduce the number microglia and neutrophils at 5 days after injury, but these differences were not significant. Together these results demonstrate that dapansutrile ameliorates inflammation after SCI and leads to beneficial effects on functional and histopathological outcomes after SCI.

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The invention, and the manner and process of making and using it, are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude the specification.