Methods for treating amyotrophic lateral sclerosis and dosing regimens therefor - Patents.com

Abstract

The present invention relates to the treatment of ALS patients with Fausudil at a dose of 60-240 mg / day in a specific therapeutic regimen that is expected to result in a 25-50% reduction in mean decline over at least three months as measured using the Revised ALS Functional Rating Scale.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 278,523, filed November 12, 2021, the disclosure of which is incorporated by reference in its entirety. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is a fatal neurodegenerative disease affecting motor neurons, resulting in progressive loss of control of voluntary movements. It is associated with degeneration of upper motor neurons and their corticospinal axonal tracts (lateral sclerosis) and loss of lower motor neurons and their axons, which leads to muscle wasting (muscle atrophy) and paralysis of voluntary muscles (Mitsumoto et al., 1998). Upper motor neurons originate from the motor areas of the cerebral cortex or brainstem and transfer motor information down to motor neurons that are directly involved in stimulating target muscles. Their dysfunction causes stiffness due to continuous muscle contractions that interfere with walking, movement, and speech. Lower motor neurons connect the brainstem and spinal cord to muscle fibers. Their dysfunction causes muscle atrophy, small localized, involuntary muscle contractions in spasms. Many individuals with ALS die of respiratory failure within 48 months of onset of symptoms, and most often within 3 to 5 years of onset.

[0003] ALS, like other neurodegenerative conditions, is believed to be caused by a combination of genetic factors, environmental factors, and age-related dysfunction. Apart from genetic factors, age and male gender increase the risk of ALS. Several studies have suggested environmental risk factors for ALS, such as smoking, body mass index, physical exercise, occupational and environmental exposure to metals, pesticides, β-methylamino-L-alanine, head injury, and viral infections. However, the causal relationship between these factors and ALS has yet to be established (Masrori 2020).

[0004] Approximately 90-96% of ALS cases are sporadic, and only 5-10% are familial due to inherited genetic mutations.

[0005] ALS has also been associated with protein inclusions in motor neurons and the CNS. Both sporadic and familial ALS have been associated with abnormally accumulated TAR DNA-binding protein 43 (TDP-43) aggregates, which are thought to spread prion-like between cells. TDP-43 is the major misfolded, mislocalized, ubiquitinated protein that constitutes the major form of neuropathological aggregates in motor neurons in ALS. TDP-43 is a DNA / RNA-binding protein that regulates RNA splicing and stability as well as microRNAs. TDP-43 normally localizes to the cell nucleus where it functions in transcription, but misfolded TDP-43 aggregates aggregate in the cytosol, resulting in loss of function of the cell nucleus that may cause transcriptional defects. It is unclear whether ALS pathogenesis is associated with loss of TDP-43 function or pathology associated with aggregates and cytoplasmic mislocalization.

[0006] Multiple molecular pathways are involved in the pathogenesis of ALS, including failure of protein homeostasis, excitotoxicity, neuroinflammation, mitochondrial dysfunction and oxidative stress, oligodendrocyte dysfunction, cytoskeletal and axonal transport defects, disturbances in RNA metabolism, nucleocytoplasmic transport defects, and DNA repair defects. Interestingly, many of the genes associated with ALS appear to be clustered in key pathways: protein quality control and degradation, RNA metabolism, and cytoskeletal and axonal transport (Masrori 2020).

[0007] Rho Kinase (ROCK) Inhibitors ALS. There have been many publications addressing the use of rho kinase inhibitors in various animal models of neurodegeneration, including ALS. Most models are deficient in that they fail to recapitulate the ALS (or other neurodegenerative disease) phenotype, or are only relevant to familial ALS, which represents only 5-10% of ALS patients. As an example, U.S. Patent No. 9,980,972 describes the use of fasudil in the SOD1 G93 mouse model, which has a mutation in the superoxide dismutase (SOD) protein. The patent claims treatment of familial early stage ALS with fasudil at 10-1200 ng per kg of body weight per day, or 1-12 mg per kg of body weight per day. No humans were treated. Furthermore, mutations in SOD are only relevant to familial ALS, which is why the claims are limited. Mice in the SOD model develop a progressive motor disorder that leads to adult-onset neurodegeneration of spinal motor neurons and paralysis. Furthermore, the original SOD1-G93A mouse (originally described in Gurney et al., 1994) has since diverged into a family of strains with different genetic backgrounds and transgene expression levels that significantly affect symptom onset and severity. As one publication stated, "Animal models have not been able to predict treatment response in humans, and there are no validated biomarkers for human ALS beyond the clinically supported diagnostic application of electromyography" (Menke 2016).

[0008] Another problem with animal models is that many of them exhibit high copy numbers of mutant alleles, i.e., they overexpress, for example, mutant SOD. This is also very different from human familial ALS, where affected patients have a mutation in one allele. Other models, such as TARDBP (TDP-43) mice that express TDP-43, also rely on overexpression approaches that do not replicate human ALS.

[0009] Fasudil was administered to three (3) human ALS patients on a compassionate use basis (Koch et al. 2020). One patient had familial ADS and the other two patients were presumed to have ADS. Patients received 30 mg of fasudil intravenously twice daily for 20 consecutive workdays (excluding weekends). There were no conclusive results beyond safety. Currently, clinical trials are underway in Germany, Switzerland, and France to infuse fasudil following the same intravenous administration and dosing schedule (Lingor et al., 2019). The trial is designed to treat three parallel arms with fasudil 15 mg twice daily, fasudil 30 mg twice daily, and a matching placebo. No updates on the trial were available in September 2021, except for a publication detailing the unexpected legal, administrative, and financial complexities of a multinational trial, in which a U.S.-based trial was proposed and added but not included. (Lingor 2021).

[0010] Other publications disclose the use of impractical routes of administration (e.g., intracerebroventricular injection) of fausudil to treat neurological and proteinopathy-related diseases, and many do not use appropriate dosing. In this regard, standard formulas exist for converting doses used in animals to the same dose in humans. Human equivalent doses (HEDs) can be calculated, for example, using Table 1 in Nair & Jacob (2016), which is the same conversion used by the US FDA.

[0011] There is a significant unmet need to provide new therapies that show benefit in non-familial ALS in humans as well as in animals with genetic mutations, which do not represent the majority of the ALS population. Summary of the Invention

[0012] The present invention contemplates the treatment of ALS patients with fasudil. In one embodiment, a method of treating a patient having amyotrophic lateral sclerosis (ALS) is provided, comprising administering a therapeutically effective amount of fasudil in the following alternating dosing regimen: (a) the patient is treated with fasudil at least three days per week for at least two weeks in a first treatment phase; (b) after the treatment period, subjecting the patient to a first treatment-free period of at least 2 weeks; (c) treating the patient in a second treatment phase, followed by subjecting the patient to a second treatment rest phase.

[0013] In one embodiment, during the first and / or second treatment phases, the patient is treated at least 5 days per week.

[0014] In another embodiment, the first and second treatment break periods are at least 1 month but less than 6 months.

[0015] In certain embodiments, the duration of the first and second treatment phases is one month, and the duration of the first and second resting phases is one month.

[0016] In some embodiments, (a)-(c) are repeated at least once, preferably two or more times.

[0017] In certain embodiments, the duration of the first and second treatment phases is one month, the duration of the first and second resting phases is one month, and this regimen (a)-(c) is repeated at least once and for the life of the patient.

[0018] In another embodiment, the dosage in the first and second treatment phases is 30-60 mg / day of fasudil, preferably 60-120 mg / day of fasudil, more preferably 120-180 mg / day of fasudil, and most preferably 180-240 mg / day of fasudil.

[0019] In one embodiment, Fasudil is administered by intravenous infusion, hi another embodiment, Fasudil is administered orally.

[0020] In one embodiment, the ALS patient to be treated has Tar DNA-binding protein 43 (TDP-43) inclusions. In certain embodiments, pathological TDP-43 is caused by sporadic mutations in the TARDBP gene that encodes TDP-43.

[0021] In certain embodiments, the ALS patient is genetically male, hi another embodiment, the ALS patient is genetically female.

[0022] In one embodiment, treatment of an ALS patient reduces the rate of decline on the ALS Functional Rating Scale-Revised (ALSFRS-R) by more than 50% when measured over a 6-12 month period.

[0023] In yet another embodiment, treatment of the ALS patient results in stabilization of the revised ALSFRS-R for at least six months.

[0024] In certain embodiments, treating an ALS patient results in a decrease in the rate of decline of at least one of the 12 domains of ALSFRS.

[0025] In another embodiment, treating an ALS patient results in reduced muscle wasting and reduced paralysis of voluntary muscles.

[0026] Yet other embodiments contemplate treating ALS patients with low static vital capacity (SVC) as predicted by the patient's gender, age, and where the patient does not exhibit bulbar symptoms.

[0027] Certain embodiments involve treating ALS patients with an ALSFRS score of ≦36.

[0028] Some embodiments include treatment of an ALS patient with fasudil hydrochloride, where the patient is also treated with riluzole and / or edaravone.

[0029] In another embodiment, an ALS patient is treated with fasudil hydrochloride, where the patient is also treated with taurursodiol and sodium phenylbutyrate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention is based on the discovery that fasudil can be used in ALS according to a particular dosing regimen.

[0031] ROCK inhibitors The method of the present invention contemplates the administration of rho kinase (ROCK) inhibitors in the treatment of a disease or condition. Two mammalian ROCK homologs are known: ROCK1 (also known as ROKβ, Rho-kinase β, or p160ROCK) and ROCK2 (also known as ROKα) (Nakagawa 1996). In humans, both ROCK1 and ROCK2 genes are located on chromosome 18. The two ROCK isoforms share 64% identity in their primary amino acid sequences, but the homology of the kinase domain is much higher (92%) (Jacobs 2006; Yamaguchi 2006). Both ROCK isoforms are serine / threonine kinases and have similar structures.

[0032] A large number of pharmacological ROCK inhibitors are known (Feng, LoGrasso, Defert, & Li, 2015). Isoquinoline derivatives are a preferred class of ROCK inhibitors. The isoquinoline derivative fasudil was the first small molecule ROCK inhibitor developed by Asahi Kasei Corporation (Tokyo, Japan). Fasudil's characteristic chemical structure consists of an isoquinoline ring linked to a homopiperazine ring via a sulfonyl group. Fasudil is a potent inhibitor of both ROCK isoforms. In vivo, fasudil undergoes hepatic metabolism to its active metabolite hydroxyfasudil (also known as M3). Other examples of isoquinoline-derived ROCK inhibitors include dimethylfasudil and ripasudil.

[0033] Other preferred ROCK inhibitors are based on the 4-aminopyridine structure. They were first developed by Yoshitomi Pharmaceutical (Uehata et al., 1997) and are exemplified by Y-27632. Still other preferred ROCK inhibitors include indazoles, pyrimidines, pyrrolopyridines, pyrazoles, benzimidazoles, benzothiazoles, benzthiophenes, benzamides, aminofurazans, quinazolines, and boron derivatives (Feng et al., 2015). Some exemplary ROCK inhibitors are shown below. [ka]

[0034] ROCK inhibitors according to the present invention may have more selective activity against either ROCK1 or ROCK2, and usually have various levels of activity against PKA, PKG, PKC, and MLCK. Some ROCK inhibitors may be highly specific for ROCK1 or ROCK2, and have much lower activity against PKA, PKG, PKC, and MLCK.

[0035] A particularly preferred ROCK inhibitor is fasudil, which may exist as a free base or a salt, and may be in the form of a hydrate, such as a hemihydrate. [ka] Hexahydro-1-(5-isoquinolinesulfonyl)-1H-1,4-diazepine monohydrochloride hemihydrate

[0036] Fasudil is a selective inhibitor of protein kinases such as ROCK, PKC, and MLCK, and treatment results in potent relaxation of vascular smooth muscle, leading to enhanced blood flow (Shibuya 2001). ROCK, a particularly important mediator of vasospasm, induces vasoconstriction by phosphorylating the myosin-binding subunit of myosin light chain (MLC) phosphatase, thereby reducing MLC phosphatase activity and enhancing vascular smooth muscle contraction. Furthermore, there is evidence that fasudil increases endothelial nitric oxide synthase (eNOS) expression by stabilizing eNOS mRNA, which contributes to increased levels of the potent vasodilator nitric oxide (NO), thereby enhancing vasodilation (Chen 2013).

[0037] Fasudil has a short half-life of about 25 minutes, but is substantially converted in vivo to its 1-hydroxy (M3) metabolite. M3 has effects similar to its fasudil parent molecule, with slightly enhanced activity and a half-life of about 8 hours (Shibuya 2001). Thus, M3 is likely responsible for most of the molecule's in vivo pharmacological activity. M3 exists as two tautomers, shown below: [ka]

[0038] The ROCK inhibitors used in the present invention, such as Fasudil, include pharmaceutically acceptable salts and hydrates.Salts that can be formed through reaction with inorganic and organic acids.These inorganic and organic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, maleic acid, maleic acid, maleic acid, oxalic acid, tartaric acid, malic acid, mandelic acid, trifluoroacetic acid, pantothenic acid, methanesulfonic acid, or para-toluenesulfonic acid.

[0039] TDP-43 and other abnormalities in ALS TDP-43 is an essential DNA / RNA binding protein that is primarily located in the cell nucleus and is ubiquitously expressed. Deletion of TARDBP (the gene encoding TDP-43) is embryonic lethal in mice.

[0040] TDP-43 is a major component of ubiquitinated and hyperphosphorylated cytoplasmic aggregates observed in postmortem tissues from ALS patients. Abnormal TDP-43 is present in approximately 97% of ALS patients, mainly in motor neurons of the cerebral cortex, but also in the spinal cord. More than 50 mutations in TARDBP are known. Generally, pathological TDP-43 in ALS is a truncated form of either 25 or 35 kD. In ALS, there is frequent mislocalization of TDP-43 from the cell nucleus to the cytoplasm, which may block cellular trafficking in motor neurons.

[0041] ALS patients also exhibit protein mutations, leading to disruption of the ubiquitin-proteasome and autophagy clearance systems, which may result in cleaved TDP-43 being inappropriately degraded. Notwithstanding the above, it is unclear whether TDP-43 contributes to ALS pathology or is a bystander to other defects, and targeting TDP-43 may be too late to prevent neurodegeneration, disease onset or progression.

[0042] Animal models of pathological TDP-43 that do not rely on artificial overexpression do not recapitulate hallmarks of ALS pathology. Motor impairments are subtle and occur at a later stage. This suggests that there are additional and unknown mechanisms contributing to ALS and that animal models of ALS, especially when TDP-43 is overexpressed, have little relevance to the pathogenesis and / or progression of the human disease.

[0043] ALS is also associated with mutations in genes encoding proteins involved in protein and membrane degradation pathways, suggesting that impaired protein clearance is pathological in ALS (and FTD). These include mutations in p62, valosin-containing protein (VCP), ubiquitin 2, and optineurin, all of which are effectors of autophagy and / or the ubiquitin proteasome system (UPS) protein degradation pathways. This suggestion is reinforced by observations with insufficiency of C9orf72.

[0044] Another genetic defect associated with some sporadic and familial ALS patients is the presence of hexanucleotide repeat expansions (HREs) of GGGGCC in the non-coding region C9orf72 gene. Such HREs occur more than 30 times more frequently in ALS, but most patients have hundreds or thousands of repeats. It is believed that the repeat expansions disrupt C9orf72 protein function, resulting in haploinsufficiency (loss of function), but also in the pathological production of abnormal proteins. The HREs in C9orf72 lead to interruptions of RNA transcripts, which then sequester RNA-binding proteins involved in transcription and splicing, resulting in protein / RNA aggregates in the cell nuclei of motor neurons and frontal cortical neurons, hippocampus, cerebellum, and spinal cord.

[0045] Mutations in genes associated with familial ALS include single gene mutations in genes selected from C9orf72, SOD1, TARDBP, FUS and TANK-binding kinase 1 (TBK1). The present disclosure excludes familial (hereditary) ALS caused by mutations in those genes.

[0046] Pharmaceutical Compositions Oral dosage forms. The pharmaceutical composition of ROCK inhibitor for oral administration may be in the form of a tablet or capsule, may be an immediate release formulation, or may be a controlled release formulation or a sustained release formulation, which may contain pharmaceutically acceptable additives such as cornstarch, mannitol, povidone, magnesium stearate, talc, cellulose, methylcellulose, carboxymethylcellulose, and similar substances. The pharmaceutical composition containing ROCK inhibitor and / or its salt may contain one or more pharmaceutically acceptable additives known in the art. Formulations include oral films, oral disintegrating tablets, effervescent tablets, and granules or beads that can be sprinkled on food or mixed with liquid as a slurry, or poured directly into the mouth and washed down.

[0047] Pharmaceutical compositions containing ROCK inhibitors, their salts and hydrates can be prepared by any method known in the pharmaceutical art. In general, such preparation methods include the step of bringing into association the ROCK inhibitor or its pharma- ceutically acceptable salt with the carrier or excipient, and / or one or more other accessory ingredients, and then, as necessary and / or desired, shaping and / or packaging the product into a desired single-dose or multi-dose unit.

[0048] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk as single unit doses and / or as a plurality of single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of active ingredient that would be administered to a subject, and / or a convenient fraction of such dosage, such as, for example, half or a third of such dosage.

[0049] The relative amounts of the active ingredient, pharma- ceutically acceptable excipients, and / or any additional components in a pharmaceutical composition of the invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as on the route by which the composition is administered. Compositions used in the methods of the invention may contain from 0.001% to 100% (w / w) active ingredient.

[0050] The pharma- ceutically acceptable additives used in the preparation of the provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils.Additives such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents, and aromatic agents may also be present in the compositions.

[0051] In certain embodiments, the pharmaceutical compositions used in the methods of the present invention may include a diluent. Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0052] In certain embodiments, the pharmaceutical composition used in the method of the present invention may include granulating and / or dispersing agents.Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, sponge, cation exchange resins, calcium carbonate, silicate, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0053] In certain embodiments, the pharmaceutical composition used in the method of the present invention may include a binder. Exemplary binders include starch (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, bread worm gum, ghatti gum, mucilage of isapol skin, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM.RTM.), and larch arabinogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0054] In certain embodiments, the pharmaceutical composition used in the method of the present invention may contain a preservative. Exemplary preservatives include antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, antiprotozoal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0055] In certain embodiments, the pharmaceutical compositions used in the methods of the present invention may contain an antioxidant. Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0056] In certain embodiments, the pharmaceutical composition used in the method of the present invention can include chelating agent.Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0057] In certain embodiments, the pharmaceutical composition may include a buffering agent together with the ROCK inhibitor or its salt.Exemplary buffering agents include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0058] In certain embodiments, the pharmaceutical compositions used in the methods of the present invention may contain a lubricant. Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0059] In other embodiments, the pharmaceutical composition containing the ROCK inhibitor or its salt is administered as a liquid dosage form.Liquid dosage forms for oral and parenteral administration include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to active ingredients, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (such as cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.In addition to inert diluents, oral compositions may contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatic agents. In certain embodiments for parenteral administration, the conjugates of the invention are mixed with a solubilizing agent such as Cremophor™, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0060] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or (a) excipients or fillers (such as starch, lactose, sucrose, glucose, mannitol, and silicic acid), (b) binders (such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), (c) humectants (such as glycerol), (d) disintegrants (such as agar, calcium carbonate, jasmine, etc.), (e) glycerol, (f) glycerol, (g) glycerol, (h) glycerol, (i) glycerol, (j) glycerol, (k) glycerol, (l ... (e) solution retarders (such as paraffin), (f) absorption enhancers (such as quaternary ammonium compounds), (g) wetting agents (such as cetyl alcohol and glycerol monostearate), (h) absorbents (such as kaolin and bentonite clay), and (i) lubricants (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof). In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0061] Some compositions of the present invention relate to sustained or controlled release formulations. These may be, for example, diffusion-controlled products, dissolution-controlled products, erosion products, osmotic pump systems, or ionic resin systems. Diffusion-controlled products include water-insoluble polymers that control the flow of water and the subsequent exit of dissolved drug from the dose. Dissolution-controlled products control the dissolution rate of the drug by using a slowly solubilizing polymer or by microencapsulation of the drug, using a variable thickness to control the release. Erosion products control the release of the drug by the erosion rate of the carrier matrix. Osmotic pump systems release the drug based on the constant influx of water across a semipermeable membrane into a reservoir containing an osmotic agent. Ion exchange resins can be used to bind the drug so that when ingested, the release of the drug is determined by the ionic environment in the gastrointestinal tract.

[0062] Parenteral dosage forms. Fasudil can be administered in parenteral dosage forms. As used herein, the term "parenteral" includes, but is not limited to, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, or infusion techniques.

[0063] Pharmaceutical compositions or formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, sealed ampoules, and vials, and may be stored in a lyophilized (lyophilized) state requiring only the addition of the sterile liquid carrier, water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0064] Parenteral pharmaceutical formulations may further contain other acceptable liquid carriers in amounts which do not render the formulation aqueous, including vegetable oils such as peanut oil, cottonseed oil, sesame oil, and the like, as well as organic solvents, PEG, propylene glycol, glycerol, and surfactants.

[0065] Parenteral formulations may further comprise at least one of any suitable auxiliary agent, including, but not limited to, diluents, crystal inhibitors, isotonicity agents, water structure forming or disrupting agents, polymers, ion pairing agents, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, and the like. Pharmaceutically acceptable auxiliary agents are preferred. Examples and methods of preparing such sterile solutions are well known in the art and are described in REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro, Ed., 18th Edition, Mack Publishing Co. (1990); Handbook of Pharmaceutical Excipients, 9 thPharmaceutical compositions and methods for treating pulmonary circulation disorders are described in well-known textbooks, such as, but not limited to, The Journal of Clinical Chemistry, vol. 10, no. 10, pp. 1111-1120, 2011. Pharmaceutical compositions and methods for treating pulmonary circulation disorders are described in ...

[0066] In one embodiment, the aqueous parenteral pharmaceutical formulation comprises at least 50% water, preferably 70% or more water.

[0067] Also provided are routes of administration and effective dosages of aqueous parenteral pharmaceutical formulations containing Fasudil.For parenteral administration, sterile suspensions and solutions are preferred.When intravenous administration is preferred, isotonic preparations are used, which generally contain suitable preservatives.The pharmaceutical composition may be parenterally administered by injection of a pharmaceutical composition containing Fasudil dissolved in an inert liquid carrier.

[0068] The pharmaceutical compositions may be prepared by dissolving or suspending the compound in a liquid carrier such that the final formulation contains from about 0.005% to 30% by weight of the compound.

[0069] The preferred route of parenteral administration is intravenous.The pharmaceutical preparations described herein may also be administered by injection.The pharmaceutical preparations described herein may also be administered by bolus dose, and may optionally be combined with administration by injection.The compounds described herein may be administered in combination with other pharmaceuticals in various protocols for the effective treatment of disease.

[0070] How to diagnose, treat, and monitor the progression of ALS The present invention contemplates fasudil, preferably fasudil hydrochloride hemihydrate, for treating ALS.

[0071] Diagnosis. ALS diagnosis can be made by clinical, electrophysiological and / or neuropathological examinations. In one embodiment, ALS diagnosis is made using the El Escorial diagnostic criteria (Brooks 1994) and is continuously updated with the Airlie House and Awaji-shima criteria (de Carvalho 2008) (Brooks 2011). The Awaji criteria proposed two modifications to the revised El Escorial. The first modification was to use both electromyography and clinical data simultaneously to determine the presence of lower motor neuron (LMN) dysfunction. The second proposed modification was to consider the possibility of fasciculations as evidence of ongoing denervation, which is equivalent in importance to the possibility of fasciculations.

[0072] In one embodiment, a diagnosis of ALS requires (1) the presence of evidence of LMN degeneration by clinical, electrophysiological or neuropathological examination; (2) the presence of upper motor neuron (UMN) degeneration by clinical examination; (3) the presence of progressive spread of symptoms or signs within one area or another as determined by medical history, clinical examination, and electrophysiological examination; and (4) the absence of electrophysiological or pathological evidence of other disease processes that may explain the observed clinical and electrophysiological signs.

[0073] Diagnostic categories include definite ALS (clinical or electrophysiological evidence of the presence of LMN and UMN signs in the bulbar region and at least two spinal regions, or the presence of LMN and UMN signs in three spinal regions), probable ALS (clinical or electrophysiological evidence of LMN and UMN signs in at least two regions, with some UMN signs necessarily rostral to the LMN signs), and possible ALS (clinical or electrophysiological signs of UMN or LMN dysfunction in only one region, or signs of UMN alone in two or more regions, or LMN rostral to the UMN signs). The present invention contemplates treating definite, probable, and possible ALS.

[0074] The diagnostic criteria for ALS are summarized in Table 1 below. [Table 1]

[0075] The clinical features of the most common manifestations of ALS, by designation and site of onset, are shown below in Table 2 (adapted from van Es 2017). [Table 2-1] [Table 2-2]

[0076] Classification of ALS phenotypes is based primarily on the relative involvement of the UMN and LMN and the regional distribution of involvement.

[0077] Diagnosis can be achieved using clinical and electrophysiological evaluation. Electrophysiological tests include, but are not limited to, electromyogram (EMG). Muscle ultrasound can detect fasciculations, which can aid in the diagnosis of ALS. In some cases, muscle biopsy is performed, which involves taking a small sample of muscle under local anesthesia.

[0078] Imaging of the brain and spinal cord with techniques including MRI to exclude other diseases, but its use to confirm ALS is less established due to the heterogeneity of ALS. Various imaging techniques are reviewed in Turner et al. 2012. The most sensitive and specific techniques to diagnose the disease are diffusion tensor MRI, MR spectroscopy, PET, a combination of multiple neuroimaging modalities, and neuroimaging with transcranial magnetic stimulation. Diffusion tensor MRI and MR spectroscopy can be used to monitor and predict disease course. (Bakulin 2019). Recently, one group reported MRI differences between ALS patients with C904f72 mutations and those with cognitive impairment (van der Burgh et al., 2020).

[0079] A diagnosis of ALS should exclude other conditions that may resemble ALS, including some forms of muscular dystrophies, a neurological condition known as spinobulbar muscular atrophy, intraspinal tumors, and a nerve-to-muscle communication disorder known as myasthenia gravis.

[0080] Strong et al. 2017 also reported revised diagnostic criteria for the diagnosis of ALS associated with frontotemporal spectrum disorder (ALS-FTSD). These criteria incorporated clinical, electrophysiological, neuropsychological, genetic and neuropathological features and recognized that ALS can exist as a pure motor syndrome but coexist with frontotemporal dementia (ALS-FTD) as defined by the Neary or Hodges criteria (Hodges 2001) (Neary 1998).

[0081] Other symptoms of motor neuron degeneration may be socially disabling and / or affect the patient's quality of life and include sialorrhea (drooling, excessive salivation, thickening of the saliva), pseudobulbar palsy, emotional lability (pathological crying, laughing, or yawning), convulsions (especially at night), spasticity, depression and anxiety, insomnia (caused by depression, convulsions, pain, and respiratory distress), constipation, and fatigue (of central and / or peripheral origin). Many patients complain of difficulty in effectively clearing bronchial secretions, including sticky sputum, and mucus accumulation is a negative prognostic factor.

[0082] Cognitive and behavioral changes are inherent components of some forms of ALS. As mentioned above, 5-15% of ALS patients also have frontotemporal dementia (FTD), and up to 50% of ALS patients have cognitive or behavioral changes within the spectrum of FTD. Disease manifestations with cognitive or behavioral changes that do not meet formal diagnostic criteria can be classified into one of three categories: ALS with behavioral disorders, ALS with executive dysfunction, and ALS non-executive dysfunction. Affective blunting and loss of empathy are the most common behavioral symptoms, while fluency, language, social cognition, and executive function are the cognitive domains most frequently affected (van Es 2017).

[0083] Treatment of ALS patients with Fasudil In one embodiment, the patient treated with oral fasudil has sporadic ALS. In another embodiment, the sporadic ALS patient has TDP-43 associated ALS.

[0084] In one embodiment, the patient treated with oral fasudil has familial ALS.

[0085] In certain embodiments, the patient has classical ALS. In another particular embodiment, the patient has bulbar onset ALS.

[0086] In one embodiment, the ALS patient does not have another proteinopathy-associated neurodegenerative disease.

[0087] In a further embodiment, the ALS patient treated with oral fasudil does not have frontotemporal dementia (FTD). In another embodiment, the sporadic ALS patient has ALS-FTSD or ALS-FTD.

[0088] In one embodiment, the ALS patient treated with oral fasudil is genetically male.

[0089] In another embodiment, the ALS patient treated with oral fasudil is between 40 and 75 years of age.

[0090] In certain embodiments, the ALS patients treated with oral fasudil are between 50 and 65 years of age.

[0091] In a further embodiment, the ALS patients treated with oral fasudil are between 20 and 39 years of age.

[0092] In one embodiment, the ALS patient is treated with oral fasudil hydrochloride hemihydrate.

[0093] According to the treatment method of the present invention, a therapeutically effective amount of ROCK inhibitor or its pharmacologic acceptable salt is administered to ALS patients once or multiple times a day.The lowest therapeutically effective amount of Fasudil is, for example, 60 mg per day, and is generally administered in two equal portions to obtain a total daily dose.The highest therapeutically effective dose can be empirically determined as the highest dose that is still effective in alleviating one or more ALS symptoms, but does not induce unacceptable levels or adverse events.Fasudil, for example, is generally not administered at a daily dose exceeding 240 mg.

[0094] One preferred dosing regimen involves treatment with Fasudil at least three days per week, followed by a treatment-free period of at least two weeks, followed by another Fasudil treatment phase followed by a second treatment-free phase.

[0095] In one particular embodiment, the patient is treated with 30-60 mg / day of fasudil at least three days per week for at least two weeks prior to a two- to four-week off-treatment phase. In one embodiment, the patient is treated five days per week. In a preferred embodiment, the patient is then treated with 30-60 mg / day of fasudil at least three days per week for a second treatment phase of at least two to four weeks. The patient is then subjected to a treatment off-phase of about one to six months, preferably one month.

[0096] In one embodiment, fasudil is administered twice daily (bid) during the treatment phase.

[0097] In certain embodiments, the patient is treated at least 5 days per week during the first and second treatment phases.

[0098] In one embodiment, the duration of the first and second treatment phases is one month.

[0099] In another embodiment, the duration of the first and second treatment rest periods is one month.

[0100] In certain embodiments, the first and second treatment phases are for one month and the first and second treatment rest periods are for one month.

[0101] In another particular embodiment, the patient is treated with 60-120 mg / day of fasudil at least 3 days per week for at least 2 weeks prior to a 2-4 week off-treatment phase. The patient is then treated with 60-120 mg / day of fasudil at least 3 days per week in a second treatment phase. The patient is then subjected to a treatment off-phase of about 1-6 months, preferably 1 month.

[0102] In one embodiment, fasudil is administered twice daily (bid) during the treatment phase.

[0103] In certain embodiments, the patient is treated at least 5 days per week during the first and second treatment phases.

[0104] In one embodiment, the duration of the first and second treatment phases is one month.

[0105] In another embodiment, the duration of the first and second treatment rest periods is one month.

[0106] In certain embodiments, the first and second treatment phases are for one month and the first and second treatment rest periods are for one month.

[0107] In a further specific embodiment, the patient is treated with 120-180 mg / day of fasudil at least 3 days per week for at least 2 weeks prior to a 2-4 week treatment rest phase. In one embodiment, the patient is treated 5 days per week. In a preferred embodiment, the patient is then treated with 120-180 mg / day of fasudil at least 3 days per week in a second treatment phase of at least 2-4 weeks. The patient is then subjected to a treatment rest phase of about 1-6 months, preferably 1 month.

[0108] In one embodiment, fasudil is administered twice daily (bid) during the treatment phase.

[0109] In certain embodiments, the patient is treated at least 5 days per week during the first and second treatment phases.

[0110] In one embodiment, the duration of the first and second treatment phases is one month.

[0111] In another embodiment, the duration of the first and second treatment rest periods is one month.

[0112] In certain embodiments, the first and second treatment phases are for one month and the first and second treatment rest periods are for one month.

[0113] In one embodiment, fasudil is administered twice daily (bid) or three times daily (tid) in an immediate release formulation. In another embodiment, fasudil is administered once daily in a sustained release formulation.

[0114] In a further specific embodiment, the patient is treated with 180-240 mg / day of fasudil at least 3 days per week for at least 2 weeks, prior to a 2-4 week treatment rest phase. In a preferred embodiment, the patient is then treated with 180-240 mg / day of fasudil at least 5 days per week in a second treatment phase. In one embodiment, the second treatment phase is at least 1 month. The patient is then subjected to a treatment rest phase of about 1-6 months, preferably 1 month.

[0115] In one embodiment, fasudil is administered twice daily (bid) or three times daily (tid) in an immediate release formulation, hi another embodiment, fasudil is administered once daily in a sustained release formulation.

[0116] In yet another specific embodiment, the patient is treated with 30-60 mg / day of fasudil at least 3 days per week for at least 2 weeks, prior to a 2-4 week treatment rest phase. In a preferred embodiment, the patient is then treated with 60-120 mg / day of fasudil at least 5 days per week in a second treatment phase. In one embodiment, the second treatment phase is at least 1 month. The patient is then subjected to a treatment rest phase of about 1-6 months, preferably 1 month.

[0117] In another particular embodiment, the patient is treated with 60-120 mg / day of fasudil at least 3 days per week for at least 2 weeks, prior to a 2-4 week treatment rest phase. In a preferred embodiment, the patient is then treated with 120-180 mg / day of fasudil at least 5 days per week in a second treatment phase. In one embodiment, the second treatment phase is at least 1 month. The patient is then subjected to a treatment rest phase of about 1-6 months, preferably 1 month.

[0118] In another particular embodiment, the patient is treated with 120-180 mg / day of fasudil at least 3 days per week for at least 2 weeks, prior to a 2-4 week treatment rest phase. In a preferred embodiment, the patient is then treated with 180-240 mg / day of fasudil at least 5 days per week in a second treatment phase. In one embodiment, the second treatment phase is at least 1 month. The patient is then subjected to a treatment rest phase of about 1-6 months, preferably 1 month.

[0119] In one embodiment, the dosage is an immediate release formulation. In another embodiment, Fasudil hydrochloride may be administered once a day using a sustained release formulation.

[0120] Treatment with a sustained release total daily dose of 180 mg of fasudil hydrochloride hemihydrate is contemplated. Typically, the sustained release dosage form will contain 180-240 mg of fasudil hydrochloride hemihydrate.

[0121] The exact duration of the first treatment phase will depend on the patient's condition and response to treatment. The most preferred methods contemplate that treatment will begin after the onset or appearance of symptoms.

[0122] It will be understood that the dosage ranges described herein provide guidance for administration of the provided pharmaceutical compositions to adults. For example, the amount administered to children or adolescents can be determined by a physician or person skilled in the art and may be lower than or the same as the amount administered to adults.

[0123] Combination therapy In some embodiments, fasudil is administered in combination with a second therapeutic agent that treats ALS or its symptoms.In some of these embodiments, the second therapeutic agent is selected from riluzole, edaravone, tetrabenazine, masitinib, tofersen, ravulizumab-wevz, mesenchymal stem cell (MSC)-neurotrophic factor (NTF) cells, AMX0035 (phenylbutyrate and taurursodiol), talampanel, tamoxifen, methylcobalamin, Aeol 10150.

[0124] In certain embodiments, ALS patients are administered fasudil in combination with riluzole or edavaron at about 50-100 mg per day. In certain embodiments, riluzole is administered at 50 mg twice daily.

[0125] Other agents may also be co-administered to treat symptoms of ALS or associated comorbidities including respiratory function, feeding, depression and anxiety, pain, dysarthria, dysphagia, sialorrhea, insomnia, behavior or mood, and constipation.

[0126] In another embodiment, fasudil can be co-administered with other agents used to treat or alleviate the symptoms of ALS. These medications include antidepressants, benzodiazepines, amyltriptyline, dextromethorphan hydrobromide / quinidine sulfate, anti-inflammatory medications, muscle relaxants (baclofen, botulinum toxin), anticonvulsants (gabapentin, sodium valproate), anticholinergics (glycopyrronium bromide), atorvastatin, lithium carbonate, avanier 07-ACR-123 (Zenvia®), SB-509, thalidomide, arimoclomol, olanzapine, memantine, tamoxifen, pioglitazone, creatine monohydrate, botulinum toxin type B, dronabinol, coenzyme Q10, escitalopram (Lexapro®), sodium phenylbutyrate, R(+) pramipexole dihydrochloride monohydrate, sodium valproate, cyclosporine, corticosteroids, and / or modafinil.

[0127] The second therapeutic agent may be administered sequentially or simultaneously.

[0128] In one embodiment, the patient is administered fasudil in combination with tetrabenzine, hi certain embodiments, the tetrabenzine is administered at a dose of 12.5-100 mg / patient / day.

[0129] In another embodiment, the patient is administered fasudil in combination with an anti-inflammatory agent.

[0130] In a further embodiment, the patient is administered fasudil in combination with agents that enhance proteasome activity, including proflavine pimozide, cyclosporine A, mifepristone, chlorpromazine, loperamide, dipyrimidol, methylbenzethonium, verapamil, ursolic acid, betulinic acid, rolipram, DPCPX, PD169316, PAP1, PA26, PA28, TCH-165, MK-886, and AM-404.

[0131] In another embodiment, the patient is administered fasudil in combination with an agent that enhances autophagy. In one embodiment, the autophagy enhancer is BRD5631, carbamazepine, rapamycin, trehalose, trifluoperazine niguldipine, metformin, lithium carbonate, sodium valproate, and ABT-737.

[0132] In a further embodiment, the patient being treated with fasudil is being treated for depression, hi a particular embodiment, the patient is being treated with an antidepressant, such as citalopram or escitalopram.

[0133] Assessment of ALS progression after fasudil treatment ALS progression is assessed by measuring progressive clinical decline using a variety of methods. One assessment is the ALS Functional Rating Scale-Revised (ALSFRS-R), which is based on crude disability measures driven by LMN dysfunction and far removed from histopathological changes and is the standard measure of ALS disease progression (Cedarbaum 1999). The ALSFRS-R consists of 12 questions totaling 48 points. Improvement in the ALSFRS-R with fasudil can be measured by change from baseline over time after treatment. The ALSFRS-R can also be used to slow progression compared to untreated ALS patients. ALS patients show an average decline in the ALSFRS-R of approximately 1 point per month. Some progress much faster and some progress much slower, but most patients will show a decline of 0.5 to 1.5 points per month. The 12 domains of the ALSFRS assess speech, salivation, swallowing, cutting, dressing and hygiene, turning in bed, walking, climbing stairs, dyspnea, orthopnea and respiratory failure.

[0134] Progression of muscle weakness may also include strength testing (muscle) to measure muscle atrophy, peripheral nerve and muscle imaging with ultrasound and MRI, respiratory function assessment (lung testing), and bulbar dysfunction testing (swallowing, tongue, lip and cheek strength).

[0135] Handheld myometry (HHD, also known as quantitative myometry) is a commonly used methodology for the assessment of muscle strength in ALS clinical trials. Electrophysiological assessments of muscles can also be used to measure disease progression. These include compound motor action potentials (CMAP) and motor unit number estimates (MUNE, MUNIX), which are nerve conduction assessments used to quantify the number of motor units innervating individual muscles. For example, isometric testing using the Tufts Quantitative Neuromuscular Exam (TQNE) or the Accurate Testing of Limb Isometric Muscle Strength (ATLIS) can be used to measure upper and lower limb muscles. (Andres 2013). The ATLIS measures isometric strength in 12 muscle groups in the arms and legs. Electrical impedance myography (EIM) is also used to assess how electrical current flows through muscles.

[0136] Bulbar dysfunction can be measured using the Iowa Oral Performance Instrument measures of tongue, lip, and cheek strength (Clark and Soloman 2012). The Sydney Swallow Questionnaire may also be useful (Wallace et al. 2000).

[0137] For the measurement of respiratory muscle function and weakness (diaphragm, bulbar, accessory muscles), forced vital capacity (FVC) is one of the most commonly used measurements in clinical trials. FVC measures the total volume of a single breath possible by a patient by measuring the amount of air expelled from a breath in the first second. It can be measured using spirometry. Other measures of lung function in ALS have also been studied, including maximum voluntary inspiratory or expiratory pressures, which are indicators of disease progression in ALS patients. Static vital capacity (SVC), which measures a patient's normal slow breathing and expiration, is also often used.

[0138] Other pulmonary measurements that may be used include maximum mid-expiratory flow and maximum cough flow, the latter of which accompanies a cough as the lungs empty. A pneumotachometer can also assess breathing strength to determine maximum inspiratory pressure (MEP).

[0139] Many patients complain of difficulty effectively clearing sticky phlegm, mucus accumulation is a negative prognostic factor, and weight loss is therefore a predictor of shorter survival in amyotrophic lateral sclerosis (ALS).

[0140] Patients with older age, bulbar onset, early respiratory insufficiency, and lower ALSFRS-R scores have a poorer prognosis.

[0141] Outcome In one embodiment, treatment of an ALS patient with fasudil reduces or reverses progressive ALS. Typically, this is measured using the ALSFRS-R, and treatment with fasudil slows the rate of decline in one or more of the 12 domains. The average rate of decline is one point per month, which should be empirically determined over a period of 2-12 months prior to fasudil treatment, and the effectiveness of fasudil treatment is then determined by assessing progression over the same period after initiation of fasudil treatment. A slowing or halting of decline based on the ALSFRS is considered successful treatment, as is reversing the decline.

[0142] In one embodiment, treatment of an ALS patient with Fasudil reduces or reverses the loss of motor neuron demyelination or deterioration. Less deterioration can be measured by techniques including imaging, such as diffusion tensor MRI, MR spectroscopy, PET, neuroimaging using transcranial magnetic stimulation, or any combination thereof.

[0143] In another embodiment, treatment of ALS patients with fasudil improves muscle deterioration (atrophy), reduces muscle paralysis or contraction, and reduces or prevents the propagation of muscle fasciculations (twitching).In a particular embodiment, treatment of ALS patients with fasudil reduces limb muscle impairment.Muscle weakness can be assessed using any method, such as, for example, strength testing, muscle strength measurement (including handheld), force transducer (strain gauge), electrophysiological evaluation, isometric testing, and peripheral nerve and muscle imaging using ultrasound and MRI.

[0144] In further embodiments, bulbar dysfunction is improved upon treatment of ALS patients with fasudil, as determined, for example, using Iowa Oral Performance Instrument measurements of tongue, lip, and cheek strength, or the Sydney Swallow Questionnaire. In certain embodiments, fasudil treatment of ALS patients improves swallowing and feeding, reduces or delays the progression of dysphagia, reduces slurred speech (dysarthria), and allows ALS patients to maintain a healthy weight. Assessments such as clinical MRI, needle EMG, Frenchay dysarthria assessment, videofluoroscopic swallowing examination (VFSE), maximum tongue pressure test, and / or EAT-10 screening tool, improvement can also be assessed by speech pathologists and nutritionists.

[0145] In another particular embodiment, treatment of ALS patients with Fasudil improves respiratory function, hi a particular embodiment, Fasudil treatment improves FVC%, oxygen saturation.

[0146] In another embodiment, treating an ALS patient reduces fatigue, improves poor balance, reduces stumbling, and improves grip strength.

[0147] In a further embodiment, improvement is measured by the improvement of 48-point ALSFRS-R rating scale score, or any subscale thereof, such as the activity of daily living (ADL) subscore, relative to the score before being treated with fasudil hydrochloride.In another embodiment, the scale is ALSAQ-40, which is a disease-specific questionnaire specifically designed to evaluate health-related quality of life in ALS patients.(Jenkinson et al.,1999).In certain embodiments, improvement occurs with a higher score from baseline.In another embodiment, a consistent score over time without decline is evidence of a slowing of ALS progression.

[0148] In another embodiment, improvement is measured by a delayed decrease in ALSFRS-R rating scale score compared to ALS patients not treated with fasudil.

[0149] In a further embodiment, treatment of ALS patients with fasudil reduces motor neuron inflammation.

[0150] In another embodiment, the present invention provides a method for reducing, reversing, or preventing accumulation of TDP-43 in an ALS patient, the method comprising administering to the subject an effective amount of fasudil or a pharma- ceutically acceptable salt thereof.

[0151] In another embodiment, TDP-43 cytoplasmic mislocalization is reduced upon treatment of ALS patients with fasudil.

[0152] In embodiments, TDP-43 aggregation, phosphorylation and / or ubiquitination is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or by about 100% upon treatment of an ALS patient with fasudil.

[0153] In other embodiments, Fasudil treatment reduces or alleviates the symptoms of ALS.

[0154] In one embodiment, treatment of ALS patients improves survival (delays death). In one embodiment, survival is extended beyond the typical two years. In further embodiments, survival is extended beyond two years, beyond three years, beyond four years, and beyond five years. In another embodiment, survival is extended by 3 months to one year.

[0155] Patient Subpopulations Certain patient subpopulations, such as renal impairment patients and / or older patients (e.g., 65 years or older), may require lower doses or sustained release formulations instead of immediate release formulations. Fasudil hydrochloride hemihydrate may have higher steady-state concentrations when administered at normal doses to patients with renal disease, and a lower dose may be required to reduce Cmax or delay the time to Cmax (increase Tmax).

[0156] Renal dysfunction occurs with age and as a result of a number of disorders, including liver cirrhosis, chronic kidney disease, acute kidney injury (e.g., due to administration of contrast agents), diabetes (type 1 or type 2), autoimmune diseases (such as lupus and IgA nephropathy), genetic diseases (such as polycystic kidney disease), nephrotic syndrome, urinary tract problems (due to conditions such as prostate enlargement, kidney stones, and some cancers), heart attack, illegal drug use and abuse, ischemic kidney conditions, urinary tract problems, high blood pressure, glomerulonephritis, interstitial nephritis, vesicoureteral, pyelonephritis, sepsis, among others. Renal dysfunction may occur with other diseases and syndromes, including non-kidney-related diseases that may occur with renal dysfunction, such as pulmonary arterial hypertension, heart failure, and cardiomyopathy, among others.

[0157] Renal function is most frequently assessed using serum (and / or urinary) creatinine. Creatinine is a breakdown product of creatine phosphate in muscle cells and is produced at a constant rate. It is excreted unchanged by the kidneys, primarily through glomerular filtration. Thus, elevated serum creatinine is a marker of renal dysfunction and is used to estimate glomerular filtration rate.

[0158] Normal levels of creatinine in blood are approximately 0.6-1.2 mg / dL for adult males and 0.5-1.1 mg / dL for adult females. If the creatinine level exceeds these figures, the subject has renal dysfunction and is therefore treatable according to the present invention. Mild renal impairment / insufficiency occurs within the range of 1.2 mg / dL-1.5 mg / dL. Moderate renal impairment / insufficiency is considered to occur at creatinine levels above 1.5 mg / dL. Severe renal impairment, including what is considered renal failure, is defined as a serum creatinine level of ≧2.0 mg / dL or the use of renal replacement therapy (such as dialysis). It is specifically contemplated to treat subjects with mild, moderate, and severe renal impairment.

[0159] Patient size is an important factor to consider when using creatinine-based estimates of renal function. The units for drug clearance are volume / time (mL / min), whereas the units for estimated GFR in chronic kidney disease are volume / time / standard size (mL / min / 1.73 m). 2 ). Generally, doses may be adjusted downwards for smaller patients (e.g., 40-50 mg per day) and upwards for larger patients (e.g., 120 mg per day). Smaller male patients will weigh about 160 pounds or less. Smaller female patients will weigh about 130 pounds or less. Patients with a body mass index of 30 or greater are considered obese.

[0160] Additionally, older patients may need to start with a lower dose and gradually increase to the recommended dose after a few days or weeks. In another embodiment, even if the anticipated titration is up to a dose of 180-240 mg / day, older patients may need a lower dose (e.g., 90 mg / day) during the treatment period.

[0161] It will be understood that the dosage ranges described herein provide guidance for administration of the provided pharmaceutical compositions to adults. For example, the amount administered to children or adolescents can be determined by a physician or person skilled in the art and may be lower than or the same as the amount administered to adults. EXAMPLES

[0162] Example 1: Treatment of ALS with Fasudil A Phase 2a Open-Label Exploratory Safety, Efficacy, and Biomarker Study of Fasudil HCl in Patients with ALS The objective of this study is to evaluate the preliminary safety, efficacy, and impact on biomarkers of oral fasudil HCl hemihydrate in patients with ALS. Patients will be cohorted into treatment groups. One group will be treated with a 60 mg / day dose of fasudil (30 mg, twice daily, immediate release tablet), 5 days per week for one month, followed by a one-month off-treatment phase. That same cohort will then be treated with 60 mg / day fasudil (30 mg, twice daily, immediate release tablet), 5 days per week for one month. This will be followed by another one-month off-treatment phase.

[0163] A second cohort is treated with a high dose of immediate release tablets at 180 mg / day (60 mg three times a day), five days a week for one month, followed by a one-month off-treatment phase. The same cohort is then treated with Faucidil at 180 mg / day, five days a week for one month. This is followed by another off-treatment phase for one month.

[0164] Patients may maintain their current dose of riluzole or edaravone. Use of more than one of the following drug classes is not permitted: long-acting nitrates, beta-blockers, or calcium channel blockers. (Note: The drug classes covered are one may be administered.)

[0165] The use of phenylbutyrate and / or tauroursodeoxycholic acid is strongly discouraged during the study and may lead to discontinuation of the study or study treatment.

[0166] This study will evaluate the effect of fasudil on change in slope of decline of the ALS Functional Rating Scale-Revised (ALSFRS-R), static vital capacity (SVC), and handheld dynamometry (HHD). Specifically, the study objectives are to: 1. To evaluate the effects of fasudil on plasma and cerebrospinal fluid (CSF) biomarkers of neurodegeneration and inflammation. 2. To evaluate the effect of fasudil on plasma biomarkers of muscle loss. 3. To measure CSF penetration of fasudil and its major metabolite (M3) in ALS patients.

[0167] Study design Approximately 20 subjects aged 18-75 years in each cohort with possible ALS, probable and laboratory-confirmed ALS, probable ALS, or definite ALS as defined by the El Escorial revised ALS diagnostic criteria will be eligible for inclusion in the study, provided that their ALSFRS-R has decreased by a mean of 0.5-1.5 points per 4 weeks at Screening 1 (see Inclusion Criteria 3 for estimation methodology). The study will enroll sufficient subjects such that approximately 20 subjects will have a mean ALSFRS-R decrease of 0.5-1.5 points per 4 weeks between Screening 1 and V3 / D1. Replacement of subjects not considered evaluable may be considered.

[0168] After consenting, participants undergo two screening assessments, which take place over an 8-week period prior to dosing with the study drug. At Screening 1 / V1 (8 weeks prior to starting dosing), ALS assessments of ALSFRS-R / SVC / HHD will be performed, as well as safety assessments. Subjects who meet the relevant inclusion / exclusion criteria will return for a second screening (Screening 2 / V2) approximately 4 weeks later, where ALS and safety assessments will again be performed. Subjects who meet the relevant Screening 2 study entry criteria will be enrolled in the study.

[0169] On Day V3 / 1, evaluations will be performed and each cohort (60 mg or 180 mg) will begin receiving study drug 5 days per week for one month, followed by a one-month off-treatment phase, in a first treatment phase, immediately followed by treatment of the same cohort with the same dose 5 days per week for one month, followed by another one-month off-treatment phase.

[0170] Participants will have an in-person visit or phone contact during the first post-treatment week to assess safety and compliance. Additional follow-up visits will occur at weeks 4, 8, and 12, during which ALS assessments of ALSFRS-R / SVC / HHD will be performed. A final visit will be conducted at week 16 (or 7±2 days after early termination) for post-treatment follow-up assessments.

[0171] Plasma biomarker collection will occur between enrollment and the start of treatment, and at weeks 4 and 12. CSF biomarker collection will occur between enrollment and the start of treatment, and at week 12.

[0172] Laboratory safety assessments and adverse events will be collected at every study visit. Subjects / caregivers will be asked to maintain a record of study drug compliance, which will be reviewed at every visit.

[0173] To minimize patient burden, the visit / test procedure may be conducted outside of the clinic (e.g., at home or other sample collection facility) and interviews may be conducted by telephone and / or telehealth, if necessary.

[0174] Duration. The maximum duration of the study is 12 weeks.

[0175] Study Evaluation Items Safety. Safety will be assessed by examining the incidence of AEs and SAEs, clinically significant physical and neurological examination abnormalities, changes in vital signs, 12-lead ECG, and hematology, blood chemistry, liver function, and urinalysis.

[0176] Efficacy. The following efficacy outcomes will be assessed: 1. Change in slope of ALSFRS-R decline during treatment versus before treatment. 2. Change in the slope of static vital capacity (SVC) decline during treatment versus before treatment. 3. Change in slope of decline in HHD measurements during treatment versus before treatment.

[0177] ALSFRS-R. The ALSFRS-R is a validated rating scale for monitoring disability progression in patients with ALS and is utilized to monitor functional changes in ALS patients. The score assesses four domains including (i) bulbar function (speech, salivation, swallowing), (ii) fine motor tasks (handwriting, cutting and utensil handling, gastrostomy status, dressing and hygiene), (iii) gross motor tasks (turning in bed, walking, climbing stairs), and (iv) respiratory function (dyspnea, orthopnea and respiratory failure).

[0178] Each item within the domain is assigned a score from 0 (complete loss of function) to 4 (normal), with a maximum score of 48 indicating preserved function.

[0179] Raters administering the ALSFRS-R must be certified by the Northeast Amyotrophic Lateral Sclerosis Consortium (NEALS). Use of alternate certification must be approved in writing by the sponsor. The ALSFRS-R should be administered by the same rater at every visit, when possible.

[0180] SVC. Respiratory function is an important predictor of survival in amyotrophic lateral sclerosis (ALS). Vital capacity is determined using the upright SVC method. SVC is measured using a study-approved, portable spirometer, and assessments are performed using a facemask. Three SVC trials are required for each study session, but up to five trials may be performed if the variability between the highest and second highest SVC for the first three trials is ≥10%. Only the best three trials will be recorded on the electronic case report form (eCRF). The highest SVC recorded will be utilized for eligibility. At least three measurable SVC trials must be completed to score SVC for all post-screening visits. Predicted SVC and percent predicted SVC values ​​are calculated using the Quanjer Global Lung Function Initiative equations.

[0181] The assessor performing the SVC must be NEALS certified. The use of alternate credentials must be approved in writing by the sponsor. The SVC should be performed by the same assessor at each visit, when possible.

[0182] Muscle strength. Muscle strength is assessed by a hand-held dynamometer (HHD). A spring-loaded device that "breaks" at a pre-set force is used to evaluate readings obtained by the HHD throughout the test. Grip force dynamometry is taken of both hands and the average force in kilograms is calculated. Measurements are taken on three occasions on each hand.

[0183] Assessors performing HHD must be certified by NEALS. Use of alternate certification must be approved in writing by the sponsor. HHD should be performed by the same assessor at each visit, when possible.

[0184] Exploratory studies. Other endpoints evaluated are the biomarkers described below: 1. Changes from pretreatment in plasma biomarkers of neurodegeneration (e.g., neurofilament light chain [NfL] and phosphorylated neurofilament heavy chain subunit [pNfH]), inflammatory markers (e.g., IFN-γ, VCAM-1, ICAM-1, IL-1, IL-6, IL-17α, TNF-α, and C1q), and markers of inflammation and neurodegeneration in neuronal and / or astrocytic exosomes (e.g., IL-6, tau, protein kinase B [AKT], and phosphorylated AKT [p-AKT]). 2. Changes from pretreatment in CSF biomarkers of axonal degeneration and / or apoptosis (e.g., tau and NfL), inflammation, and markers of drug target association (e.g., phosphorylated tau [p-tau], p-NfL PTEN, and AKT / p-AKT species). 3. Changes from pretreatment in plasma biomarkers of muscle loss (e.g., actin, myosin, myosin light chain, troponin, titin, myozenin, alpha-actinin, nebulin, cofilin 2, tropomyosin 2, creatine kinase, myoglobin, sarcospan, integrin alpha 7, agrin, laminin 211, collagen IV, collagen VI, and collagen fragments). 4. The ratio of CSF fasudil / M3 metabolite concentrations to plasma fasudil / M3 metabolite concentrations measured after 24 weeks of treatment.

[0185] The CSF is analyzed for biomarkers of neurodegeneration, which may include, but are not limited to, the following: NfL Phosphorylated tau species (e.g., P-tau181, pS202, pS386, Thr245, Thr377, Ser409) Phosphorylated NfL species (e.g., Ser26 and Ser57) Total tau fragment levels Other tau species ·Inflammation markers (IFN-γ, VCAM-1, ICAM-1, IL-1, IL-6, IL-17α, TNF-α, C1q) Other markers of target engagement (e.g., PTEN and AKT / p-AKT)

[0186] result ALSFRS-R. Fasudil treatment at 180 mg / day is expected to result in approximately a 35-50% reduction in mean decline over at least 3 months as measured by the ALS Functional Rating Scale-Revised (ALSFRS-R).

[0187] In one embodiment, the decline in ALSFRS-R is reduced by an average of 0.5 to 1.5 points (global) per 4 weeks (when using the change between ALSFRS-R at Screening 1 and the most recent ALSFRS-R measure at least 12 weeks prior to Screening 1). If previous values ​​are not available, the rate of decline may be estimated as follows: (48 value at Screening) / [estimated number of months between Screening and onset of ALS symptoms (weakness and / or dysarthria, and / or dysphagia).

[0188] SVC. Decreasing slope as the slope correlates with disease progression. This is likely to be a rate of decline of around 20-50% per month. In another embodiment, the rate of decline averages less than 2.5-3.0% per month. In a particular embodiment, the rate of respiratory depression slows by an average of 1.5 percentage points per month.

[0189] Muscle strength. Handheld dysmetria (HHD) is a measure of muscle strength, and scores decline as ALS progresses. Treatment with fasudil is expected to reduce the expected decline in muscle strength that occurs in ALS patients.

[0190] Biomarkers. Fasudil treatment is expected to reduce the presence and / or levels of biomarkers associated with neurodegeneration and axonal degeneration, and to reduce the amount of biomarkers associated with muscle loss by 25-50%. Alternatively, treatment with fasudil is expected to reduce the presence and / or levels of biomarkers associated with neurodegeneration and axonal degeneration, and to reduce the presence of biomarkers associated with muscle loss, compared to those reported for patients not receiving fasudil.

[0191] References Andres PL, English R, Mendoza M, et al. Developing normalized strength scores for neuromuscular research. Muscle Nerve 2013;47:177-182. Bakulin, I.S. et al., Structural and Functional Neuroimaging in Amyotrophic Lateral Sclerosis. Human Physiology. 2018; 44: 844-854. Brooks BR, El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases / Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial "Clinical limits of amyotrophic lateral sclerosis" J Neurol Sci. 1994 Jul; 124 Suppl():96-107. Brooks BR, Miller RG, Swash M, Munsat TL, El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. World Federation of Neurology Research Group on Motor Neuron Diseases. Amyotroph Lateral Scler Other Motor Neuron Disord. 2000 Dec; 1(5):293-9. 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[0192] The disclosure of each reference mentioned herein is incorporated herein by reference in its entirety.

Claims

1. A pharmaceutical composition for treating amyotrophic lateral sclerosis, comprising a therapeutically effective amount of fasudil or a pharmaceutically acceptable salt thereof, (a) fasudil is administered to the patient at least three days per week for at least two weeks in the first treatment phase; (b) after said first treatment phase, subjecting the patient to a first treatment rest period of at least 2 weeks; (c) after the first treatment-free period, fasudil is administered to the patient in a second treatment phase; (d) after said second treatment phase, subjecting the patient to a second treatment rest phase; Pharmaceutical compositions.

2. 10. The pharmaceutical composition of claim 1, wherein fasudil is administered to the patient at least three days per week during the second treatment phase.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the second treatment phase is for at least 2 weeks.

4. 4. The pharmaceutical composition of claim 3, wherein the first treatment phase and the second treatment phase are for one month.

5. The pharmaceutical composition of claim 1, wherein the patient is treated at least 5 days per week during the first and second treatment phases.

6. The pharmaceutical composition of claim 1, wherein the first and second treatment rest periods are at least 1 month but less than 6 months.

7. The pharmaceutical composition described in claim 1, wherein the duration of the first and second treatment phases is one month, and the duration of the first and second treatment rest phases is one month.

8. The pharmaceutical composition of claim 1, wherein (a) to (d) are repeated at least once.

9. The pharmaceutical composition of claim 1, wherein (a) to (d) are repeated throughout the patient's life.

10. The pharmaceutical composition of claim 1, wherein 30 to 60 mg / day of fasudil is administered in the first and second treatment phases.

11. The pharmaceutical composition of claim 1, wherein 60 to 120 mg / day of fasudil is administered in the first and second treatment phases.

12. The pharmaceutical composition of claim 1, wherein 120 to 180 mg / day of fasudil is administered in the first and second treatment phases.

13. The pharmaceutical composition of claim 1, wherein 80 to 240 mg / day of fasudil is administered in the first and second treatment phases.

14. The pharmaceutical composition of claim 1, wherein the treatment is by intravenous infusion.

15. The pharmaceutical composition of claim 1, wherein fasudil is administered orally.

16. The pharmaceutical composition of claim 1, wherein the patient has classical ALS.

17. The pharmaceutical composition of claim 1, wherein the patient has ALS with frontotemporal dementia (ALS-FTD).

18. The pharmaceutical composition of claim 1, wherein the patient has only lower motor neuron involvement.

19. The pharmaceutical composition of claim 1, wherein the patient has only upper motor neuron involvement.

20. The pharmaceutical composition of claim 1, wherein the ALS patient has Tar DNA-binding protein 43 (TDP-43) inclusion bodies.

21. The pharmaceutical composition of claim 1, wherein the treatment results in a 50% reduction in decline over at least 3 months as measured by the ALS Functional Rating Scale-Revised (ALSFRS-R).

22. The pharmaceutical composition of claim 1, wherein fasudil is administered in two or three equal doses throughout the day.

23. The pharmaceutical composition of claim 1, wherein fasudil is administered in a sustained release formulation.