Methods for treating ALS with a RHO kinase inhibitor based on use of neurofilament light chain biomarker

Abstract

A method of treating amyotrophic lateral sclerosis (ALS), includes: determining for a patient diagnosed with ALS, a pre-treatment amount of neurofilament light chain (NfL) in the patient's serum or plasma; orally administering to the patient a first rho kinase inhibitor in a predetermined amount for a predetermined period of time; determining a post-treatment amount of NfL in the patient's serum or plasma; when the post-treatment of amount of NfL is determined to be lower than the pre-treatment amount of NfL, orally administering to the patient a second rho kinase inhibitor in a therapeutically effective amount for treating ALS; and when the post-treatment of amount of NfL is determined to be not lower than the pre-treatment amount of NfL, withholding administration of the second rho kinase inhibitor to the patient.

Description

METHODS FOR TREATING ALS WITH A RHO KINASE INHIBITOR BASED ON USE OF NEUROFILAMENT LIGHT CHAIN BIOMARKERCROSS REFERENCE

[0001] This application claims priority to U.S. Provisional Application No. 63 / 569,474, filed March 25, 2024, the contents of which are incorporated herein by reference in its entirety.BACKGROUND1. Field

[0002] The disclosure relates generally to methods for treating amyotrophic lateral sclerosis (ALS) and more specifically to methods for treating ALS with a rho kinase inhibitor that is based on the collection and quantification of neurofilament light chain (NfL).2. Description of Related Art

[0003] Amyotrophic Lateral Sclerosis (ALS), commonly known as LouGehrig’s disease, is a fatal neurodegenerative disease that affects motor neurons, resulting in a progressive loss of control of voluntary movements. It is associated with degeneration of upper motor neurons and their corticospinal axonal tracts (lateral sclerosis) and associated with the loss of lower motor neurons and their axons, which leads to muscle wasting (amyotrophy) and paralysis of voluntary muscles (Mitsumoto et al., 1998). Upper motor neurons originate in the motor region of the cerebral cortex or brain stem and move motor information underneath motor neurons that are directly responsible for stimulation of the target muscle. Their dysfunction causes stiffness due to continuous muscle contraction that interferes with walking, movement and speech. Lower motor neurons connect the brainstem and spinal cord to muscle fibers. Their dysfunction causes muscle atrophy, spasmssmall, local, involuntary muscle contraction. Many individuals with ALS die from respiratory failure within 48 months from the onset of symptoms and most within 3 to 5 years from onset.

[0004] ALS is thought to be caused by a combination of genetic factors, environmental factors, and aging-related dysfunction, similar to other neurodegenerative conditions. Apart from genetic factors, age and male sex increase the risk for ALS. Several studies have suggested environmental risk factors for ALS, such as smoking, body mass index, physical exercise, occupational and environmental exposures to metals, pesticides, p-methylamino-L-alanine, head injury, and viral infections. However, the causal relationship of these factors with ALS remains to be established (Masrori 2020).

[0005] About 90-96% of ALS cases are sporadic, with only 5-10% being familial due to inherited gene mutations.

[0006] ALS is also associated with protein inclusions in motor neurons and the CNS. Both sporadic and familial ALS are associated with abnormal accumulation TAR DNA-binding protein 43 (TDP-43) aggregates, which is thought to spread in a prion-like manner between cells. TDP-43 is the primary misfolded, mis-localized, ubiquitinated protein composing 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 and microRNA. TDP-43 normally localizes to the nucleus where it functions in transcription, but misfolded TDP-43 aggregates in the cytosol, leading to a nuclear loss-of-function that might cause transcription deficits. It is unclear whether ALS pathogenesis is linked to loss of TDP-43 function or the pathology associated with the aggregates and cytoplasmic mis-localization.

[0007] Multiple molecular pathways have been implicated in the pathogenesis of ALS, such as failure of proteostasis, excitotoxicity, neuroinflammation,mitochondrial dysfunction and oxidative stress, oligodendrocyte dysfunction, cytoskeletal disturbances and axonal transport defects, disturbed RNA metabolism, nucleocytoplasmic transport deficits and impaired DNA repair. Interestingly, many of the genes associated with ALS appear to cluster in key pathways: protein quality control and degradation, RNA metabolism, and cytoskeletal and axonal transport (Masrori 2020).

[0008] Rho Kinase (ROCK) Inhibitors ALS. There are a number of 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 reproduce the ALS (or other neurodegenerative disease) phenotype, or are pertinent only to familial ALS which is only 5-10% of ALS patients. As one example, U.S.9,980,972 describes using fasudil in the SOD1 G93 mouse model, which harbors a mutation in the superoxide dismutase (SOD) protein. This patent claims treating familial, early-stage ALS with fasudil at 10-1200 ng / kg body weight per day or 1-12 mg / kg body weight per day. No humans were treated. Further, mutations in SOD are associated only with familial ALS which is why the claims are limited. The mice in the SOD model develop adult-onset neurodegeneration of spinal motor neurons and progressive motor deficits leading to paralysis. Further, 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, which significantly affect the onset and severity of symptoms. 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). W

[0009] Another problem with the animal models is that many of them exhibit a high copy number of the mutant allele, i.e. , they overexpress e.g., mutant SOD. Thisis vastly different from even human familial ALS, where afflicted patients have a mutation in one allele. Other models, such as TARDBP (TDP-43) mice that display TDP-43, also rely on overexpression approaches that do not replicate human ALS.

[0010] 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 two other patients had probable ADS. Patients were dosed with 30 mg of intravenously administered fasudil twice daily over 20 consecutive working days (not weekends). There were no conclusive results beyond safety. Currently, there are clinical trials in progress in Germany, Switzerland and France for infusion of fasudil according to the same intravenous administration and dosing schedule. (Lingor et al., 2019). The trial is designed to treat three parallel groups: fausdil 15 mg twice daily, fasudil 30 mg twice daily, and matching placebo. No updates on this trial were available in September 2021 except for a publication detailing the unanticipated legal, administrative and financial complexities of a multi-national trial to which U.S.- based trials were proposed added but were not. (Lingor 2021 ).

[0011] Other publications disclose using unrealistic routes of administration (e.g., intraventricular injection) of fausdil for treatment of neurological and proteinopathy-associated 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 dose (HED) can be calculated, for example, using Table 1 of Nair & Jacob (2016), which are the same conversions used by the US FDA.

[0012] Other publications describe the potential use of serum neurofilaments as prognostic biomarkers for ALS and additionally hinted at the possibility for use as potential pharmacodynamic biomarkers without showing specific treatment effects on neurofilament levels (Benatar 2020).

[0013] There exists a significant unmet need to provide new, therapies that show benefit in both familial and non-familial ALS in humans, not just animals with the genetic mutations that do not recapitulate most of the ALS population. There exists an additional significant unmet need to tailor these therapies based on reliable biomarker indicatiors.SUMMARY

[0014] A method of treating amyotrophic lateral sclerosis (ALS), may include: determining for a patient diagnosed with ALS, a pre-treatment amount of neurofilament light chain (NfL) in the patient’s serum or plasma; orally administering to the patient a first rho kinase inhibitor in a predetermined amount for a predetermined period of time; determining a post-treatment amount of NfL in the patient’s serum or plasma; when the post-treatment of amount of NfL is determined to be lower than the pre-treatment amount of NfL, orally administering to the patient a second rho kinase inhibitor in a therapeutically effective amount for treating ALS; and when the post-treatment of amount of NfL is determined to be not lower than the pre-treatment amount of NfL, withholding administration of the second rho kinase inhibitor to the patient.

[0015] The predetermined period of time may be less than or equal to twelve months.

[0016] The predetermined period of time may be between one month and six months.

[0017] The first rho kinase inhibitor and the second rho kinase inhibitor may be different compounds.

[0018] The first rho kinase inhibitor and the second rho kinase inhibitor may be a same compound.

[0019] Each of the first rho kinase inhibitor and the second rho kinase inhibitor may include: fasudil (1-(5-isoquinolinesulfonyl)homopiperazine); a fasudil derivative selected from: hydroxy-fasudil (1-(1-hydroxyl-5- isoquinolinesulfonyl)homopiperazine), and dimethyl-fasudil ((S)-(+)-2-Methyl-1-[(4- methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1 H-1 ,4-diazepine); or a pharmaceutically acceptable salt thereof.

[0020] The predetermined amount may be at least 90 mg / day.

[0021] The predetermined amount may be at least 300 mg / day.

[0022] The predetermined amount may be at least 320 mg / day.

[0023] The method may further include: prior to determining the pre-treatment amount of NFL in the patient’s serum or plasma, diagnosing the patient with ALS by satisfying at least one of (i) El Escorial Revised ALS diagnostic criteria, and (ii) Awaji-shima diagnostic criteria.

[0024] The therapeutically effective amount may be at least 90 mg / day.

[0025] The therapeutically effective amount may be at least 300 mg / day.

[0026] The therapeutically effective amount may be at least 320 mg / day.

[0027] A method of treating amyotrophic lateral sclerosis (ALS) may include: determining for a patient diagnosed with ALS, a first pre-treatment amount of neurofilament light chain (NfL) in the patient’s serum or plasma at a first time; determining for the patient, a second pre-treatment amount of NfL in the patient’s serum or plasma at a second time, the second time being after the first time; determining whether the second pre-treatment amount is less than, equal to, or greater than the first pre-treatment amount; when the second pre-treatment amount of NfL is determined to be greater than or equal to the first pre-treatment amount, orally administering to the patient a rho kinase inhibitor in a therapeutically effective amount for treating ALS; and when the second pre-treatment amount of NfL isdetermined to be less than the first pre-treatment amount, withholding administration of the rho kinase inhibitor to the patient.

[0028] The method may further include: prior to determining the pre-treatment amount of NFL in the patient’s serum or plasma at the first time, diagnosing the patient with ALS by satisfying at least one of (i) El Escorial Revised ALS diagnostic criteria, and (ii) Awaji-shima diagnostic criteria.

[0029] The diagnosing the patient with ALS may be by satisfying the Awaji- shima diagnostic criteria. The patient diagnosed with ALS may be presymptom atic. The orally administering to the patient the rho kinase inhibitor in the therapeutically effective amount for treating ALS is performed only when the second pre-treatment amount of NfL is determined to be greater the first pre-treatment amount.

[0030] The second time may be at least one month after the first time. The orally administering to the patient the rho kinase inhibitor in the therapeutically effective amount for treating ALS may be performed only when the second pretreatment amount of NfL is determined to be at least 1% greater the first pretreatment amount.

[0031] The diagnosing the patient with ALS may be by satisfying the Awaji- shima diagnostic criteria. The patient diagnosed with ALS may be diagnosed with familial ALS.

[0032] The therapeutically effective amount may be at least 300 mg / day.

[0033] The therapeutically effective amount may be at least 320 mg / day.DETAILED DESCRIPTION

[0034] In designing treatments for Treatments for amyotrophic lateral sclerosis (ALS), whether sporadic or familial in nature, it is desirable to base decisions regarding treatment based (i) on the verifiable presence / absence of disease and / or(ii) the degree to which disease may be present at a given time. Accordingly, the identification of a reliable biomarker for ALS may be useful for such treatment decisions.

[0035] A biomarker is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biomarkers used in the medical field, may be part of a clinical toolset categorized by their clinical applications. The four main classes are molecular, physiologic, histologic and radiographic biomarkers. All four types of biomarkers have a clinical role in narrowing or guiding treatment decisions and follow a sub-categorization of being either predictive, prognostic, or diagnostic.

[0036] Predictive molecular, cellular, or imaging biomarkers that pass validation can serve as a method of predicting clinical outcomes. Predictive biomarkers are used to help optimize ideal treatments, and often indicate the likelihood of benefiting from a specific therapy. Diagnostic biomarkers that meet a burden of proof can serve a role in narrowing down diagnosis. This can lead to diagnosis that are significantly more specific to individual patients. A prognostic biomarker provides information about the patients overall outcome, regardless of any treatment or therapeutic intervention.

[0037] An ideal biomarker for use in decisions relating to ALS treatment would have all three predictive, prognostic, and diagnostic characteristics. One potential biomarker that may have such predictive, prognostic, and diagnostic characteristics is Neurofilament light chain (NfL).

[0038] NfL is a neuronal cytoplasmic protein expressed in myelinated axons. NfL may be present in an individual’s cerebral spinal fluid (CSF) and blood inproportion to a degree of axonal damage in a variety of neurological disorders, including inflammatory, neurodegenerative, traumatic and cerebrovascular diseases (e.g., ALS). Evidence of axonal damage (even prior to the onset of clinical symptoms) may allow for a predictive biomarker role of NfL with respect to ALS. Evidence of a threshold amount of axonal damage may allow for a prognostic biomarker role of NfL with respect to ALS. Evidence regarding the degree / extent of axonal damage may allow for a prognostic biomarker role of NfL with respect to ALS. This predictive, prognostic, and diagnostic evidence may be used to inform decisions to (i) initiate ALS treatment, (ii) maintain ALS treatment, (iii) adjust ALS treatment, and / or (iv) cease ALS treatment.

[0039] We have surprisingly discovered that administration of a rho kinase to an ALS patient results in a reduction of NfL in the majority of, but not all patients. Because NfL is strongly associated with outcomes, it is reasonable to conclude that NfL responders will also be clinical responders. The idea is to treat only those patients who will respond (ie, have a reduction in NfL). It takes a very large study and / or a long time to see a clinical response (eg, ALSFRS-R), but NfL responds quickly, within months and with small numbers.

[0040] A variety of methods may be used to obtain NfL biomarker levels from a patient. As mentioned above, NfL may be sampled from a patient’s blood or CSF.

[0041] For example, blood may be collected (e.g., in serum-separating BD vacutainers) and allowed to clot (e.g., upright at room temperature for 1-2 hours). The clotted blood may then undergo centrifugation (e.g., 1 ,750 g for 10minutes at 4°C) to allow for separation of serum that may be aliquoted (e.g., into cryogenic sterile freestanding conical microtubes and stored (e.g., at -80°C) for subsequent quantification of NfL.

[0042] In another example, CSF may be collected (e.g., in polypropylene tubes). The CSF may then undergo centrifugation (e.g., 1 ,750 g for 10 minutes at 4°C) and then be aliquoted (e.g., into polypropylene cryogenic sterile freestanding conical microtubes. The aliquoted centrifuged CSF may then be stored (e.g., by freezing within about 30 minutes of collection, and stored at -80°C) for subsequent quantification of NfL

[0043] Serum and CSF neurofilament concentrations may be quantified by a variety of different assay devices (e.g. the Simoa® NfL LDT produced by Quanterix®).

[0044] In some embodiments, levels of NfL in patients who have already been diagnosed with ALS may be used to determine if treatment with a rho kinase inhibitor is effective and / or should be continued.

[0045] For example, in a patient who has been diagnosed with ALS (e.g., familial ALS), a pre-treatment amount of NfL may be determined by collecting the patient’s serum or plasma and quantifying the amount / concentration of NfL. The pre-treatment amount of NfL may be designated as “pre-treatment” because the collection of the patient’s plasma occurs prior to any treatment with a rho kinase inhibitor.

[0046] After the pre-treatment amount of NfL has been determined as a baseline, the patient may be orally administered a first rho kinase inhibitor in a predetermined amount for a predetermined period of time.

[0047] The first rho kinase inhibitor may be any pharmaceutically acceptable rho kinase (ROCK) inhibitor such as fasudil (1-(5- isoquinolinesulfonyl)homopiperazine), hydroxy-fasudil (1 -(1 -hydroxyl-5- isoquinolinesulfonyl)homopiperazine), dimethyl-fasudil ((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1 H-1 ,4-diazepine), another fasudil derivative, and / or a pharmaceutically acceptable salt thereof.

[0048] 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 Chemical Industry (Tokyo, Japan). The characteristic chemical structure of fasudil consists of an isoquinoline ring, connected via a sulphonyl group to a homopiperazine ring. Fasudil is a potent inhibitor of both ROCK isoforms. In vivo, fasudil is subjected to hepatic metabolism to its active metabolite hydroxyfasudil (aka, M3). Other examples of isoquinoline derived ROCK inhibitors include dimethylfasudil and ripasudil.

[0049] Other preferred ROCK inhibitors are based on based on 4- aminopyridine structures. These were first developed by Yoshitomi Pharmaceutical(Uehata et al., 1997) and are exemplified by Y-27632. Still other preferred ROCK inhibitors include indazole, pyrimidine, pyrrolopyridine, pyrazole, benzimidazole, benzothiazole, benzathiophene, benzamide, aminofurazane, quinazoline, and boron derivatives (Feng et al., 2015). Some exemplary ROCK inhibitors are shown below: a

[0050] ROCK inhibitors according to the invention may have more selective activity for either ROCK1 (aka ROKp, Rho-kinase p, or p160ROCK) or ROCK2 (akaROKa) and will usually have varying levels of activity on PKA, PKG, PKC, andMLCK. Some ROCK inhibitors may be highly specific for ROCK1 or ROCK2 and have much lower activity against PKA, PKG, PKC, and MLCK.

[0051] A particularly preferred ROCK inhibitor is fasudil. Fasudil may exist as a free base or salt and may be in the form of a hydrate, such as a hemihydrate.Hexahydro-1 -(5-isoquinolinesulfonyl)-1 H-1 ,4-diazepine monohydrochloride hemihydrate

[0052] Fasudil is a selective inhibitor of protein kinases, such as ROCK, PKC and MLCK and treatment results in a potent relaxation of vascular smooth muscle, resulting in enhanced blood flow (Shibuya 2001). A particularly important mediator of vasospasm, ROCK induces vasoconstriction by phosphorylating the myosin- binding subunit of myosin light chain (MLC) phosphatase, thus decreasing MLC phosphatase activity and enhancing vascular smooth muscle contraction. Moreover, there is evidence that fasudil increases endothelial nitric oxide synthase (eNOS) expression by stabilizing eNOS mRNA, which contributes to an increase in the level of the potent vasodilator nitric oxide (NO), thereby enhancing vasodilation (Chen 2013).

[0053] Fasudil has a short half-life of about 25 minutes, but it is substantially converted in vivo to its 1 hydroxy (M3) metabolite. M3 has similar effects 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 the bulk of the in vivo pharmacological activity of the molecule after oral administration of fasudil to humans. It is important to note the oral administration in rodents results in a mixture of fasudil and M3 in the blood and not solely M3. Likewise, iv administration in humans also results in a mixture of fasudil and M3. Thus, oral administration to rodents is similar to iv administration to humans, both of which are different than oral administration to humans. M3 exists as two tautomers, depicted below:

[0054] The ROCK inhibitors described herein, such as fasudil, include pharmaceutically acceptable salts and hydrates. Salts that may be formed via reaction with inorganic and organic acid. Those inorganic and organic acids are included as following: hydrochloric acid, hydrobromide acid, hydriodic acid, sulphuric acid, nitric acid, phosphoric acid, acetic acid, maleic acid, maleic acid, maleic acid, oxalic acid, oxalic acid, tartaric acid, malic acid, mandelic acid, trifluoroacetic acid, pantothenic acid, methane sulfonic acid, or para-toluenesulfonic acid.

[0055] As described above, oral administration of the first rho kinase inhibitor may be in a predetermined amount for a predetermined time. For example, the firstrho kinase inhibitor may be administered in any therapeutically effective amount for any therapeutically effective amount of time. In some examples, the predetermined amount may be at least 90 mg / day, at least 180 mg / day, at least 240 mg / day, at least 300 mg / day, or at least 320 mg / day. The predetermined amounts may be administered based on TID dosing using and immediate release formulation. However, other dosage amounts, dosing frequencies, and release formulations may be used. Higher dosage amounts, for example 320 mg / day may provide better health outcomes without substantial negative side effects. Previously it was thought that dosages above 240 mg / day may result in undesirable renal side effects.However, we have recently found that higher dosage amounts, such as 320 mg / day, does not damage the kidneys when administered TID due to the fast drug half life. In some examples, the predetermined amount of time may be less than or equal to twelve months, or between one and six months.

[0056] Pharmaceutical compositions of ROCK inhibitors usable in the are generally oral and may be in the form of tablets or capsules and may be immediate- release formulations or may be controlled- or extended-release formulations, which may contain pharmaceutically acceptable excipients, such as corn starch, mannitol, povidone, magnesium stearate, talc, cellulose, methylcellulose, carboxymethylcellulose and similar substances. A pharmaceutical composition comprising a ROCK inhibitor and / or a salt thereof may comprise one or more pharmaceutically acceptable excipients, which are known in the art. Formulations include oral films, orally 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 to be washed down.

[0057] Pharmaceutical compositions containing ROCK inhibitors, salts and hydrates thereof can be prepared by any method known in the art of pharmaceutics.In general, such preparatory methods include the steps of bringing a ROCK inhibitor or a pharmaceutically acceptable salt thereof into association with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping, and / or packaging the product into a desired single- or multi-dose unit.

[0058] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0059] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition used in accordance with the methods of the present invention may comprise between 0.001% and 100% (w / w) active ingredient.

[0060] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

[0061] The pharmaceutical composition used in the methods described herein may comprise 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, cornstarch, powdered sugar, and mixtures thereof.

[0062] The pharmaceutical composition used in the methods described herein may comprise a granulating and / or dispersing agent. Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0063] The pharmaceutical composition used in the methods described herein may comprise a binding agent. Exemplary binding agents include starch (e.g., cornstarch 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, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM. RTM.), and larch arabogalactan), alginates,polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0064] The pharmaceutical composition used in the methods described herein invention may comprise a preservative. Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan 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.

[0065] The pharmaceutical composition used in the methods described herein may comprise 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.

[0066] The pharmaceutical composition used in the methods described herein may comprise a chelating agent. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. 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.

[0067] The pharmaceutical composition may comprise a buffering agent together with the ROCK inhibitor or the salt thereof. Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, 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 mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0068] The pharmaceutical composition used in the methods described herein may comprise a lubricating agent. Exemplary lubricating agents 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.

[0069] The pharmaceutical composition of containing a ROCK inhibitor or salt thereof may be administered as a liquid dosage form. Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates of the invention are mixed with solubilizing agents such as Cremophor™, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0070] 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, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, 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 include a buffering agent.

[0071] Some compositions relate to extended- 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 comprise a water-insoluble polymer which controls the flow of water and the subsequent egress of dissolved drug from the dosage from. Dissolution-controlled products control the rate of dissolution of the drug by using a polymer that slowly solubilizes or by microencapsulation of the drug - using varying thicknesses to control release. Erosion products control release of drug by the erosion rate of a carrier matrix. Osmotic pump systems release a drug based on the constant inflow of water across a semi permeable membrane into a reservoir which contains an osmotic agent. Ion exchange resins can be used to bind drugs such that, when ingested, the release of drug is determined by the ionic environment within the gastrointestinal tract.

[0072] In accordance with the treatment methods described herein, a therapeutically effective amount of a ROCK inhibitor or a pharmaceutically acceptable salt thereof may be administered to the ALS patient one or more times a day. The lowest therapeutically effective amount of fasudil, for example, is 90 mg per day, generally administered in 2 to 3 equal portions to obtain the full daily dose. The highest therapeutically effective dose may be determined empirically as the highest dose that remains effective in alleviating one or more ALS symptoms, but does not induce an unacceptable level or adverse events.

[0073] One preferred dosing regimen involves the treatment with 60 mg of fasudil hydrochloride hemihydrate three times per day using an oral immediate- release formulation, for a total daily dose of 180 mg. Other daily doses will range from 90 mg to 180 mg per day b.i.d. A further dosing regimen involves the treatment with 90 mg of fasudil hydrochloride hemihydrate only two times per day using animmediate-release formulation, for a total daily dose of 180 mg. In other embodiments, fasudil hydrochloride may be administered three times a day using an immediate-release formulation at 180 mg / day, 240 mg / day, or about 320 mg / day. In certain embodiments, fasudil hydrochloride may be administered three times a day using an immediate-release formulation at 306 mg / day or 324 mg / day. Based on ROCK inhibitory activity, one skilled in the art can readily extrapolate the provided dosing ranges for fasudil to other ROCK inhibitors.

[0074] Another example involves the treatment with 90-360 mg of fasudil hydrochloride hemihydrate once per day in an extended release dosage form. Treatment with an extended release total daily dose of 180 mg fasudil hydrochloride hemihydrate is preferred. Generally an extended release dosage form will contain between 180 and 360 mg of fasudil hydrochloride hemihydrate.

[0075] It will be appreciated that dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0076] In some examples, fasudil may be administered combination with a second therapeutic agent that treats ALS or symptoms thereof. In some of such 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, methylcobalamine, Aeol 10150.

[0077] In one example, the ALS patient may be administered fasudil in combination with riluzole or edavarone at about 50 to 100 mg day. In a specific embodiment, riluzole is administered 50 mg twice daily.

[0078] Other agents can be co-administered to treat symptoms of ALS or associated comorbidities, including respiratory function, eating, depression and anxiety, pain, dysarthria, dysphagia, sialorrhoea, insomnia, behavior or mood, and constipation can also be co-administered.

[0079] In another example, fasudil can be co-administered with other agents that have been used to treat or mitigate symptoms of ALS. Such agents include antidepressants, benzodiazepines, amiltriptyline, dextromethorphan hydrobromide / quinidine sulfate, anti-inflammatories, muscle relaxants (baclofen, botulinum toxin), anticonvulsants (gabapentin, sodium valproate), anti-cholinergic drugs (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, cyclosporin, corticosteroids, and / or modafinil.

[0080] The second therapeutic agent may be to be administered sequentially or simultaneously.

[0081] In another example, the patient may be administered fasudil in combination with an anti-inflammatory.

[0082] In a further example, the patient may be administered fasudil in combination with an agent that enhances proteasome activity. Such agents include proflavine pimozide, cyclosporin A, mifepristone, chlorpromazine, loperamide, dipyrimidole, methylbenzethonium, verapamil, ursolic acid, betulinic acid, rolipram, DPCPX, PD169316, PAP1 , PA26, PA28, TCH-165, MK-886, and AM-404.

[0083] In another example, the patient may be administered fasudil in combination with an agent that enhances autophagy. In one embodiment, theautophagy enhancer is BRD5631 , carbamazepine, rapamycin, trehalose, trifluoperazine niguldipine, metformin, lithium carbonate, sodium valproate, and ABT- 737.

[0084] In a further example the patient treated with fasudil may be being treated for depression. In a specific example, the patient is treated with an antidepressant such as citalopram or escitalopram.

[0085] After the predetermined period of time has passed, a post-treatement amount of NfL may be determined by again collecting the patient’s serum or plasma and quantifying the amount / concentration of NfL

[0086] The pre-treatment amount of NfL may and post-treatment amount of NfL may then be compared to ascertain the patient’s responsiveness to treatment with the first rho kinase inhibitor (i.e., the effectiveness of treatment).

[0087] For example, when the post-treatment of amount of NfL is determined to be lower than the pre-treatment amount of NfL, the treatment with the first rho kinase inhibitor may be seen to be effective at reducing axonal damage, and consequently effective at treating the patient’s ALS. In this example, since the first rho kinase inhibitor was seen to be effective at treating ALS, the patient may be orally administered a second rho kinase inhibitor in a therapeutically effective amount for treating ALS.

[0088] The second rho kinase inhibitor may be any pharmaceutically acceptable ROCK inhibitor such as fasudil, hydroxy-fasudil, dimethyl-fasudil, another fasudil derivative, and / or a pharmaceutically acceptable salt thereof. The second rho kinase inhibitor may be the same as the first rho kinase inhibitor or may be different than the first rho kinase inhibitor.

[0089] The therapeutically effective amount may be administered in any therapeutically effective amount such as at least 90 mg / day, at least 180 mg / day, at least 240 mg / day, or at least 360 mg / day.

[0090] The patient’s progress after receiving the second rho kinase inhibitor may be further analyzed by obtaining subsequent amount(s) of NfL and further treatment with a ROCK inhibitor may be continued, increased, decreased, or ceased based on such subsequent amount(s) of NfL. For example, subsequent treatment with a ROCK inhibitor may be increased in dosage if the patient is becoming less responsive to lower dosage. Additionally, subsequent treatment may be ceased if treatment is no longer effective at lowering or maintaining NfL levels.

[0091] In another example, after the pre-treatment amount of NfL may and post-treatment amount of NfL have been compared, if the the post-treatment of amount of NfL is determined to be not lower than the pre-treatment amount of NfL, the treatment with the first rho kinase inhibitor may be seen to not be effective at reducing axonal damage, and consequently not be effective at treating the patient’s ALS. In this example, since the first rho kinase inhibitor was seen to be not effective at treating ALS, subsequent treatment with a second rho kinase inhibitor may be withheld from the patient.

[0092] As described above, the method for treatment of ALS is for patients who have already been diagnosed with ALS (whether familial or non-familial). Diagnosis of ALS can be done by clinical, electrophysiological and / or neuropathologic examination. In one embodiment, ALS diagnosis is made using El Escorial diagnostic criteria (Brooks 1994) successively updated in Airlie House and Awaji-shima criteria (de Carvalho 2008) (Brooks 2011). The Awaji criteria proposed two changes to the revised El Escorial. The first change was to use both electromyography and clinical data simultaneously to determine the presence oflower motor neuron (LMN) dysfunction. The second proposed change was to consider fasciculation potentials as evidence of ongoing denervation, equivalent in importance to fibrillation potentials.

[0093] In one example, ALS diagnosis requires: (1 ) the presence of evidence of LMN degeneration by clinical, electrophysiological or neuropathological examination, (2) presence of upper motor neuron (UMN) degeneration by clinical examination, (3) presence of progressive spread of symptoms or signs within a region or other regions, as determined by history, clinical examination or electrophysiological tests, and (4) absence of electrophysiological or pathological evidence of other disease processes that might explain the observed clinical and electrophysiological signs.

[0094] Diagnostic categories include: definite ALS (clinical or electrophysiological evidence by the presence of LMN as well as 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 by 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 UMN signs alone in two or more regions or LMN rostral to UMN signs). The invention contemplates treating definite, probably and possible ALS.

[0095] Diagnostic criteria for ALS are summarized in Table 1 below:Table 1 :

[0096] Clinical characteristics of the most common presentations of ALS, by designation and site of onset, are shown below in Table 2 (adapted from van Es 2017).Table 2:Signs of UMN or LMN, or both, in multiple regions at presentation Patients who fulfil the diagnostic criteria for both ALS and FTDALS = amyotrophic lateral sclerosis; FTD = frontotemporal dementia; LMN = lower motor neuron: UMN = upper motor neuron

[0097] Classification according to ALS phenotype is mainly based on the relative UMN versus LMN involvement and the regional distribution of involvement.

[0098] Diagnosis can be achieved using clinical and electrophysiological assessments. Electro-physiological testing includes without limitation electromyography (EMG). Ultrasound of the muscles can detect fasciculations that can aid in the diagnosis of ALS. In some instances, a muscle biopsy, which involves taking a small sample of muscle under local anesthesia, is performed.

[0099] Imaging of the brain and spinal cord by techniques including MRI to rule out other disease but use to confirm ALS is less established due to the heterogeneity of ALS. Various imaging techniques are reviewed in T urner et al. 2012. The most sensitive and specific techniques to diagnose the disease are diffusion-tensor MRI, MR spectroscopy, PET, a combination of several neuroimaging methods, and neuroimaging with transcranial magnetic stimulation. Diffusion-tensor MRI and MR spectroscopy can be used to monitor and predict the disease course. (Bakulin 2019). Recently, one group reported MRI differences between ALS patients with C904f72 mutations and with impaired cognition, (van der Burgh et al., 2020).

[0100] Other conditions should be ruled out in an ALS diagnosis. Among the conditions that resemble ALS are some forms of muscular dystrophy, the neurologic conditions known as spinal-bulbar muscular atrophy, intraspinal tumor, the nerve-to- muscle transmission disorder known as myasthenia gravis.

[0101] Strong et al. 2017 also reported revised diagnostic criteria for diagnosing ALS associated with frontotemporal spectrum disorder (ALS-FTSD). These criteria, which incorporated clinical, electrophysiological, neuropsychological, genetic and neuropathological characteristics, recognized that ALS could exist as a pure motor syndrome but that it can coexist with a frontotemporal dementia (ALS- FTD) as defined by the Neary or Hodges criteria (Hodges 2001 ) (Neary 1998).

[0102] Other symptoms of the motor neuron degeneration may be socially disabling and / or affect the patient’s quality of life include sialorrhoea (drooling,excessive salivation, thickened saliva), pseudobulbar emotional lability (pathological weeping, laughing, or yawning), cramps (especially at night), spasticity, depression and anxiety, insomnia (caused by depression, cramps, pain, and respiratory distress), constipation, and fatigue (of central and / or peripheral origin). Many patients report difficulties in effectively clearing bronchial secretions including tenacious sputum, and mucus accumulation is a negative prognostic factor.

[0103] Cognitive and behavioral changes are an intrinsic component of some forms of ALS. As mentioned above, 5-15% of patients with ALS also have frontotemporal dementia (FTD), and up to 50% of patients with ALS have cognitive or behavioral changes within the spectrum of FTD. Disease presentations with cognitive or behavioral changes that do not fulfil formal diagnostic criteria can be grouped into 1 of 3 categories: ALS with behavioral impairment; ALS with executive dysfunction; and ALS non-executive dysfunction. Apathy and loss of sympathy are the most common behavioral symptoms, while fluency, language, social cognition, and executive function are the cognitive domains that are most often affected (van Es 2017).

[0104] In some embodiments, levels of NfL in patients who have already been diagnosed with ALS may be used to determine if the disease is progressing and / or whether treatment with a rho kinase inhibitor should be initiated.

[0105] For example, in a patient who has been diagnosed with ALS (e.g., familial ALS), a first pre-treatment amount of NfL may be determined at a first time by collecting the patient’s serum or plasma and quantifying the amount / concentration of NfL. The first pre-treatment amount of NfL may be designated as “pre-treatment” because the collection of the patient’s plasma occurs prior to any treatment with a rho kinase inhibitor.

[0106] After a predetermined amount of time, a second pre-treatment amountof NfL may be determined at a second time by again collecting the patient’s serum or plasma and quantifying the amount / concentration of NfL. The second pre-treatment amount of NfL (similar to the first pre-treatment amount of NfL) may be designated as “pre-treatment” because the collection of the patient’s plasma occurs prior to any treatment with a rho kinase inhibitor.

[0107] After the first and second pre-treatment amounts of NfL have been determined, the amounts may be compared to determine whether the second pretreatment amount is less than, equal to, or greater than the first pre-treatment amount. This determination may involve the use of a coefficient of variation. The coefficient of variation is equal to the standard deviation divided by the mean.Inclusion of the coefficient of variation in the determination when comparing the first pre-treatment amount and the second pre-treatment amount can help conclude whether an observation of change is real. The coefficient of variation may account for a certain amount of statistical error that can lead to variation between samples. NfL amounts may vary by as much as 5% or 7% and still be considered to be within the coefficient of variation. For example, if the first pre-treatment amount of NfL and second pre-treatment amount of NfL differ by only 3%, this would be considered within the coefficient of variation and the first pre-treatment amount of NfL would be considered to be equal to the second pretreatment amount of NfL.

[0108] The comparison between he first pre-treatment amount of NfL may and second pre-treatment amount of NfL may be used to ascertain if the disease is progressing and / or whether treatment with a rho kinase inhibitor should be initiated.

[0109] For example, when the second pre-treatment amount of NfL is determined to be greater than or equal to the first pre-treatment amount, the axonal damage may be seen to be stable or increasing, and consequently the patient’s ALS may be seen to be stable or worsening. In this example, since the patient’s ALS maybe seen to be stable or worsening, the patient may be orally administered a rho kinase inhibitor in a therapeutically effective amount for treating ALS.

[0110] The rho kinase inhibitor may be any pharmaceutically acceptable ROCK inhibitor such as fasudil, hydroxy-fasudil, dimethyl-fasudil, another fasudil derivative, and / or a pharmaceutically acceptable salt thereof.

[0111] The therapeutically effective amount may be administered in any therapeutically effective amount such as at least 90 mg / day, at least 180 mg / day, at least 240 mg / day, or at least 360 mg / day.

[0112] In another example, when the second pre-treatment amount of NfL is determined to be greater than the first pre-treatment amount, the axonal damage may be seen to be increasing, and consequently the patient’s ALS may be seen to be worsening. In this example, since the patient’s ALS may be seen to be worsening, the patient may be orally administered a rho kinase inhibitor in a therapeutically effective amount for treating ALS.

[0113] The patient’s progress after receiving the rho kinase inhibitor may be further analyzed by obtaining subsequent amount(s) of NfL and further treatment with a ROCK inhibitor may be continued, increased, decreased, or ceased based on such subsequent amount(s) of NfL. For example, subsequent treatment with a ROCK inhibitor may be increased in dosage if the patient is becoming less responsive to lower dosage. Additionally, subsequent treatment may be ceased if treatment is no longer effective at lowering or maintaining NfL levels.

[0114] In another example, when the second pre-treatment amount of NfL is determined to be less than or equal to the first pre-treatment amount, the axonal damage may be seen to be stable or decreasing, and consequently the patient’sALS may be seen to be stable or improving. In this example, since the patient’s ALS may be seen to be stable or improving, treatment with a rho kinase inhibitor may bewithheld from the patient.

[0115] In another example, when the second pre-treatment amount of NfL is determined to be less than the first pre-treatment amount, the axonal damage may be seen to be decreasing, and consequently the patient’s ALS may be seen to be improving. In this example, since the patient’s ALS may be seen to be improving, treatment with a rho kinase inhibitor may be withheld from the patient.

[0116] Even though particular combinations of features are recited in the claims and / or disclosed in the specification, these combinations are not intended to limit the disclosure of possible implementations. In fact, many of these features may be combined in ways not specifically recited in the claims and / or disclosed in the specification. Although each dependent claim listed below may directly depend on only one claim, the disclosure of possible implementations includes each dependent claim in combination with every other claim in the claim set.

[0117] No element, act, or instruction used herein should be construed as critical or essential unless explicitly described as such. Also, as used herein, the articles “a” and “an” are intended to include one or more items, and may be used interchangeably with “one or more.” Furthermore, as used herein, the term “set” is intended to include one or more items (e.g., related items, unrelated items, a combination of related and unrelated items, etc.), and may be used interchangeably with “one or more.” Where only one item is intended, the term “one” or similar language is used. Also, as used herein, the terms “has,” “have,” “having,” or the like are intended to be open-ended terms. Further, the phrase “based on” is intended to mean “based, at least in part, on” unless explicitly stated otherwise.

[0118] Expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list. For example, the expression, "at least one of a, b, and c,"should be understood as including only a, only b, only c, both a and b, both a and c, both b and c, all of a, b, and c, or any variations of the aforementioned examples.

[0119] While such terms as “first,” “second,” etc., may be used to describe various elements, such elements must not be limited to the above terms. The above terms may be used only to distinguish one element from another.

Claims

What is Claimed is:1 . A method of treating amyotrophic lateral sclerosis (ALS), comprising: determining for a patient diagnosed with ALS, a pre-treatment amount of neurofilament light chain (NfL) in the patient’s serum or plasma; orally administering to the patient a first rho kinase inhibitor in a predetermined amount for a predetermined period of time; determining a post-treatment amount of NfL in the patient’s serum or plasma; when the post-treatment of amount of NfL is determined to be lower than the pre-treatment amount of NfL, orally administering to the patient a second rho kinase inhibitor in a therapeutically effective amount for treating ALS; and when the post-treatment of amount of NfL is determined to be not lower than the pre-treatment amount of NfL, withholding administration of the second rho kinase inhibitor to the patient.

2. The method of claim 1 , wherein the predetermined period of time is less than or equal to twelve months.

3. The method of claim 2, wherein the predetermined period of time is between one month and six months.

4. The method of claim 1 , wherein the first rho kinase inhibitor and the second rho kinase inhibitor are different compounds.

5. The method of claim 1 , wherein the first rho kinase inhibitor and the second rho kinase inhibitor are a same compound.

6. The method of claim 1 , wherein each of the first rho kinase inhibitor and the second rho kinase inhibitor comprises: fasudil (1 -(5-isoquinolinesulfonyl)homopiperazine); a fasudil derivative selected from:hydroxy-fasudil (1-(1-hydroxyl-5- isoquinolinesulfonyl)homopiperazine), and dimethyl-fasudil ((S)-(+)-2-Methyl-1-[(4-methyl-5- isoquinolinyl)sulfonyl]-hexahydro-1 H-1 ,4-diazepine); or a pharmaceutically acceptable salt thereof.

7. The method of claim 1 , wherein the predetermined amount is at least 90 mg / day.

8. The method of claim 1 , wherein the predetermined amount is at least 300 mg / day.

9. The method of claim 1 , wherein the predetermined amount is at least 320 mg / day.

10. The method of claim 1 , further comprising: prior to determining the pre-treatment amount of NFL in the patient’s serum or plasma, diagnosing the patient with ALS by satisfying at least one of (i) El Escorial Revised ALS diagnostic criteria, and (ii) Awaji-shima diagnostic criteria.11 . The method of claim 1 , wherein the therapeutically effective amount is at least 90 mg / day.

12. The method of claim 1 , wherein the therapeutically effective amount is at least 300 mg / day.

13. The method of claim 1 , wherein the therapeutically effective amount is at least 320 mg / day.

14. A method of treating amyotrophic lateral sclerosis (ALS), comprising: determining for a patient diagnosed with ALS, a first pre-treatment amount of neurofilament light chain (NfL) in the patient’s serum or plasma at a first time;determining for the patient, a second pre-treatment amount of NfL in the patient’s serum or plasma at a second time, the second time being after the first time; determining whether the second pre-treatment amount is less than, equal to, or greater than the first pre-treatment amount; when the second pre-treatment amount of NfL is determined to be greater than or equal to the first pre-treatment amount, orally administering to the patient a rho kinase inhibitor in a therapeutically effective amount for treating ALS; and when the second pre-treatment amount of NfL is determined to be less than the first pre-treatment amount, withholding administration of the rho kinase inhibitor to the patient.

15. The method of claim 14, further comprising: prior to determining the pre-treatment amount of NFL in the patient’s serum or plasma at the first time, diagnosing the patient with ALS by satisfying at least one of (i) El Escorial Revised ALS diagnostic criteria, and (ii) Awaji-shima diagnostic criteria.

16. The method of claim 15, wherein the diagnosing the patient with ALS is by satisfying the Awaji-shima diagnostic criteria, wherein the patient diagnosed with ALS is presymptomatic, and wherein the orally administering to the patient the rho kinase inhibitor in the therapeutically effective amount for treating ALS is performed only when the second pre-treatment amount of NfL is determined to be greater the first pre-treatment amount.

17. The method of claim 16, wherein the second time is at least one month after the first time, andwherein the orally administering to the patient the rho kinase inhibitor in the therapeutically effective amount for treating ALS is performed only when the second pre-treatment amount of NfL is determined to be at least 1 % greater the first pretreatment amount.

18. The method of claim 15, wherein the diagnosing the patient with ALS is by satisfying the Awaji-shima diagnostic criteria, and wherein the patient diagnosed with ALS is diagnosed with familial ALS.

19. The method of claim 14, wherein the therapeutically effective amount is at least 300 mg / day.

20. The method of claim 14, wherein the therapeutically effective amount is at least 320 mg / day.

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