Fasudil for use in treating amyotrophic lateral sclerosis

Abstract

Disclosed herein are formulations of fasudil and methods for using the same to treat neurological conditions such as ALS.

Description

Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin FASUDIL FOR USE IN TREATING AMYOTROPHIC LATERAL SCLEROSIS STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0001] This invention was made with Government support under CReATe Consortium(CReATe; U54 NS092091, NIH 3U54NS092091-04S2) awarded by National Institutes ofHealth. The Government has certain rights in the invention.BACKGROUND

[0002] Neurodegenerative diseases, characterized by the progressive loss of structure orfunction of neurons, affect numerous people around the world. For example, amyotrophic lateral sclerosis, also known as ALS, is a disease that affects nerve cells in the brain and spinal cord, eventually causing loss of muscle control. Early death is common as the loss of muscle control eventually hinders vital functions, such as breathing. INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification areherein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. SUMMARY OF THE INVENTION

[0004] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron loss at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for motor neuron loss at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits an at least 5% decrease in motor neuron loss at the second timepoint as compared to the first timepoint.

[0005] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron loss at a first timepoint; b) administering to the subject a -1-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for motor neuron loss at a second timepoint, wherein thesecond timepoint occurs at least 50 days after step b), and wherein the subject exhibits an atleast 5% decrease in motor neuron loss at the second timepoint as compared to the firsttimepoint.

[0006] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating thesubject for motor neuron dysfunction at a first timepoint; b) administering to the subject apharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for motor neuron dysfunction at a second timepoint,wherein the second timepoint occurs at least 50 days after step b), and wherein the subjectexhibits an at most 20% decrease in motor neuron dysfunction at the second timepoint ascompared to the first timepoint.

[0007] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating thesubject for motor neuron dysfunction at a first timepoint; b) administering to the subject apharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twicea day; and c) evaluating the subject for motor neuron dysfunction at a second timepoint,wherein the second timepoint occurs at least 50 days after step b), and wherein the subjectexhibits an at most 20% decrease in motor neuron dysfunction at the second timepoint ascompared to the first timepoint.

[0008] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for a biomarker at a first timepoint; b) administering to the subject a pharmaceuticalcomposition wherein the pharmaceutical composition comprises 15 mg of fasudil or apharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; andc) evaluating the subject for the biomarker at a second timepoint, wherein the secondtimepoint occurs at least 50 days after step b), and wherein the subject exhibits a change of atleast 5% in the biomarker at the second timepoint as compared to the first timepoint.

[0009] In some embodiments disclosed herein are methods treating amyotrophic lateral-2-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin sclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for a biomarker at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for the biomarker at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits a change of at least 5% in the biomarker at the second timepoint as compared to the first timepoint.

[0010] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron loss at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twicea day; c) administering to the subject a pharmaceutical composition wherein thepharmaceutical composition comprises a therapeutically-effective amount of a second agent; and d) evaluating the subject for motor neuron loss at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits an at least 5%decrease in motor neuron loss at the second timepoint as compared to the first timepoint.

[0011] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron loss at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twicea day; c) administering to the subject a pharmaceutical composition wherein thepharmaceutical composition comprises a therapeutically-effective amount of a second agent; and d) evaluating the subject for motor neuron loss at a second timepoint, wherein the secondtimepoint occurs at least 50 days after step b), and wherein the subject exhibits an at least 5%decrease in motor neuron loss at the second timepoint as compared to the first timepoint.

[0012] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating thesubject for motor neuron dysfunction at a first timepoint; b) administering to the subject apharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; c) administering to the subject a pharmaceutical composition wherein the -3-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin pharmaceutical composition comprises a therapeutically-effective amount of a second agent; and d) evaluating the subject for motor neuron dysfunction at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits an atmost 20% decrease in motor neuron dysfunction at the second timepoint as compared to thefirst timepoint.

[0013] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron dysfunction at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; c) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises a therapeutically-effective amount of a second agent;and d) evaluating the subject for motor neuron dysfunction at a second timepoint, wherein thesecond timepoint occurs at least 50 days after step b), and wherein the subject exhibits an atmost 20% decrease in motor neuron dysfunction at the second timepoint as compared to thefirst timepoint.

[0014] In some embodiments, disclosed herein are method of treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for a biomarker at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for the biomarker at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits a change of at least 5% in the biomarker at the second timepoint as compared to the first timepoint.

[0015] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for a biomarker at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or apharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; andc) evaluating the subject for the biomarker at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits a change of at least 5% in the biomarker at the second timepoint as compared to the first timepoint.

[0016] In some embodiments, disclosed herein are method of treating amyotrophic lateral-4-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin sclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for a biomarker at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for the biomarker at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits a change of at least 5% in the biomarker at the second timepoint as compared to the first timepoint.

[0017] In some embodiments disclosed herein are methods treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, wherein the method comprises: a) evaluating the subject for a biomarker at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; c) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises a therapeutically-effective amount of a second agent; and d) evaluating the subject for the biomarker at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits a change of at least 5% in the biomarker at the second timepoint as compared to the first timepoint. FIGURE DESCRIPTIONS

[0018] Figure 1 shows the scheme of an illustrative phase 2 trial using a placebo bid, 15 mgof fasudil bid, or 30 mg fasudil bid. Measured endpoints occurred at end of treatment (V20), Day 90 (V22), and Day 180 (V23). BID = bis in die (twice daily). V = visit.

[0019] Figure 2 shows motor unit number index (MUNIX) evaluation, providing an estimateof the number of functional lower motor neurons in a muscle at different treatment pointsincluding prior to first treatment, at the end of the treatment period, day 60 and day 180 in theper-protocol population (PPP) comprising all patients who received at least 80% of the scheduled treatment doses (total 113 patients).

[0020] Figure 3 shows slow vital capacity evaluation as compared to prior to the firsttreatment in the PPP group.

[0021] Figure 4 shows the scheme of an illustrative phase 2 trial including patient numbersfor screening, randomization, treatment, and follow-up.

[0022] Figure 5 shows safety and tolerability outcomes. Panel A shows the proportion ofpatents for whom the treatment was safe or tolerable in each treatment arm. Panel B shows -5-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin differences in proportions of patients for whom the treatment was safe or tolerable between both active arms and placebo with 95% confidence intervals.

[0023] Figure 6 shows efficacy outcomes. Panel A shows Kaplan-Meier survival curves foroverall survival time until death in the intention-to-treat population at the given time-points.Panels B-D show estimated point difference from baseline for ALSFRS-R (AmyotrophicLateral Sclerosis Functional Rating Scale-Revised) total score (Panel B), predicted SVC (slow vital capacity) (Panel C), and MUNIX (Motor Unit Number Index) megascore 10 (comprising the abductor pollicis brevis (APB), abductor digiti minimi (ADM), biceps brachii (BB), tibialis anterior (TA), extensor digitorum brevis (EDB) muscles on both sides) (Panel D) in the intention-to-treat population (118 patients) at the given time-points. Error bars represent 95% CI. P-values according to MMRM.

[0024] Figure 7 shows mean heart rate, systolic and diastolic arterial blood pressure for thedifferent treatment groups at given time-points before and after start of the study drug infusion in the morning and in the afternoon.

[0025] Figure 8 shows results from pairwise contrast tests of the difference from baseline inthe predicted slow vital capacity (SVC) between the treatment groups at each follow-up visit in all subgroups. Expected marginal mean estimates of differences in differences from baseline for the SVC are shown with 95% confidence intervals and p values testing the nullhypothesis of no difference for the post-hoc subgroup analyses in the intention-to-treatpopulation.

[0026] Figure 9 shows results from pairwise contrast tests of the ratios to baseline inneurofilament light chain (NfL) between the treatment groups at each follow-up visit in all subgroups. Expected marginal mean estimates of ratios in ratios to baseline are shown with 95% confidence intervals and p values testing the null hypothesis of no difference for the post-hoc subgroup analyses in the intention-to-treat population.

[0027] Figure 10 shows levels of fasudil, hydroxyfasudil and riluzole in plasma andcerebrospinal fluid (CSF) of the patients in the indicated treatment arms of the intention-to- treat population.

[0028] Figure 11 illustrates a summary of fasudil safety and tolerability data following 30and 60 mg administration. It is a poster entitled: “Safety, tolerability and efficacy of the rho kinase inhibitor fasudil in ALS (ROCK-ALS): a phase 2, randomised, double-blind, placebo- controlled trial” authored by the inventor. The “Background” section reads: “Fasudil is a small molecule inhibitor of the rho-associated kinase (ROCK) and is approved for the -6-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin treatment of subarachnoid hemorrhage. In preclinical studies, fasudil attenuatesneurodegeneration, modulates neuroinflammation, and fosters axonal regeneration. 1-7,9”The “Methods” section reads: “This randomized, double-blind, placebo-controlled, phase 2 clinical trial was conducted at 19 sites in Germany, France, and Switzerland. Adult patients with at least probable ALS (revised El Escorial criteria), a disease duration of 6-24 months,and a slow vital capacity (SVC) of >65% of normal were included. Subjects were randomlyassigned (1:1:1) to receive 30 mg (15 mg twice daily) fasudil, 60 mg (30 mg twice daily) fasudil, or placebo intravenously over 20 treatment days. Follow-up assessments were performed at 45, 90 and 180 days after treatment initiation. Primary endpoints were tolerability during the treatment period and safety until the end of the study. All subjects who received at least one study drug dose were part of the intention-to-treat (ITT)-analysis.10”

[0029] The “Findings” section comprises Figures 4, 5 and 6 and reads: “Safety andtolerability outcomes: In the ITT population (118 participants), there were no significant differences between groups in regard to tolerability (estimated proportion of patients without event for placebo: 0.93 (95% Cl: 0.80 to 0.99], fasudil 30 mg: 1.00 (0.90 to 1.00] fasudil 60 mg: 0.90 [0.76 to 0.97]) and safety (placebo: 1.00 [0.90 to 1.00], 30 mg fasudil: 1.00 [0.89to 1.00], fasudil 60 mg: 1.00 [0.90 to 1.00]). Serious adverse events (total 40 in 30 / 118patients (25.4%)) and adverse events were distributed equally between the treatment groupsand mainly related to ALS disease progression. No serious adverse events or deaths wereattributed to the treatment. Efficacy outcomes: In the secondary outcomes, motor unitnumber index (MUNIX) showed a significantly reduced decline for fasudil 60 mg at 26 and 90 days, and for fasudil 30 mg at 90 days after treatment start. There was a directional difference for a reduced SVC decline favoring fasudil 60 mg at day 26, which was significant in a post-hoc subgroup analysis of females at all time points. ALSFRS-R (Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised), ALSAQ-5 (Amyotrophic Lateral Sclerosis Assessment Questionnaire 5), ECAS (Edinburgh Cognitive and Behavioural ALSScreen) and survival were not different between treatment groups. Overall, the findings of theROCK-ALS study suggest that fasudil 30 and 60 mg intravenously is safe and tolerable and may have an effect on motor unit number preservation in patients with ALS,which argues for further investigation of this drug as disease-modifying drug in ALS.13” The“Interpretation and added value” section reads: “This study is the first randomised, placebo-controlled trial designed to assess the safety, tolerability, and efficacy of the ROCK inhibitor fasudil for the treatment of patients with ALS. Fasudil treatment was well tolerated and safe -7-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin in ALS patients. MUNIX evaluation suggests a slower loss of motor units in fasudil-treated patients compared to placebo, suggesting a benefit of fasudil to be explored in further clinical trials. This trial also shows that the neurophysiological MUNIX assessment can beutilised in multicentric treatment trials and may be more sensitive to change than scale-basedmeasures, such as the well-established ALSFRS-R.8, 11, 12” The numbered references are listedafter the Examples.

[0030] Figure 12 shows percentage of muscles with new spreading events over time acrosstreatment groups and MUNIX cut-off thresholds. The figure displays the proportion ofmuscles with newly defined "spreading events" at three timepoints (baseline, day 90, and day 180) for each treatment group: placebo (light grey), fasudil 15 mg (black), and fasudil 30 mg (dark grey). Spreading was defined using three cut-off thresholds (10%, 20%, and 30%) and is illustrated by line type (solid for 10%, dashed for 20%, dotted for 30%). At day 90, asignificant difference between groups was observed for the 20% and 30% cut-offs (globalChi-square test: p=0.003 and p=0.0005, respectively), with pairwise comparisons indicating a significantly lower proportion of newly affected muscles in the fasudil 30 mg group versus placebo (p=0.007 and p=0.0006, respectively), and for the fasudil 15 mg group versus placebo at the 30% cut-off (p=0.004). No significant group differences were observed at day 180 for any cut-off level. Vertical annotations to the left indicate pairwise Fisher exact test p- values at day 90 for the 30% cut-off.

[0031] Figure 13 shows percentage of limbs with new spreading events over time acrosstreatment groups and MUNIX cut-off thresholds. This figure presents the proportion of limbs exhibiting newly affected muscles (“spreading events”) at three timepoints—baseline, day 90, and day 180—across treatment groups: placebo (light grey), fasudil 15 mg (black), andfasudil 30 mg (dark grey). A limb was determined to be affected if at least one muscle withinthe limb met the spreading criteria based on MUNIX changes. Three cut-off thresholds (10%, 20%, and 30%) were applied, illustrated by line style (solid = 10%, dashed = 20%, dotted =30%). An overall increase in affected limbs over time is observed in all groups, nostatistically significant differences between treatment arms were found at day 90 (global Chi- square test: p=0.4 for 10% cut-off, p=0.85 for 20%, and p=0.27 for 30%). DETAILED DESCRIPTION OF THE INVENTION

[0032] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a rareneuromuscular disorder, and people of all races and ethnic backgrounds are affected. ALS is -8-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin most commonly diagnosed in people between 40 and 60 years of age, and men are affected slightly more often than women. One or two out of 100,000 people develop ALS each year and estimated 5,000 people in the United States are diagnosed with the disease each year. ALS is a progressive disease characterized by rapidly progressive weakness, muscle atrophy and fasciculation, spasticity, dysarthria, dysphagia, and respiratory compromise. It is a neurodegenerative movement disorder caused by the degeneration of neurons located in the ventral horn of the spinal cord and the cortical neurons. Unable to function, the muscles weaken and atrophy. Cognitive function is generally spared for most patients, although thereis significant overlap with frontotemporal dementia, particularly in patients who have aC9orf72 co-pathology. ˜30-50% of patients also show subtle cognitive changes which can berevealed by detailed neuropsychological testing.

[0033] ALS is a fatal disorder characterized by subtle onset of focal weakness, typically inthe limbs but approximately one third of cases also in bulbar muscles. ALS progresses toparalysis of almost all skeletal muscles. Significant clinico-pathological and genetic overlap exists between ALS and frontotemporal lobar dementia (FTLD). In ALS, death from respiratory paralysis is common within five years. The cellular pathology is focal at onset and spreads in a pattern and suggests successive involvement of contiguous neuronal populations. Death of motor neurons occurs in conjunction with deposition of aggregated proteins in motoneurons and oligodendrocytes, and neuroinflammation. Most cases of ALS are sporadic(sALS).In about 10% of all cases, patients report a family history for the disorder (familialALS, fALS).

[0034] TDP-43 (Transactivating response element DNA binding protein 43 kDa)accumulates in cytoplasm of motor neurons in most cases of ALS. TDP-43 is a nuclear RNA-binding protein involved in several aspects of RNA processing that actively shuttles betweenthe nucleus and cytoplasm. In ALS and frontotemporal dementia, TDP-43 translocates to the cytoplasm. However, such cytoplasmic mislocalization is common in neuronal injury orstress. TDP-43-positive inclusions represent secondary pathology in some neurodegenerativedisorders. Possible mechanisms of death of motor neurons in ALS include a) glutamate- mediated excitotoxicity; b) decrease in neurotrophic factors (BDNF, GDNF) and associated signaling; c) mitochondrial alterations and oxidative damages; and d) abnormalities in cytoskeletal proteins resulting in neuronal atrophy and death.

[0035] Fasudil is a potent Rho-kinase (ROCK) inhibitor and vasodilator that can beadministered for treatment of ALS by mediating neuronal survival, and which is currently-9-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin approved for the treatment of subarachnoid hemorrhage. Disclosed herein are methods of treatment of ALS comprising administering fasudil or a pharmaceutically acceptable salt thereof. Compounds of the Disclosure

[0036] Described herein are compositions of fasudil or a pharmaceutically-acceptable saltthereof. Fasudil is a nonspecific RhoA / ROCK inhibitor that can have an inhibitory effect onprotein kinases. Fasudil is also a calcium channel antagonist that primarily acts through theinhibition of the Rho-kinase signaling pathway. This action allows for vasodilation through the activation of myosin phosphatase. Fasudil is an isoquinoline substituted with a (1,4-diazepan-1-yl)sulfonyl group at position 5. Fasudil has a molecular formula of C14H17N3O2S and a structure of:

[0037] Described herein are also fasudil derivatives or a pharmaceutically-acceptable saltthereof. In some embodiments, the fasudil derivative is 1-(6-hydroxyl-5- isoquinolinesulfonyl)homopiperazine (hydroxyl-fasudil). In some embodiments, the fasudil derivative is (S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4- diazepine (dimethyl-fasudil). Pharmaceutically-Acceptable Salts

[0038] The invention provides the use of pharmaceutically-acceptable salts of any compounddescribed herein. Pharmaceutically-acceptable salts include, for example, acid-addition salts and base-addition salts. The acid that is added to the compound to form an acid-addition salt can be an organic acid or an inorganic acid. A base that is added to the compound to form a base-addition salt can be an organic base or an inorganic base. In some embodiments, a pharmaceutically-acceptable salt is a metal salt. In some embodiments, a pharmaceutically- acceptable salt is an ammonium salt.

[0039] Metal salts can arise from the addition of an inorganic base to a compound of the-10-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin invention. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. In some embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium,strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.

[0040] In some embodiments, a metal salt is a lithium salt, a sodium salt, a potassium salt, acesium salt, a cerium salt, a magnesium salt, a manganese salt, an iron salt, a calcium salt, astrontium salt, a cobalt salt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt,or a zinc salt.

[0041] Ammonium salts can arise from the addition of ammonia or an organic amine to acompound of the invention. In some embodiments, the organic amine is triethyl amine, diisopropyl amine, ethanol amine, diethanol amine, triethanol amine, morpholine, N- methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrazole, piprazole, imidazole, or pyrazine.

[0042] In some embodiments, an ammonium salt is a triethyl amine salt, a diisopropyl aminesalt, an ethanol amine salt, a diethanol amine salt, a triethanol amine salt, a morpholine salt, an N-methylmorpholine salt, a piperidine salt, an N-methylpiperidine salt, an N- ethylpiperidine salt, a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrazole salt, a piprazole salt, an imidazole salt, or a pyrazine salt.

[0043] Acid addition salts can arise from the addition of an acid to a compound of theinvention. In some embodiments, the acid is organic. In some embodiments, the acid is inorganic. In some embodiments, the acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, gentisinic acid, gluconic acid, glucaronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid.

[0044] In some embodiments, the salt is a hydrochloride salt, a hydrobromide salt, ahydroiodide salt, a nitrate salt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactate salt, a salicylate salt, a tartrate salt, an ascorbate salt, a gentisinate salt, a gluconate salt, a glucaronate salt, a saccharate salt, a formate salt, a benzoate salt, a glutamate salt, a pantothenate salt, an acetate salt, a propionate salt, a butyrate salt, a fumarate -11-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin salt, a succinate salt, a methanesulfonate salt, an ethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt, a citrate salt, an oxalate salt, or a maleate salt.

[0045] In some embodiments, the salt is a hydrochloride salt, forming fasudil hydrochloride.Fasudil hydrochloride has a chemical formula of C14H17N3O2S • HCl and a structure of:

[0046] In some embodiments, the salt is a hydrochloride salt, forming fasudil hydrochloridehydrate. Fasudil hydrochloride has a chemical formula of C14H17N3O2S • HCl• ½ H2O and astructure of:Formulations

[0047] In some embodiments, fasudil or a pharmaceutically-acceptable salt thereof is presentin a formulation in an amount of from about 0.1 mg to about 100 mg, from about 10 mg to about 100 mg, from about 10 mg to about 80 mg, from about 20 mg to about 100 mg, from about 20 mg to about 60 mg, from about 20 mg to about 40 mg, from about 1 mg to about 20 mg, from about 40 mg to about 50 mg, from about 0.1 mg to about 1 mg, from about 0.5 mg to about 1 mg, from about 0.5 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, or from about 95 mg to about 100 mg. -12-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0048] In some embodiments, fasudil or a pharmaceutically-acceptable salt thereof is presentin a formulation in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, or about 100 mg. In some embodiments, fasudil or a pharmaceutically-acceptable salt thereof is present in a formulation in an amount of 15 mg. In some embodiments, fasudil or a pharmaceutically-acceptable salt thereof is present in a formulation in an amount of 30 mg. In some embodiments, fasudil or a pharmaceutically-acceptable salt thereof is present in a formulation in an amount of 60 mg.

[0049] A formulation disclosed herein can be stable for about 1 day, about 2 days, about 3days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about one year. A formulation disclosed herein can be stable, for example, at about 0 °C, about 5 °C, about 10 °C, about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 60 °C, about 70 °C, or about 80 °C.

[0050] A pharmaceutical composition of the invention can be a combination of anypharmaceutical compounds described herein with other chemical components, such ascarriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, or excipients. Pharmaceutical compositions can be formulated using one or more -13-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizinphysiologically-acceptable carriers comprising excipients and auxiliaries, which facilitateprocessing of the active compounds into preparations that can be used pharmaceutically. Formulations can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising a compound described herein can be manufactured, for example, by mixing, dissolving, emulsifying, processes.

[0051] The pharmaceutical compositions can include at least one pharmaceutically-acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically-acceptable salt form. The methods and pharmaceutical compositions described herein include the use of crystalline forms (also known as polymorphs), and active metabolites of these compounds having the same type of activity.

[0052] A pharmaceutical composition can comprise one or more carriers. Non-limitingexamples of pharmaceutically-acceptable carriers include saline solution, Ringer's solution, and dextrose solution. The pH of the solution can be from about 5 to about 8. The pH of the solution can be from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the fasudil or a pharmaceutically-acceptable salt thereof, where the matrices are in the form of shaped articles, such as films, liposomes, microparticles, and microcapsules.

[0053] A pharmaceutical composition can comprise one or more fillers. Non-limitingexamples of fillers can include lactose, sucrose, magnesium stearate, glucose, cellulose, and calcium carbonate. In some embodiments, a filler can be cellulose (e.g., microcrystalline cellulose).

[0054] In some embodiments, a filler (e.g., a cellulose or microcrystalline cellulose) ispresent in a composition in an amount of present in a formulation in an amount of from about 0.1 mg to about 100 mg, from about 10 mg to about 100 mg, from about 10 mg to about 80 mg, from about 20 mg to about 100 mg, from about 20 mg to about 60 mg, from about 20 mgto about 40 mg, from about 1 mg to about 20 mg, from about 40 mg to about 50 mg, fromabout 0.1 mg to about 1 mg, from about 0.5 mg to about 1 mg, from about 0.5 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, about 75 mg to about 80 mg, -14-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg toabout 95 mg, or from about 95 mg to about 100 mg.

[0055] In some embodiments, a filler (e.g., cellulose or microcrystalline cellulose) is presentin a formulation in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg,about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, or about 100 mg.

[0056] Pharmaceutical formulations can include additional carriers, thickeners, diluents,buffers, preservatives, and surface-active agents in addition to the compounds disclosed herein. An excipient can fill a role as simple and direct as being an inert filler, or an excipient as used herein can be part of a pH stabilizing system or coating to insure delivery of the ingredients safely to another desired target. Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice ofPharmacy, Nineteenth Ed (Easton, Pa. Mack Publishing Company, 1995); Hoover, JohnE., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman,H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.(Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

[0057] A pharmaceutical composition comprising fasudil, a derivative thereof, or apharmaceutically-acceptable salt thereof can be an intravenous formulation. For example, a -15-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizincomposition can be in a liquid dosage form suitable for an intravenous infusion. In someembodiments, a composition can be an isotonic solution suitable for an intravenous infusion.

[0058] In practicing the methods of treatment or use provided herein, therapeutically-effective amounts of the compounds described herein are administered in pharmaceutical compositions to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal such as a human. A therapeutically-effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.

[0059] The disclosed methods include administration of a fasudil or a pharmaceutically-acceptable salt thereof, in combination with a pharmaceutically-acceptable carrier. The carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0060] The fasudil or a pharmaceutically-acceptable salt thereof as described herein can beformulated into pharmaceutical compositions composed of one or more pharmaceutically-acceptable carriers. See e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W.Martin Mack Pub. Co., Easton, Pa., which discloses carriers and methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compound described herein and which is incorporated by reference herein.

[0061] The disclosed methods relate to intravenous administration of fasudil or apharmaceutically-acceptable salt thereof as part of a pharmaceutical composition. In some embodiments, a method of treating a disease of a subject by administering the intravenous formulation is provided. The method comprises providing an intravenous formulation as described herein having an effective amount of fasudil or a pharmaceutically-acceptable salt, solvate, anomer, hydrate, or prodrug thereof, and administering the formulation to a subjectto treat the diseased state.Pharmaceutical Compositions

[0062] Pharmaceutical compositions containing the compounds described herein can beadministered for prophylactic or therapeutic treatments. In therapeutic applications, the compositions can be administered to a subject already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition, or to cure, heal, improve, reduce, lessen, or ameliorate the disease or condition. Compounds can also be administered to lessen or reduce a likelihood of developing, -16-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin contracting, or worsening a condition. Amounts effective for this use can vary based on the severity and course of the disease or condition, previous therapy, the subject's health status,weight, response to the drugs, and the judgment of the treating physician. In someembodiments, compositions comprising fasudil or a pharmaceutically acceptable salt thereof can attenuate neurodegeneration, modulate neuroinflammation, and foster axonal regeneration.

[0063] Compounds and compositions of the invention can be packaged as a kit. In someembodiments, the invention provides a kit comprising a compound disclosed herein, or apharmaceutically-acceptable salt thereof, and written instructions on use of the kit in thetreatment of a condition described herein. In some embodiments, the invention provides a kit comprising a compound disclosed herein, or a pharmaceutically-acceptable salt thereof, and written instructions on use of the kit in the treatment of a condition described herein.

[0064] The compounds described herein can be administered before, during, or after theoccurrence of a disease or condition, and the timing of administering the compositioncontaining a compound can vary. For example, the compounds can be used as a prophylacticand can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen or reduce a likelihood of the occurrence of the disease or condition. Thecompounds and compositions can be administered to a subject during or as soon as possibleafter the onset of the symptoms. The administration of the compounds can be initiated within the first 48 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms. The initial administration can be via any route practical, such as by any route described herein using any formulation described herein.

[0065] A compound can be administered as soon as is practical after the onset of a disease orcondition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months. In some embodiments,the length of time a compound can be administered in a course of treatment can be about 1day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about one month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 -17-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 4 months, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 5 months, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months about 23 months, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years. The length of treatment can vary for each subject.

[0066] Pharmaceutical compositions described herein can be in unit dosage forms suitablefor single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosagecan be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged injectables, vials, or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with or without a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.

[0067] A fasudil compound described herein can be present in a composition in a range offrom about 1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, from about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, from about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, from about 95 mg to about 100 mg, from about 100 mg to about 125 mg,from about 125 mg to about 150 mg, from about 150 mg to about 175 mg, from about 175mg to about 200 mg, from about 200 mg to about 225 mg, from about 225 mg to about 250 mg, or from about 250 mg to about 300 mg.

[0068] A fasudil compound described herein can be present in a composition in an amount ofabout 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, -18-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, or about 300 mg.

[0069] A fasudil compound described herein can be administered at least once a week, atleast once every other day, at least once a day, at least twice a day, or at least three times a day. Treatment of Subjects with Neurodegenerative Disease

[0070] The invention discloses methods for treating a subject afflicted a neurodegenerativedisease. A neurodegenerative disease is a progressive disease in which cells of the central nervous system stop working and / or die. Non-limiting examples of neurodegenerative diseases include Alzheimer’s disease, Parkinson’s disease, prion disease, Amyotrophic lateral sclerosis (ALS), motor neuron disease, Huntington’s disease, dementia, spinal muscular atrophy, and spinocerebellar ataxia.

[0071] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is aneurodegenerative disease that results in the progressive loss of motor neurons that controlvoluntary muscles. Different types of ALS can be classified by the types of motor neuronsthat are affected – upper motor neurons (e.g., in the motor cortex of the brain) or lower motorneurons (e.g., neurons of the spinal cord).

[0072] Non-limiting examples of possible subjects for administration include the following.Subjects can be humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rats, mice, and guinea pigs. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, and infants.

[0073] The subject can be a human. Treatment can include treating a human in a clinical trial.A treatment can comprise administering to a subject a pharmaceutical composition comprising one or more of fasudil or a pharmaceutically-acceptable salt thereof, as described throughout the disclosure.

[0074] In some embodiments, the invention provides fasudil, a derivative thereof, or apharmaceutically-acceptable salt thereof for use in treatment of a neurodegenerative disease (e.g., ALS). In some embodiments, the invention provides fasudil, a derivative thereof, or a pharmaceutically-acceptable salt thereof for use in the manufacture of a medicament for the -19-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin treatment of a neurodegenerative disease (e.g., ALS).

[0075] In some embodiments, the disclosure provides administering of a second therapeuticagent. In some embodiments, the disclosure provides administering therapeutically-effectiveamounts of the second agent. In some embodiments, the second agent can be administered prior to the administration of fasudil, a derivative thereof, or a pharmaceutically-acceptablesalt thereof. In some embodiments, the second agent can be administered after theadministration of fasudil, a derivative thereof, or a pharmaceutically-acceptable salt thereof. In some embodiments, the second agent can be administered concurrently with the administering of fasudil. In some embodiments, the second agent comprises riluzole. In some embodiments, the second agent comprises edaravone. In some embodiments, the second agent comprises tofersen. In some embodiments, the second agent comprises nuedexta. In some embodiments, the second agent comprises jacifusen. In some embodiments, the second agent comprises sodium phenylbutyrate. In some embodiments, the second agent comprises taurursodiol.

[0076] Treatment of a neurodegenerative disease (e.g., ALS) can result in reducing muscleweakness 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%, atleast about 95%, or at least about 100%. Treatment of a neurodegenerative disease (e.g.,ALS) can result in reducing muscle weakness by about 5% to about 6%, by about 5% toabout 7%, by about 5% to about 8%, by about 5% to about 9%, by about 5% to about 10%, by about 5% to about 11%, by about 5% to about 12%, by about 5% to about 13%, by about 5% to about 14%, by about 5% to about 15%, by about 5% to about 16%, by about 5% toabout 17%, by about 5% to about 18%, by about 5% to about 19%, or by about 5% to about20%. Treatment of a neurodegenerative disease (e.g., ALS) can result in reducing muscle weakness by up to 1%, by up to 2%, by up to 3%, by up to 4%, by up to 5%, by up to 6%, by up to 7%, by up to 8%, by up to 9%, by up to 10%, by up to 11%, by up to 12%, by up to 13%, by up to 14%, by up to 15%, by up to 16%, by up to 17%, by up to 18%, by up to 19%,by up to 20%, by up to 25%, by up to 30%, by up to 35%, by up to 40%, by up 45%, by up to50%, by up to 50%, by up to 55%, by up to 60%, by up to 65%, by up to 70%, by up to 75%, by up to 80%, by up to 85%, by up to 90%, by up to 95%, or by up to 100%.

[0077] Treatment of a neurodegenerative disease (e.g., ALS) can result in reducing motor-20-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizinneuron loss or dysfunction by at least about 5%, at least about 10%, at least about 15%, atleast 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%, atleast about 90%, at least about 95%, or at least about 100%. Treatment of aneurodegenerative disease (e.g., ALS) can result in reducing motor neuron loss ordysfunction by about 5% to about 6%, by about 5% to about 7%, by about 5% to about 8%,by about 5% to about 9%, by about 5% to about 10%, by about 5% to about 11%, by about 5% to about 12%, by about 5% to about 13%, by about 5% to about 14%, by about 5% toabout 15%, by about 5% to about 16%, by about 5% to about 17%, by about 5% to about18%, by about 5% to about 19%, or by about 5% to about 20%. Treatment of aneurodegenerative disease (e.g., ALS) can result in reducing motor neuron loss ordysfunction by up to 1%, by up to 2%, by up to 3%, by up to 4%, by up to 5%, by up to 6%,by up to 7%, by up to 8%, by up to 9%, by up to 10%, by up to 11%, by up to 12%, by up to 13%, by up to 14%, by up to 15%, by up to 16%, by up to 17%, by up to 18%, by up to 19%, by up to 20%, by up to 25%, by up to 30%, by up to 35%, by up to 40%, by up 45%, by up to 50%, by up to 50%, by up to 55%, by up to 60%, by up to 65%, by up to 70%, by up to 75%, by up to 80%, by up to 85%, by up to 90%, by up to 95%, or by up to 100%.

[0078] Treatment of a neurodegenerative disease (e.g., ALS) can be measured usingevaluations of motor neuron loss. Non-limiting examples of evaluations of motor neuron losscan be using a motor unit number index (MUNIX), using a slow vital capacity (SVC) test, ora test of muscle strength, such as a hand grip test, for example with a hand-held dynamometer Evaluation of motor neuron loss can comprise a first timepoint wherein a measurement is taken prior to treatment in order to establish a baseline or control. Evaluation of motor neuron loss can comprise a second timepoint. Evaluation of motor neuron loss can comprise subsequent timepoints including but not limited to a third, a fourth, a fifth, a sixth, a seventh, an eighth, a ninth, a tenth, an eleventh, a twelfth, a thirteenth, a fourteenth, a fifteenth, a sixteenth, a seventeenth, an eighteenth, a nineteenth, a twentieth, a twenty-first, a twenty- second, a twenty-third, a twenty-fourth, a twenty-fifth, a twenty-sixth, a twenty-seventh, a twenty-eighth, a twenty-ninth, or a thirtieth timepoint.

[0079] In some embodiments, the second timepoint occurs at least about 1 day, at least about2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at -21-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months or more after the first timepoint.

[0080] Treatment of a neurodegenerative disease (e.g., ALS) can be indicated or quantifiedthrough a MUNIX. MUNIX provides an estimate of the number of functional lower motor neurons (LMNs) in a muscle and is monitored on multiple muscles and over time to provide alongitudinal window into neuronal health across the body. In some embodiments, MUNIXcan be analyzed at the single muscle level. In some embodiments, MUNIX can be analyzed atproximal muscles. In some embodiments, MUNIX can be analyzed at distal muscles. Non-limiting examples of muscles that can be analyzed by MUNIX include: abductor digiti minimi, abductor pollicis brevis, biceps brachii, tibialis anterior, and exterior dig. brevis. Decreasing MUNIX is a marker of loss of LMNs and thus disease progression. Changes in MUNIX score are detectable earlier in ALS than functional changes and are correlated withthe future rate of disease progression. In some embodiments, MUNIX can be used to analyzedisease spread to one or more muscles over time.

[0081] Treatment of a neurodegenerative disease (e.g., ALS) can be indicated or quantifiedby a subject having a MUNIX score of at least about 1% higher, at least about 2% higher, at least about 3% higher, at least about 4% higher, at least about 5% higher, at least about 6% higher, at least about 7% higher, at least about 8% higher, at least about 9% higher, at least about 10% higher, at least about 11% higher, at least about 12% higher, at least about 13% higher, at least about 14% higher, at least about 15% higher, at least about 16% higher, atleast about 17% higher, at least about 18% higher, at least about 19% higher, at least about20% higher, at least about 21% higher, at least about 22% higher, at least about 23% higher, at least about 24% higher, at least about 25% higher, at least about 30% higher, at least about 35% higher, at least about 40% higher, at least about 45% higher, at least about 50% higher, at least about 55% higher, at least about 60% higher, at least about 65% higher, at least about 70% higher, at least about 75% higher, at least about 80% higher, at least about 85% higher, -22-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin at least about 90% higher, at least about 95% higher, at least about 100% higher, or more as compared to a MUNIX score of a placebo treatment.

[0082] Treatment of a neurodegenerative disease (e.g., ALS) can be indicated or quantifiedthrough a SVC test. A slow vital capacity test measures the maximum amount of air exhaledin a relaxed expiration. Decreasing SVC is a marker of loss of LMNs and thus diseaseprogression. Changes in SVC are detectable earlier in ALS than functional changes and arecorrelated with the future rate of disease progression.

[0083] Treatment of a neurodegenerative disease (e.g., ALS) can be indicated or quantifiedby a subject having a SVC of at least about 1% higher, at least about 2% higher, at least about 3% higher, at least about 4% higher, at least about 5% higher, at least about 6% higher, at least about 7% higher, at least about 8% higher, at least about 9% higher, at least about 10% higher, at least about 11% higher, at least about 12% higher, at least about 13% higher, at least about 14% higher, at least about 15% higher, at least about 16% higher, at least about 17% higher, at least about 18% higher, at least about 19% higher, at least about 20% higher, at least about 21% higher, at least about 22% higher, at least about 23% higher, at least about 24% higher, at least about 25% higher, at least about 30% higher, at least about 35% higher, at least about 40% higher, at least about 45% higher, at least about 50% higher, at least about55% higher, at least about 60% higher, at least about 65% higher, at least about 70% higher,at least about 75% higher, at least about 80% higher, at least about 85% higher, at least about 90% higher, at least about 95% higher, at least about 100% higher, or more as compared to a SVC of a placebo treatment.

[0084] Treatment of a neurodegenerative disease (e.g., ALS) can be indicated or quantifiedthrough functional scales. In some embodiments, the functional scale comprises the the Revised ALS Functional Rating Scale (ALSFRS-R). The ALSFRS-R encompasses 12 prompts that are grouped into four domains to assess bulbar symptoms, limb and trunk functionality, respiratory symptoms, and the need for percutaneous endoscopic gastrostomy, non-invasive ventilation, or tracheostomy with invasive ventilation. In some embodiments, the ALSFRS-R scale can be assessed only for the bulbar symptoms. In some embodiments, the ALSFRS-R scale can be assessed only for the respiratory symptoms. In someembodiments, the functional scale comprises the Edinburgh Cognitive and Behavioral ALSScreen (ECAS). The ECAS incorporates a range of cognitive tests to assess cognitive impairment in ALS. In some embodiments, the ECAS assesses cognitive impairment in ALS- specific domains. In some embodiments, the ECAS assesses cognitive impairment in ALS- -23-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin non-specific domains.

[0085] In some embodiments, treatment of a neurodegenerative disease can compriseassessing biomarker and target engagement following administration of a pharmaceutical composition. In some embodiments, the assessing comprises measuring the levels of fasudil, a metabolite, or a derivative thereof in a biological sample obtained from a subject. In some embodiments, the assessing comprises measuring the activity of ROCK in a biologicalsample obtained from a subject following administration of fasudil. In some embodiments,the assessing comprises measuring the amount of neurofilaments in a biological sampleobtained from a subject following administration of fasudil. In some embodiments, theassessing comprises measuring the amount of glial fibrillary acidic protein (GFAP) in abiological sample obtained from a subject following administration of fasudil. In some embodiments, the assessing comprises measuring the amount of urinary neurotrophic receptor p75 extracellular domain in a biological sample obtained from a subject following administration of fasudil. In some embodiments, the assessing comprises measuring TDP-43 mislocalizaton in a biological sample obtained from a subject following administration offasudil. In some embodiments, the biological sample can be a serum sample. In someembodiments, the biological sample can be a plasma sample. In some embodiments, thebiological sample can be a sample of cerebrospinal fluid (e.g., obtained by lumbar puncture).In some embodiments, the biological sample can be a urine sample. In some embodiments,the biological sample can be a saliva sample. In some embodiments, the biological sample can be a tear sample.

[0086] In some embodiments, treatment of a neurodegenerative disease can compriseassessing safety and tolerability following administration of a pharmaceutical composition. In some embodiments, treatment of a neurodegenerative disease can comprise assessing survival following administration of a pharmaceutical composition. In some embodiments, treatment of a neurodegenerative disease can comprise assessing mutation status before, during, or after administration of a pharmaceutical composition. In some embodiments, treatment of aneurodegenerative disease can comprise assessing C9orf72 mutation status beforeadministration of a pharmaceutical composition. In some embodiments, treatment of a neurodegenerative disease can comprise performing cardiovascular monitoring before, during, or after administration of a pharmaceutical composition. In some embodiments, the cardiovascular monitoring can include measuring pulse. In some embodiment, the cardiovascular can comprise measuring systolic and diastolic blood pressure. In some -24-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin embodiments, treatment of a neurodegenerative disease can comprise measuring body weight of a subject before, during, or after administration of a pharmaceutical composition. In someembodiments, treatment of neurodegenerative disease can comprise measuring serum creatinekinase levels before, during, or after administration of a pharmaceutical composition.

[0087] In some embodiments, treatment of a neurodegenerative disease can compriseevaluating a biomarker in a subject. In some embodiments, evaluation of a biomarker can occur before, during, or after administration of a pharmaceutical composition. In some embodiments, evaluation of a biomarker can comprise measuring a parameter obtained from a subject administered a pharmaceutical composition. In some embodiments, evaluation of a biomarker can comprise measuring a parameter in a biological sample obtained from a subject administered a pharmaceutical composition. In some embodiments, the parameter can comprise vital signs (e.g., pulse, blood pressure) of a subject. In some embodiments,evaluation of a biomarker can comprise measure the level of a parameter in a subjectadministered a pharmaceutical composition. In some embodiments, evaluation of a biomarker can comprise measuring the level of a parameter in a biological sample obtained from a subject administered a pharmaceutical composition. In some embodiments, the parameter can comprise laboratory parameters from a biological sample obtained from a subject administered a pharmaceutical composition. In some embodiments, the parameter can be hemoglobin. In some embodiments, the parameter can be erythrocytes. In some embodiments, the parameter can be mean corpuscular volume. In some embodiments, the parameter can be mean corpuscular hemoglobin. In some embodiments, the parameter can be mean corpuscular hemoglobin concentration. In some embodiments, the parameter can be thrombocytes. In some embodiments, the parameter can be leukocytes. In some embodiments, the parameter can be creatinine. In some embodiments, the parameter can be glucose. In some embodiments, the parameter can be aspartate transaminase. In some embodiments, the parameter can be alanine transaminase. In some embodiments, the parameter can be gamma glutamyl transpeptidase. In some embodiments, the parameter can be creatin kinase. In some embodiments, the parameter can be neurofilament light chain (NfL). In some embodiments, the parameter can be Rho kinase activity. In some embodiments, the parameter can be phosphorylated neurofilament heavy chain. In some embodiments, the parameter can be glial fibrillary acidic protein (GFAP). In some embodiments, the parameter can be urate. In some embodiments, the parameter can be soluble p75 extracellular region. In some embodiments, the parameter can be chromogranin -25-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin A. In some embodiments, the parameter can be CHIT1. In some embodiments, the parameter can be UCHL1. In some embodiments, the parameter can be GPNMB. In some embodiments, the parameter can be SPP1. The skilled person is aware how to measure these parameters.

[0088] Fasudil for use in a method of treating ALS in a subject is also disclosed, whereinfasudil is administered during a treatment period, wherein the treatment period is from 2 to 6weeks, wherein 150 mg to 630 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 5 to 7 days in each week of the treatment period. Optionally, the treatment period is followed by a drug holiday period in which fasudil is notadministered. In some embodiment, the drug holiday period is from 1 week to 3 months. Atreatment period is a period of time in which a drug, such as fasudil, is administered. A drugholiday period is deliberate interruption of the therapy, here fasudil administration, for adefined period.

[0089] In some embodiments, the subject exhibits an increased MUNIX score after thetreatment period as compared to a MUNIX score of a subject undergoing placebo treatment, if determined. In some embodiment, the subject exhibits an increased MUNIX score after the drug holiday period as compared to a MUNIX score of a subject undergoing placebo treatment, if determined. In preferred embodiments, the MUNIX score is increased by at least3 %, preferably at least 4%, more preferably at least 5%, more preferably at least 6%, morepreferably at least 7%, more preferably at least 8%, more preferably at least 9%, morepreferably at least 10%, more preferably at least 12%, more preferably at least 14%, morepreferably at least 16%, more preferably at least 18%, and even more preferably at least 20%;and / or at most 100%. The MUNIX score can be determined as disclosed herein and asdemonstrated in the Example section. The expressions “after the treatment period” and “afterthe drug holiday period” as used herein are to be understood as referring to the time point at which a determination, measurement, or assessment is conducted immediately following theconclusion of the respective period. For example, if the period is 4 weeks (28 days), thedetermination is intended to be performed on the same day that the period ends (e.g., day 28) or within 1 to 2 days thereafter. This ensures that the timing of the determination closely reflects the state resulting from the completed period, thereby providing accurate and relevant data for the intended evaluation.

[0090] In some embodiments, the subject exhibits an at most 20% decrease in MUNIX scoreafter the treatment period as compared to before initiation of fasudil administration, ifdetermined. In some embodiments, the subject exhibits an at most 20% decrease in MUNIX-26-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin score after the drug holiday period as compared to before initiation of fasudil administration.In preferred embodiments, the subject exhibits an at most 18% decrease in MUNIX scoreafter the treatment period and / or the drug holiday period as compared to before initiation of fasudil administration, in particular an at most 17% decrease, preferably an at most 16%decrease, more preferably an at most 15% decrease, more preferably an at most 14%decrease, more preferably an at most 13% decrease, more preferably an at most 12%decrease, more preferably an at most 11% decrease, more preferably an at most 10%decrease, more preferably an at most 8% decrease, more preferably an at most 6% decrease,more preferably an at most 4% decrease and most preferably an at most 2% decrease. Asdemonstrated herein, the skilled person may determine the MUNIX score before initiation offasudil administration and after the treatment period and / or the drug holiday period and by comparing these two value, the skilled person is able to determine the percentage of decrease.

[0091] In some embodiments, the subject exhibits an increased SVC after the treatmentperiod and / or the drug holiday period compared to an SVC of a subject undergoing placebo treatment, if determined. As disclosed herein, the skilled person is capable of determining SVC, which is a commonly known method in the field. Furthermore, the skilled person is aware that SVC decreases with the progression of ALS. It is preferred that said increased SVC is increased by at least 1 %, preferably at least 2%, more preferably at least 3%, more preferably at least 4%, even more preferably at least 5%, even more preferably at least 6%, even more preferably at least 7%, even more preferably at least 8%, even more preferably atleast 9%, even more preferably at least 10%, even more preferably at least 12%, even morepreferably at least 14%, even more preferably at least 16%, even more preferably at least18%, and most preferably at least 20 %; and / or at most 100%.

[0092] The skilled person is aware that SVC decreases with the progression of ALS. In someembodiment, the subject exhibits a SVC, which is reduced by at most 10 % after the treatment period and / or the drug holiday period compared to the SVC measured beforeinitiation of fasudil administration, if determined. In these embodiments, it is preferred thatthe SVC is reduced by at most 9.5 %, more preferably at most 9%, even more preferably atmost 8.5 %, even more preferably at most 8 %, even more preferably at most 7.5 %, evenmore preferably at most 7 %, even more preferably at most 6.5 %, and most preferably atmost 6 %.In some embodiments, the subject exhibits a lower number of newly affected muscles afterthe treatment period and / or the drug holiday period as compared to a subject undergoing -27-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizinplacebo treatment, wherein the number of newly affected muscles is determined by MUNIXscore, wherein a muscle is determined to be affected if the MUNIX score decreases by atleast 10% relative to the MUNIX score of the contralateral muscle or the MUNIX score ofthe same muscle prior to the treatment period. It is preferred that the MUNIX score decreases by at least 20%. It is even more preferred that the MUNIX score decreases by at least 30%. Example 2 provides data on the determination of MUNIX scores in patients of newly affected muscles. In some embodiments, the subject treated with fasudil exhibits lower number of newly affected limbs as compared to number of newly affected limbs in a subject undergoingplacebo treatment, wherein the limb is determined to be affected if at least one muscle of thatlimb is affected, wherein the number of newly affected muscles is determined by MUNIXscore, wherein a muscle is determined to be affected if the MUNIX score decreases by atleast 10% relative to the MUNIX score of the contralateral muscle or the MUNIX score ofthe same muscle prior to the treatment period. It is preferred that the MUNIX score decreasesby at least 20%. It is even more preferred that the MUNIX score decreases by at least 30%. Example 2 provides data on the determination of MUNIX scores in patients of newly affected limbs.

[0093] In some embodiments, the subject exhibits an increased muscle strength compared toa muscle strength of a subject undergoing placebo treatment, if determined. As known in theart, muscle strength deteriorates with progression of ALS disease. In some embodiments, themuscle strength is increased by at least 5%, more preferably at least 7%, even more preferably at least 10 % compared to a muscle strength of a subject undergoing placebo treatment. The skilled person knows how to determine muscle strength. For example, the skilled person can measure muscle strength by a hand grip test. The skilled person is aware of how a hand grip test is carried out. Ideally, the hand grip test is carried out using a hand-held dynamometer.

[0094] In some embodiment, the subject exhibits a decrease in the concentration of abiomarker after the treatment period and / or the drug holiday period compared to before initiation of fasudil administration, if determined. The skilled person is aware of ALS biomarkers. Exemplary biomarkers have been measured herein. Ideally the biomarker isselected from one or more of NfL, GFAP, p75ECD, creatine kinase, CHIT1, GPNMB,UCHL1, and SPP1. Preferably, the biomarker is selected from one or more of NfL, GFAP, p75ECD, and creatine kinase. The skilled person is aware how to measure these biomarkers. -28-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, UniversitätsmedizinIn general, the higher the concentration of one or more of these biomarkers, the faster theprogression of ALS. Hence, a decrease in the concentration of the biomarker indicates thatdisease progression is slowed down.

[0095] In some embodiments, the treatment period is from 3 to 6 weeks. In some preferredembodiments, the treatment period is from 4 to 6 weeks. Ideally, the treatment period is around 4 weeks.

[0096] In some embodiments, 200 mg to 600 mg of fasudil is administered per week duringthe treatment period, preferably 240 mg to 550 mg of fasudil is administered per week during the treatment period, more preferably 270 mg to 450 mg of fasudil is administered per weekduring the treatment period, more preferably 280 mg to 420 mg of fasudil is administered perweek during the treatment period, and more preferably 300 mg to 400 mg of fasudil is administered per week during the treatment period.

[0097] In some embodiments, fasudil is administered on 5, 6 or 7 days in each week of thetreatment period. In some preferred embodiments, fasudil is administered on 5 days in each week of the treatment period. In preferred some embodiments, fasudil is administered on 7 days in each week of the treatment period.

[0098] In some embodiments, fasudil is administered intravenously, orally orsubcutaneously. The skilled person is aware from the prior art that fasudil has been administered intravenously and orally. It is preferred that fasudil is administered intravenously or orally.

[0099] In some embodiments, fasudil is administered in a pharmaceutical composition duringthe treatment period, wherein the pharmaceutical composition comprises 15 to 45 mg offasudil or a pharmaceutically acceptable salt, ideally, 15 mg, 30 mg or 45 mg of fasudil or apharmaceutically acceptable salt thereof, wherein said pharmaceutical composition is administered twice a day or three times a day on 5 to 7 days in each week of the treatmentperiod. It is preferred that the pharmaceutical composition is administered twice a day on 5 to7 days in each week of the treatment period.

[0100] In some embodiments, the treatment period is followed by a drug holiday period. Theskilled person is able to determine an optimal drug holiday period. The advantage of a drug holiday period is that the subject can rest from the frequent administration of the drug. Even though fasudil was very well tolerated during the clinical trial disclosed herein, a drugholiday offers the advantage of a resting or recovering period for the subject. Said drug-29-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizinholiday period is, for example, from 2 weeks to 2 months, preferably from 3 weeks to 1.5months. Ideally, the drug holiday period is around 1 month.

[0101] In some embodiments, a fixed dose of fasudil is administered. In some embodiments,the amount of fasudil is not adjusted based on weight of the subject.

[0102] In some embodiments, a therapeutically-effective amount of a second agent is alsoadministered, preferably wherein the second agent comprises riluzole, edaravone, tofersen, nuedexta, or jacifusen. Modes of sequential or co-administration are disclosed above and apply equally to these embodiments.

[0103] In some embodiments, fasudil is administered during a treatment period, wherein thetreatment period is from 4 to 5 weeks, wherein around 300 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 5 days in each week of the treatment period. Ideally, a next treatment period starts immediately after the end of the initial treatment period. In these embodiments, fasudil is administered constantly. This has the advantage that a chronic disease is treated constantly. Based on the very positive safety and tolerability outcomes of this clinical trial, it is expected that it is safe and tolerable for patients to chronically be administered with fasudil.

[0104] In some embodiments, fasudil is administered during a treatment period, wherein thetreatment period is from 4 to 5 weeks, wherein around 450 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 5 days in each week of the treatment period. Ideally, a next treatment period starts immediately after the end of the initial treatment period.

[0105] In some embodiments, fasudil is administered during a treatment period, wherein thetreatment period is from 4 to 5 weeks, wherein around 420 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 7 days in each week of the treatment period. Ideally, a next treatment period starts immediately after the end of the initial treatment period.

[0106] In some embodiments, fasudil is administered during a treatment period, wherein thetreatment period is from 4 to 5 weeks, wherein around 630 mg of fasudil is administered perweek during the treatment period, wherein fasudil is administered on 7 days in each week ofthe treatment period. Ideally, a next treatment period starts immediately after the end of the initial treatment period.

[0107] In some embodiments, fasudil is administered during a treatment period, wherein thetreatment period is from 4 to 5 weeks, wherein around 300 mg of fasudil is administered per -30-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin week during the treatment period, wherein fasudil is administered on 5 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month.

[0108] In some embodiments, fasudil is administered during a treatment period, wherein thetreatment period is from 4 to 5 weeks, wherein around 450 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 5 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month.

[0109] In some embodiments, fasudil is administered during a treatment period, wherein thetreatment period is from 4 to 5 weeks, wherein around 420 mg of fasudil is administered perweek during the treatment period, wherein fasudil is administered on 7 days in each week ofthe treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month.

[0110] In some embodiments, fasudil is administered during a treatment period, wherein thetreatment period is from 4 to 5 weeks, wherein around 630 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 7 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month. The inventors conducted a randomized, placebo-controlled Phase 2 clinical trial (ROCK- ALS, NCT03792490) which demonstrated that fasudil, a Rho kinase inhibitor, is safe and well-tolerated in patients with amyotrophic lateral sclerosis (ALS). The results also suggestthat fasudil may exert beneficial neuroprotective effects by slowing disease progression (seeExamples 1 and 2). Specifically, treatment with fasudil was associated with a reduced loss of motor units, as measured by the MUNIX score, and a lower number of newly affected muscles and limbs compared to the placebo group. These findings represent the first clinical evidence supporting fasudil as a treatment for ALS.

[0111] The invention is further described in the following embodiments.EMBODIMENTS

[0112] Embodiment 1. A method for treating amyotrophic lateral sclerosis (ALS) in a subjectin need thereof, wherein the method comprises: a) evaluating the subject for motor neuron loss at a first timepoint; -31-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for motor neuron loss at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits an at least 5% decrease in motor neuron loss at the second timepoint as compared to the first timepoint.

[0113] Embodiment 2. The method of embodiment 1, wherein the administering occurs for atmost 26 days.

[0114] Embodiment 3. The method of embodiment 1, wherein the administering occurs for atmost 21 days.

[0115] Embodiment 4. The method of embodiment 1, wherein the administering occurs for atmost 14 days.

[0116] Embodiment 5. The method of any one of embodiments 1-4, wherein the evaluatingthe subject for motor neuron loss at the first timepoint comprises using motor unit numberindex (MUNIX) and the evaluating the subject for motor neuron loss at the second timepointcomprises using motor unit number index (MUNIX).

[0117] Embodiment 6. The method of any one of embodiments 1-4, wherein the evaluatingthe subject for motor neuron loss at the first timepoint comprises using slow vital capacity(SVC) and the evaluating the subject for motor neuron loss at the second timepoint comprises using slow vital capacity (SVC).

[0118] Embodiment 7. The method of any one of embodiments 1-6, wherein the secondtimepoint occurs at least 100 days after step (b).

[0119] Embodiment 8. The method of any one of embodiments 1-6, wherein the secondtimepoint occurs at least 150 days after step (b).

[0120] Embodiment 9. The method of any one of embodiments 1-8, wherein the evaluatingthe subject for motor neuron loss at the second timepoint identifies an at least 10% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0121] Embodiment 10. The method of any one of embodiments 1-8, wherein the evaluatingthe subject for motor neuron loss at the second timepoint identifies an at least 20% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint. -32-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0122] Embodiment 11. The method of any one of embodiments 1-8, wherein the evaluatingthe subject for motor neuron loss at the second timepoint identifies an at least 30% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0123] Embodiment 12. The method of any one of embodiments 1-11, wherein the subject isa human.

[0124] Embodiment 13. The method of any one of embodiments 1-12, further comprisingadministering a therapeutically-effective amount of a second agent.

[0125] Embodiment 14. The method of embodiment 13, wherein the second agent comprisesriluzole.

[0126] Embodiment 15. The method of embodiment 13, wherein the second agent comprisesedaravone.

[0127] Embodiment 16. The method of embodiment 13, wherein the second agent comprisestofersen.

[0128] Embodiment 17. The method of embodiment 13, wherein the second agent comprisesnuedexta.

[0129] Embodiment 18. The method of embodiment 13, wherein the second agent comprisesjacifusen.

[0130] Embodiment 19. The method of embodiment 13, wherein the second agentcomprises sodium phenylbutyrate.

[0131] Embodiment 20. The method of embodiment 13, wherein the second agent comprisestaurursodiol.

[0132] Embodiment 21. The method of any one of embodiments 1-20, further comprisingevaluating the subject for a biomarker.

[0133] Embodiment 22. The method of embodiment 21, wherein the evaluating of thebiomarker occurs before, during, or after step b).

[0134] Embodiment 23. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron loss at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and -33-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin c) administering to the subject a pharmaceutical composition wherein thepharmaceutical composition comprises a therapeutically-effective amount of a second agent; and d) evaluating the subject for motor neuron loss at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits an at least 5% decrease in motor neuron loss at the second timepoint as compared to the first timepoint.

[0135] Embodiment 24. The method of embodiment 23, wherein the administering of thesecond agent occurs prior to the administering of fasudil.

[0136] Embodiment 25. The method of embodiment 23, wherein the administering of thesecond agent occurs after the administering of fasudil.

[0137] Embodiment 26. The method of embodiment 23, wherein the administering of thesecond agent occurs concurrently with the administering of fasudil.

[0138] Embodiment 27. The method of any one of embodiments 23-26, wherein theadministering of b) occurs for at most 26 days.

[0139] Embodiment 28. The method of any one of embodiments 23-26, wherein theadministering of b) occurs for at most 21 days.

[0140] Embodiment 29. The method of any one of embodiments embodiment 23-26, whereinthe administering of b) occurs for at most 14 days.

[0141] Embodiment 30. The method of any one of embodiments 23-29, wherein theevaluating the subject for motor neuron loss at the first timepoint comprises using motor unitnumber index (MUNIX) and the evaluating the subject for motor neuron loss at the second timepoint comprises using motor unit number index (MUNIX).

[0142] Embodiment 31. The method of any one of embodiments 23-29, wherein theevaluating the subject for motor neuron loss at the first timepoint comprises using slow vitalcapacity (SVC) and the evaluating the subject for motor neuron loss at the second timepoint comprises using slow vital capacity (SVC).

[0143] Embodiment 32. The method of any one of embodiments 23-31, wherein the secondtimepoint occurs at least 100 days after step (b).

[0144] Embodiment 33. The method of any one of embodiments 23-31, wherein the secondtimepoint occurs at least 150 days after step (b).

[0145] Embodiment 34. The method of any one of embodiments 23-33, wherein theevaluating the subject for motor neuron loss at the second timepoint identifies an at least 10%-34-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0146] Embodiment 35. The method of any one of embodiments 23-33, wherein theevaluating the subject for motor neuron loss at the second timepoint identifies an at least 20% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0147] Embodiment 36. The method of any one of embodiments 23-33, wherein theevaluating the subject for motor neuron loss at the second timepoint identifies an at least 30% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0148] Embodiment 37. The method of any one of embodiments 23-36, wherein the subjectis a human.

[0149] Embodiment 38. The method of any one of embodiments 23-37, wherein the secondagent comprises riluzole.

[0150] Embodiment 39. The method of any one of embodiments 23-37, wherein the secondagent comprises edaravone.

[0151] Embodiment 40. The method of any one of embodiments 23-37, wherein the secondagent comprises tofersen.

[0152] Embodiment 41. The method of any one of embodiments 23-37, wherein the secondagent comprises nuedexta.

[0153] Embodiment 42. The method of any one of embodiments 23-37, wherein the secondagent comprises jacifusen.

[0154] Embodiment 43. The method of any one of embodiments 23-37, wherein the secondagent comprises sodium phenylbutyrate.

[0155] Embodiment 44. The method of any one of embodiments 23-37, wherein the secondagent comprises taurursodiol.

[0156] Embodiment 45. The method of any one of embodiments 23-44, further comprisingevaluating the subject for a biomarker.

[0157] Embodiment 46. The method of embodiment 45, wherein the evaluating of thebiomarker occurs before, during, or after step b).

[0158] Embodiment 47. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for a biomarker at a first timepoint; -35-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for the biomarker at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits a change of at least 5% in the biomarker at the second timepoint as compared to the first timepoint.

[0159] Embodiment 48. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for a biomarker at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) administering to the subject a pharmaceutical composition, wherein thepharmaceutical composition comprises a therapeutically-effective amount of a second agent; d) evaluating the subject for the biomarker at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits a change of at least 5% in the biomarker at the second timepoint as compared to the first timepoint.

[0160] Embodiment 49. The method of embodiment 47 or 48, wherein the biomarkercomprises neurofilament light chain (NfL).

[0161] Embodiment 50. The method of embodiment 47 or 48, wherein the biomarkercomprises glial fibrillary acidic protein (GFAP).

[0162] Embodiment 51. The method of embodiment 47 or 48, wherein the biomarkercomprises urate.

[0163] Embodiment 52. The method of embodiment 47 or 48, wherein the biomarkercomprises p75 extracellular region.

[0164] Embodiment 53. The method of embodiment 47 or 48, wherein the biomarkercomprises chromogranin A.

[0165] Embodiment 54. The method of embodiment 47 or 48, wherein the biomarkercomprises CHIT1. -36-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0166] Embodiment 55. The method of embodiment 47 or 48, wherein the biomarkercomprises UCHL1.

[0167] Embodiment 56. The method of embodiment 47 or 48, wherein the biomarkercomprises GPNMB.

[0168] Embodiment 57. The method of embodiment 47 or 48, wherein the biomarkercomprises SPP1.

[0169] Embodiment 58. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron loss at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for motor neuron loss at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits an at least 5% decrease in motor neuron loss at the second timepoint as compared to the first timepoint.

[0170] Embodiment 59. The method of embodiment 58, wherein the administering occurs forat most 26 days.

[0171] Embodiment 60. The method of embodiment 58, wherein the administering occurs forat most 21 days.

[0172] Embodiment 61. The method of embodiment 58, wherein the administering occurs forat most 14 days.

[0173] Embodiment 62. The method of any one of embodiments 58-61, wherein theevaluating the subject for motor neuron loss at the first timepoint comprises using motor unit number index (MUNIX) and the evaluating the subject for motor neuron loss at the second timepoint comprises using motor unit number index (MUNIX).

[0174] Embodiment 63. The method of any one of embodiments 58-61, wherein theevaluating the subject for motor neuron loss at the first timepoint comprises using slow vital capacity (SVC) and the evaluating the subject for motor neuron loss at the second timepoint comprises using slow vital capacity (SVC).

[0175] Embodiment 64. The method of any one of embodiments 58-63, wherein the secondtimepoint occurs at least 100 days after step (b). -37-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0176] Embodiment 65. The method of any one of embodiments 58-63, wherein the secondtimepoint occurs at least 150 days after step (b).

[0177] Embodiment 66. The method of any one of embodiments 58-65, wherein theevaluating the subject for motor neuron loss at the second timepoint identifies an at least 10% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0178] Embodiment 67. The method of any one of embodiments 58-65, wherein theevaluating the subject for motor neuron loss at the second timepoint identifies an at least 20% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0179] Embodiment 68. The method of any one of embodiments 58-65, wherein theevaluating the subject for motor neuron loss at the second timepoint identifies an at least 30% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0180] Embodiment 69. The method of any one of embodiments 58-68, wherein the subjectis a human.

[0181] Embodiment 70. The method of any one of embodiments 58-69, further comprisingadministering a therapeutically-effective amount of a second agent.

[0182] Embodiment 71. The method of embodiment 70, wherein the second agent comprisesriluzole.

[0183] Embodiment 72. The method of embodiment 70, wherein the second agent comprisesedaravone.

[0184] Embodiment 73. The method of embodiment 70, wherein the second agent comprisestofersen.

[0185] Embodiment 74. The method of embodiment 70, wherein the second agent comprisesnuedexta.

[0186] Embodiment 75. The method of embodiment 70, wherein the second agent comprisesjacifusen.

[0187] Embodiment 76. The method of embodiment 70, wherein the second agent comprisessodium phenylbutyrate.

[0188] Embodiment 77. The method of embodiment 70, wherein the second agent comprisestaurursodiol. -38-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0189] Embodiment 78. The method of any one of embodiments 58-77, further comprisingevaluating the subject for a biomarker.

[0190] Embodiment 79. The method of embodiment 78, wherein the evaluating of thebiomarker occurs before, during, or after step b).

[0191] Embodiment 80. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron loss at a first timepoint; b) administering to the subject a pharmaceutical composition wherein thepharmaceutical composition comprises 30 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) administering to the subject a pharmaceutical composition wherein thepharmaceutical composition comprises a therapeutically-effective amount of a second agent; and d) evaluating the subject for motor neuron loss at a second timepoint, wherein thesecond timepoint occurs at least 50 days after step b), and wherein the subject exhibits an at least 5% decrease in motor neuron loss at the second timepoint as compared to the first timepoint.

[0192] Embodiment 81. The method of embodiment 80, wherein the administering of thesecond agent occurs prior to the administering of fasudil.

[0193] Embodiment 82. The method of embodiment 80, wherein the administering of thesecond agent occurs after the administering of fasudil.

[0194] Embodiment 83. The method of embodiment 80, wherein the administering of thesecond agent occurs concurrently with the administering of fasudil.

[0195] Embodiment 84. The method of any one of embodiments 80-83, wherein theadministering of fasudil occurs for at most 26 days.

[0196] Embodiment 85. The method of any one of embodiments 80-83, wherein theadministering of fasudil occurs for at most 21 days.

[0197] Embodiment 86. The method of any one of embodiments embodiment 80-83, whereinthe administering of fasudil occurs for at most 14 days.

[0198] Embodiment 87. The method of any one of embodiments 80-86, wherein theevaluating the subject for motor neuron loss at the first timepoint comprises using motor unitnumber index (MUNIX) and the evaluating the subject for motor neuron loss at the secondtimepoint comprises using motor unit number index (MUNIX). -39-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0199] Embodiment 88. The method of any one of embodiments 80-86, wherein theevaluating the subject for motor neuron loss at the first timepoint comprises using slow vital capacity (SVC) and the evaluating the subject for motor neuron loss at the second timepoint comprises using slow vital capacity (SVC).

[0200] Embodiment 89. The method of any one of embodiments 80-88, wherein the secondtimepoint occurs at least 100 days after step (b).

[0201] Embodiment 90. The method of any one of embodiments 80-88, wherein the secondtimepoint occurs at least 150 days after step (b).

[0202] Embodiment 91. The method of any one of embodiments 80-90, wherein theevaluating the subject for motor neuron loss at the second timepoint identifies an at least 10% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0203] Embodiment 92. The method of any one of embodiments 80-90, wherein theevaluating the subject for motor neuron loss at the second timepoint identifies an at least 20% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0204] Embodiment 93. The method of any one of embodiments 80-90, wherein theevaluating the subject for motor neuron loss at the second timepoint identifies an at least 30% decrease in motor neuron loss as compared to the evaluating the subject for motor neuron loss at the first timepoint.

[0205] Embodiment 94. The method of any one of embodiments 80-93, wherein the subjectis a human.

[0206] Embodiment 95. The method of any one of embodiments 80-94, wherein the secondagent comprises riluzole.

[0207] Embodiment 96. The method of any one of embodiments 80-94, wherein the secondagent comprises edaravone.

[0208] Embodiment 97. The method of any one of embodiments 80-94, wherein the secondagent comprises tofersen.

[0209] Embodiment 98. The method of any one of embodiments 80-94, wherein the secondagent comprises nuedexta.

[0210] Embodiment 99. The method of any one of embodiments 80-94, wherein the secondagent comprises jacifusen. -40-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0211] Embodiment 100. The method of any one of embodiments 80-94, wherein the secondagent comprises sodium phenylbutyrate.

[0212] Embodiment 101. The method of any one of embodiments 80-94, wherein the secondagent comprises taurursodiol.

[0213] Embodiment 102. The method of any one of embodiments 80-101, further comprisingevaluating the subject for a biomarker.

[0214] Embodiment 103. The method of embodiment 102, wherein the evaluating of thebiomarker occurs before, during, or after step b).

[0215] Embodiment 104. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for a biomarker at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceuticallyacceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for the biomarker at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits a change of at least 5% in the biomarker at the second timepoint as compared to the first timepoint.

[0216] Embodiment 105. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for a biomarker at a first timepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceuticallyacceptable salt thereof, wherein the administering occurs twice a day; and c) administering to the subject a pharmaceutical composition, wherein thepharmaceutical composition comprises a therapeutically-effective amount of asecond agent; d) evaluating the subject for the biomarker at a second timepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits a change of at least 5% in the biomarker at the second timepoint as compared to the first timepoint.

[0217] Embodiment 106. The method of embodiment 104 or 105, wherein the biomarkercomprises neurofilament light chain (NfL). -41-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0218] Embodiment 107. The method of embodiment 104 or 105, wherein the biomarkercomprises glial fibrillary acidic protein (GFAP).

[0219] Embodiment 108. The method of embodiment 104 or 105, wherein the biomarkercomprises urate.

[0220] Embodiment 109. The method of embodiment 104 or 105, wherein the biomarkercomprises p75 extracellular region.

[0221] Embodiment 110. The method of embodiment 104 or 105, wherein the biomarkercomprises chromogranin A.

[0222] Embodiment 111. The method of embodiment 104 or 105, wherein the biomarkercomprises CHIT1.

[0223] Embodiment 112. The method of embodiment 104 or 105, wherein the biomarkercomprises UCHL1.

[0224] Embodiment 113. The method of embodiment 104 or 105, wherein the biomarkercomprises GPNMB.

[0225] Embodiment 114. The method of embodiment 104 or 105, wherein the biomarkercomprises SPP1.

[0226] Embodiment 115. Fasudil for use in a method of treating ALS in a subject, whereinfasudil is administered during a treatment period, wherein the treatment period is from 2 to 6 weeks, wherein 150 mg to 630 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 5 to 7 days in each week of the treatment period, optionally followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is from 1 week to 3 months.

[0227] Embodiment 116. Fasudil for the use according to embodiment 115, wherein thesubject exhibits an increased MUNIX score after the treatment period and / or the drug holiday period as compared to a MUNIX score of a subject undergoing placebo treatment, preferably wherein the MUNIX score is increased by at least 3 %, more preferably at least 4%, morepreferably at least 5%, more preferably at least 6%, more preferably at least 7%, morepreferably at least 8%, more preferably at least 9%, more preferably at least 10%, morepreferably at least 12%, more preferably at least 14%, more preferably at least 16%, morepreferably at least 18%, and even more preferably at least 20%; and / or at most 100%.

[0228] Embodiment 117. Fasudil for the use according to embodiments 115 or 116, whereinthe subject exhibits an at most 20% decrease in MUNIX score after the treatment periodand / or the drug holiday period as compared to before initiation of fasudil administration, -42-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin preferably an at most 18% decrease, more preferably an at most 17% decrease, morepreferably an at most 16% decrease, more preferably an at most 15% decrease, morepreferably an at most 14% decrease, more preferably an at most 13% decrease, morepreferably an at most 12% decrease, more preferably an at most 11% decrease, morepreferably an at most 10% decrease, more preferably an at most 8% decrease, morepreferably an at most 6% decrease, more preferably an at most 4% decrease and most preferably an at most 2% decrease.

[0229] Embodiment 118. Fasudil for use according to any of embodiment 115-117, whereinthe subject exhibits a lower number of newly affected muscles after the treatment period and / or the drug holiday period as compared to a subject undergoing placebo treatment, wherein the number of newly affected muscles is determined by MUNIX score, wherein a muscle is determined to be affected if the MUNIX score decreases by at least 10%, preferably by at least 20%, and more preferably by at least 30% relative to the MUNIX score of thecontralateral muscle or the MUNIX score of the same muscle prior to the treatment period.

[0230] Embodiment 119. Fasudil for use according to embodiment 118, wherein the subjecttreated with fasudil exhibits lower number of newly affected limbs as compared to number of newly affected limbs in a subject undergoing placebo treatment, wherein the limb isdetermined to be affected if at least one muscle of that limb is affected,wherein the number of newly affected muscles is determined by MUNIX score, wherein a muscle is determined to be affected if the MUNIX score decreases by at least 10%, preferablyby at least 20%, and more preferably by at least 30% relative to the MUNIX score of thecontralateral muscle or the MUNIX score of the same muscle prior to the treatment period.

[0231] Embodiment 120. Fasudil for the use according to any of embodiments 115 or 117,wherein the subject exhibits an increased SVC after the treatment period and / or the drug holiday period compared to an SVC of a subject undergoing placebo treatment, preferably wherein said increased SVC is increased by at least 1 %, preferably at least 2%, morepreferably at least 3%, more preferably at least 4%, even more preferably at least 5%, evenmore preferably at least 6%, even more preferably at least 7%, even more preferably at least8%, even more preferably at least 9%, even more preferably at least 10%, even morepreferably at least 12%, even more preferably at least 14%, even more preferably at least16%, even more preferably at least 18%, and most preferably at least 20 %; and / or at most100%. -43-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0232] Embodiment 121. Fasudil for the use according to any one of embodiments 115 to120, wherein the subject exhibits a SVC, which is reduced by at most 10 % after the treatment period and / or the drug holiday period compared to the SVC measured before initiation of fasudil administration, preferably at most 9.5 %, more preferably at most 9%,even more preferably at most 8.5 %, even more preferably at most 8 %, even more preferablyat most 7.5 %, even more preferably at most 7 %, even more preferably at most 6.5 %, andmost preferably at most 6 %.

[0233] Embodiment 122. Fasudil for the use according to any one of embodiments 115 to121, wherein the subject exhibits an increased muscle strength compared to a muscle strengthof a subject undergoing placebo treatment, preferably wherein the muscle strength is increased by at least 5%, more preferably at least 7%, even more preferably at least 10 %, more preferably wherein muscle strength is measured by a hand grip test, more preferably wherein the hand grip test is carried out using a hand-held dynamometer.

[0234] Embodiment 123. Fasudil for the use according to any one of embodiments 115 to122, wherein the subject exhibits a decrease in the concentration of a biomarker after the treatment period and / or the drug holiday period compared to before initiation of fasudil administration, preferably wherein the biomarker is selected from one or more of NfL, GFAP, p75ECD, and creatine kinase.

[0235] Embodiment 124. Fasudil for the use according to any one of embodiments 115 to123, wherein the treatment period is from 3 to 6 weeks, more preferably wherein thetreatment period is from 4 to 6 weeks, and even more preferably wherein the treatment periodis around 4 weeks.

[0236] Embodiment 125. Fasudil for the use according to any one of embodiments 115 to124, wherein 200 mg to 600 mg of fasudil is administered per week during the treatment period, preferably wherein 240 mg to 520 mg of fasudil is administered per week during the treatment period, more preferably wherein 270 mg to 450 mg of fasudil is administered perweek during the treatment period, more preferably wherein 280 mg to 420 mg of fasudil isadministered per week during the treatment period, and more preferably wherein 300 mg to 400 mg of fasudil is administered per week during the treatment period.

[0237] Embodiment 126. Fasudil for the use according to any one of embodiments 115 to125, wherein fasudil is administered on 5, 6 or 7 days in each week of the treatment period

[0238] Embodiment 127. Fasudil for the use according to any one of embodiments 115 to126, wherein fasudil is administered on 5 days in each week of the treatment period. -44-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0239] Embodiment 128. Fasudil for the use according to any one of embodiments 115 to126, wherein fasudil is administered on 7 days in each week of the treatment period.

[0240] Embodiment 129. Fasudil for the use according to any one of embodiments 115 to128, wherein fasudil is administered intravenously, orally or subcutaneously, preferably wherein fasudil is administered intravenously or orally.

[0241] Embodiment 130. Fasudil for the use according to any one of embodiments 115 to129, wherein fasudil is administered in a pharmaceutical composition during the treatment period, wherein the pharmaceutical composition comprises 15 mg, 30 mg or 45 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein said pharmaceutical composition is administered twice a day or three times a day on 5 to 7 days in each week of the treatment period, preferably twice a day on 5 to 7 days in each week of the treatment period.

[0242] Embodiment 131. Fasudil for the use according to any one of embodiments 115 to130, wherein the drug holiday period is from 2 week to 2 months, preferably from 3 weeks to 1.5 months, and more preferably around 1 month.

[0243] Embodiment 132. Fasudil for the use according to any one of embodiments 115 to131, wherein a fixed dose of fasudil is administered.

[0244] Embodiment 133. Fasudil for the use according to any one of embodiments 115 to133, wherein a therapeutically-effective amount of a second agent is also administered,preferably wherein the second agent comprises riluzole, edaravone, tofersen, nuedexta, or jacifusen.

[0245] Embodiment 134. Fasudil for the use according to any one of embodiments 115 to133, wherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 300 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 5 days in each week of the treatment period, preferably wherein the next treatment period starts immediately after the end of the last treatment period.

[0246] Embodiment 135. Fasudil for the use according to any one of embodiments 115 to133, wherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 450 mg of fasudil is administered per week during thetreatment period, wherein fasudil is administered on 5 days in each week of the treatmentperiod, preferably wherein the next treatment period starts immediately after the end of the last treatment period. -45-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0247] Embodiment 136. Fasudil for the use according to any one of embodiments 115 to133, wherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 420 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 7 days in each week of the treatment period, preferably wherein the next treatment period starts immediately after the end of the last treatment period.

[0248] Embodiment 137. Fasudil for the use according to any one of embodiments 115 to133, wherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 630 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 7 days in each week of the treatment period, preferably wherein the next treatment period starts immediately after the end of the last treatment period.

[0249] Embodiment 138. Fasudil for the use according to any of embodiments 115 to 133,wherein fasudil is administered during a treatment period, wherein the treatment period isfrom 4 to 5 weeks, wherein around 300 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 5 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month.

[0250] Embodiment 139. Fasudil for the use according to any of embodiments 115 to 133,wherein fasudil is administered during a treatment period, wherein the treatment period isfrom 4 to 5 weeks, wherein around 450 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 5 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month.

[0251] Embodiment 140. Fasudil for the use according to any of embodiments 115 to 133,wherein fasudil is administered during a treatment period, wherein the treatment period isfrom 4 to 5 weeks, wherein around 420 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 7 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month.

[0252] Embodiment 141. Fasudil for the use according to any of embodiments 115 to 133,wherein fasudil is administered during a treatment period, wherein the treatment period isfrom 4 to 5 weeks, wherein around 630 mg of fasudil is administered per week during the -46-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin treatment period, wherein fasudil is administered on 7 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month.

[0253] Embodiment 142. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron dysfunction at a first timepoint;b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for motor neuron dysfunction at a second timepoint,wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits an at most 20% decrease in motor neuron dysfunction atthe second timepoint as compared to the first timepoint.

[0254] Embodiment 143. The method of embodiment 142, wherein the administering occursfor at most 26 days.

[0255] Embodiment 144. The method of embodiment 142, wherein the administering occursfor at most 21 days.

[0256] Embodiment 145. The method of embodiment 142, wherein the administering occursfor at most 14 days.

[0257] Embodiment 146. The method of any one of embodiments 142-145, wherein theevaluating the subject for motor neuron dysfunction at the first timepoint comprises usingmotor unit number index (MUNIX) and the evaluating the subject for motor neurondysfunction at the second timepoint comprises using motor unit number index (MUNIX).

[0258] Embodiment 147. The method of any one of embodiments 142-145, wherein theevaluating the subject for motor neuron dysfunction at the first timepoint comprises usingslow vital capacity (SVC) and the evaluating the subject for motor neuron dysfunction at thesecond timepoint comprises using slow vital capacity (SVC).

[0259] Embodiment 148. The method of any one of embodiments 142-147, wherein thesecond timepoint occurs at least 100 days after step (b).

[0260] Embodiment 149. The method of any one of embodiments 142-147, wherein thesecond timepoint occurs at least 150 days after step (b).

[0261] Embodiment 150. The method of any one of embodiments 142-149, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an at-47-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizinmost 15% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0262] Embodiment 151. The method of any one of embodiments 142-149, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 10% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0263] Embodiment 152. The method of any one of embodiments 142-149, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 5% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0264] Embodiment 153. The method of any one of embodiments 142-152, wherein thesubject is a human.

[0265] Embodiment 154. The method of any one of embodiments 142-153, furthercomprising administering a therapeutically-effective amount of a second agent.

[0266] Embodiment 155. The method of embodiment 154, wherein the second agentcomprises riluzole.

[0267] Embodiment 156. The method of embodiment 154, wherein the second agentcomprises edaravone.

[0268] Embodiment 157. The method of embodiment 154, wherein the second agentcomprises tofersen.

[0269] Embodiment 158. The method of embodiment 154, wherein the second agentcomprises nuedexta.

[0270] Embodiment 159. The method of embodiment 154, wherein the second agentcomprises jacifusen.

[0271] Embodiment 160. The method of embodiment 154, wherein the second agentcomprises sodium phenylbutyrate.

[0272] Embodiment 161. The method of embodiment 154, wherein the second agentcomprises taurursodiol.

[0273] Embodiment 162. The method of any one of embodiments 142-161, furthercomprising evaluating the subject for a biomarker.

[0274] Embodiment 163. The method of embodiment 162, wherein the evaluating of thebiomarker occurs before, during, or after step b). -48-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0275] Embodiment 164. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron dysfunction at a first timepoint;b) administering to the subject a pharmaceutical composition wherein thepharmaceutical composition comprises 15 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein the administering occurs twice a day; and c) administering to the subject a pharmaceutical composition wherein thepharmaceutical composition comprises a therapeutically-effective amount of a second agent; and d) evaluating the subject for motor neuron dysfunction at a second timepoint,wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits an at least 5% decrease in motor neuron dysfunction at thesecond timepoint as compared to the first timepoint.

[0276] Embodiment 165. The method of embodiment 164, wherein the administering of thesecond agent occurs prior to the administering of fasudil.

[0277] Embodiment 166. The method of embodiment 164, wherein the administering of thesecond agent occurs after the administering of fasudil.

[0278] Embodiment 167. The method of embodiment 164, wherein the administering of thesecond agent occurs concurrently with the administering of fasudil.

[0279] Embodiment 168. The method of any one of embodiments 164-167, wherein theadministering of b) occurs for at most 26 days.

[0280] Embodiment 169. The method of any one of embodiments 164-167, wherein theadministering of b) occurs for at most 21 days.

[0281] Embodiment 170. The method of any one of embodiments embodiment 164-167,wherein the administering of b) occurs for at most 14 days.

[0282] Embodiment 171. The method of any one of embodiments 164-170, wherein theevaluating the subject for motor neuron dysfunction at the first timepoint comprises usingmotor unit number index (MUNIX) and the evaluating the subject for motor neurondysfunction at the second timepoint comprises using motor unit number index (MUNIX).

[0283] Embodiment 172. The method of any one of embodiments 164-170, wherein theevaluating the subject for motor neuron dysfunction at the first timepoint comprises usingslow vital capacity (SVC) and the evaluating the subject for motor neuron dysfunction at thesecond timepoint comprises using slow vital capacity (SVC). -49-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0284] Embodiment 173. The method of any one of embodiments 164-172, wherein thesecond timepoint occurs at least 100 days after step (b).

[0285] Embodiment 174. The method of any one of embodiments 164-172, wherein thesecond timepoint occurs at least 150 days after step (b).

[0286] Embodiment 175. The method of any one of embodiments 164-174, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 15% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0287] Embodiment 176. The method of any one of embodiments 164-174, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 10% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0288] Embodiment 177. The method of any one of embodiments 164-174, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 5% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0289] Embodiment 178. The method of any one of embodiments 164-177, wherein thesubject is a human.

[0290] Embodiment 179. The method of any one of embodiments 164-178, wherein thesecond agent comprises riluzole.

[0291] Embodiment 180. The method of any one of embodiments 164-179, wherein thesecond agent comprises edaravone.

[0292] Embodiment 181. The method of any one of embodiments 164-179, wherein thesecond agent comprises tofersen.

[0293] Embodiment 182. The method of any one of embodiments 164-179, wherein thesecond agent comprises nuedexta.

[0294] Embodiment 183. The method of any one of embodiments 164-179, wherein thesecond agent comprises jacifusen.

[0295] Embodiment 184. The method of any one of embodiments 164-179, wherein thesecond agent comprises sodium phenylbutyrate.

[0296] Embodiment 185. The method of any one of embodiments 164-179, wherein thesecond agent comprises taurursodiol. -50-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0297] Embodiment 186. The method of any one of embodiments 164-185, furthercomprising evaluating the subject for a biomarker.

[0298] Embodiment 187. The method of embodiment 186, wherein the evaluating of thebiomarker occurs before, during, or after step b).

[0299] Embodiment 188. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron dysfunction at a first timepoint;b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceuticallyacceptable salt thereof, wherein the administering occurs twice a day; and c) evaluating the subject for motor neuron dysfunction at a second timepoint,wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits an at most 20% decrease in motor neuron dysfunction atthe second timepoint as compared to the first timepoint.

[0300] Embodiment 189. The method of embodiment 188, wherein the administeringoccurs for at most 26 days.

[0301] Embodiment 190. The method of embodiment 188, wherein the administeringoccurs for at most 21 days.

[0302] Embodiment 191. The method of embodiment 188, wherein the administeringoccurs for at most 14 days.

[0303] Embodiment 192. The method of any one of embodiments 188-191, wherein theevaluating the subject for motor neuron dysfunction at the first timepoint comprises usingmotor unit number index (MUNIX) and the evaluating the subject for motor neurondysfunction at the second timepoint comprises using motor unit number index (MUNIX).

[0304] Embodiment 193. The method of any one of embodiments 188-191, wherein theevaluating the subject for motor neuron dysfunction at the first timepoint comprises usingslow vital capacity (SVC) and the evaluating the subject for motor neuron dysfunction at thesecond timepoint comprises using slow vital capacity (SVC).

[0305] Embodiment 194. The method of any one of embodiments 188-193, wherein thesecond timepoint occurs at least 100 days after step (b).

[0306] Embodiment 195. The method of any one of embodiments 188-193, wherein thesecond timepoint occurs at least 150 days after step (b). -51-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0307] Embodiment 196. The method of any one of embodiments 188-195, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 15% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0308] Embodiment 197. The method of any one of embodiments 188-195, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 10% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0309] Embodiment 198. The method of any one of embodiments 188-195, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 5% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0310] Embodiment 199. The method of any one of embodiments 188-198, wherein thesubject is a human.

[0311] Embodiment 200. The method of any one of embodiments 188-199, furthercomprising administering a therapeutically-effective amount of a second agent.

[0312] Embodiment 201. The method of embodiment 200, wherein the second agentcomprises riluzole.

[0313] Embodiment 202. The method of embodiment 200, wherein the second agentcomprises edaravone.

[0314] Embodiment 203. The method of embodiment 200, wherein the second agentcomprises tofersen.

[0315] Embodiment 204. The method of embodiment 200, wherein the second agentcomprises nuedexta.

[0316] Embodiment 205. The method of embodiment 200, wherein the second agentcomprises jacifusen.

[0317] Embodiment 206. The method of embodiment 200, wherein the second agentcomprises sodium phenylbutyrate.

[0318] Embodiment 207. The method of embodiment 200, wherein the second agentcomprises taurursodiol.

[0319] Embodiment 208. The method of any one of embodiments 188-207, furthercomprising evaluating the subject for a biomarker. -52-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0320] Embodiment 209. The method of embodiment 208, wherein the evaluating of thebiomarker occurs before, during, or after step b).

[0321] Embodiment 210. A method for treating amyotrophic lateral sclerosis (ALS) in asubject in need thereof, wherein the method comprises: a) evaluating the subject for motor neuron dysfunction at a firsttimepoint; b) administering to the subject a pharmaceutical composition wherein the pharmaceutical composition comprises 30 mg of fasudil or a pharmaceuticallyacceptable salt thereof, wherein the administering occurs twice a day; and c) administering to the subject a pharmaceutical composition wherein thepharmaceutical composition comprises a therapeutically-effective amount of a second agent; and d) evaluating the subject for motor neuron dysfunction at a secondtimepoint, wherein the second timepoint occurs at least 50 days after step b), and wherein the subject exhibits an at most 20% decrease in motor neurondysfunction at the second timepoint as compared to the first timepoint.

[0322] Embodiment 211. The method of embodiment 210, wherein the administering ofthe second agent occurs prior to the administering of fasudil.

[0323] Embodiment 212. The method of embodiment 210, wherein the administering ofthe second agent occurs after the administering of fasudil.

[0324] Embodiment 213. The method of embodiment 210, wherein the administering ofthe second agent occurs concurrently with the administering of fasudil.

[0325] Embodiment 214. The method of any one of embodiments 210-213, wherein theadministering of b) occurs for at most 26 days.

[0326] Embodiment 215. The method of any one of embodiments 210-213, wherein theadministering of b) occurs for at most 21 days.

[0327] Embodiment 216. The method of any one of embodiments embodiment 210-213,wherein the administering of b) occurs for at most 14 days.

[0328] Embodiment 217. The method of any one of embodiments 210-216, wherein theevaluating the subject for motor neuron dysfunction at the first timepoint comprises usingmotor unit number index (MUNIX) and the evaluating the subject for motor neurondysfunction at the second timepoint comprises using motor unit number index (MUNIX).-53-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0329] Embodiment 218. The method of any one of embodiments 210-216, wherein theevaluating the subject for motor neuron dysfunction at the first timepoint comprises usingslow vital capacity (SVC) and the evaluating the subject for motor neuron dysfunction at thesecond timepoint comprises using slow vital capacity (SVC).

[0330] Embodiment 219. The method of any one of embodiments 210-218, wherein thesecond timepoint occurs at least 100 days after step (b).

[0331] Embodiment 220. The method of any one of embodiments 210-218, wherein thesecond timepoint occurs at least 150 days after step (b).

[0332] Embodiment 221. The method of any one of embodiments 210-220, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 15% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0333] Embodiment 222. The method of any one of embodiments 210-220, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 10% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0334] Embodiment 223. The method of any one of embodiments 210-220, wherein theevaluating the subject for motor neuron dysfunction at the second timepoint identifies an atmost 5% decrease in motor neuron dysfunction as compared to the evaluating the subject formotor neuron dysfunction at the first timepoint.

[0335] Embodiment 224. The method of any one of embodiments 210-223, wherein thesubject is a human.

[0336] Embodiment 225. The method of any one of embodiments 210-224, wherein thesecond agent comprises riluzole.

[0337] Embodiment 226. The method of any one of embodiments 210-224, wherein thesecond agent comprises edaravone.

[0338] Embodiment 227. The method of any one of embodiments 210-224, wherein thesecond agent comprises tofersen

[0339] Embodiment 228. The method of any one of embodiments 210-224, wherein thesecond agent comprises nuedexta.

[0340] Embodiment 229. The method of any one of embodiments 210-224, wherein thesecond agent comprises jacifusen. -54-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0341] Embodiment 230. The method of any one of embodiments 210-224, wherein thesecond agent comprises sodium phenylbutyrate.

[0342] Embodiment 231. The method of any one of embodiments 210-224, wherein thesecond agent comprises taurursodiol.

[0343] Embodiment 232. The method of any one of embodiments 210-231, furthercomprising evaluating the subject for a biomarker.Embodiment 233. The method of embodiment 232, wherein the evaluating of thebiomarker occurs before, during, or after step b). EXAMPLES EXAMPLE 1: Treatment of ALS using Fasudil

[0344] This randomized, double-blind, placebo-controlled, phase 2 clinical trial wasconducted at 19 sites. Adult patients with at least probable ALS (revised El Escorial criteria), a disease duration of 6-24 months, and a slow vital capacity (SVC) of >65% of normal were included. Subjects were randomly assigned (in a 1:1:1 ratio) to receive 30 mg (15 mg twicedaily) fasudil, 60 mg (30 mg twice daily) fasudil, or placebo intravenously for 20 treatmentdays over a four week period as an add-on therapy to riluzole. Follow-up assessments wereperformed at 45, 90, and 180 days after treatment initiation. Primary endpoints were tolerability during the treatment period and safety until the end of the study. All subjects whoreceived at least one study drug dose were part of the intention-to-treat (ITT)-analysis.Changes in ALS Functional Rating Scale revised (ALSFRS-R), SVC % predicted, ALS Assessment Questionnaire (ALSAQ-5), Edinburgh Cognitive and Behavioral ALS Screen (ECAS), Motor Unit Number Index (MUNIX), survival, as well as safety during the treatment period were also evaluated. A schematic of the trial design can be found in FIG.1.

[0345] Patients

[0346] Patients aged 18 to 80 years with clinical probable, laboratory supported probable ordefinite ALS (sporadic or familial) according to the revised El Escorial criteria and a disease duration of more than 6 and less than 24 months were included. A co-medication with riluzole 50 mg twice daily was compulsory. The predicted slow vital capacity (SVC) had to be >65% of normal. Exclusion criteria comprised tracheostomy or assisted ventilation during the preceding three months, gastrostomy, known arterial hypotension (<90 / 60 mmHg) and a -55-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin patient or family history for intracranial bleeding, intracerebral aneurysms or Moyamoya disease (Table 1). Table 1: Inclusion and Exclusion Criteria of the ROCK-ALS Trial-56-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin

[0347] A total of 120 patients were enrolled. In the ITT population (118 participants), nosignificant differences were observed between groups in regard to safety (estimatedproportion of patients without event for placebo: 1.00 [95% CI: 0.91 to 1.00], fasudil 30 mg: 1.00 [0.89 to 1.00], fasudil 60 mg: 1.00 [0.90 to 1.00]) and tolerability (estimated proportion of patients without event for placebo: 0.93 [0.81 to 0.99], fasudil 30 mg: 1.00 [0.90 to 1.00], fasudil 60 mg: 0.90 [0.76 to 0.97]).

[0348] FIG. 4 shows the trial profile including the numbers of patients in screening,randomization, treatment and follow-up of the ROCK-ALS trial. Two patients who wererandomized to the placebo group withdrew their consent before the start of treatment. Patientswho received at least one dose of the study drug were included in the intention-to-treat population (ITT; total 118 patients). Five patients discontinued treatment due to the given reasons. The per-protocol population (PPP) comprises all patients who received at least 80% of the scheduled treatment doses (total 113 patients).

[0349] Patients were randomly assigned in a 1:1:1 allocation ratio to receive intravenousfasudil 15 mg, fasudil 30 mg, or matching placebo twice daily. Stratification occurred bygeographical region and type of onset (bulbar, spinal). Participants, medical personnelinvolved in the intervention or in assessing outcomes as well as those analyzing the data were masked to group assignment.

[0350] Treatment

[0351] Fasudil hydrochloride hydrate (15 mg / mL ampoules) was used. Intravenouscompositions contained either 30 mg fasudil (2x 1 mL fasudil), 15 mg fasudil (1 mL fasudil and 1 mL sodium chloride [NaCl] 0.9%), or placebo (2x 1 mL NaCl 0.9%), all of which were diluted in 100 mL NaCl 0.9% prior to intravenous administration. The high fasudil dosage (60 mg (2 x 30 mg) for 20 days) corresponds to the highest cumulative licensed dosage of fasudil hydrochloride. Intravenous compositions were infused twice daily over 45 minutes using a CE-certified infusion pump at an interval of 7 ± 1 hours between administrations. Blood pressure and heart rate were captured before and at 0, 10, 20, 30, 45, and 60 minutes after infusion start. A total of 24 study visits (V0-V23) were planned. Screening and written informed consent were performed on V0, up to 42 days before the baseline visit at V1. From V1 to V20, patients received trial medication on 20 consecutive working days, excluding weekends and holidays. Three follow-up visits (V21, V22, and V23) took place at 45, 90, and180 days after baseline, respectively. Safety and tolerability were assessed at each visit.Efficacy readouts were acquired at baseline (V1, day 0), day 26 (V20), day 90 (V22), and day -57-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin 180 (V23) after treatment start. On the last day of treatment (V20) a lumbar puncture was performed if no contraindications applied. For pharmacokinetic analyses, blood plasma was obtained. Simultaneous quantification of fasudil, hydroxyfasudil and riluzole was performedon these samples by LC-MS / MS. Whole genome sequencing and C9orf72 expansion PCRwas performed to detect disease-causing ALS mutations.

[0352] The co-primary outcomes were safety from baseline to day 180 and tolerability ofintravenous fasudil during the treatment period. The treatment was considered safe for an individual patient if no drug-related serious adverse events (SAE) were recorded until 180 days after initiation of the 20-day treatment period. The treatment with fasudil was considered tolerable if participants did not discontinue treatment due to suspected drug- related adverse events (AE).

[0353] Secondary efficacy outcomes were the survival time (time until death ortracheostomy / PAV) and the change of revised ALS Functional Rating Scale-revised (ALSFRS-R), ALS Assessment Questionnaire (ALSAQ-5), Edinburgh Cognitive andBehavioral ALS Screen (ECAS), Motor Unit Number Index (MUNIX) and predicted slowvital capacity (SVC) from baseline to day 26, day 90, and day 180. For MUNIX analysis, separately trained and certified raters measured the biceps brachii (BB), abductor pollicis brevis (APB), abductor digiti minimi (ADM), tibialis anterior (TA), and extensor digitorum brevis (EDB) muscles. The MUNIX measurements were calculated as a ratio to baseline (day 1) to ensure comparability across muscles. The lower limit of detection (LLOD) was considered as one-fifth of the published 5% quantiles. Values below LLOD were set a priori to 50% of LLOD. A further secondary outcome was safety from baseline to end of treatment.

[0354] Additional outcomes were the change of serum neurofilament light chain (NfL),serum glial fibrillary acidic protein (GFAP) (Simoa immunoassay on a HD-X, Quanterix,Lexington, MA), serum creatine kinase (CK), urinary neurotrophin receptor p75 extracellular domain (p75ECD) (determined by ELISA), and body weight from baseline to day 26 and day 180.

[0355] It was estimated that a sample size of 102 patients (e.g., 34 patients per treatmentgroup) yielded sufficiently narrow confidence intervals (CI) for the difference in proportionsbetween the placebo group and the treatment groups for both primary endpoints, theproportion of patients without significant drug intolerance, and the proportion of patientswithout drug-related substantial adverse effects. Under the assumption of no difference between each of the two treatment groups and the placebo group, the half-width of the 95% -58-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin CI for the difference in proportions was at most 0·24. High proportions of tolerability and safety were expected so the CI became narrower. Adjusting for dropout of 15%, the study aimed to recruit a total number of 120 patients (e.g., 40 patients per treatment group).

[0356] Statistical Analysis

[0357] The primary analyses of safety and tolerability were carried out on the intention-to-treat population (ITT), which included all patients that entered the treatment phase. For the purpose of the tolerability analyses, in a first analysis subjects who discontinued during the treatment period were considered as worst case, e.g., no drug tolerability. Exact two-sided 95% CIs for the difference between treatment arms in proportions of tolerability were calculated as the intersection of the two one-sided 97.5% CIs derived using the inductive method. In an additional analysis of tolerability, patients who dropped out during the treatment period with an explicitly not drug-related reason were considered as competing events. Proportions at day 26 were estimated with 95% confidence interval using the Aalen- Johansen estimator. Differences between treatment arms were estimated with 95% confidence interval using the method from Scosyrev. Using the time to first drug-related substantial adverse effect, the proportions free of any drug-related substantial adverse effect at day 180 were assessed using Kaplan-Meier estimates and 95% CIs for the differences between treatment arms were calculated using the beta product confidence procedure for right censored data.

[0358] Efficacy outcomes ALSFRS-R, ALSAQ-5, ECAS, SVC, and MUNIX through day180 as well as exploratory outcomes were also analyzed in the ITT by means of Gaussian linear models for repeated measures (so-called MMRM) with treatment group, time (days 26, 90, and 180), treatment-by-time interaction, region, stratum of onset, and sex as factors and baseline measurements of the outcome as well as pre-trial estimated ALSFRS-R progression (change per month) as covariates. Least squares (expected marginal) mean changes from baseline were reported for the treatment groups with 95% CI as well as the difference between the least squares treatment group means with 95% CI and p-value testing the null hypothesis of no treatment effect.

[0359] For ALSFRS-R, SVC, NfL, and body weight subgroup analyses for subgroups by sex(male and female), by stratum of onset (spinal, bulbar), and by pre-study disease progression speed (faster and slower, split at the median) were conducted.

[0360] The Kaplan-Meier method was applied to estimate the survival probabilities in eachgroup. In this phase 2a trial, p-values were not adjusted for multiple testing and were -59-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin descriptive only; p-values smaller or equal to 0.05 are referred to a statistically significant. Sample size calculations were done using nQuery 4.0, statistical analyses were performed in R (version 4.2.3).

[0361] Safety results were reviewed by an independent data safety and monitoring boardevery three months during the trial with recommendations to the sponsor whether to continue, modify or terminate the trial.

[0362] Results

[0363] A total of 120 participants were enrolled into the ROCK-ALS trial. Two patientswithdrew their consent before treatment initiation. Thus 118 out of 120 patients received thetreatment composition and were included in the ITT for assessment of the primary andsecondary endpoints. Of those, 39 were randomly assigned to the 60 mg fasudil group, 35 to the 30 mg fasudil group and 44 to the placebo group (FIG.4) after stratification to bulbar (39patients; 33.1%) and spinal onset. 113 out of 118 patients received at least 80% of thetreatment composition and were considered as per-protocol population (PPP). The baselinecharacteristics of the patients were similar among the three arms (Table 2). Table 2: Demographic and Clinical Patient Characteristics at Baseline-60-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, UniversitätsmedizinData are given for the intention-to-treat population as median (range) or n (%). The “other class 4 / 5 mutations” were detected in the following genes: TARDP (c.881G>T (p.Gly294Val) (class 5) in one male patient with sporadic ALS in the fasudil 60 mg group), SOD1 (c.341T>C (p.Ile114Thr) (class 5) in one female patient with familial ALS in the fasudil 60 mg group) and NEK1 (two patients with sporadic ALS, one male in the placebo group (c.379C>T (p.Arg127Ter), class 5) and one female in the fasudil 60 mg group (c.1791del (p.Phe597LeufsTer32), class 4). BMI=Body mass index; ALSFRS-R=Amyotrophic Lateral Sclerosis Functional Rating Scale Revised. NfL=Neurofilament Light chain; MUNIX=Motor Unit Number Index; ALSAQ- 5=Amyotrophic Lateral Sclerosis assessment questionnaire-5; ECAS=Edinburgh Cognitive and Behavioral Amyotrophic Lateral Sclerosis Screen.

[0364] No significant difference was observed between the treatment groups regardingserious adverse events and adverse events, which were mainly related to ALS disease progression. No serious adverse events or deaths were attributed to the treatment. In the secondary outcomes, motor unit number index (MUNIX) showed a significantly reduced decline for fasudil 60 mg at 26 and 90 days, and for fasudil 30 mg at 90 days after treatment start. ALSFRS-R, ALSAQ-5, ECAS, survival, and safety during the treatment period were not significantly different between treatment groups.

[0365] Because no treatment-related substantially adverse effects were recorded in any of thetreatment groups up to day 180, the treatment was considered safe for all participants (co- primary endpoint safety; Table 3). The second co-primary endpoint, treatment tolerability, was met by 41 out of 44 patients (0.93; 95% CI: 0.81 to 0.99) in the placebo group, by 35 out -61-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin of 35 patients (1.00; 95% CI: 0.90 to 1.00) in the fasudil 30 mg group, and by 35 out of 39 patients (0.90; 95% CI: 0.76 to 0.97) in the fasudil 60 mg group. When dropouts unrelated to the treatment were not considered worst case, e.g., intolerability events, but were modelled as competing events instead, treatment intolerability occurred in 4.55% (95% CI: 0% to 11.7%) in the placebo group, in 0% (95% CI: 0% to 5.2%) in the fasudil 30 mg group and in 7.69%(95% CI: 0% to 16.8%) in the fasudil 60 mg group. No significant differences were observedbetween groups (FIG.5). Table 3: Primary and Secondary EndpointsData are given as proportions and differences in proportions for tolerability and safety endpoints, Kaplan-Meier based survival estimates, and expected marginal mean estimates of differences from baseline or ratios to baseline, as indicated. All analyses performed in the intention-to-treat population (118 patients). Values in square brackets give 95% confidence intervals. ALSFRS-R=Amyotrophic Lateral Sclerosis Functional RatingScale Revised. ALSAQ-5=Amyotrophic Lateral Sclerosis assessment questionnaire-5; ECAS=EdinburghCognitive and Behavioral Amyotrophic Lateral Sclerosis Screen; MUNIX=Motor Unit Number Index, presented here as the MUNIX megascore 10 comprising the following muscles on both sides: abductor pollicis brevis (APB), abductor digiti minimi (ADM), biceps brachii (BB), tibialis anterior (TA), extensor digitorum -62-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin brevis (EDB).

[0366] All adverse events and substantial adverse events leading to discontinuation of thestudy drug were similar between the groups without significant differences (Table 4). A total of 352 adverse events were reported in 92 out of 118 patients (78.0 %). Overall, 89 adverse events (25.3%) were deemed at least possibly related to the fasudil composition. The mostcommon non-serious adverse events were injuries (72, 36 of which were falls), headache(45), gastrointestinal disorders (41, 6 of which were dysphagia), respiratory disorders (37, 11of which were dyspnea and 10 of which were respiratory failures), and infections (34). Atotal of 40 substantial adverse events in 30 out of 120 (25·4%) patients were recorded, butnone of them related to the treatment composition. The most common substantial adverseevent was respiratory failure (7), followed by gastrostomy (5), pneumonia (4) and dysphagia(4). Two substantial adverse events were related to the lumbar puncture procedure. No suspected serious adverse reaction was reported (Table 3). Table 4: Adverse Events-63-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, UniversitätsmedizinData present all adverse events during the 6 months trial period and are given for the intention-to-treat population as n or n (%). No p-values are given here since statistical testing of safety and tolerability was already performed for the primary endpoints and did not reveal any significant differences. Serious adverse events by system organ class may contain multiple organ class listings for a single serious adverse event, thus the total number of events here may be higher than the actual number of serious adverse events.

[0367] Safety during the treatment period was separately assessed as a secondary outcome,which also did not reveal any safety events related to the treatment composition in anytreatment group and therefore no significant differences between treatment groups (Table 3). Because fasudil has vasodilatory effects, the trial protocol required blood pressure monitoring during and after drug infusion. No clinically significant changes of the arterial blood pressureand heart rate were observed in either group (FIG. 7 and Table 5). Survival until the end ofthe trial did not significantly differ among the treatment groups (Table 3 and FIG. 6 (PanelA)). There were two deaths in the placebo group, four deaths in the fasudil 30 mg group andtwo deaths in the fasudil 60 mg group (Table 4). Most deaths were related to respiratoryfailure as part of disease progression, none were judged to be related to the study drug or study procedures. Table 5: Vital Signs During Study Drug Infusion-64-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, UniversitätsmedizinSystolic and diastolic arterial blood pressure and heart rate are given as mean ± standard deviation for the different treatment groups at given time-points before and after the study drug infusion in the morning and in the afternoon.

[0368] All efficacy analyses are also shown on the ITT. The expected marginal means for thedifference in the ALSFRS-R score from baseline to day 180 were -6.5 (95% CI: -8.16 to - 4.84) for placebo, -7.94 (95% CI: -9.721 to -6.17) for fasudil 30 mg, and -6.85 (95% CI: - -65-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin 8.63 to -5.07) for fasudil 60 mg. This was not significantly different between groups, and there were no significant differences at days 26 and day 90 (Table 3 and FIG.6 (Panel B)). Post-hoc analyses of the bulbar and respiratory subscores (Q1-3 and Q10-12, respectively) showed no significant differences.

[0369] The subjective health status of the patients was measured by ALSAQ-5 and thecognitive and behavioral status was assessed by ECAS, both of which did not showsignificant differences between treatment groups at any time point (Table 3).

[0370] The respiratory function, assessed by SVC, declined over time in all groups. Adirectional difference was observed for a reduced SVC decline favoring fasudil 60 mg at day 26: expected marginal means of the difference in calculated SVC in % from baseline were - 8.34 (95% CI: -15.24 to -1.44) for placebo, -2.87 (95% CI: -10.27 to 4.54) for fasudil 30 mg, and -0.075 (95% CI: -7.68 to 7.53) for fasudil 60 mg (p=0.067 for fasudil 60 mg vs. placebo). Also, at day 90 and day 180, the expected marginal means of the SVC showed higher values for fasudil 60 mg compared to placebo, although not significantly different between groups(Table 3 and FIG. 6 (Panel C)). In a post-hoc subgroup analysis of female and male patients,there was a significantly slower SVC decline in females in the fasudil 60 mg group at all time-points (FIG.8).

[0371] Due to potentially greater sensitivity to functional decline compared to ALSFRS-R,MUNIX was analyzed across five muscles on each side, summarized as MUNIX megascore 10. At day 26, the expected marginal means for the ratio relative to baseline were 0.94 (95% CI: 0.89 to 0.99) for placebo, 0.98 (95% CI: 0.93 to 1.04) for fasudil 30 mg, and 1.01 (95% CI: 0.95 to 1.07) for fasudil 60 mg. Pairwise contrast tests showed significant differences between the placebo and fasudil 60 mg groups (p=0.015). At day 90, the expected marginal means for the ratio relative to baseline were 0.79 (95% CI: 0.73 to 0.86) for placebo, 0.89(95% CI: 0.81 to 0.97) for fasudil 30 mg, and 0.89 (95% CI: 0.81 to 0.97) for fasudil 60 mg.Here, pairwise contrast tests showed significant differences between placebo and fasudil 30 mg (p = 0.042) as well as placebo and fasudil 60 mg (p=0.038). No significant differencesbetween groups were observed at day 180 (Table 3 and FIG. 6 (Panel D)). A prespecifiedmegascore 8 analysis (excluding the BB due to frequent technical challenges analyzing this muscle) largely resembled the megascore 10 results (Table 6). Table 6: Secondary Outcomes Absolute Values -66-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin-67-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, UniversitätsmedizinData are given as median (range) in the intention-to-treat population. ALSFRS-R=Amyotrophic Lateral Sclerosis Functional Rating Scale Revised. ALSAQ-5=Amyotrophic Lateral Sclerosis assessment questionnaire- 5; ECAS=Edinburgh Cognitive and Behavioral Amyotrophic Lateral Sclerosis Screen; MUNIX=Motor Unit Number Index, presented here as the MUNIX megascore 10 comprising the following muscles on both sides: abductor pollicis brevis (APB), abductor digiti minimi (ADM), biceps brachii (BB), tibialis anterior (TA), extensor digitorum brevis (EDB) and the MUNIX megascore 8 with the same muscles except for the BB.

[0372] Several exploratory analyses were performed to characterize the molecular effects ofROCK-inhibition. There were no significant effects of fasudil treatment on serum NfL in thepre-specified analysis (Table 7 and Table 8). However, a directional NfL decline wasobserved in the fasudil 60 mg group from 126 ng / ml at baseline to 108 ng / ml at day 180whereas NfL levels increased slightly in the other groups. A post-hoc analysis showed asignificant effect of fasudil 60 mg at day 180 on the ratio of NfL values relative to baseline in the bulbar subgroup (1.92; 95% CI: 1.26 to 2.92; p = 0.003) (FIG.9). CK in serum and p75ECDin urine were not significantly different between groups. Interestingly, serum GFAP was reduced significantly on day 180 in the fasudil 60 mg group (0.75; 95% CI: 0.60 to 0.95) compared to placebo (0.90; 95% CI: 0.75 to 1.08; p=0.041) (Table 7 and Table 8). Body weight decreased in all groups over the study period, but was less pronounced in the fasudil treated groups, with a directional benefit for fasudil 60 mg especially at day 90 (p=0.074 for fasudil 60 mg vs. placebo) (Table 7). Table 7: Exploratory Outcomes-68-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, UniversitätsmedizinData are given for the intention-to-treat population as expected marginal mean estimates of differences frombaseline or ratios to baseline with 95% confidence intervals. NfL=Neurofilament light chain (in serum); GFAP=Glial fibrillary acidic protein (in serum); p75ECD=neurotrophin receptor p75 extracellular domain (in urine); CK=creatine kinase (in serum). Table 8: Exploratory Outcomes Absolute Values-69-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, UniversitätsmedizinData are given as median (range) in the intention-to-treat population. NfL=Neurofilament Light chain (in serum); GFAP=Glial Fibrillary Acid Protein (in serum); p75ECD=neurotrophin receptor p75 extracellular domain (in urine); CK=creatine kinase (in serum).

[0373] To understand whether fasudil acts in a differential manner on patients with fast orslow progression, post-hoc subgroup analyses were performed after dichotomizingprogression rate into faster (> 0.58 pts. / month) and slower progressors (< 0.58 pts / month). None of the results were specific to these subgroups (FIG. 8 and FIG. 9).

[0374] Plasma levels of fasudil and its active metabolite hydroxyfasudil were assessed at thefirst and last day of drug treatment. While fasudil was almost non-detectable due to its fast metabolism, levels of hydroxyfasudil were expectedly higher in the fasudil 60 mg group than in the fasudil 30 mg group and were non-detectable in the placebo group. On day 26, lumbar puncture was performed in 67 patients and CSF was analyzed for fasudil, hydroxyfasudil and riluzole. Corresponding to the serum results, fasudil levels were nearly undetectable in the CSF. Hydroxyfasudil was detected in all fasudil-treated subjects (fasudil 30 mg: 5.8 ± 2.0 µg / L; fasudil 60mg: 9.4 ± 4.5 µg / L; p=0.001), but in none of the placebo-treated subjects. To rule out an effect of fasudil by interaction with riluzole, riluzole levels were determined before treatment, at day 26, and day 180. No significant changes in riluzole levels were observed between groups and levels remained stable over time (FIG. 10 and Table 9).Table 9: Plasma Levels of Fasudil and Hydroxyfasudil-70-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, UniversitätsmedizinData are given as n (%), mean ± standard deviation, or as expected marginal mean estimates of differences from baseline with 95% confidence intervals in the intention-to-treat population.

[0375] Although the current label of fasudil limited the treatment period and the maximalcumulative dosage (1200 mg), the study was designed to assess immediate and potential long-term effects on safety and efficacy up to 180 days after treatment start. To modify the disease early, we included patients with a short interval from onset of weakness to baseline (median 14 months), but we did not enrich the population for fast progressors, thus the median progression rate of 0.58 points loss in ALSFRS-R per month was rather low. This made it more challenging to recognize small therapeutic effects following the short treatment period.

[0376] The treatment with fasudil was well tolerated and safe with no treatment-relatedsubstantial adverse events. The adverse events were mainly attributed to disease progression, not differing between treatment groups. No acute effects on blood pressure or heart rate were found during treatment with fasudil.

[0377] In this phase 2 trial, fasudil treatment over 20 days did not result in significant effectson the ALSFRS-R, survival, the ALSAQ-5, or the ECAS, which was not unexpected because of the limited treatment duration. However, a directional difference favoring fasudil in the SVC was determined, which was significant in a post-hoc analysis for females. In addition toits established efficacy in pulmonary hypertension, fasudil was previously shown to exert positive effects on lung vascular remodeling, lung smooth muscle, endothelial cells, and on pulmonary inflammation. Given that respiratory failure is by far the most common cause of death in ALS, this finding may be of particular interest. The data suggests that some effects of fasudil may be more sex-specific than others.

[0378] MUNIX was suggested to be more sensitive than ALSFRS-R to monitor diseaseprogression in ALS, detecting changes already in presymptomatic muscles and predicting the -71-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin rate of disease progression already earlier. Therefore, MUNIX may be more apt to detect subtle therapeutic effects, as expected by a short-term treatment with a putatively neuroprotective drug like fasudil. Here, there was found to be a significant attenuation of MUNIX decline (corresponding to the loss of motor units) at day 90 for the fasudil 30 mg group and at days 26 and 90 for the fasudil 60 mg group. This effect was dose-dependent and suggests a disease-modifying effect of fasudil. At day 180, this effect was no longer sustained. Preclinical studies have shown that fasudil improves motoneuron survival, induces axonal regeneration and inhibits the pathological activation of microglia. In vitro, effects on microglia occurred within hours.

[0379] Because such effects would lead to a better preservation of motoneurons, it isplausible that an attenuated decay of motor units is among the earliest signs of a therapeutic effect exerted by fasudil. As the ALSFRS-R reflects a functional composite of different muscle groups, the modulation of this scale would require a stronger effect size and may be insensitive to short-term treatment.

[0380] Pharmacokinetic analyses demonstrated that the active metabolite, hydroxyfasudil,was dose-dependently present in the CSF. Although the treatment regimens are not directly comparable, the high fasudil dosage (60 mg per day) resulted in CSF levels (9.4 ± 4.5 µg / L) that were similar to those achieved in the SOD1.G93A mouse model (8.8 ± 3.0 µg / L) with the fasudil concentration that showed significant effects on survival. Higher doses of fasudil could result in a stronger clinical effect. However, as all patients in this trial were treated with riluzole and riluzole levels in serum and CSF were not altered by fasudil, a riluzole-mediated effect of fasudil can largely be excluded.

[0381] Although significant effects for MUNIX were observed, the molecular biomarkersNfL and p75ECDwere not different between groups, even though the absolute NfL values showed a directional difference in the fasudil 60 mg group from baseline to day 180. Interestingly, there was a significant decrease in GFAP at day 180 in the fasudil 60 mg group compared to control, potentially implying a decrease in astrogliosis, which was also one of the strongest fasudil effects in the SOD1.G93A mice.

[0382] Conclusion

[0383] Overall, results suggest that fasudil 30 and 60 mg intravenously is safe and tolerableand may have an effect on motor unit number preservation in patients with ALS.

[0384] In the secondary outcomes, motor unit number index (MUNIX) showed asignificantly reduced decline for fasudil 60 mg at 26 and 90 days, and for fasudil 30 mg at 90 -72-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizindays after treatment start. Fasudil slowed the change in MUNIX-megascore 8 at the end ofthe treatment period and for at least 60 days post-treatment, reaching statistical significance atboth end of treatment and day 90 in the PPP group. See FIG. 2.

[0385] A directional difference was also observed for a reduced slow vital capacity (SVC)decline favoring fasudil 60 mg at day 26. The difference was significant in a post-hoc subgroup analysis of females at all time points. ALSFRS-R, ALSAQ-5, ECAS and survival were not different between treatment groups.

[0386] Results from both the MUNIX and SVC data indicated that fasudil treatmentprotected the motor units, resulting in a measurably larger number of functional neurons after90 days, and suggesting a slowing of ALS disease progression through slower loss of motorunits. Fasudil treatment was also considered well tolerated and safe in ALS patients.

[0387] Serious adverse events (40 in 30 out of 118 patients (25.4%)) and adverse events weredistributed equally between the treatment groups and mainly related to ALS diseaseprogression. No serious adverse events or deaths were attributed to the treatment.

[0388] Fasudil treatment was well tolerated and safe in ALS patients. MUNIX evaluationsuggests a slower loss of motor units in fasudil-treated patients compared to placebo, suggesting a benefit of fasudil. EXAMPLE 2: Spreading analyses

[0389] In this Example, additional results on the effect of fasudil on spreading of muscleweakness are quantified by the motor unit number index (MUNIX) assessment.Study Design and Participants

[0390] ROCK-ALS was a phase 2, randomised, double-blind, placebo-controlled trialevaluating two different doses of fasudil as an add-on therapy to riluzole in patients with amyotrophic lateral sclerosis (ALS). The trial was conducted at 19 centres across Germany, France, and Switzerland. The study protocol was approved by the relevant ethics committees and regulatory authorities in each participating country. Lead approval in Germany was obtained from the Ethics Committee of the University of Göttingen (approval number 31 / 5 / 18). The trial was registered at ClinicalTrials.gov (NCT03792490) and Eudra-CT (2017- 003676-31). The study is now completed.

[0391] Adults aged 18 to 80 years with a diagnosis of probable, laboratory-supportedprobable, or definite ALS according to the revised El Escorial criteria, and a disease duration of more than 6 months but less than 24 months, were eligible. Co-treatment with oral riluzole -73-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin (50 mg twice daily) was mandatory. Inclusion required a predicted slow vital capacity (SVC) of >65%. Key exclusion criteria included tracheostomy or assisted ventilation in the preceding 3 months, gastrostomy, known arterial hypotension (<90 / 60 mm Hg), and personal or family history of intracranial bleeding, intracerebral aneurysm, or Moyamoya disease. Allparticipants provided written informed consent prior to enrolment.Intervention

[0392] Fasudil hydrochloride hydrate (15 mg / mL ampoules; Eril, Asahi Kasei Pharma,Tokyo, Japan) was administered intravenously after dilution in 100 mL of 0.9% sodium chloride. Participants received either 30 mg fasudil (2 × 1 mL fasudil), 15 mg fasudil (1 mL fasudil + 1 mL NaCl), or placebo (2 × 1 mL NaCl). The investigational product was infused twice daily over 45 minutes using a CE-certified infusion pump, with a 6–8 hour interval between doses. The high-dose fasudil group received a cumulative dose of 60 mg / day for 20 days, the maximum licensed cumulative dose. Outcomes

[0393] Secondary efficacy endpoints of the study included survival time (defined as time todeath, tracheostomy, or permanent assisted ventilation) and changes from baseline to days 26, 90, and 180 in the following assessments: ALS Functional Rating Scale–Revised (ALSFRS- R), ALS Assessment Questionnaire (ALSAQ-5), Edinburgh Cognitive and Behavioural ALSScreen (ECAS), Motor Unit Number Index (MUNIX), and predicted SVC. This Examplepresents the results obtained by MUNIX assessment.

[0394] MUNIX is a non-invasive, rapid, and technically straightforward electrophysiologicalmethod used to estimate the number and size of motor units in individual muscles. It can be applied to any proximal or distal muscle from which a compound muscle action potential (CMAP) can be elicited through supramaximal nerve stimulation. The procedure consists ofthree steps: (1) recording the CMAP using standard motor nerve conduction techniques; (2)recording surface interference pattern (SIP) electromyography signals during varying levels of voluntary isometric contraction; and (3) calculating the MUNIX value through regression analysis of CMAP and SIP signal parameters. The method yields an “ideal case motor unit count” (ICMUC), from which MUNIX and the Motor Unit Size Index (MUSIX) are derived. Measurements are performed under standardized filter settings, and signal quality is manuallyreviewed to exclude artifacts or tremor. MUNIX has demonstrated good intra- and inter-raterreliability and is particularly suited for multicenter longitudinal studies such as ALS trials, especially when combining multiple muscles into a composite “megascore” to track disease -74-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin progression.

[0395] In this study, MUNIX was measured in five muscles on each side (biceps brachii,abductor pollicis brevis, abductor digiti minimi, tibialis anterior, and extensor digitorum brevis) and summarized as the MUNIX megascore 10. Assessments were performed by certified raters trained in MUNIX methodology. MUNIX values were measured for each muscle at baseline and at the end of the 20-day treatment period. Follow-up MUNIX assessments were conducted at day 90 and day 180 post-baseline.

[0396] For MUNIX, the lower limit of detection (LLOD) was defined as one-fifth of thepublished 5% quantiles. Values below the LLOD were imputed as 50% of the LLOD. To evaluate disease spreading, all 10 muscles were assessed. A baseline MUNIX score was defined as the mean of measurements at baseline and day 21. A muscle was classified as "affected" if it met one of the following criteria: (1) MUNIX value below the published 5% quantile; (2) ≥10%, 20%, or 30% reduction compared to the contralateral side; or (3) ≥10%,20%, or 30% decline compared to the prior measurement. Once classified as affected, amuscle remained so for the duration of the study.

[0397] Analyses were conducted both at the level of individual muscles and by limb, where alimb (upper or lower, left or right) was deemed affected if any muscle in that limb wasaffected. Statistical Analysis

[0398] Descriptive statistics were calculated for the number of newly affected muscles at day90 and day 180. Group comparisons were performed using the Chi-square test. Wheresignificant overall differences were detected, post-hoc pairwise comparisons were conducted(Fasudil 15 mg vs placebo; Fasudil 30 mg vs placebo). A two-sided significance level of 0.05 was used throughout. Statistical analyses were performed using R Studio Version 2024.09.1. Results

[0399] At baseline, MUNIX data were available for 98 of 118 enrolled participants. Follow-up data were available for 89 participants at day 90 and for 70 participants at day 180. Thiscorresponds to 836 individual muscles with at least one follow-up measurement: 309 musclesin the placebo group, 253 muscles in the 15 mg fasudil group, and 274 muscles in the 30 mg fasudil group.

[0400] To assess disease progression, three relative change thresholds (≥10%, ≥20%, or≥30%) were applied to identify affected muscles. A muscle was classified as “affected” if its-75-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin MUNIX value fell below these thresholds relative to either its contralateral counterpart or its previous measurement, thereby exceeding the expected re-measurement variance.

[0401] Across all thresholds and treatment arms, the number of newly affected musclesincreased progressively from baseline to day 90, and again from day 90 to day 180 (seeFigure 12). Using the 20% threshold, the number of unaffected muscles at baseline was:^ 115 in the placebo group^ 90 in the fasudil 15 mg group^ 103 in the fasudil 30 mg group

[0402] At day 90, the number of newly affected muscles differed significantly betweengroups (global Chi-square test: p = 0.003). Post hoc pairwise comparisons revealed asignificantly lower number of newly affected muscles in the fasudil 30 mg group compared toplacebo (Fisher’s exact test: p = 0.007). The effect was replicated using the 30% threshold,where baseline unaffected muscle counts were: ^124 in the placebo group^ 98 in the fasudil 15 mg group^ 114 in the fasudil 30 mg group

[0403] At day 90, a significant group difference was again observed (global Chi-square test:p = 0.0005). Post hoc analysis showed fewer newly affected muscles in both fasudil groupscompared to placebo: ^Fasudil 30 mg vs placebo: p = 0.0006^ Fasudil 15 mg vs placebo: p = 0.004

[0404] A dose-dependent reduction in muscle involvement was observed numerically (seeFigure 12).

[0405] For the 10% threshold, a similar trend was observed: fasudil treatment groupsexhibited fewer newly affected muscles at day 90 (global Chi-square: p = 0.15 at day 90; p =0.42 at day 180).

[0406] In addition to the analysis at the individual muscle level, disease progression was alsoassessed at the limb level. Each limb (upper left, upper right, lower left, and lower right) wasclassified as affected if any one of its corresponding muscles met the criteria for beingaffected, based on the previously described thresholds (10%, 20%, or 30%).

[0407] At the limb level, a continued increase in the number of newly affected limbs wasobserved between day 90 and day 180 and a trend toward fewer newly affected limbs in the-76-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin fasudil-treated groups compared to placebo was noted, suggesting a potential dose-dependent effect (Figure 13).

[0408] Overall, the data suggest that treatment with fasudil has the potential to slow diseaseprogression in amyotrophic lateral sclerosis (ALS). This is supported by a reduced rate of newly affected muscles and limbs, as measured by MUNIX assessments, in subjects treated with 30 mg (15 mg twice daily) or 60 mg (30 mg twice daily) of fasudil, compared to those receiving a placebo.

[0409] References1. Lingor, P., Teusch, N., Schwarz, K., Mueller, R., Mack, H., Bähr, M., & Mueller, B. K.(2007). Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo. Journal ofNeurochemistry, 103(1), 181–189. https: / / doi.org / 10.1111 / j.1471-4159.2007.04756.x2. Lingor, P., Tönges, L., Pieper, N., Bermel, C., Barski, E., Planchamp, V., & Bähr, M. (2008).ROCK inhibition and CNTF interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells. Brain, 131(1), 250–263. https: / / doi.org / 10.1093 / brain / awm2843. Tönges, L., Günther, R., Suhr, M., Jansen, J., Balck, A., Saal, K., Barski, E., Nientied, T.,Götz, A. A., Koch, J., Mueller, B. K., Weishaupt, J. H., Sereda, M. W., Hanisch, U., Bähr, M., & Lingor, P. (2014). Rho kinase inhibition modulates microglia activation and improves survival in a model of amyotrophic lateral sclerosis. Glia, 62(2), 217–232. https: / / doi.org / 10.1002 / glia.226014. Takata, M., Tanaka, H., Kimura, M., Nagahara, Y., Tanaka, K., Kawasaki, K., Seto, M.,Tsuruma, K., Shimazawa, M., & Hara, H. (2013). Fasudil, a rho kinase inhibitor, limits motor neuron loss in experimental models of amyotrophic lateral sclerosis. British Journal of Pharmacology, 170(2), 341–351. https: / / doi.org / 10.1111 / bph.122775. Satoh, S., Ikegaki, I., Kawasaki, K., Asano, T., & Shibuya, M. (2014). Pleiotropic Effects ofthe Rho-kinase Inhibitor Fasudil After Subarachnoid Hemorrhage: A Review of Preclinical and Clinical Studies. Current Vascular Pharmacology, 12(5), 758–765. https: / / doi.org / 10.2174 / 15701611126661406131158136. Koch, J. C., Tatenhorst, L., Roser, A.-E., Saal, K.-A., Tönges, L., & Lingor, P. (2018). ROCKinhibition in models of neurodegeneration and its potential for clinical translation. Pharmacology & Therapeutics, 189, 1–21. https: / / doi.org / 10.1016 / j.pharmthera.2018.03.0087. Koch, J. C., Tönges, L., Barski, E., Michel, U., Bähr, M., & Lingor, P. (2014). ROCK2 is amajor regulator of axonal degeneration, neuronal death and axonal regeneration in the CNS. Cell Death & Disease, 5(5), e1225–e1225. https: / / doi.org / 10.1038 / cddis.2014.1918. Neuwirth, C., Barkhaus, P. E., Burkhardt, C., Castro, J., Czell, D., de Carvalho, M.,Nandedkar, S., Stålberg, E., & Weber, M. (2017). Motor Unit Number Index (MUNIX) detects motor neuron loss in pre-symptomatic muscles in Amyotrophic Lateral Sclerosis. ClinicalNeurophysiology, 128(3), 495–500. https: / / doi.org / 10.1016 / j.clinph.2016.11.026 -77-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin9. Günther, R., Balck, A., Koch, J. C., Nientiedt, T., Sereda, M., Bähr, M., Lingor, P., & Tönges,L. (2017). Rho Kinase Inhibition with Fasudil in the SOD1G93A Mouse Model ofAmyotrophic Lateral Sclerosis—Symptomatic Treatment Potential after Disease Onset. Frontiers in Pharmacology, 8. https: / / doi.org / 10.3389 / fphar.2017.0001710. Lingor, P., Weber, M., Camu, W., Friede, T., Hilgers, R., Leha, A., Neuwirth, C., Günther, R.,Benatar, M., Kuzma-Kozakiewicz, M., Bidner, H., Blankenstein, C., Frontini, R., Ludolph, A., & Koch, J. C. (2019). ROCK-ALS: Protocol for a Randomized, Placebo-Controlled, Double- Blind Phase IIa Trial of Safety, Tolerability and Efficacy of the Rho Kinase (ROCK) Inhibitor Fasudil in Amyotrophic Lateral Sclerosis. Frontiers in Neurology, 10. https: / / doi.org / 10.3389 / fneur.2019.0029311. Requardt, M. V., Görlich, D., Grehl, T., & Boentert, M. (2021). Clinical Determinants ofDisease Progression in Amyotrophic Lateral Sclerosis—A Retrospective Cohort Study. Journal of Clinical Medicine, 10(8), 1623. https: / / doi.org / 10.3390 / jcm1008162312. Risi, B., Cotti Piccinelli, S., Gazzina, S., Labella, B., Caria, F., Damioli, S., Poli, L., Padovani,A., & Filosto, M. (2023). Prognostic Usefulness of Motor Unit Number Index (MUNIX) in Patients Newly Diagnosed with Amyotrophic Lateral Sclerosis. Journal of Clinical Medicine, 12(15), 5036. https: / / doi.org / 10.3390 / jcm1215503613. Wolff, A. W., Peine, J., Höfler, J., Zurek, G., Hemker, C., & Lingor, P. (2024). SAFE-ROCK:A Phase I Trial of an Oral Application of the ROCK Inhibitor Fasudil to Assess Bioavailability, Safety, and Tolerability in Healthy Participants. CNS Drugs, 38(4), 291–302. https: / / doi.org / 10.1007 / s40263-024-01070-7 -78-

Claims

Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin CLAIMS1. Fasudil for use in a method of treating ALS in a subject, wherein fasudil is administeredduring a treatment period, wherein the treatment period is from 2 to 6 weeks, wherein 150 mg to 630 mg of fasudil is administered per week during the treatment period,wherein fasudil is administered on 5 to 7 days in each week of the treatment period, optionally followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is from 1 week to 3 months.

2. Fasudil for the use according to claim 1, wherein the subject exhibits an increasedMUNIX score after the treatment period and / or the drug holiday period as compared to a MUNIX score of a subject undergoing placebo treatment, preferably wherein the MUNIX score is increased by at least 3 %, more preferably at least 4%, more preferably at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably atleast 8%, more preferably at least 9%, more preferably at least 10%, more preferably atleast 12%, more preferably at least 14%, more preferably at least 16%, more preferablyat least 18%, and even more preferably at least 20%; and / or at most 100%.

3. Fasudil for the use according to claims 1 or 2, wherein the subject exhibits an at most20% decrease in MUNIX score after the treatment period and / or the drug holiday periodas compared to before initiation of fasudil administration, preferably an at most 18% decrease, more preferably an at most 17% decrease, more preferably an at most 16% decrease, more preferably an at most 15% decrease, more preferably an at most 14%decrease, more preferably an at most 13% decrease, more preferably an at most 12%decrease, more preferably an at most 11% decrease, more preferably an at most 10%decrease, more preferably an at most 8% decrease, more preferably an at most 6%decrease, more preferably an at most 4% decrease and most preferably an at most 2% decrease.

4. Fasudil for use according to any of the preceding claims, wherein the subject exhibits alower number of newly affected muscles after the treatment period and / or the drugholiday period as compared to a subject undergoing placebo treatment,wherein the number of newly affected muscles is determined by MUNIX score, wherein amuscle is determined to be affected if the MUNIX score decreases by at least 10%,preferably by at least 20%, and more preferably by at least 30% relative to the MUNIX score of the contralateral muscle or the MUNIX score of the same muscle prior to the treatment period. -79-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin5. Fasudil for use according to claim 4, wherein the subject treated with fasudil exhibitslower number of newly affected limbs as compared to number of newly affected limbsin a subject undergoing placebo treatment, wherein the limb is considered affected if at least one muscle of that limb is affected, wherein the number of newly affected muscles is determined by MUNIX score, wherein amuscle is determined to be affected if the MUNIX score decreases by at least 10%,preferably by at least 20%, and more preferably by at least 30% relative to the MUNIX score of the contralateral muscle or the MUNIX score of the same muscle prior to the treatment period.

6. Fasudil for the use according to any of the preceding claims, wherein the subject exhibitsan increased SVC after the treatment period and / or the drug holiday period compared to an SVC of a subject undergoing placebo treatment, preferably wherein said increased SVC is increased by at least 1 %, preferably at least 2%, more preferably at least 3%, more preferably at least 4%, even more preferably at least 5%, even more preferably at least 6%, even more preferably at least 7%, even more preferably at least 8%, even more preferably at least 9%, even more preferably at least 10%, even more preferably at least12%, even more preferably at least 14%, even more preferably at least 16%, even morepreferably at least 18%, and most preferably at least 20 %; and / or at most 100%.

7. Fasudil for the use according to any one of the preceding claims, wherein the subjectexhibits a SVC, which is reduced by at most 10 % after the treatment period and / or the drug holiday period compared to the SVC measured before initiation of fasudil administration, preferably at most 9.5 %, more preferably at most 9%, even more preferably at most 8.5 %, even more preferably at most 8 %, even more preferably atmost 7.5 %, even more preferably at most 7 %, even more preferably at most 6.5 %, andmost preferably at most 6 %.

8. Fasudil for the use according to any one of the preceding claims, wherein the subjectexhibits an increased muscle strength compared to a muscle strength of a subjectundergoing placebo treatment, preferably wherein the muscle strength is increased by at least 5%, more preferably at least 7%, even more preferably at least 10 %, more preferably wherein muscle strength is measured by a hand grip test, more preferably wherein the hand grip test is carried out using a hand-held dynamometer; and / or wherein the subject exhibits a decrease in the concentration of a biomarker after the treatment period and / or the drug holiday period compared to before initiation of fasudil administration, preferably wherein the biomarker is selected from one or more of NfL,GFAP, p75ECD, and creatine kinase. -80-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin9. Fasudil for the use according to any one of the preceding claims, wherein the treatmentperiod is from 3 to 6 weeks, more preferably wherein the treatment period is from 4 to 6 weeks, and even more preferably wherein the treatment period is around 4 weeks.

10. Fasudil for the use according to any one of the preceding claims, wherein 200 mg to 600mg of fasudil is administered per week during the treatment period, preferably wherein 240 mg to 520 mg of fasudil is administered per week during the treatment period, morepreferably wherein 270 mg to 450 mg of fasudil is administered per week during the treatment period, more preferably wherein 280 mg to 420 mg of fasudil is administeredper week during the treatment period, and more preferably wherein 300 mg to 400 mg of fasudil is administered per week during the treatment period; and / or wherein fasudil is administered on 5, 6 or 7 days in each week of the treatment period.

11. Fasudil for the use according to any one of the preceding claims, wherein fasudil isadministered intravenously, orally or subcutaneously, preferably wherein fasudil is administered intravenously or orally; and / orwherein fasudil is administered in a pharmaceutical composition during the treatment period, wherein the pharmaceutical composition comprises 15 mg, 30 mg or 45 mg of fasudil or a pharmaceutically acceptable salt thereof, wherein said pharmaceutical composition is administered twice a day or three times a day on 5 to 7 days in each week of the treatment period, preferably twice a day on 5 to 7 days in each week of thetreatment period.

12. Fasudil for the use according to any one of the preceding claims, wherein the drug holidayperiod is from 2 week to 2 months, preferably from 3 weeks to 1.5 months, and more preferably around 1 month.

13. Fasudil for the use according to any one of the preceding claims, wherein a fixed dose offasudil is administered.

14. Fasudil for the use according to any one of the preceding claims, wherein atherapeutically-effective amount of a second agent is also administered, preferablywherein the second agent comprises riluzole, edaravone, tofersen, nuedexta, or jacifusen.

15. Fasudil for the use according to any one of the preceding claims, wherein fasudil isadministered during a treatment period, wherein the treatment period is from 4 to 5 weeks, wherein around 300 mg of fasudil is administered per week during the treatment -81-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizin period, wherein fasudil is administered on 5 days in each week of the treatment period, preferably wherein the next treatment period starts immediately after the end of the last treatment period; orwherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 450 mg of fasudil is administered per week duringthe treatment period, wherein fasudil is administered on 5 days in each week of the treatment period, preferably wherein the next treatment period starts immediately after the end of the last treatment period; orwherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 420 mg of fasudil is administered per week duringthe treatment period, wherein fasudil is administered on 7 days in each week of thetreatment period, preferably wherein the next treatment period starts immediately after the end of the last treatment period; orwherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 630 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 7 days in each week of the treatment period, preferably wherein the next treatment period starts immediately after the end of the last treatment period; orwherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 300 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 5 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month; orwherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 450 mg of fasudil is administered per week duringthe treatment period, wherein fasudil is administered on 5 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month; orwherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 420 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 7 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month; or -82-Georg-August-Universität Göttingen 30A-163414 Stiftung Öffentlichen Rechts, Universitätsmedizinwherein fasudil is administered during a treatment period, wherein the treatment periodis from 4 to 5 weeks, wherein around 630 mg of fasudil is administered per week during the treatment period, wherein fasudil is administered on 7 days in each week of the treatment period, followed by a drug holiday period in which fasudil is not administered, wherein the drug holiday period is around 1 month. -83-

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