Method of treating patients with notch3 mutations

Abstract

Disclosed is a method of treating patients with NOTCH3 mutation with rho kinase inhibitors. Aberrant NOTCH3 signaling is a pathological actor in small vessel cerebrovascular diseases, including CADASIL. A preferred aspect relates to treating patients orally with the rho kinase inhibitor fasudil or its active metabolite hydroxyfasudil.

Description

[0001] Method of Treating Patients with NOTCH3 Mutations

[0002] Background

[0003] NOTCH3. The neurogenic locus notch homolog protein 3, N0TCH3, is encoded by the N0TCH3 gene, which is located on chromosome 19 between bands 13.2 and 13.1 (19p 13) and consists of 33 exons that encode a 2,321 amino acid protein. It is a human homologue of the notch protein, first identified in fruit flies as a regulator of development. In humans, it is expressed almost exclusively in vascular smooth muscle cells (VSMCs).

[0004] The notch protein is a transmembrane receptor, consisting of an intracellular domain, a transmembrane domain and an extracellular end domain. The ligand binds the extracellular domain, and this results in the proteolytic detachment of the intracellular domain, which translocates to the nucleus where it modulates transcription of certain target genes. The protein plays a key role in the function and survival of VSMCs and is thought to be essential for the maintenance of blood vessels.

[0005] NOTCH3 and CADASIL. Mutations in the N0TCH3 gene can lead to small vessel disease (SVD) in humans, including cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a condition caused by mutations altering the number of cysteine residues in the extracellular domain. These changes cause an odd number of cysteines, leading to a misfolding of the receptor, enhanced formation of oligomers, and ECD aggregation facilitated by crosslinking of sulfhydryl groups. The mutations may cause a thickening in the walls of small and medium sized vessels and impede blood flow.

[0006] Most pathogenic N0TCH3 variants lead to an odd number of cysteines, and affect the 34 epidermal growth factor-like repeats (EGFrs). These 34 regions span residues 40 to 1373 of NOTCH3. Each region spans about 40 amino acid-long modular protein subunits, and each region contains a conserved number and position of six cysteine residues forming three disulfide bridges. The mutations disrupt disulfide bonding and lead to pathogenic aggregation, which is considered to be a gain of function. Most pathogenic mutations in NOTCH3 are located in exon 4, followed by exons 3, 5, 6, and 11, but mutations in any region can be pathogenic. Studies have reported that mutations in EGFrs 1-6 are associated with a higher risk of strokes. (Rutten 2019). Another study reported that mutations EGFrs 18- 34 seemed to be as severe as those having mutations in EGFRs 1-6, but found a lower stroke risk in patients having NOTCH3 mutations in EGFrs 10-17. (Cho 2022). Other types ofNOTCH3 mutations, including cysteine-sparing missense mutations, frameshift and nonsense mutations have also been identified. (Muino et al. 2017) These occur at relatively high frequency in the non-CADASIL small vessel disease population, suggesting a broader role for NOTCH3 mutations in cerebrovascular disease. (Schoemaker 2021).

[0007] CADASIL. CADASIL-causing NOTCH3 mutations lead to progressive degeneration of VSMCs. CADASIL is characterized by the intravascular accumulation of granular osmiophilic material (GOM) around VSMCs and pericytes. GOMs are made up of the notch protein extracellular domain and other extracellular matrix proteins such as clusterin and collagen 18 al / endostatin. The pathological link between GOMs and the CADASIL disease state remains unclear. Likewise, the role of aberrant notch signaling and transcriptional modulation remain largely unelucidated.

[0008] CADASIL is a rare genetic disorder with an estimated prevalence of 1.3-4.1 per 100,000 among Caucasians. However, large human genomic studies are finding a much higher prevalence of NOTCH3 variants in the general population. Pathogenic mutations in CADASIL are autosomal dominant; the presence of only one mutation in one of both alleles is the cause of the pathology. With the advent of broad genetic testing, more patients with CADASIL are being identified. However, a significant genotype-phenotype correlation is still lacking.

[0009] In addition to NOTCH3 mutations, magnetic resonance imaging (MRI) may show widespread, confluent white matter hyperintensities on T2-weighted MRI images, with hyperintense lesions often evident in the basal ganglia, thalamus, and pons. (Stojanov 2015). The initial stage of the disease in the majority of cases involves the anterior temporal lobe and external capsule, with relative sparing of the occipital and orbitofrontal subcortical white matter, subcortical U-fibers, and cortex. Cerebral microhemorrhages may be present. On progression, cerebral atrophy is present, and this is associated with cognitive decline.

[0010] The clinical presentation of CADASIL can vary, with some patients presenting with all and some presenting with only a subset of the constellation of symptoms, with severity and progression varying widely. (Yamamoto 2023). The most common initial symptom of CADASIL is recurrent migraine with aura, which typically begins in early adulthood with an average onset of around age 30, although they can start a decade earlier. The aura may include visual disturbances, such as flashing lights or blind spots, as well as sensory or language disturbances. Women with CADASIL and migraine with aura tend to have symptom onset earlier than affected men.

[0011] Recurrent subcortical ischemic strokes or transient ischemic attacks (TIA) generally begin in CADASIL patients around ages 50-59. These may present as sudden weakness, numbness, or paralysis on one side of the body, difficulty speaking, or vision changes. MRI shows basal ganglia and confluent white matter lesions. Sensory, sensory and cognitive impairments, with or without psychiatric disorders. Progressive cognitive decline usually becomes evident around ages 50-59, and may include problems with memory, attention, and executive function. Early features of vascular cognitive impairment (VCI) in CADASIL include executive dysfunction and slowing of information processing speed and may also include verbal fluency and ideational praxis. Recall, orientation, and receptive language skills are largely preserved early in the stages - deficits in working memory and episodic long-term memory tend to develop later.

[0012] Eventually, CADASIL may lead to subcortical dementia, usually after about age 60. Other symptoms may include mood and psychiatric disturbances, gait disturbance, urinary incontinence, pseudobulbar palsy, epileptic seizures and acute-onset reversible encephalopathy called “CADASIL coma” affects about 10% of patients. MRI shows diffuse leukoencephalopathy and multiple infarcts of basal ganglia at this stage.

[0013] While the natural history of CADASIL is variable, a large majority of patients experience at least one stroke or TIA and a large majority of those experience multiple ischemic events. Nearly all patients experience progressive cognitive decline and many of them develop dementia. More significant ischemic events are significantly associated with more profound cognitive decline. CADASIL patients also tend to have a shortened lifespan.

[0014] CADASIL is a devastating condition because it affects adults in their prime years with the age of onset about 30 years, it is progressive, it is commonly associated with depression and psychiatric disorders, and there are no available treatments (Hack 2019). Care of CADASIL patients is directed at managing symptoms and risk factors, such as hypertension and smoking. Acute strokes may be managed with tPA (recombinant tissue plasminogen activator; Alteplase), depending on an individualized assessment. Migraines are treated with traditional analgesics, but more rarely vasoconstrictors, like triptans or ergot derivates are used. Anti -hypertensive, anti -convul sants, and anti-depressants may be used for migraine prevention. Depression and other psychiatric issues may be managed pharmaceutically. None of these approaches address the underlying vascular condition caused by the underlying N0TCH3 mutation and none halts or slows the progression of the disease.

[0015] In at least one report, CADASIL was hypothesized to be triggered by COVID-19 in a patient with a N0TCH3 mutation. (Rajendran 2021). In another, CADASIL was worsened by COVID-19 infection. (Rosenblum 2022).

[0016] Fasudil and NOTCH3 mutations. There is a report in the literature of using fasudil, a rho kinase inhibitor, to treat several patients with white matter vascular disease Kamei (1996) reported on using fasudil in two patients with wandering due to VaD. The patients were treated by the investigator for wandering following participating in a chronic stroke study where they were treated with fasudil. One patient was diagnosed with Binswanger-type cerebral infarction, confirmed by MRI imaging. The other patient was diagnosed with sequelae of cerebral bleeding and multiple lacunar infarctions, confirmed by MRI, and a diagnosis of “lacunar dementia” (a synonym of Binswanger’s). The authors reported that the patients, who had exhibited wayfinding symptoms (getting lost) improved for the duration of treatment, returning each time treatment was stopped. There is no evidence that the work of Kamei in subcortical vascular dementia can be extrapolated to patients having NOTCH 3 mutations and / or CADISIL.

[0017] Transgenic mice harboring the CADASIL-associated R169C mutation were found to exhibit gain-of-function signaling and hypercontractility of pulmonary arteries exposed to 5-HT and ET1, an effect inhibited by fasudil. (Morris 2019). However, this model. Another group reported that fasudil ameliorated aberrant vascular responses (reduced induced vasorelaxation) and signaling when incubated in vessels extracted the same R169C transgenic mouse model. (Neves 2019). However, this model does not appear to faithfully recapitulate human disease because the cerebrovascular dysfunction in the mouse model is associated with upregulation of voltage-dependent potassium (Kvl) channels, blunted membrane depolarization, and reduced myogenic tone due to increased levels of metallopeptidase inhibitor TIMP3, which have not been demonstrated in human disease.

[0018] Neves 2019 further showed that NOTCH3 and ROCK2 expression (along with other ER- stress proteins) was increased in VSMCs from human CADASIL patients, and that incubating CADASIL VSMCs with fasudil ameliorated activation of cytoskeletal regulatory proteins. Neves used 10 pM FAS in vitro (2.9 pg / ml). This is not a physiologically relevant concentration.

[0019] Because no fasudil was administered to either the humans or the mice the connection to treating CADASIL in humans using fasudil is tenuous.

[0020] There are no disease modifying therapies for small vessel disease including CADASIL.

[0021] There is an ongoing need to treat patients with NOTCH3 mutations who have or could be at risk of developing CADISIL or another small vessel disease.

[0022] Summary of the Invention

[0023] Provided is a method of treating a human patient having a mutation in the NOTCH3 gene, comprising administering to said patient a therapeutically effective amount of oral fasudil or hydroxy fasudil.

[0024] In one embodiment, the patient has been diagnosed with CADASIL. In another embodiment, the patient has not been diagnosed with CADASIL.

[0025] In one embodiment, the patient has been diagnosed with cerebral small vessel disease.

[0026] In a further embodiment, the NOTCH3 mutation is a gain of function mutation that results in odd number of cysteines in an extracellular domain.

[0027] In another embodiment, the mutation is not a gain of function mutation. In specific embodiment, the NOTCH3 mutation is a cysteine-sparing mutation.

[0028] In one embodiment, the patient has at least one white matter hyperintensity.

[0029] In another embodiment, the patient has granular osmiophilic material.

[0030] In one embodiment, the patient has had at least one stroke or a transient ischemic attack.

[0031] In another embodiment, the patient has had at least one stroke or a transient ischemic attack at least 90 days before treatment begins. In a further embodiment, the patient has a pre-existing cerebral infarction. In a specific embodiment, the pre-existing infarction is from a lacunar ischemic stroke. In another specific embodiment, any prior cerebral hemorrhage was micro-hemorrhage.

[0032] In one embodiment, the patient shows evidence of abnormally reduced cerebrovascular reactivity, neurovascular coupling, or regional or global cerebral hypoperfusion.

[0033] In another embodiment, the patient shows evidence of abnormally increased vascular myogenic tone.

[0034] In an embodiment, the patient has vascular mild cognitive impairment.

[0035] In another embodiment, the patient has vascular dementia.

[0036] In a specific embodiment, the N0TCH3 variant is an EGFr 1-6 pathogenic variant; an EGFr 7-34 pathogenic variant; or an EGFr 18-34 pathogenic variant.

[0037] Certain embodiments involve treating patients with high risk NOTCH3C" mutations. In one particular embodiment high risk patients are treated in the absence of any evidence of small vessel disease. In another embodiment, high risk patients are treated if they have at least one additional risk factor of small vessel disease, such as hypertension, smoking, diabetes or hypercholesterolemia. High risk patients will be treated if they have a Fazekas score of at least 1 in the deep or periventricular white matter.

[0038] Certain other embodiments involve treating patients with medium risk NOTCH3C" mutations. In one specific embodiment, medium risk patients are only treated if they have at least one additional risk factor of small vessel disease, such as hypertension, smoking, diabetes or hypercholesterolemia and / or they have a Fazekas score of at least 1 in the deep or periventricular white matter and / or they are displaying one or more signs or symptoms of CADASIL, including evidence of subcortical ischemic events, cognitive impairment, migraine with aura, and / or psychiatric disturbances (e.g., mood disturbances; apathy).

[0039] Still further embodiments involve treating patients with low risk NOTCH3C" mutations. In one specific embodiment, medium risk patients are only treated if they have a Fazekas score of at least 1 in the deep or periventricular white matter. In another embodiment, low risk patient are only treated if they have either (a) at least one additional risk factor of small vessel disease, such as hypertension, smoking, diabetes or hypercholesterolemia and / or (b) they are displaying one or more signs or symptoms of CADASIL, including evidence of subcortical ischemic events, cognitive impairment, migraine with aura, and / or psychiatric disturbances (e.g., mood disturbances; apathy) and (c) they have a Fazekas score of at least 1 in the deep or periventricular white matter.

[0040] Additional embodiments contemplate treating patients with unknown risk NOTCH3C" mutations. In one specific embodiment, unknow risk patients are only treated if they a have additional significant findings consistent with NOTCH3-associated small vessel disease, such as evidence of GOMs and a Fazekas score of at least 1 in the deep or periventricular white matter. In another embodiment, low risk patient are only treated if they have at least one additional risk factor of small vessel disease, such as hypertension, smoking, diabetes or hypercholesterolemia and they have a Fazekas score of at least 1 in the deep or periventricular white matter, and they will generally displaying one or more signs or symptoms of CADASIL, including evidence of subcortical ischemic events, cognitive impairment, migraine with aura, and / or psychiatric disturbances (e.g., mood disturbances; apathy).

[0041] In an embodiment, the patient does not have an R169C N0TCH3 mutation.

[0042] In a further embodiment, the patient is treated with therapeutically effective amount of a second vasoactive pharmaceutical agent.

[0043] In one specific embodiment, the second vasoactive agent is selected from the group consisting of an angiotensin converting enzyme inhibitor, an angiotensin receptor blocker, a calcium channel blocker, a phosphodiesterase inhibitor and a nitrate.

[0044] In an embodiment, the patient suffers from migraines. In a specific embodiment, the migraines occur at least once per month. In another specific embodiment, the patient experience migraines is female.

[0045] In another embodiment, the patient treated is under 40 years of age and suffers from migraines. In a specific embodiment, the patient under 40 is female.

[0046] In one embodiment, the patient is between 40 and 50 years of age.

[0047] In a further embodiment, the patient is over 50 years of age. In a specific embodiment the patient over 50 years of age does not have dementia. Detailed Description

[0048] The invention relates to the use of fasudil or its metabolite M3 to treat the cerebrovascular effects of mutations in N0TCH3 in human patients.

[0049] N0TCH3 mutations

[0050] In one embodiment, patients treated with fasudil or M3 in accordance with the disclosure will have at least one mutation in N0TCH3. In a specific embodiment, the patient will have confirmation of a N0TCH3 mutation prior to initiating the inventive treatment.

[0051] The mRNA sequence and the amino acid sequence for human N0TCH3 are provided in GenBank Accession No. NM_000435.

[0052] The N0TCH3 mutation may be an EGFr 1-6 pathogenic variant or it may be an EGFr 7-34 pathogenic variant. In one embodiment, the patient has EGFr 1-6 mutation. In another embodiment, the patient having the EGFr 1-6 mutation has an earlier-onset of stroke and a lower survival than those in other EGFrs.

[0053] In another embodiment the patient has a N0TCH3 mutation EGFrs 18-34

[0054] In another embodiment, the patient has a N0TCH3 mutation in EGFrs 10-17.

[0055] In one embodiment, the NOTCH3 mutations will result in an odd number of cysteines in the extracellular domain. In another embodiment, the NOTCH3 mutation is a gain of function mutation.

[0056] In one embodiment, the mutation is a missense mutation related to a cysteine residue in one or more of the 34 EGFr on the NOTCH3 extracellular domain.

[0057] These cysteine-affecting mutations, aka NOTCH3C" mutations, are annotated in the literature several different ways, but generally follow the format of specifying the wild-type amino acid residue, followed by the amino acid position and then the mutant amino acid. As understood by one of ordinary skill in the art, there are standard three-letter and one letter systems for naming amino acids. Thus, C49Y specifies that the mutation results in the cysteine at position 49 (following the N-terminal to C-terminal convention) has been replaced by a tyrosine. The same mutation may be designated as Cys43Tyr or some similar naming variant. It will also be recognized by one of ordinary skill that other standard notations may be used, for example “p.” as a prefix to denote changes in the protein sequence. Thus, C49Y, Cys43Tyr and p.Cys43Tyr are equivalent.

[0058] In one embodiment, exemplary N0TCH3 mutations are set forth in the below Table 1 :

[0059] Table 1

[0060] * Human Gene Mutation Data Base - Cardiff University In another embodiment, NOTCH3 mutations may be at least one of the following: C43S, C43G, C55S, R61W, C65Y, C67S, G73S, R75P, C76R, D80G, C82F, G85C, R90C, C93Y, P109T, R110C, S126C, C128F, R133C, C134R, C134Y, R141C, F142C, C144Y, Y150C, R153C, S154C, S154C, R169C, C174R, R182C, C183R, C185S, Y189C, C194S, C194R, C201R, P203H, R207C, C212Y, C212R, R213K, C222R, C224Y, C224R; C233R, D239N, C240Y, C245S, C245R, C245Y, C251Y, C251R, C311G, R322C, C323S, C323Y, R332C, C338R, C349S, C355S, C379S, C379F, C402S, C408G, C408Y; C419W; R421C, R427C, C428Y, C428R, C440G, C440Y, R449C, C457S, G460C, Y465C, C466Y, S476C, G481C, C493S, C493R, C504G, C516F, C522S, R544C, G528C, R558C, P572L, R578C, R587C, R592C, C597R, C597S, C597W, R607C, C608Y, C624S, R635C, R640C, R717C, G942C, R951C, G953C, C977G, R985C, R1006C, A1020P, Y1021C, R1031C, C1061Y, Y1069C, R1076C, R1100C, Y1106C, C1119Y, R1143C, R1190C, R1201C, C1202S, R1210C, C1222G, R1231C, R1242C, C1250G, A1604T, C1897T, and C1222G.

[0061] In a specific embodiment, the patient having a NOTCH3 mutation has a cysteine-sparing mutations. Cysteine-sparing mutations that do not result in a removal or addition of a cysteine and may not result in a gain of function pathology. In a specific embodiment, the cysteine-sparing mutation is at least one of R61W, G73S, R75P, D80G, P109T, P203H, R213K, D239N, P572L, A1020P, and A1604T.

[0062] In other embodiments, the NOTCH3 mutation in the patient is a null mutation, a homozygous mutation, or an in-frame insertion / deletion mutation. Examples of homozygous mutations include R544C. Examples of duplications include duplication of Glu434 to Leu436 and an inframe heterozygous 15 bp duplication in exon 7 resulting in insertion of five extra amino acids at positions 353 to 357, including cysteine 355. Duplications in exon 28 have ALSO been reported. Deletions reported include deletions from A88 to G91, and from T312 to G316 ofNOTCH3.

[0063] In another embodiment, the NOTCH3 mutation is a splice site mutation, including one resulting from an intronic deletion such as c.341-26_24delAAC that results in an insertion of 25 amino acids.

[0064] High Risk Mutations. NOTCH3C" mutations in EGFr domains 1-6, 8, 11 and 26 are of particular interest. Patients with these “high risk” mutations are not only very likely to get cerebral small vessel disease they are likely to get CADASIL. Patients with these mutations may be treated according to the invention with very little further evidence of disease - they may be treated without any additional signs or symptoms (i.e., prophylactically) or even without risk factors like hypertension, smoking, diabetes or hypercholesterolemia. Typically, high risk patients will be treated based on the presence of a mutation and a Fazekas score of at least 1 in the deep or periventricular white matter.

[0065] The following NOTCH3C" mutations are high risk and fall into the high-risk category:

[0066] Cys43Gly, Cys43Tyr, Cys43Phe, Cys43Arg, Cys49Tyr, Cys49Phe, Cys49Arg, Gly53Cys, Arg54Cys, Cys55Arg, Cys55Gly, Cys55Ser, Ser60Cys, Cys65Tyr, Cys65Ser, Cys67Ser, Trp71Cys, Cys76Arg, Cys76Tyr, Cys76Trp, Cys82Phe, Gly85Cys, Cys87Arg, Cys87Tyr, Cys87Phe, Arg90Cys, Cys93Phe, Cys93Gly, Cys93Tyr, CyslO6Arg, CyslO6Trp, CyslO8Arg, CyslO8Gly, CyslO8Phe, CyslO8Tyr, CyslO8Ser, Argl lOCys, Cysl l7Arg, Cysl l7Tyr, Cysl l7Phe, Cysl l7Thr, Cysl l7Trp, Serl l8Cys, Cysl23Phe, Cysl23Thr, Serl26Cys, Cysl28Phe, Argl33Cys, Cysl34Tyr, Cysl34Gly, Cysl34Trp, Argl41Cys, Phel42Cys, Cysl44Ser, Cysl44Phe, Cysl44Tyr, Cysl44Trp, Cysl44Thr, Serl45Cys, Cysl46Arg, Cysl46Trp, Cysl46Tyr, Glyl49Cys, Tyrl50Cys, Argl53Cys, Serl54Cys, Cysl55Trp, Cysl55Ser, Cysl55Tyr, Trpl56Cys, Cysl62Ser, Cysl62Trp, Cysl62Tyr, Glyl65Cys, Argl69Cys, Hisl70Cys, Glyl71Cys, Cysl74Arg, Cysl74Tyr, Serl80Cys, Argl82Cys, Cysl83Arg, Cysl83Tyr, Cysl83Ser, Cysl83Phe, Cysl85Arg, Cysl85Ser, Cysl85Tyr, Cysl85Gly, Tyrl89Cys, Cysl94Arg, Cysl94Ser, Cysl94Tyr, Cysl94Phe, Cys201Arg, Cys201Ser, Cys201Tyr, Cys206Ser, Arg207Cys, Cys212Arg, Cys212Trp, Cys212Ser, Cys212Tyr, Cys222Gly, Cys222Arg, Cys222Ser, Cys222Tyr, Cys222Thr, Cys222Phe, Cys224Arg, Cys224Tyr, Phe228Cys, Cys233Ser, Cys233Arg, Cys233Tyr, Cys233Trp, Cys240Tyr, Cys240Ser, Cys245Arg, Cys245Ser, Cys245Tyr, Cys251Arg, Cys251Tyr, Cys251Ser, Tyr258Cys, Cys260Arg, Cys260Phe, Cys260Tyr, Cys262Arg, Cys318Phe, Cys323Ser, Cys323Tyr, Cys323Trp, Cys329Tyr, Arg332Cys, Ser335Cys, Tyr337Cys, Cys338Arg, Cys338Ser, Cys340Phe, Cys349Ser, Cys435Arg, Cys435Tyr, Cys435Thr, Cys435Phe, Cys440Gly, Cys440Arg, Cys446Ser, Cys446Gly, Cys446Phe, Cys446Tyr, Arg449Cys, Cys455Arg, Cys455Phe, Cys455Tyr, Cys457Ser, Tyr465Cys, Cys466Tyr, Trpl003Cys, Cysl004Tyr, Cysl004Gly, Argl006Cys, Cysl009Phe, CyslO15Arg, CyslO15Trp, TyrlO21Cys, CyslO22Gly, and ArglO31Cys.

[0067] Medium Risk Mutations. NOTCH3C" mutations in EGFr domains 9, 10, 12-15, 17, 25, 27 and 32 are of significant interest. Patients with these “medium risk” mutations are at substantial risk of getting cerebral small vessel disease, they are at risk of getting CADASIL. Patients with these mutations may be treated according to the invention with Fazekas score of at least 1 in the deep or periventricular white matter. These patients are more likely to be treated if they have one or more risk factors, such as hypertension, smoking, diabetes or hypercholesterolemia. They would be even more likely to be treated if displaying signs or symptoms of CADASIL, including evidence of subcortical ischemic events, cognitive impairment, migraine with aura, and / or psychiatric disturbances (e.g., mood disturbances; apathy).

[0068] The following NOTCH3C" mutations are medium risk and fall into the medium-risk category:

[0069] Cys355Ser, Cys360Tyr, Cys377Gly, Cys377Tyr, Cys379Tyr, Cys379Phe, Cys379Ser, Gly382Cys, Cys388Arg, Cys388Tyr, Cys395Arg, Ser396Cys, Cys402Ser, Cys408Arg, Cys408Gly, Cys408Tyr, Cys408Trp, Ser414Cys, Cys417Arg, Cys419Ser, Cys419Arg, Cys419Trp, Gly420Cys, Arg421Cys, Arg427Cys, Cys428Arg, Cys428Tyr, Ser476Cys, Gly481Cys, Cys484Tyr, Cys484Arg, Ser492Cys, Cys493Ser, Cys493Arg, Cys495Tyr, Cys495Gly, Gly498Cys, Cys504Arg, Cys504Gly, Cys511Arg, Cys511Tyr, Cys511Phe, Cys516Phe, Cys516Tyr, Cys522Ser, Cys522Trp, Gly528Cys, Cys531Gly, Cys531Tyr, Arg532Cys, Cys533Ser, Cys542Arg, Cys542Tyr, Arg544Cys, Cys549Ser, Cys549Tyr, Cys549Arg, Cys551Arg, Cys554Phe, Gly557Cys, Arg558Cys, Cys559Trp, Cys568Tyr, Tyr574Cys, Arg578Cys, Cys579Tyr, Arg587Cys, Arg592Cys, Gly595Cys, Cys597Arg, Cys597Ser, Cys597Tyr, Tyr604Cys, Cys606Arg, Cys606Tyr, Cys606Ser, Cys606Phe, Arg607Cys, Cys608Tyr, Cys608Trp, Ser664Cys, Gly667Cys, Ser671Cys, Cys672Tyr, Arg680Cys, Cys971Tyr, Gly975Cys, Cys977Ser, Phe984Cys, Arg985Cys, Cys986Arg, Cys986Gly, Cys988Tyr, Cys988Arg, Cys988Phe, Gly994Cys, Cys997Gly, Cys997Ser, CyslO55Tyr, GlylO58Cys, CyslO61Arg, CyslO61Tyr, Glyl l65Cys, SerlO67Cys, TyrlO69Cys, Cysl070Ser, CyslO72Tyr, ArglO72Cys, ArglO76Cys, Cysl250Arg, Cysl250Gly, Cysl261Arg, Cysl261Tyr, Cysl261Trp, Argl262Cys, Glyl266Cys, Cysl275Ser, Cysl277Tyr, and Glyl283Cys.

[0070] Low Risk Mutations. NOTCH3C" mutations in EGFr domains 16, 18-20, 23, 24, 28-3 land 33 are of some interest. Patients with these “low risk” mutations are at risk of getting cerebral small vessel disease, they have a small risk of getting CADASIL. Patients with these mutations may be treated according to the invention with Fazekas score of at least 1 in the deep or periventricular white matter, but generally more will be required. For instance, they will have one or more risk factors, such as hypertension, smoking, diabetes or hypercholesterolemia and / or they will be displaying one or more signs or symptoms of CADASIL, including evidence of subcortical ischemic events, cognitive impairment, migraine with aura, and / or psychiatric disturbances (e.g., mood disturbances; apathy).

[0071] The following NOTCH3C" mutations are low risk and fall into the low-risk category:

[0072] Cys629Arg, Cys634Gly, Arg635Cys, Arg640Cys, Cys645Tyr, Cys654Gly, Cys654Tyr, Tyr710Cys, Arg717Cys, Cys720Tyr, Arg728Cys, Cys729Gly, Ser740Cys, Cys743Tyr, Cys749Tyr, Cys758Tyr, Arg767Cys, Cys769Tyr, Cys775Arg, Arg785Cys, Cys796Phe, Cys796Ser, Cys796Tyr, Cys798Tyr, Trp802Cys, Cys891Gly, Cys910Tyr, Cys910Arg, Cys912Ser, Tyr916Cys, Cys928Tyr, Ser932Cys, Cys933Gly, Cys939Ser, Cys939Trp, Gly942Cys, Cys948Phe, Cys950Gly, Arg951Cys, Gly953Cys, Cys959Ser, CyslO99Phe, Argl lOOCys, Cysl lO8Arg, Cysl l lOArg, Cysl l lOSer, Cysl l l9Tyr, Cysl l31Trp, Cysl l37Arg, Argl l43Cys, Tyrl l44Cys, Cysl l48Phe, Cysl l57Arg, Cysl l76Tyr, Cysl l82Trp, Argl l90Cys, Cysl l91Ser, Cysl l93Tyr, Argl201Cys, Cysl202Ser, Argl210Cys, Cysl222Gly, Cysl222Ser, Cysl222Arg, Argl231Cys, Cysl232Trp, Argl242Cys, Cysl250Arg, Cysl250Gly, Argl291Cys, Cysl293Phe, Cysl293Trp, Cysl298Phe, Cysl313Gly, Cysl313Ser, Cysl315Phe, Cysl315Trp, Cysl315Tyr, Cysl324Ser, and Cysl324Tyr.

[0073] NOTCH3C" mutations in EGFr domains 7, 21, 22, and 34 are of unknown significance in relation to the development of SVD or CADASIL and these mutations would only be treated incidental to significant clinical evidence of SVD and additional findings suggesting the involvement of N0TCH3 in the pathology of the disease, such as the presence of GOMs.

[0074] The following NOTCH3C" mutations are of unknown low risk and fall into the unknown-risk category:

[0075] Cys291Tyr, Cys311Ser, Phe833Cys, Cys846Tyr, Cys858Trp, Gly861Cys, Cys873Arg, Arg886Cys, Cysl350Gly, Argl360Cys, and Cysl372Trp.

[0076] CADASIL

[0077] In a specific embodiment, the patient with the N0TCH3 mutation has been diagnosed with CADASIL. CADASIL is suspected based on age (40-50), symptoms (including recurrent TIA or strokes, migraine, depression, emotional disturbance, pseudobulbar paralysis, mild cognitive impairment), family history, and MRI showing white matter lesions compatible with the disease (Yamamoto 2023). CADASIL diagnosis is confirmed by DNA testing of blood samples for characteristic mutations in the NOTCH3 gene or by identifying granular osmiophilic material (GOM) inclusions on a skin biopsy.

[0078] In another embodiment, the patient harboring a NOTCH3 mutation has not been diagnosed with CADASIL. In one embodiment, the patient has been diagnosed with cerebral small vessel disease (SVD).

[0079] In a further embodiment, the patient has granular osmiophilic material (GOM) on biopsy.

[0080] In one embodiment, the patient does not suffer from hypertension.

[0081] In another embodiment, the patient will have had at least one stroke or transient ischemic attack prior to treatment. In a specific embodiment, the patient may have had at least one lacunar stroke and / or at least one microbleed in the periventricular or deep white matter. Treatment of such patients with cerebral ischemic or hemorrhagic events with fasudil generally will not be in the acute phase of the ischemic event and, therefore, treatment will begin 30, 60 or 90 days or more after symptomatic resolution of the ischemic or hemorrhagic event.

[0082] In one embodiment, the CADASIL patient is East Asian. CADASIL patients from East Asia have a greater risk of intracerebral hemorrhage (ICH) than those in Caucasian populations.

[0083] In another embodiment, the patient treated with fasudil may have mild cognitive impairment. Patients with dementia will usually be excluded as they are likely to be beyond the point at which treatment may be effective.

[0084] In an embodiment, the patient does not exhibit wandering as wandering is not a typical symptom of the patients for whom treatment is contemplated.

[0085] In one embodiment, the patients to be treated according to the disclosed methods will have at least one white matter hyperintensity on imaging.

[0086] In some embodiments, the white lesions will be graded as having mild (punctate), moderate (some confluence) or severe white matter disease (large confluent areas). In an embodiment, the imaging is performed using MRI.

[0087] In specific embodiment, the MRI white matter lesions may be graded according to the Fazekas score and will have a minimum score of 1, 2 or 3 according to the following grading:

[0088] Periventricular white matter (PVWM)

[0089] 0 = absent

[0090] 1 = “caps” or pencil-thin lining

[0091] 2 = smooth “halo”

[0092] 3 = irregular periventricular signal extending into the deep white matter

[0093] Deep white matter (DWM)

[0094] 0 = absent

[0095] 1 = punctate foci

[0096] 2 = beginning confluence

[0097] 3 = large confluent areas

[0098] In one embodiment, only the deep white matter scale is applied, for a maximum score of 3.

[0099] In another embodiment, the full score is used, the patients will have a Fazekas score of 4, 5 or even 6.

[0100] In another embodiment, the two scales are presented separately. Thus, patients may have a PVWM score of 0, 1, 2 or 3 and a DWM score of 0, 1, 2 or 3, but both scores may not be 0. In a specific embodiment, exemplary scores, denoting PVWM:DWM, include 1 : 1, 0:2, 1 :2 and the like.

[0101] In another embodiment, patients with white lesions are graded using the Lesion Segmentation Tool (LST) ( ppUed- tiptms.de / isthpu)). This method automatically segments white matter signal abnormalities in the form of hyperintensities in the T2-weighted fluid-attenuated inversion recovery (FLAIR) MRI sequence. (Schmidt 2011). In a specific embodiment, the proposed cut-off value to classify low and high Fazekas WMSA burden is 0.00496 for LST. (Cedres 2020).

[0102] In yet another embodiment, patients with white lesions are evaluated using FreeSurfer software (surfer.nmr.mgh.harvard.edu) (Fischl 2002). This method is increasingly used to automatically segment WMSA in the form of hypointensities in the T1 -weighted MRI sequence. Hypointense WMSA seems to be related to poorer white matter integrity as compared to hyperintense white matter signal abnormalities. In a specific embodiment, the proposed cut-off value to classify low and high Fazekas WMSA burden is 0.00321 for FreeSurfer WMSA. (Cedres 2020).

[0103] In an embodiment, the patient may show evidence of cerebral hemodynamic dysfunction, such as abnormally reduced cerebrovascular reactivity, or regional or global cerebral hypoperfusion.

[0104] In another embodiment, the patient may show evidence of abnormally increased vascular myogenic tone.

[0105] In a further embodiment, the patient typically will have elevated rho kinase activity. In one embodiment, the ROCK2 activity is elevated.

[0106] In an embodiment, the patient may have mild vascular cognitive impairment (VCI) or vascular dementia (VaD). Mild VCI is impairment in at least one cognitive domain, and mild to no impairment in instrumental activities of daily living (IADL) or basic activities of daily living ADL, respectively (independent of the motor / sensory sequelae of the vascular event). VaD (aka major VCI) is clinically significant deficits of sufficient severity in at least one cognitive domain (deficits may be present in multiple domains) and severe disruption to IADLS / ADLS (independent of the motor / sensory sequelae of the vascular event). IADLS are those activities that allow an individual to live independently in a community; the major domains of IADLs include cooking, cleaning, transportation, laundry, and managing finances. In contrast with IADLs, ADLs are necessary for basic functional living; the major domains of ADLs are feeding, dressing, bathing, and walking.

[0107] In another embodiment, the patient may have decreased executive function or reduced processing speed. In a specific embodiment, the patient with cognitive decline an impaired working memory is male.

[0108] In a further embodiment, the patient may have memory deficits, including in working memory and long-term memory. N0TCH3 mutations in other diseases

[0109] N0TCH3 truncating mutations and deletions or insertions have also been reported in other diseases although the causation is unclear. Cysteine-sparing N0TCH3 mutations A198E, G284A, and A284T have been associated with Alzheimer’s and other dementias. (Patel 2019). NOTCH3 mutations Hl 70R, P496L, V764A, H1133Q, V1183M, L1518M, D1823N, and V1952M have been associated with age-related cerebral small vessel disease. (Schmidt 2011).

[0110] In one embodiment, dementia patients harboring the foregoing NOTCH3 mutations are treated with the fasudil or M3 according to the disclosure.

[0111] Patients with lateral meningocele syndrome (LMS) have mutations in the last NOTCH3 coding exon. One patient had a 26 bp deletion G2154fsTer78), two carried the same single base pair insertion (c.6692_93insC, p.P223 IfsTerl 1), and three individuals had a nonsense point mutation at c.6247A > T (pK2083*), C.6663C > G (p.Y2221*) or C.6732C > A, (p.Y2244*).

[0112] In another embodiment, LMS patients are treated with fasudil or M3 according to the disclosed method.

[0113] NOTCH3 mutations also have been linked to myofibromatosis, which is a common proliferative fibrous tumor of childhood. The tumors have features of both differentiated fibroblasts and smooth muscle cells (myofibroblasts). Mutation at residue L1519P of NOTCH3 was associated with this disorder. (U.S. 10,155,995).

[0114] Other pericytic tumors including myopericytomas, myopericytomatoses, angioleiomyomas, and glomus tumors have been associated with NOTCH3 mutations. The mutations include but are not limited to A1480S / T, D1481N, G1482S, T1490A, E1491K, G1494S, and V1512A, which are all in exon 25.

[0115] In an embodiment, patients having pericytic tumors harboring NOTCH3 mutations are treated with fasudil or M3 according to the present disclosure. NOTCH3 overexpression and fusion are also linked to metastasis in breast cancer, including estrogen receptor positive breast tumors. In addition, expression of activated NOTCH3 was shown to promote choroid tumors, retinal glial tumors, and invasive glioma formation in mice.

[0116] Fasudil

[0117] The disclosed methods contemplate the administration of the rho kinase (ROCK) inhibitor fasudil or its metabolite M3 in the treatment of a disease or condition associated with mutated N0TCH3. Two mammalian ROCK homologs are known, R0CK1 (aka ROKP, Rho-kinase P, or pl60ROCK) and R0CK2 (aka ROKa) (Nakagawa 1996). In humans, the genes for both R0CK1 and R0CK2 are located on chromosome 18. The two ROCK isoforms share 64% identity in their primary amino acid sequence, whereas the homology in the kinase domain is even higher (92%) (Jacobs 2006; Yamaguchi 2006). Both ROCK isoforms are serine / threonine kinases and have a similar structure.

[0118] The isoquinoline derivative fasudil was the first small molecule ROCK inhibitor developed by Asahi Chemical Industry (Tokyo, Japan). The characteristic chemical structure of fasudil consists of an isoquinoline ring, connected via a sulphonyl group to a homopiperazine ring. Fasudil is a potent inhibitor of both ROCK isoforms.

[0119] In vivo, fasudil is subjected to hepatic metabolism to its active metabolite hydroxyfasudil (aka, M3). Other examples of isoquinoline derived ROCK inhibitors include dimethylfasudil, ripasudil, Y-27632, and Y39983.

[0120] Fasudil Hydroxyfasudil

[0121] The oral administration of fasudil, in contrast to intravenous, results in nearly 100% conversion to M3, whereas intravenous administration results in a mixture of the parent fasudil and the metabolite M3. While both fasudil and M3 are potent ROCK inhibitors, they do not have the same specificity on the two different ROCK isoforms and they do not have the same specificity against off-target kinases, like protein kinase G. Thus, the administration of intravenous fasudil is not predictive of the administration of oral fasudil. Moreover, rodents metabolize oral fasudil differently from humans and they are exposed to a mixture of fasudil and M3 after oral administration. In other words, oral fasudil in rodents mimics intravenous fasudil in humans, but is different from oral fasudil in humans.

[0122] Fasudil has been reported to have a number of potentially beneficial effects in conditions of the CNS, although no there is no Level 1 evidence of treatment effect in any condition, meaning any potential medical application remains speculative.

[0123] The hemodynamic effects of fasudil are mediated by two principal mechanisms. It inhibits calcium-independent, ROCK-mediated phosphorylation of myosin light chain phosphatase, a principal player in vascular myogenic tone. Thus, in situations where ROCK activity is elevated, it can provide an additional vasodilatory response on top of vasodilators that rely on calcium mobilization (e.g., CO, NO, PDE inhibitors, calcium channel blockers). In addition, ROCK elevation is often responsible for endothelial dysfunction by inhibiting the expression and activity of endothelial nitric oxide synthase. Hence, fasudil can improve both the vasodilatory signal coming from the endothelium and the dynamics of the response by the vascular smooth muscle cells.

[0124] As used herein, the term fasudil means the free base, salts, hydrates, ion pairs, polymorphs and metabolites thereof, such as hydroxyfasudil (aka, M3), also including the free base, salts, hydrates, polymorphs and ion pairs of metabolites. Specific dosing information refers to fasudil hydrochloride hemihydrate and molar equivalents of other forms or metabolites.

[0125] Dosage and Administration In accordance with the treatment methods, a therapeutically effective amount of fasudil or a pharmaceutically acceptable salt thereof, such as fasudil hydrochloride hemihydrate, for administration one or more times a day may comprise from about 10 mg to about 1000 mg.

[0126] Fasudil hydrochloride hemihydrate, for example, is suitably administered in a daily amount of about 10 mg to about 500 mg, about 10 mg to about 400 mg, about 10 mg to about 200 mg, about 10 mg to about 100 mg, about 20 mg to about 10 mg.

[0127] One dosing regimen involves the treatment with 25, 30, 40, 60 or 80 mg of fasudil hydrochloride hemihydrate three times per day using an immediate-release formulation, for a total daily dose of 70 to 240 mg per day. Most preferred dosing exceeds a daily dose of 70 mg, with most preferred ranges for daily dosing being 90 to 240 mg administered in two or three equal amounts during the day.

[0128] A particularly preferred daily dose is 180 mg / day administered 60 mg TID immediate release tablets. Fasudil is most preferably administered orally in accordance with the foregoing using an immediate release formulation. In another embodiment, fasudil is administered in a dose of 240 mg / day administered 80 mg TID immediate-release tablets.

[0129] Another embodiment involves the treatment with 90-360 mg of fasudil hydrochloride hemihydrate once per day in an extended-release dosage form. Treatment with an extended- release total daily dose of 90 mg fasudil hydrochloride hemihydrate once per day is preferred.

[0130] In one embodiment, the fasudil is administered to the patient in a dose from about 90 to 240 mg / day as an immediate release formulation.

[0131] In another embodiment, the fasudil is administered to the patient in an extended-release formulation, wherein the maximum plasma concentration and area under the curve in plasma over 24 hours do not exceed by more than 15% what is obtained using a dosing regimen of 70 to 240 mg / day as an immediate release formulation.

[0132] Methods of administering compositions according to the invention would generally be continued for at least one day. Some preferred methods treat for up to 30 days or up to 60 days or even up to 90 days or even more. Treatment for more than 60 days is preferred and treatment for at least 6 months is particularly preferred. The precise duration of treatment will depend on the patient’s condition and response to treatment. Pharmaceutical Compositions

[0133] In a preferred embodiment, fasudil is administered in an oral dosage form. Pharmaceutical compositions of fasudil or its salts or hydrates for oral administration may be in the form of tablets or capsules and may be immediate-release formulations or may be controlled- or extended-release formulations, which may contain pharmaceutically acceptable excipients, such as com starch, mannitol, povidone, magnesium stearate, talc, cellulose, methylcellulose, carboxymethylcellulose and similar substances. The oral pharmaceutical composition comprising fasudil or a salt or hydrate thereof may comprise one or more pharmaceutically acceptable excipients, which are known in the art.

[0134] Formulations also include oral films, orally disintegrating tablets, effervescent tablets and granules or beads that can be sprinkled on food or mixed with liquid as a slurry or poured directly into the mouth to be washed down.

[0135] In one embodiment, the fasudil is administered in a taste-masked oral formulation. An example of a bitterness masker that can bind a rho kinase inhibitor is an ion exchange resin, for example, as disclosed in U.S. Application Serial Nos. 18 / 171,007; 18 / 171,070; and 18 / 171,077.

[0136] In another embodiment, the fasudil is orally administered in the form of liposomes.

[0137] Pharmaceutical compositions containing fasudil, or salts or hydrates thereof can be prepared by any method known in the art of pharmaceutics. In general, such preparatory methods include the steps of bringing fasudil or a pharmaceutically acceptable salt or hydrate thereof into association with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping, and / or packaging the product into a desired single- or multi-dose unit.

[0138] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition used in accordance with the methods of the present invention may comprise between 0.001% and 100% (w / w) active ingredient.

[0139] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Excipients such as coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

[0140] In certain embodiments, the pharmaceutical composition used in the methods of the present invention may comprise a diluent. Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

[0141] In certain embodiments, the pharmaceutical composition used in the methods of the present invention may comprise a granulating and / or dispersing agent. Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, crosslinked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0142] In certain embodiments, the pharmaceutical composition used in the methods of the present invention may comprise a binding agent. Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM.RTM.), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0143] In certain embodiments, the pharmaceutical composition used in the methods of the present invention may comprise a preservative. Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0144] In certain embodiments, the pharmaceutical composition used in the methods of the present invention may comprise an antioxidant. Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0145] In certain embodiments, the pharmaceutical composition used in the methods of the present invention may comprise a chelating agent. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. In certain embodiments, the pharmaceutical composition may comprise a buffering agent together fasudil or salt or hydrate thereof. Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0146] In certain embodiments, the pharmaceutical composition used in the methods of the present invention may comprise a lubricating agent. Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0147] In other embodiments, the pharmaceutical composition of containing fasudil or salt or hydrate thereof will be administered as a liquid dosage form. Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0148] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

[0149] Some compositions of the invention relate to extended- or controlled-release formulations. These may be, for example, diffusion-controlled products, dissolution-controlled products, erosion products, osmotic pump systems or ionic resin systems. Diffusion-controlled products comprise a water-insoluble polymer which controls the flow of water and the subsequent egress of dissolved drug from the dosage from. Dissolution-controlled products control the rate of dissolution of the drug by using a polymer that slowly solubilizes or by microencapsulation of the drug - using varying thicknesses to control release. Erosion products control release of drug by the erosion rate of a carrier matrix. Osmotic pump systems release a drug based on the constant inflow of water across a semi permeable membrane into a reservoir which contains an osmotic agent. Ion exchange resins can be used to bind drugs such that, when ingested, the release of drug is determined by the ionic environment within the gastrointestinal tract.

[0150] Combination Therapy

[0151] The inventive methods can also employ fasudil in combination with one or more other vasoactive agents. These may be administered with fasudil in a single, fixed dose combination composition or fasudil and the other vasoactive agent may be administered as separate compositions but as part of a common treatment regimen. Exemplary other vasoactive agents include angiotensin converting enzyme (ACE) inhibitors, an angiotensin receptor blocker (ARB), calcium channel blockers (CCBs), phosphodiesterase (PDE) inhibitors and nitrates ARBs include, but are not limited to, valsartan, losartan, azilsartan, irbesartan, olmesartan, telmisartan, and fimasartan,

[0152] ACE inhibitors include, but are not limited to, alacepril, captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril, cilazapril, and fosinopril

[0153] CCBs include, but are not limited to, amlodipine, aranidipine, azelnidipine, bamidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, fendiline, gallopamil, verapamil and diltiazem.

[0154] PDE is include, but are not limited to, sildenafil, tadalafil, vardenafil, udenafil, avanafil, dipyridamole, amrinone, milrinone, enoximone, anagrelide, cilostazol and pimobendane.

[0155] Nitrates include, but are not limited to, amyl nitrite, glyceryl trinitrate, isosorbide mononitrate (with or without hydralazine), nitroglycerin and pentaerythritol tetranitrate.

[0156] The inventive methods can also employ fasudil in combination with one or more other cognitive agents such as the cholinesterase inhibitors (e.g., donepezil, galantamine, rivastigmine) or memantine.

[0157] The inventive methods can also employ fasudil in combination with one or more other antimigraine agents including beta-blockers, triptans including sumatriptan, naratriptan, zolmitriptan, rizatriptan, acetaminophen with codeine or hydrocodone, morphine, tramadol, propranolol, amitriptyline, pizotifen, acetazolamide, and simple analgesics such as aspirin, acetaminophen, and ibuprofen.

[0158] Assessing Outcomes of Fasudil Treatment

[0159] The following measures may be used to assess patients treated with fasudil or M3 according to the disclosure. A patient will typically have a deficit in one or more of these assessments prior to treatment. Treatment will generally improve performance on at least one of these assessments. Typical improvements are in 5%, 10%, 15%, 20%, or 25% or more. Other improvements can be mor descriptive, such as severe may be come moderate or moderate may become mild or mild may become asymptomatic. In some cases, assessment scores or categories will stabilize or the rate of deterioration is slowed relative to expectation or control patients.

[0160] One of ordinary skill in the art will be familiar with these measures and others that are useful in assessing patient deficits and outcomes of treatment. The invention contemplates improvements in one or more of the below assessments with fasudil or M3 treatment.

[0161] Imaging. In one embodiment, patients with N0TCH3 mutations can be monitored he MRI and other imaging described above. White matter burden, as assessed by MRI, increases over time, and is linked with cognitive and functional decline.

[0162] In an embodiment, patients treated according to the invention may be seen to have a slowing of the rate of white matter lesion accumulation. Treated patients may also experience a reduction in the frequency of lacunar strokes and other cerebral ischemic events relative to their own clinical history or relative to an untreated cohort.

[0163] In another embodiment, patients with white lesions may be diagnosed or monitored using diffusion tensor imaging, which measures water diffusion inside and outside cerebral lesions. Diffusion-tensor imaging detects ultrastructural tissue damage even in areas that appear normal in conventional MRI. The mean diffusivity was found to be the main predictor of clinical progression in patients with CADASIL. (Holtmannspotter 2005).

[0164] Other methods of assessment commonly used by providers include CT angiography,

[0165] Neuropsychological and disability assessments. The Modified Rankin Scale is a disability scale often used in patients with small vessel disease, including CADASIL. It is a sevenpoint scale (0-6), ranging from no symptoms to dead:

[0166] 0 - No symptoms.

[0167] 1 - No significant disability. Able to carry out all usual activities, despite some symptoms.

[0168] 2 - Slight disability. Able to look after own affairs without assistance, but unable to carry out all previous activities. 3 - Moderate disability. Requires some help, but able to walk unassisted.

[0169] 4 - Moderately severe disability. Unable to attend to own bodily needs without assistance, and unable to walk unassisted.

[0170] 5 - Severe disability. Requires constant nursing care and attention, bedridden, incontinent.

[0171] 6 - Dead.

[0172] The Barthel Index can also be used to monitor disease progression. Barthel Index is a 100- point scale that measures activities of daily living. A score of 100 is fully independent. Feeding (0-10 points), bathing (0-5 points), grooming (0-5 points), dressing (0-10 points), bowel control (0-10 points), bladder control (0-10 points), toilet use (0-10 points), transfers (bed to chair and back) (0-15 points), mobility on level surfaces (0-15 points), and stairs (0- 10 points).

[0173] Tests of working memory useful for assessing outcomes also include the Digit Span test, which involves the immediate recall of digit strings, forwards and backwards (WAIS-III 1997).

[0174] The Wechsler Memory Scale III (WMS-III 1997) or WMS-IV can also be used. The memory indices are: Auditory Immediate, Visual Immediate, Immediate Memory, Auditory Delayed, Visual Delayed, Auditory Recognition Delayed, General Memory and Working Memory. Long term memory tests on the WMS include the Logical Memory test and the Visual Reproduction test. The Logical Memory test measures the immediate and delayed recall of short stories. It can also be used to monitor disease progression. The Visual Reproduction test measures the immediate and delayed reproduction of line drawings.

[0175] The Brief Memory and Executive Test (BMET) is a cognitive screening tool designed and previously validated to be sensitive to the cognitive deficit seen in sporadic SVD and shown to be sensitive to the cognitive deficit in CADASIL. (Brookes 2015). The test comprises 8 tasks (domains) that provide measures that form 2 subscales and an overall score. The first subscale, Executive Functioning and Processing Speed, is calculated using the tasks Letter- Number Matching, Motor Sequencing, Letter Sequencing, and Number-Letter Sequencing. The second subscale, Orientation and Memory, is made up of the Orientation, 5-item Repetition, 5-item Recall, and 5-item Recognition tasks. The measures from each task are transformed into scales that have a maximum of 2, giving a maximum total of 8 on each subscale and 16 overall. These scorings are age-adjusted, with each measure adjusted for the age of the participant.

[0176] Processing speed in N0TCH3 mutation patients can be assessed by the BIRT Memory and Information Processing Battery, the Digit Symbol test, and the Grooved Pegboard test. The BIRT involves the timed cancellation of the second highest of five two-digit numbers (Coughlin 2007). The Digit Symbol test is a timed transcoding test (WAIS-III 1997). The Grooved Pegboard involves picking up, rotating and placing small pegs while being timed (Matthews 1964, Dawson 2010). The DSST is a paper-and-pencil cognitive test presented on a single sheet of paper that requires a subject to match symbols to numbers according to a key located on the top of the page. The subject copies the symbol into spaces below a row of numbers. The number of correct symbols within the allowed time, usually 90 to 120 seconds, constitutes the score.

[0177] The DSST measures a range of cognitive operations. DSST may prove to be a practical and effective method for use in clinical practice to detect decrements in cognitive processing over time. Good performance on the DSST requires intact motor speed, attention, and visuoperceptual functions, including scanning and the ability to write or draw (i.e., basic manual dexterity). Performance might also be affected by associative learning. For example, if pairings are rapidly learned following the first few trials, performance speed will improve because the subject will not need to refer to the key to check the accuracy of each pairing. The decision to consciously engage in this learning strategy to improve performance speed calls for the executive functions of planning and strategizing. Working memory, another executive function, is likely required to hold in mind the task rules and for the continual updating of required symbol-digit pairs. CADASIL patients score approximately 1 SD below normal on the DSST.

[0178] Symbol coding paradigms comparable to the DSST (with different symbols) are included as subtests in the Brief Assessment of Cognition in Schizophrenia and the Repeatable Battery for the Assessment of Neuropsychological Status. The Symbol Digit Modalities Test uses essentially the same method, except in reverse; instead of drawing symbols that match digits, the subject is required to write the matching digit in the blank.9 This version of the coding paradigm has the advantage that it can be easily altered to allow oral responses in disorders that affect motor functioning. Computerized tests offer another option for monitoring change over time, for example the Cambridge Neuropsychological Test Auto-mated Battery developed by Cambridge Cognition, Cognigram, a version of the Cogstate Brief Battery, and THINC-IT (a freely available computerized test). (Jaeger 2018)

[0179] Executive Function in NOTCH3 mutation patients can be tested using the Trail Making Test (the entire test or each individual part A or B), the DSST, the SL-Verbal Fluency test and the modified Wisconsin Card-Sorting Test. The Trail Making Test involves sequencing alternating letters and numbers using pen and paper (Reitan 1955, Mitrushina 2005; eatspeakthink.com / wp-content / uploads / 2018 / 06 / IowaTrailMaking. pdf). The SL-Verbal Fluency tests the ability of the subject to generate words beginning with certain letters in a certain period of time (Delis 2001). The modified Wisconsin Card-Sorting Test involves asking the subject to sort a set of cards without instructions; they are given feedback on their performance and then adapt their strategy (Nagahama 2003). It tests the flexibility and the ability to learn the rules of the game. The Stroop test can also assess executive function. It assesses the reaction to conflicting stimuli, just as the word red written in blue letters.

[0180] Cognitive assessments for dementia also can be used including the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MocA). The maximum score for the MMSE is 30. A score of 25 or higher is classed as normal. If the score is 24 or below, the result is usually considered to be abnormal, indicating possible cognitive impairment. In one embodiment, the patient may be selected for treatment based on their MMSE, and / or their MMSE will improve with treatment according to the invention. The maximum score for the MoCA is 30. A score of 26 or higher is classed as normal. If the score is below 26, the result is usually considered to be abnormal, indicating possible cognitive impairment. In one embodiment, the patient may be selected based on their MoCA and / or their MoCA will improve with treatment according to the invention.

[0181] Migraines. Migraines in patients with NOTCH3 mutations can be assessed in a number of ways. One measure is frequency (i.e., how many times per day / week / month / year). Another is severity. Severity may be assessed on simple visual-analog scale (e.g., 0-10 or 0-100) or using a functional assessment, such as The Functional Disability Scale (FDS). The FDS asks subjects to rate the performance of daily activities using 4 response options ranging from 0 (no disability, able to function normally) to 3 (severely impaired, cannot do all or most things, bed rest may be necessary). The Migraine Specific Quality of Life Questionnaire or The Activity Impairment in Migraine Diary (AIM-D) may also be used to assess impact on functionality.

[0182] In one embodiment, treatment of the patient with fasudil or M3 reduces the frequency of migraines and / or severity of the migraines.

[0183] Vascular assessments. In addition, patients may show improvements upon treatment with fasudil or M3 on hemodynamic parameters, such are cerebrovascular reactivity (the ability of the blood vessels to dilate and constrict in response to stimuli) or cerebral hypoperfusion. Improvements may also be seen in blood vessel patency. In another embodiment, patients with hypertension may improve (lower blood pressure or return to normal).

[0184] In another embodiment, retinal vascular density (VD) measured using optical coherence tomography with angiography (OCTA) has been suggested as a potential marker of intracerebral vascular changes. Specifically, a reduction in retinal VD at a younger age is associated with CADASIL patients. Fasudil or M3 treatment may prevent VD reduction.

[0185] In a further embodiment, coronary symptoms including revealed diffuse vessel wall irregularities, vascular thickening, and mixed non-calcified / calcified coronary lesions with intermediate stenosis will improve with treatment.

[0186] Caregiver assessment. Caregiver assessments also can be used to monitor progression of N0TCH3 patients. NPI-Q. In one Embodiment, the patient having NOTCH3-associated small vessel disease treated with the ROCK inhibitor such as fasudil to is assessed using the Neuropsychiatric Inventory Questionnaire (NPI-Q).

[0187] The NPI-Q is designed to be a self-administered questionnaire completed by informants about patients for whom they care. Each of the twelve NPI-Q domains contains a survey question that reflects cardinal symptoms of that domain. Initial responses to each domain question are “Yes”(present) or “No” (absent). Thus, a total score of 36 points would indicate maximum severity in all parameters considered.

[0188] If the response to the domain question is “No,” the informant goes to the next question. If “Yes,” the informant then rates both the Severity of the symptoms present within the last month on a 3 -point scale and the associated impact of the symptom manifestations on them (i.e. Caregiver Distress) using a five-point scale. The NPI-Q provides symptom Severity and Distress ratings for each symptom reported, and total Severity and Distress scores reflecting the sum of individual domain scores. The NPI-Q Distress ratings have a maximum score of 60 (a score of 5 on all questions).

[0189] Example

[0190] Approximately 40 patients diagnosed as having CADASIL are recruited meeting the following

[0191] Inclusion Criteria:

[0192] • Patients 30-65 years of age

[0193] • Diagnosis of CADASIL based on clinical symptoms, MRI, and genetic analysis. Specifically, patients having a known or new mutation in the NOTCH3 gene responsible for an odd number of cysteine residues in at least one EGFR domain OR having positive skin biopsy showing typical granular osmiophilic material (GOM) in the vascular wall of small vessels with electron microscopy. Patients without a NOTCH3 sequence at inclusion will have it sequenced as part of the study.

[0194] • An alternate to this criterion is: Diagnosis of CADASIL as documented by:

[0195] (a) genetic testing of the NOTCH3 gene showing the presence of a high- or medium-risk NOTCH3 mutation per Hack (2023) and Fazekas deep or superficial white matter score >

[0196] OR

[0197] (b) genetic testing of the NOTCH3 gene showing the presence of a low-risk NOTCH3 mutation per Hack (2023), Fazekas deep or superficial white matter score >1 and at least one of the following clinical signs / symptoms (in the opinion of the investigator and following review and approval by the Sponsor:

[0198] • Subcortical ischemic events; and / or

[0199] • Cognitive impairment; and / or

[0200] 1To be confirmed by a screening MRI, unless a standard of care MRI obtained within previous X months is available. Migraine with aura

[0201] • MMSE >18

[0202] • Fazekas Grade 1-3

[0203] • Ability to comply with study procedures

[0204] Exclusion Criteria. Patients will be excluded if they have any of the following:

[0205] • A symptomatic stroke in the preceding 90 days

[0206] • Any significant neurological disease / conditions other than CADASIL

[0207] • Focal lesions that may be responsible for an impaired cognitive status (e.g. prior large vessel stroke, infectious disease, space-occupying lesion, normal pressure hydrocephalus)

[0208] • Any other diseases / conditions that may affect compliance with the protocol, such as unmanaged psychiatric conditions or alcohol or substance abuse

[0209] • Inability to tolerate MRI scanning

[0210] A physical examination, vital signs and standard laboratory assessments will be conducted at each study visit. A gluteal muscle biopsy will be taken at baseline and 6 months.

[0211] Patients will be randomized to either placebo or fasudil treatment. On day one, patients will come to the clinic and have a baseline MRI sequence performed. This will include structural sequences to assess white matter burden, examine brain structures, such as the Virchow Robins spaces and to provide a baseline for assessments of changes, such as atrophy, at later time points. In addition, sequences will be performed to assess regional and total cerebral blood flow (arterial spin labeling), reactivity to CO2 (blood oxygenation level dependent MRI), reactivity in response to a visual or motor stimulus (blood oxygenation level dependent MRI), 2D Phase Contrast will be used to look at pulsatility (carotid, CSF and veins) and diffusivity in the white matter tracts (diffusion weighted imaging). After the first MRI session, patient are treated with 60 mg of fasudil or matching placebo orally and one hour after treatment, the functional MRI scans are repeated.

[0212] One day to one month after the first MRI visit, patients return to the clinic. The pre-treatment functional MRI scans are performed before and after treatments again, but treatment assignments are reversed from the first MRI visit. Patients will remain on this treatment assignment for 12 months, with patients on drug taking 60 mg fasudil orally, three times per day for a daily dose of 180 mg. Patients in the placebo group will receive matching placebo.

[0213] Patients will return to the clinic at one month, three months and six months. Structural MRI will be repeated at 3 months and 6 months. Functional MRI will be repeated at all visits. Physical and laboratory assessments will be done at each visit, along with standard cognitive testing to include the Trail-Making Test B and the Wisconsin Card-Sorting Test for executive function and the Digit Symbol Substitution Test for cognition including processing speed and / or executive function.

[0214] The population shows an improvement in gray matter cerebral perfusion, along with improvements in white matter cerebral perfusion and overall cerebral perfusion that become significant at the one-month visit (after 3 scans) in the drug treatment arm, as compared to the placebo arm. Enhanced reactivity to CO2 and visual or motor stimulus is observed with drug treatment. Improvements in processing speed are evidence in the drug group after a single treatment and persist out to the 6-month visit. Executive function in the drug treated group is stable through 6 months, whereas the placebo group deteriorates; executive function in the treatment group is better than the placebo group at 6 months. Biopsies show a decrease in rho kinase activity in smooth muscle cells, along with activation of markers of survival / anti - apoptosis in the drug treated group as compared to the placebo group, suggesting that fasudil is alleviating apoptotic stress on the vasculature. This is supported by MRI pulsatility measurements that indicate less vessel stiffness in the drug treated group as compared to the placebo group.

[0215] List of References

[0216] All the references herein and below are incorporated herein by reference in their entireties.

[0217] Brookes RL et al., The Brief Memory and Executive Test (BMET) for detecting vascular cognitive impairment in small vessel disease: a validation study. BMC Med. 2015; 13:51.

[0218] Cedres N et al., Predicting Fazekas scores from automatic segmentations of white matter signal abnormalities. Aging. 2020; 12(1): 894-901.

[0219] Cho et al., Association of NOTCH3 Variant Position With Stroke Onset and Other Clinical Features Among Patients With CADASIL. Neurology. 2022; 99(5): e430-439

[0220] Coughlan AK, Oddy M, Crawford JR. The BIRT Memory and Information Processing Battery (B-MIPB). Wakefield, UK: The Brain Injury Rehabilitation Trust (BIRT); 2007.

[0221] Dawson JD, Uc EY, Anderson SW, Johnson AM, Rizzo M. Neuropsychological predictors of driving errors in older adults. J Am Geriatr Soc. 2010; 58: 1090-1096. doi: 10.1111 / j .1532- 5415.2010.02872.x PMID: 20487082

[0222] Delis DC, Kaplan E, Kramer JH. Delis-Kaplan Executive Function Scale (D-KEFS). San Antonio, TX: The Psychological Corporation; 2001.

[0223] Fischl B et al., Whole brain segmentation: automated labeling of neuroanatomical structures in the human brain. Neuron. 2002; 33(3): 341-55.

[0224] Hack RJ et al., CADASIL (2019) In M. P. Adam, H. H. Ardinger, R.A. Pagon, S. E. Wallace, L. J. H. Bean, K. Stephens, & A. Amemiya (Eds.), GeneReviews((R)). University of Washington, Seattle.

[0225] Holtmannspotter M, Peters N, Opherk C, Martin D, Herzog J, Bruckmann H, et al. Diffusion magnetic resonance histograms as a surrogate marker and predictor of disease progression in CADASIL: a two-year follow-up study. Stroke. 2005;36:2559-2565.

[0226] Jacobs M, Hayakawa K, Swenson L, Bellon S, Fleming M, Taslimi P, Doran J, The structure of dimeric ROCK I reveals the mechanism for ligand selectivity. J Biol Chem. 2006; 281(1): 260-68.

[0227] Kamei S, Oishi M, Takasu T. 1996a. Evaluation of fasudil hydrochloride treatment forwandering symptoms in cerebrovascular dementia with 3 IP-magnetic resonance spectroscopy and Xe-computed tomography. Clin Neuropharmacol. 1996; 19:428-38.

[0228] Matthews CG, Klove H. Instruction manual for the Adult Neuropsychology Test battery.

[0229] University of Wisconsin Medical School; 1964.

[0230] Mitrushina M, Boone KB, Razani J, D’Elia LF. Handbook of Normative Data for Neuropsychological Assessment. 2nd ed. USA: Oxford University Press; 2005.

[0231] Morris HE et al., Notch3 signalling and vascular remodelling in pulmonary arterial hypertension. Clin Sci (Lond). 2019; 133(24): 2481-2498. Morris, Hypertension. 2019; 74:AP145.

[0232] Muino E et al., Systematic Review of Cysteine-Sparing NOTCH3 Missense Mutations in Patients with Clinical Suspicion of CADASIL. Int J Mol Sci. 2017;18(9): 1964.

[0233] Nagahama Y, Okina T, Suzuki N, Matsuzaki S, Yamauchi H, Nabatame H, et al. Factor structure of a modified version of the Wisconsin card sorting test: an analysis of executive deficit in Alzheimer’s disease and mild cognitive impairment. Dement Geriatr Cogn Disord. 2003; 16: 103-112. doi: 10.1159 / 000070683 PMID: 12784035.

[0234] Neves KB et al., ER stress and Rho kinase activation underlie the vasculopathy of CADASIL. JCI Insight. 2019; 4(23): el31344.

[0235] Patel D et al., Association of Rare Coding Mutations With Alzheimer Disease and Other Dementias Among Adults of European Ancestry. JAMA Netw Open. 2019; 2(3): el91350.

[0236] Rajendran I et al., A case of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) presenting as post-infectious manifestation of SARS-CoV-2 infection. BJR case reports. 2021; 7(3):

[0237] Reitan RM. The relation of the trail making test to organic brain damage. J Consult Psychol. 1955; 19:393-394. PMID: 13263471.

[0238] Rosenblum JS et al., Acute worsening of CADASIL in a patient with COVID-19 infection: illustrative case. J Neurosurg Case Lessons. 2022; 4(26): CASEE22413.

[0239] Rutten et al., The effect of NOTCH3 pathogenic variant position on CADASIL disease severity: NOTCH3 EGFr 1-6 pathogenic variant are associated with a more severe phenotype and lower survival compared with EGFr 7-34 pathogenic variant. Genet Med. 2019; 21(3): 676-82.

[0240] Schmidt H et al., Genetic variants of the NOTCH3 gene in the elderly and magnetic resonance imaging correlates of age-related cerebral small vessel disease. Brain. 2011; 134(Ptl 1): 3384-97.

[0241] Schoemaker D and Arboleda-Velasquez JF. Notch3 Signaling and Aggregation as Targets for the Treatment of CADASIL and Other NOTCH3 -Associated Small-Vessel Diseases. Am J Pathol. 2021; 191(11): 1856-70.

[0242] Stojanov D et al., Imaging characteristics of cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL). Bosn J Basic Med Sci. 2015; 15(1): 1-8.

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[0246] U.S. 10,155,995

Claims

Claims1. A method of treating a human patient having a mutation in the N0TCH3 gene, comprising administering to said patient a therapeutically effective amount of oral fasudil or hydroxyfasudil.

2. The method of claim 1, wherein the patient has been diagnosed with CADASIL.

3. The method of claim 1, wherein the patient has not been diagnosed with CADASIL.

4. The method of claim 1, wherein the patient has been diagnosed with cerebral small vessel disease.

5. The method of any of the preceding claims, wherein the N0TCH3 mutation is a gain of function mutation that results in odd number of cysteines in an extracellular domain.

6. The method according to any of the preceding claims wherein the mutation is not a gain of function mutation.

7. The method any one of the preceding claims, wherein the patient has at least one white matter hyperintensity.

8. The method any one of the preceding claims, wherein the patient has a Fazekas score of at least 1 in the deep or periventricular white matter.

9. The method any one of the preceding claims, wherein the patient has a deficit on the DSST or SDDT.

10. The method of any of the preceding claims, wherein the patient has granular osmiophilic material.

11. The method of any of the preceding claims, wherein the patient has had at least one stroke or a transient ischemic attack.

12. The method of claim 9, wherein the patient has a pre-existing cerebral infarction.

13. The method of claim 10, wherein the pre-existing infarction is from a lacunar ischemic stroke and any prior cerebral hemorrhage was micro-hemorrhage.

14. The method of claim 9, wherein the patient has had at least one stroke or a transient ischemic attack at least 90 days before treatment begins.

15. The method of any of the preceding claims, wherein the patient shows evidence of abnormally reduced cerebrovascular reactivity, neurovascular coupling, or regional or global cerebral hypoperfusion.

16. The method of any of the preceding claims, wherein the patient shows evidence of abnormally increased vascular myogenic tone.

17. The method of any of the preceding claims, wherein the patient has vascular mild cognitive impairment.

18. The method of any of the preceding claims, wherein the patient has vascular dementia.

19. The method of claim any of the preceding claims wherein the NOTCH3 variant is an EGFr 1-6 pathogenic variant.

20. The method of any of the preceding claims, wherein the NOTCH3 variant is an EGFr 7- 34 pathogenic variant.

21. The method of any of the preceding claims, wherein the NOTCH3 variant is an EGFr 18- 34 pathogenic variant.

22. The method of any of the preceding claims, wherein the NOTCH3 variant is a high risk variant.

23. The method of any of the preceding claims, wherein the NOTCH3 variant is a medium risk variant.

24. The method of any of the preceding claims, wherein the NOTCH3 variant is a low risk variant.

25. The method of any of the preceding claims, wherein the NOTCH3 variant is an unknown risk variant.

26. The method of any of the preceding claims, wherein the patient is treated with therapeutically effective amount of a second vasoactive pharmaceutical agent.

27. The method of claim 20, wherein the second vasoactive agent is selected from the group consisting of an angiotensin converting enzyme inhibitor, an angiotensin receptor blocker, a calcium channel blocker, a phosphodiesterase inhibitor and a nitrate.

28. The method of any of the preceding claims, wherein the patient suffers from migraines.

29. The method of claim 22, wherein the migraines occur at least once per month.

30. The method of claim 22, wherein the patient is female.

31. The method of any of the preceding claims, wherein the patient does not have an R169C NOTCH3 mutation.

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