Methods for treating patients with NOTCH3 mutations

Abstract

A method for treating patients with NOTCH3 mutations using a rho kinase inhibitor has been disclosed. Abnormal NOTCH3 signaling is a pathological factor in cerebral small vessel diseases, including CADASIL. A preferred embodiment relates to treating patients orally with the rho kinase inhibitor fasudil or its active metabolite hydroxyfasudil.

Description

[Background technology]

[0001] NOTCH3. The neural gene locus Notch homolog protein 3, NOTCH3, is encoded by the NOTCH3 gene, located on chromosome 19 between bands 13.2 and 13.1 (19p13), and consists of 33 exons encoding a 2,321-amino acid protein. It is the human homolog of the Notch protein and was first identified in fruit flies as a developmental regulator. In humans, it is expressed almost exclusively in vascular smooth muscle cells (VSMCs).

[0002] The Notch protein is a transmembrane receptor consisting of an intracellular domain, a transmembrane domain, and an extracellular terminal domain. The ligand binds to the extracellular domain, resulting in the intracellular domain being cleaved by proteolysis, translocated to the nucleus, and regulating the transcription of specific target genes. The protein plays a crucial role in the function and survival of vasculoskeletal cells (VSMCs) and is considered essential for maintaining blood vessels.

[0003] NOTCH3 and CADASIL. Mutations in the NOTCH3 gene can lead to small vessel diseases (SVDs) in humans, including autosomal dominant arteriovenous disease (CADASIL), which involves subcortical infarction and leukoencephalopathy. These changes result in an odd number of cysteine ​​residues, leading to receptor misfolding, enhanced oligomer formation, and ECD aggregation facilitated by sulfhydryl group crosslinking. Mutations can thicken the walls of small and medium-sized vessels, impeding blood flow.

[0004] Most pathogenic NOTCH3 variants result in an odd number of cysteine ​​residues and affect 34 epidermal growth factor-like repeat sequences (EGFr). These 34 regions span residues 40–1373 of NOTCH3. Each region spans a modular protein subunit approximately 40 amino acids long, and each region contains a conserved number and position of six cysteine ​​residues that form three disulfide crosslinks. Mutations disrupt these disulfide bonds, leading to pathogenic aggregation, which is thought to be a gain-of-function process. 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 EGFr1–6 are associated with a higher risk of stroke (Rutten 2019). Another study reported that mutations in EGFr18-34 appear to be as severe as those in EGFr1-6, but patients with NOTCH3 mutations in EGFr10-17 have a lower risk of stroke (Cho 2022).

[0005] Other types of NOTCH3 mutations have also been identified, including cysteine-preserving missense, frameshift, and nonsense mutations (Muino et al. 2017). These occur relatively frequently in non-CADASIL small vessel disease populations, suggesting a broader role for NOTCH3 mutations in cerebrovascular disease (Schoemaker 2021).

[0006] CAADASIL. NOTCH3 mutations that cause CADASIL result in progressive degeneration of VSMCs. CADASIL is characterized by the intravascular accumulation of granular osmiumophilic material (GOM) around VSMCs and pericytes. GOM consists of the extracellular domain of the Notch protein, as well as other extracellular matrix proteins such as clatherin and collagen 18α1 / endostatin. The pathological link between GOM and the pathogenesis of CADASIL remains unclear. Similarly, the role of abnormal Notch signaling and transcriptional regulation remains largely unknown.

[0007] CADASIL is a rare genetic disorder with an estimated prevalence of 1.3–4.1 per 100,000 Caucasians. However, large-scale human genome studies have found that the prevalence of the NOTCH3 variant is much higher in the general population. The pathogenic mutation in CADASIL is autosomal dominant, meaning that the presence of only one mutation in one of the two alleles is the cause of the pathology. The advent of widespread genetic testing has led to the identification of more CADASIL patients. However, a significant correlation between genotype and phenotype has yet to be established.

[0008] In addition to NOTCH3 mutations, magnetic resonance imaging (MRI) may show widespread fused white matter hyperintensity on T2-weighted MRI images, with hyperintensity lesions often prominent in the basal ganglia, thalamus, and pons (Stojanov 2015). In the early stages of the disease in the majority of cases, the anterior temporal lobe and external capsule are involved, while the subcortical white matter, subcortical U fibers, and cortex of the occipital and orbitofrontal lobes are relatively preserved. Cerebral microhemorrhages may be present. As the disease progresses, brain atrophy is present, which is associated with cognitive decline.

[0009] The clinical manifestations of CADASIL are diverse, with some patients exhibiting all symptoms and others only a portion of the symptom set, and the severity and progression vary greatly (Yamamoto 2023). The most common initial symptom of CADASIL is episodic migraines with aura, which typically begin in early adulthood with an average onset of around 30 years, but can also begin a decade earlier. The aura may include visual disturbances such as flashing lights or blind spots, as well as sensory or speech disturbances. Women with CADASIL and migraines with aura tend to experience an earlier onset than affected men.

[0010] Recurrent subcortical ischemic stroke or transient ischemic attack (aHUS) typically begins in CADASIL patients aged 50–59 years. These may present as sudden weakness, paralysis, or palsy on one side of the body, dysarthria, or visual alterations. MRI shows lesions in the basal ganglia and fusion white matter. Sensory, perceptual, and cognitive impairments, with or without psychiatric impairment. Progressive cognitive decline usually becomes apparent around age 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 slow information processing speed, as well as impaired verbal fluency and ideation. Recall, orientation, and receptive language skills are largely preserved in the early stages, but deficits in working memory and episodic long-term memory tend to develop later.

[0011] Ultimately, CADASIL can lead to subcortical dementia, typically occurring after the age of 60. Other symptoms include mood and mental disorders, gait disturbances, urinary incontinence, pseudobulbar palsy, epileptic seizures, and acute-onset reversible encephalopathy known as "CADASIL coma," which affects about 10% of patients. MRI at this stage reveals diffuse leukoencephalopathy and multiple infarcts in the basal ganglia.

[0012] The natural course of CADASIL varies, but the majority of patients experience at least one stroke or TIA, and the majority experience multiple ischemic events. Almost all patients experience progressive cognitive decline, and many develop dementia. More severe ischemic events are significantly associated with more severe cognitive decline. Patients with CADASIL also tend to have a shorter lifespan.

[0013] CADASIL is a devastating condition because it affects middle-aged adults, typically at around age 30, is progressive, commonly associated with depression and psychiatric disorders, and has no available treatments (Hack 2019). Care for CADASIL patients is focused on managing symptoms and risk factors, such as hypertension and smoking. Acute stroke may be managed with tPA (recombinant tissue plasminogen activator; alteplase), depending on individual assessment. Migraines are treated with conventional analgesics, although vasoconstrictors such as triptans or ergo derivatives may be used in rare cases. Antihypertensives, anticonvulsants, and antidepressants may be used for migraine prevention. Depression and other psychiatric problems may be managed pharmacologically. None of these approaches address the underlying vascular condition caused by the underlying NOTCH3 mutation and do not halt or slow disease progression.

[0014] In at least one report, CADASIL was hypothesized to be induced by COVID-19 in patients with the NOTCH3 mutation (Rajendran 2021). In another embodiment, CADASIL was exacerbated by COVID-19 infection (Rosenblum 2022).

[0015] Fasudil and NOTCH3 mutations. There are reports in the literature of the use of fasudil, a rho kinase inhibitor, to treat several patients with white matter vascular disease. Kamei (1996) reported using fasudil in two patients with wandering due to VaD. The patients were treated for wandering by the principal investigator after participating in a chronic stroke study treated with fasudil. One patient was diagnosed with Binswanger cerebral infarction, confirmed by MRI. The other patient was diagnosed with sequelae of cerebral hemorrhage and multiple lacunar infarcts, confirmed by MRI, and diagnosed with "lacunar dementia" (a synonym for Binswanger). The authors reported that patients exhibiting wayfinding symptoms improved during treatment and returned each time treatment was discontinued. There is no evidence that Kamei's study in subcortical vascular dementia can be extrapolated to patients with NOTCH3 mutations and / or CADISIL.

[0016] Transgenic mice carrying the CADASIL-associated R169C mutation were found to exhibit gain-of-function signaling and hyperconstriction in pulmonary arteries exposed to 5-HT and ET1, an effect inhibited by fasudil (Morris 2019). However, this is a model. Another group reported that fasudil improved abnormal vascular responses (reduced induced vasodilation) and signaling when incubated in blood vessels extracted from the same R169C transgenic mouse model (Neves 2019). However, this model does not appear to faithfully reproduce human disease because cerebrovascular dysfunction in the mouse model is associated with upregulation of voltage-dependent potassium (Kv1) channels, blunting of membrane depolarization, and reduced myogenic tone due to increased levels of the metallopeptidase inhibitor TIMP3, which has not been demonstrated in human disease.

[0017] Neves 2019 further showed that in VSMCs from human CADASIL patients, the expression of NOTCH3 and ROCK2 (along with other ER stress proteins) was increased, and culturing CADASIL VSMCs with fasudil improved the activation of cytoskeletal regulatory proteins. Neves used 10 μM FAS (2.9 μg / ml) in vitro. This is not a physiologically relevant concentration.

[0018] Since fasudil was not administered to either humans or mice, the relevance of fasudil to the treatment of CADASIL in humans is weak.

[0019] There is no disease-modifying therapy for small vessel diseases including CADASIL. There is a continuing need to treat patients with NOTCH3 mutations who have or are at risk of developing CADISIL or another small vessel disease.

Summary of the Invention

[0020] A method for treating a human patient having a mutation in the NOTCH3 gene is provided, the method comprising administering to the patient a therapeutically effective amount of oral fasudil or hydroxyfasudil.

[0021] In one embodiment, the patient is diagnosed with CADASIL. In another embodiment, the patient is not diagnosed with CADASIL.

[0022] In one embodiment, the patient is diagnosed with cerebral small vessel disease.

[0023] In a further embodiment, the NOTCH3 mutation is a gain-of-function mutation that results in an odd number of cysteines in the extracellular domain.

[0024] In another embodiment, the mutation is not a gain-of-function mutation. In certain embodiments, the NOTCH3 mutation is a cysteine-conserved mutation.

[0025] In one embodiment, the patient has at least one white matter hyperintense region.

[0026] In another embodiment, the patient has a granular osmiophilic substance.

[0027] In one embodiment, the patient has had at least one stroke or transient ischemic attack.

[0028] In another embodiment, the patient has had at least one stroke or transient ischemic attack at least 90 days prior to the start of treatment.

[0029] In a further embodiment, the patient has an existing cerebral infarction. In certain embodiments, the existing infarction is due to a lacunar infarction. In another specific embodiment, any prior cerebral hemorrhage was a microbleed.

[0030] In one embodiment, the patient shows evidence of cerebral vascular reactivity, abnormal reduction of neurovascular coupling, or local or global cerebral hypoperfusion.

[0031] In another embodiment, the patient shows evidence of abnormal increase in vasomyogenic tone.

[0032] In one embodiment, the patient has vascular mild cognitive impairment.

[0033] In another embodiment, the patient has vascular dementia.

[0034] In certain embodiments, the NOTCH3 variant is an EGFr1 - 6 pathogenic variant, an EGFr7 - 34 pathogenic variant, or an EGFr18 - 34 pathogenic variant.

[0035] Certain specific embodiments are high - risk NOTCH3 cysThis includes treating patients with mutations. In one particular embodiment, high-risk patients are treated even if there is no evidence of small vessel disease. In another embodiment, high-risk patients are treated if they have at least one additional risk factor for small vessel disease, such as hypertension, smoking, diabetes, or hypercholesterolemia. High-risk patients are treated if they have at least one Fazekas score of 1 in deep or periventricular white matter.

[0036] Certain other embodiments of the medium-risk NOTCH3 cys This includes treating patients with mutations. In one particular embodiment, intermediate-risk patients are treated only if they have at least one additional risk factor for small vessel disease, such as hypertension, smoking, diabetes, or hypercholesterolemia, and / or have at least one Fazekas score of 1 in deep or periventricular white matter, and / or exhibit one or more signs or symptoms of CADASIL, including evidence of subcortical ischemic events, cognitive impairment, migraine with aura, and / or psychiatric disorders (e.g., mood disorders; apathy).

[0037] In further embodiments, a low-risk NOTCH3 cys This includes treating patients with mutations. In one particular embodiment, intermediate-risk patients are treated only if they have at least one Fazekas score of 1 in the deep or periventricular white matter. In another embodiment, low-risk patients are treated only if they (a) have at least one additional risk factor for small vessel disease, such as hypertension, smoking, diabetes, or hypercholesterolemia, and / or (b) exhibit one or more signs or symptoms of CADASIL, including evidence of subcortical ischemic events, cognitive impairment, migraine with aura, and / or psychiatric disorders (e.g., mood disorders; apathy), and (c) have at least one Fazekas score of 1 in the deep or periventricular white matter.

[0038] Additional embodiments include NOTCH3, which addresses unknown risks. cysThe aim is to treat patients with mutations. In one particular embodiment, patients at unknown risk are treated only if they have additional significant findings consistent with NOTCH3-related small vessel disease, such as evidence of GOM and at least one Fazekas score of 1 in the deep or periventricular white matter. In another embodiment, low-risk patients are treated only if they have at least one additional risk factor for small vessel disease, such as hypertension, smoking, diabetes, or hypercholesterolemia, and have at least one Fazekas score of 1 in the deep or periventricular white matter, and would generally exhibit one or more signs or symptoms of CADASIL, including evidence of subcortical ischemic events, cognitive impairment, migraine with aura, and / or psychiatric disorders (e.g., mood disorders; apathy).

[0039] In one embodiment, the patient does not have the R169C NOTCH3 mutation.

[0040] In a further embodiment, the patient is treated with a therapeutically effective dose of a second vasoactive drug.

[0041] In one particular embodiment, the second vasoactive agent is selected from the group consisting of angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, phosphodiesterase inhibitors, and nitrates.

[0042] In one embodiment, the patient has migraines. In a particular embodiment, the migraines occur at least once a month. In another particular embodiment, the patient experiencing migraines is female.

[0043] In another embodiment, the patient being treated is under 40 years old and has migraines. In a specific embodiment, the patient under 40 years old is female.

[0044] In one embodiment, the patient is 40 to 50 years old.

[0045] In further embodiments, the patient is over 50 years old. In certain embodiments, the patient over 50 years old does not have dementia. [Modes for carrying out the invention]

[0046] This invention relates to the use of fasudil or its metabolite M3 for treating cerebrovascular effects of mutations in NOTCH3 in human patients.

[0047] NOTCH3 mutation In one embodiment, a patient treated with fasudil or M3 according to this disclosure has at least one mutation in NOTCH3. In a particular embodiment, the patient has confirmation of a NOTCH3 mutation before initiating treatment according to the present invention.

[0048] The mRNA and amino acid sequences of human NOTCH3 are available under GenBank accession number NM_000435.

[0049] The NOTCH3 mutation may be an EGFr1-6 pathogenic variant or an EGFr7-34 pathogenic variant. In one embodiment, the patient has an EGFr1-6 mutation. In another embodiment, the patient with the EGFr1-6 mutation has an earlier onset of stroke and lower survival than those with other EGFr mutations.

[0050] In another embodiment, the patient has a NOTCH3 mutation in EGFr18-34.

[0051] In another embodiment, the patient has a NOTCH3 mutation in EGFr10-17.

[0052] In one embodiment, the NOTCH3 mutation results in an odd number of cysteine ​​molecules in the extracellular domain. In another embodiment, the NOTCH3 mutation is a gain-of-function mutation.

[0053] In one embodiment, the mutation is a missense mutation related to one or more cysteine ​​residues in the 34 EGFr extracellular domains of NOTCH3.

[0054] These are mutations that affect cysteine, commonly known as NOTCH3. cys Mutations are annotated in several different ways in the literature, but generally follow a format that identifies the wild-type amino acid residue, followed by the amino acid position, and then the mutant amino acid. As will be understood by those skilled in the art, there are standard three-letter and one-letter systems for naming amino acids. Thus, C49Y indicates that the mutation replaces the cysteine ​​at position 49 (following the convention of N-terminus to C-terminus) with tyrosine. The same mutation may be designated as Cys43Tyr or several similar naming variants. It will also be recognized by those skilled in the art that other standard notations, such as "p.", can be used as a prefix to indicate a change in the protein sequence. Thus, C49Y, Cys43Tyr, and p.Cys43Tyr are equivalent.

[0055] In one embodiment, exemplary NOTCH3 mutations are shown in Table 1 below. [Table 1]

[0056] In another embodiment, the NOTCH3 mutation 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, C174 R, R182C, C183R, C185S, Y189C, C194S, C194R, C201R, P203H, R207C, C212Y, C212R, R213K, C222R, C224Y, C224R; C233R, D239 N, 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.

[0057] In certain embodiments, patients with the NOTCH3 mutation have a cysteine ​​preservation mutation. A cysteine ​​preservation mutation that does not result in cysteine ​​removal or addition and may not result in gain-of-function pathology. In certain embodiments, the cysteine ​​preservation mutation is at least one of R61W, G73S, R75P, D80G, P109T, P203H, R213K, D239N, P572L, A1020P, and A1604T.

[0058] In other embodiments, NOTCH3 mutations in patients are null mutations, homozygous mutations, or in-frame insertion / deletion mutations. An example of a homozygous mutation is R544C. Examples of duplications include duplication of Glu434–Leu436 and an in-frame heterozygous 15bp duplication in exon 7, resulting in the insertion of five extra amino acids, including cysteine ​​355, at positions 353–357. Duplication of exon 28 has also been reported. Reported deletions include deletions of NOTCH3 A88–G91 and T312–G316.

[0059] In another embodiment, NOTCH3 mutations are splice site mutations, including mutations resulting from intron deletions such as c.341-26_24delAAC, which leads to the insertion of 25 amino acids.

[0060] High-risk mutation. NOTCH3 in EGFr domains 1-6, 8, 11 and 26. cys The mutations are of particular interest. Patients with these “high-risk” mutations are not only very likely to develop cerebral small vessel disease, but also highly likely to develop CADASIL. Patients with these mutations may be treated according to the present invention with little further evidence of disease, and they may be treated without additional signs or symptoms (i.e., prophylactically), or without risk factors such as hypertension, smoking, diabetes, or hypercholesterolemia. Typically, high-risk patients are treated based on the presence of mutations in deep or periventricular white matter and at least one Fazekas score.

[0061] The following NOTCH3 cys The mutation is high-risk and is classified as a high-risk category: 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、Cys106Arg、Cys106Trp、Cys108Arg、Cys108Gly、Cys108Phe、Cys108Tyr、Cys108Ser、Arg110Cys、Cys117Arg、Cys117Tyr、Cys117Phe、Cys117Thr、Cys117Trp、Ser118Cys、Cys123Phe、Cys123Thr、Ser126Cys、Cys128Phe、Arg133Cys、Cys134Tyr、Cys134Gly、Cys134Trp、Arg141Cys、Phe142Cys、Cys144Ser、Cys144Phe、Cys144Tyr、Cys144Trp、Cys144Thr、Ser145Cys、Cys146Arg、Cys146Trp、Cys146Tyr、Gly149Cys、Tyr150Cys、Arg153Cys、Ser154Cys、Cys155Trp、Cys155Ser、Cys155Tyr、Trp156Cys、Cys162Ser、Cys162Trp、Cys162Tyr、Gly165Cys、Arg169Cys、His170Cys、Gly171Cys、Cys174Arg、Cys174Tyr、Ser180Cys、Arg182Cys、Cys183Arg、Cys183Tyr、Cys183Ser、Cys183Phe、Cys185Arg、Cys185Ser、Cys185Tyr、Cys185Gly、Tyr189Cys、Cys194Arg、Cys194Ser、Cys194Tyr、Cys194Phe、Cys201Arg、Cys201Ser、Cys201Tyr、Cys206Ser、Arg207Cys、Cys212Arg、Cys212Trp、Cys212Ser、Cys212Tyr、Cys222Gly, Cys222Arg, Cys222Ser, Cys222Tyr, Cys222Thr, Cys222Phe, Cys224Arg, Cys224Tyr, Ph e228Cys, Cys233Ser, Cys233Arg, Cys233Tyr, Cys233Trp, Cys240Tyr, Cys240Ser, Cys245Arg, Cys24 5Ser, Cys245Tyr, Cys251Arg, Cys251Tyr, Cys251Ser, Tyr258Cys, Cys260Arg, Cys260Phe, Cys260Ty r, Cys262Arg, Cys318Phe, Cys323Ser, Cys323Tyr, Cys323Trp, Cys329Tyr, Arg332Cys, Ser335Cys, T yr337Cys, Cys338Arg, Cys338Ser, Cys340Phe, Cys349Ser, Cys435Arg, Cys435Tyr, Cys435Thr, Cys4 35Phe, Cys440Gly, Cys440Arg, Cys446Ser, Cys446Gly, Cys446Phe, Cys446Tyr, Arg449Cys, Cys455A rg, Cys455Phe, Cys455Tyr, Cys457Ser, Tyr465Cys, Cys466Tyr, Trp1003Cys, Cys1004Tyr, Cys1004G ly, Arg1006Cys, Cys1009Phe, Cys1015Arg, Cys1015Trp, Tyr1021Cys, Cys1022Gly, and Arg1031Cys. ,

[0062] Intermediate-risk mutation. NOTCH3 in EGFr domains 9, 10, 12-15, 17, 25, 27, and 32. cysThe mutations are very interesting. Patients having these "moderate-risk" mutations have a significant risk of developing cerebral small vessel disease and a risk of developing CADASIL. Patients having these mutations can be treated according to the present invention if they have a 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. These patients are even more likely to be treated if they present symptoms or signs of CADASIL, including evidence of subcortical ischemic events, cognitive impairment, migraine with aura, and / or mental disorders (e.g., mood disorders, apathy).

[0063] The following NOTCH3 cys The mutations are of moderate risk and are classified into the moderate-risk category: 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、Cys1055Tyr、Gly1058Cys, Cys1061Arg, Cys1061Tyr, Gly1165Cys, Ser1067Cys, Tyr1069Cys, Cys1070Ser, Cys1072Tyr, Arg1072Cys, Arg1076Cys, Cys1250Arg, Cys1250Gly, Cys1261Arg, Cys1261Tyr, Cys1261Trp, Arg1262Cys, Gly1266Cys, Cys1275Ser, Cys1277Tyr, and Gly1283Cys.

[0064] Low-risk mutation. NOTCH3 in EGFr domains 16, 18-20, 23, 24, 28-31, and 33. cys The mutations are somewhat interesting. Patients with these “low-risk” mutations are at risk of developing cerebral small vessel disease, but have a low risk of developing CADASIL. Patients with these mutations may be treated according to the invention with at least a Fazekas score of 1 in deep or periventricular white matter, but generally more is required. For example, a patient has one or more risk factors such as hypertension, smoking, diabetes, or hypercholesterolemia, and / or presents with one or more signs or symptoms of CADASIL, including evidence of subcortical ischemic events, cognitive impairment, migraine with aura, and / or psychiatric disorders (e.g., mood disorders, apathy).

[0065] The following NOTCH3 cys The mutation is low-risk and falls into the low-risk category: 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、Cys1099Phe、Arg1100Cys、Cys1108Arg、Cys1110Arg、Cys1110Ser、Cys1119Tyr、Cys1131Trp、Cys1137Arg、Arg1143Cys、Tyr1144Cys、Cys1148Phe、Cys1157Arg、Cys1176Tyr、Cys1182Trp、Arg1190Cys、Cys1191Ser、Cys1193Tyr、Arg1201Cys、Cys1202Ser、Arg1210Cys、Cys1222Gly、Cys1222Ser、Cys1222Arg、Arg1231Cys、Cys1232Trp、Arg1242Cys、Cys1250Arg、Cys1250Gly、Arg1291Cys、Cys1293Phe、Cys1293Trp、Cys1298Phe、Cys1313Gly、Cys1313Ser、Cys1315Phe、Cys1315Trp、Cys1315Tyr、Cys1324Ser、およびCys1324Tyr。

[0066] EGFrドメイン7、21、22、および34におけるNOTCH3 cysThe mutations are not known to be associated with the development of SVD or CADASIL, and these mutations should only be treated if there is significant clinical evidence of SVD and additional findings suggesting the involvement of NOTCH3 in the pathology of the disease, such as the presence of GOM.

[0067] The following NOTCH3 cys The mutation is an unknown low-risk mutation and is classified into an unknown risk category: Cys291Tyr, Cys311Ser, Phe833Cys, Cys846Tyr, Cys858Trp, Gly861Cys, Cys873Arg, Arg886Cys, Cys1350Gly, Arg1360Cys, and Cys1372Trp.

[0068] CADASIL In certain embodiments, patients with NOTCH3 mutations are diagnosed with CADASIL. CADASIL is suspected based on age (40–50 years), symptoms (including recurrent transient ischemic attacks or strokes, migraines, depression, mood disorders, pseudobulbar palsy, and mild cognitive impairment), family history, and MRI showing white matter lesions consistent with the disease (Yamamoto 2023). A CADASIL diagnosis is confirmed by DNA testing of a blood sample for characteristic mutations in the NOTCH3 gene, or by identifying granular osmiumophilic material (GOM) inclusions in a skin biopsy.

[0069] In another embodiment, a patient carrying the NOTCH3 mutation is not diagnosed with CADASIL. In one embodiment, the patient is diagnosed with cerebral small vessel disease (SVD).

[0070] In a further embodiment, the patient has granular osmiumophilic material (GOM) at the time of biopsy.

[0071] In one embodiment, the patient does not have hypertension.

[0072] In another embodiment, the patient may have experienced 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 with fasudil in such patients with cerebral ischemia or hemorrhagic events is generally not performed in the acute phase of the ischemic event; therefore, treatment is initiated 30, 60, or 90 days or later after the symptomatic resolution of the ischemic or hemorrhagic event.

[0073] In one embodiment, CADASIL patients are East Asian. East Asian CADASIL patients have a higher risk of intracerebral hemorrhage (ICH) than Caucasian patients.

[0074] In another embodiment, patients treated with fasudil may have mild cognitive impairment. Patients with dementia are usually excluded because they are likely past the point at which treatment may be effective.

[0075] In one embodiment, the patient does not exhibit wandering because it is not a typical symptom of the patient for whom treatment is intended.

[0076] In one embodiment, a patient treated according to the method of the present disclosure has at least one white matter high-signal area in the image.

[0077] In some embodiments, white lesions are graded as having mild (punctate), moderate (some fusion), or severe white matter disease (large fused areas).

[0078] In one embodiment, imaging is performed using MRI.

[0079] In certain embodiments, MRI white matter lesions may be evaluated according to the Fazekas score, having a minimum score of 1, 2, or 3 according to the following evaluation: Periventricular white matter (PVWM) No orders 1 = "Cap" or thin inner layer like a pencil 2 = A smooth "halo" 3 = Irregular periventricular signals extending into the deep white matter. Deep white matter (DWM) No orders 1 = Punctate lesions 2 = Start of fusion 3 = Large Fusion Area

[0080] In one embodiment, only the deep white matter scale is applied to a maximum score of 3. In another embodiment, all scores are used, and the patient has a Fazekas score of 4, 5, or 6.

[0081] In another embodiment, the two scales are presented separately. Thus, a patient 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 particular embodiment, exemplary scores indicating PVWM:DWM include 1:1, 0:2, 1:2, etc.

[0082] In another embodiment, patients with white lesions are graded using the Lesion Segmentation Tool (LST) (applied-statistics.de / lst.html). This method automatically segments white matter signal abnormalities in the form of high-signal morphology in T2-weighted water-suppressed inversion-recovery (FLAIR) MRI sequences (Schmidt 2011). In a specific embodiment, a proposed cutoff value for classifying low Fazekas and high FazekasWMSA loading is 0.00496 relative to LST (Cedres 2020).

[0083] 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 low signal intensity in T1-weighted MRI sequences. Low signal intensity WMSA appears to be associated with poor white matter integrity compared to high signal intensity white matter signal abnormalities. In a particular embodiment, a proposed cutoff value for classifying low Fazekas and high Fazekas WMSA loading is 0.00321 relative to FreeSurfer WMSA (Cedres 2020).

[0084] In one embodiment, the patient may show evidence of cerebral hemodynamic dysfunction, such as abnormally reduced cerebrovascular responsiveness or localized or global cerebral hypoperfusion.

[0085] In another embodiment, the patient may show evidence of an abnormal increase in vascular muscle tone.

[0086] In further embodiments, patients typically have increased rho kinase activity. In one embodiment, ROCK2 activity is elevated.

[0087] In one embodiment, a patient may have mild vascular cognitive impairment (VCI) or vascular dementia (VaD). Mild VCI is an impairment in at least one cognitive domain (unrelated to motor / sensory sequelae of a vascular event) that is mild to no impairment in instrumental activities of daily living (IADL) or basic activities of daily living (ADL), respectively. VaD (also known as major VCI) is a sufficiently severe, clinically significant impairment in at least one cognitive domain (the deficit may be present in multiple domains) that significantly disrupts IAD / ADL (unrelated to motor / sensory sequelae of a vascular event). IADL are activities that enable an individual to live independently in a community, and the main domains of IADL include cooking, cleaning, transporting, laundry, and financial management. In contrast to IADL, ADL are necessary for basic functional living, and the main domains of ADL are eating, dressing, bathing, and walking.

[0088] In another embodiment, the patient may have impaired executive function or reduced processing speed. In a particular embodiment, the patient with cognitive impairment and working memory impairment is male.

[0089] In further embodiments, the patient may have memory impairments, including working memory and long-term memory.

[0090] NOTCH3 mutations in other diseases NOTCH3 truncation mutations, deletions, or insertions have also been reported in other diseases, but the causes are unknown. Cysteine ​​preservation NOTCH3 mutations A198E, G284A, and A284T are associated with Alzheimer's disease and other dementias (Patel 2019). NOTCH3 mutations H170R, P496L, V764A, H1133Q, V1183M, L1518M, D1823N, and V1952M are associated with age-related cerebral small vessel disease (Schmidt 2011).

[0091] In one embodiment, dementia patients carrying the aforementioned NOTCH3 mutation are treated with fasudil or M3 according to this disclosure.

[0092] Patients with lateral meningocele syndrome (LMS) have mutations in the exon encoding the last NOTCH3. One patient had a 26bp deletion (G2154fsTer78), two had the same single base pair insertion (c.6692_93insC, p.P2231fsTer11), and three had nonsense point mutations in c.6247A>T(pK2083*), c.6663C>G(p.Y2221*), or c.6732C>A(p.Y2244*).

[0093] In another embodiment, LMS patients are treated with fasudil or M3 according to the disclosed method.

[0094] NOTCH3 mutations are also associated with myofibromatosis, a common proliferative fibrous tumor in children. The tumors exhibit characteristics of both differentiated fibroblasts and smooth muscle cells (myofibroblasts). Mutations at residue L1519P of NOTCH3 were associated with this disorder (U.S. Patent No. 10,155,995).

[0095] Myopericytoma, myopericytomatose, angioleiomyoma, and other pericytic tumors, including glomus tumors, are associated with NOTCH3 mutations. Mutations include, but are not limited to, A1480S / T, D1481N, G1482S, T1490A, E1491K, G1494S, and V1512A, all located in exon 25.

[0096] In one embodiment, a patient with a pericytic tumor carrying a NOTCH3 mutation is treated with fasudil or M3 in accordance with this disclosure.

[0097] Overexpression and fusion of NOTCH3 have also been associated with metastasis of breast cancer, including estrogen receptor-positive mammary tumors. Furthermore, activated NOTCH3 expression has been shown to promote the formation of choroidal tumors, retinal glial tumors, and invasive gliomas in mice.

[0098] Fasgil The method described herein involves the administration of the rho kinase (ROCK) inhibitor fasudil or its metabolite M3 in the treatment of diseases or conditions associated with mutated NOTCH3. Two mammalian ROCK homologs are known: ROCK1 (also known as ROKβ, Rho-kinase β, or p160ROCK) and ROCK2 (also known as ROKα) (Nakagawa 1996). In humans, both ROCK1 and ROCK2 genes are located on chromosome 18. The two ROCK isoforms share 64% identity in their primary amino acid sequences, but the homology of their kinase domains is even higher (92%) (Jacobs 2006; Yamaguchi 2006). Both ROCK isoforms are serine / threonine kinases and have similar structures.

[0099] The isoquinoline derivative Fasudil was the first small molecule ROCK inhibitor developed by Asahi Kasei Corporation (Tokyo, Japan). Fasudil's characteristic chemical structure consists of an isoquinoline ring bonded to a homopiperazine ring via a sulfonyl group. Fasudil is a potent inhibitor of both ROCK isoforms.

[0100] In vivo, fasudil is metabolized in the liver to its active metabolite, hydroxyfasudil (also known as M3). Other examples of isoquinoline-derived ROCK inhibitors include dimethylfasudil, ripasudil, Y-27632, and Y39983. [ka]

[0101] In contrast to intravenous administration, oral administration of fasudil results in nearly 100% conversion to M3, while intravenous administration results in a mixture of the parent fasudil and its metabolite M3. Although both fasudil and M3 are potent ROCK inhibitors, they do not have the same specificity for the two different ROCK isoforms, nor for off-target kinases such as protein kinase G. Therefore, intravenous administration of fasudil is not a predictor of oral administration. Furthermore, rodents metabolize oral fasudil differently than humans and 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 differs from oral fasudil in humans.

[0102] While fasudil has been reported to have many potentially beneficial effects in CNS conditions, there is no evidence of Level 1 therapeutic effect in any of these conditions, meaning that any potential medical applications remain speculative.

[0103] The hemodynamic effects of fasudil are mediated by two main mechanisms. It inhibits the calcium-independent ROCK-mediated phosphorylation of myosin light chain phosphatase, a major factor in vasomyogenic tone. Therefore, in situations where ROCK activity is elevated, it can provide an additional vasodilatory response in addition to calcium-mobilization-dependent vasodilators (e.g., CO, NO, PDE inhibitors, calcium channel blockers). Furthermore, elevated ROCK is often involved in endothelial dysfunction by inhibiting the expression and activity of endothelial nitric oxide synthase. Thus, fasudil can improve both the dynamics of vasodilatory signals originating from the endothelium and the response by vascular smooth muscle cells.

[0104] As used herein, the term fasudil means the free base, salt, hydrate, ion pair, polymorph and its metabolites, such as hydroxyfasudil (also known as M3), and also includes the ion pairs of the free base, salt, hydrate, polymorph and metabolite. Specific dosage information refers to the molar equivalents of fasudil hydrochloride hemihydrate and other forms or metabolites.

[0105] Dosage and administration Depending on the treatment method, the therapeutically effective dose of fasudil or a pharmaceutically acceptable salt thereof, such as fasudil hydrochloride hemihydrate, for administration once or more days may contain approximately 10 mg to approximately 1000 mg.

[0106] For example, fasudil hydrochloride hemihydrate is preferably administered in daily doses of approximately 10 mg to 500 mg, approximately 10 mg to 400 mg, approximately 10 mg to 200 mg, approximately 10 mg to 100 mg, and approximately 20 mg to 10 mg.

[0107] One dosing regimen involves treatment with fasudil hydrochloride hemihydrate three times daily at doses of 25, 30, 40, 60, or 80 mg, with a total daily dose of 70–240 mg, using an immediate-release formulation. The most preferred dose is greater than 70 mg per day, with the most preferred range for daily doses being 90–240 mg, administered in two or three equal doses per day.

[0108] A particularly preferred daily dose is 180 mg / day, administered in the form of an immediate-release tablet with 60 mg TID. Fasudil is most preferably administered orally using an immediate-release formulation as described above. In another embodiment, fasudil is administered at a dose of 240 mg / day, administered in the form of an immediate-release tablet with 80 mg TID.

[0109] Another embodiment involves treatment with a sustained-release dosage form, using 90 to 360 mg of fasudil hydrochloride hemihydrate once daily. Treatment with a sustained-release dosage form once daily totaling 90 mg of fasudil hydrochloride hemihydrate per day is preferred.

[0110] In one embodiment, fasudil is administered to the patient as an immediate-release formulation at a dose of approximately 90-240 mg / day.

[0111] In another embodiment, fasudil is administered to the patient in a sustained-release formulation, and the maximum plasma concentration and the area under the plasma curve over 24 hours do not exceed by more than 15% of those obtained using a 70-240 mg / day dosing regimen as an immediate-release formulation.

[0112] The method of administering the composition according to the present invention is generally continued for at least one day. Some preferred methods involve treatment for up to 30 days, or up to 60 days, or even up to 90 days, or even longer. Treatment for more than 60 days is preferred, and treatment for at least 6 months is particularly preferred. The exact duration of treatment depends on the patient's condition and response to treatment.

[0113] Pharmaceutical composition In preferred embodiments, fasudil is administered in oral dosage form. Pharmaceutical compositions of fasudil or a salt or hydrate for oral administration may be in the form of tablets or capsules, immediate-release formulations, controlled-release formulations, or sustained-release formulations, which may contain pharmaceutically acceptable excipients, such as corn starch, mannitol, povidone, magnesium stearate, talc, cellulose, methylcellulose, carboxymethylcellulose, and similar substances. Pharmaceutical compositions containing fasudil or a salt or hydrate may also contain one or more pharmaceutically acceptable excipients known in the art.

[0114] The formulations also include oral films, orally disintegrating tablets, effervescent tablets, and granules or beads that can be sprinkled on food, mixed with liquids as a slurry, or poured directly into the mouth and swallowed.

[0115] In one embodiment, fasudil is administered in a taste-masked oral formulation. Examples of bitterness maskers that can bind to RHO kinase inhibitors are ion exchange resins, for example, disclosed in U.S. Patent Applications No. 18 / 171,007, 18 / 171,070, and 18 / 171,077.

[0116] In another embodiment, fasudil is administered orally in the form of liposomes.

[0117] Pharmaceutical compositions comprising fasudil or a salt or hydrate thereof can be prepared by any method known in the pharmaceutical art. Generally, such preparation methods include the steps of associating fasudil or a pharmaceutically acceptable salt or hydrate thereof with a carrier or additive and / or one or more other minor components, and then, if necessary and / or desirable, forming and / or packaging the product into desired single-dose or multi-dose units.

[0118] Pharmaceutical compositions may be prepared, packaged, and / or sold in bulk as single unit doses and / or as multiple single unit doses. As used herein, “unit dose” refers to a specific amount of a pharmaceutical composition containing a given amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject, and / or a convenient proportion of such dose, such as half or one-third of such dose.

[0119] The relative amounts of the active ingredient, pharmaceutically acceptable additives, and / or any additional ingredients in the pharmaceutical composition of the present invention vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route through which the composition is administered. The composition used according to the method of the present invention may contain 0.001% to 100% (w / w) of the active ingredient.

[0120] Examples of pharmaceutically acceptable additives used in the manufacture of the provided pharmaceutical composition include inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and / or oils. Additives such as colorants, coatings, sweeteners, flavorings, and fragrances may also be present in the composition.

[0121] In certain embodiments, the pharmaceutical composition used in the method of the present invention may include a diluent. Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium lactose phosphate, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0122] In certain embodiments, the pharmaceutical composition used in the method of the present invention may include granulators and / or dispersants. Exemplary granulators and / or dispersants include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0123] In certain embodiments, the pharmaceutical composition used in the method of the present invention may include a binder. Exemplary binders include starch (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic rubbers (e.g., acacia, sodium alginate, Irish moss extract, panwar gum, gatti gum, isapol bark mucus, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate (VEEGUM.RTM.), and larch arabinogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylate, wax, water, alcohol, and / or mixtures thereof.

[0124] In certain embodiments, the pharmaceutical composition used in the method of the present invention may contain a preservative. Examples of preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoal preservatives, alcoholic preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0125] In certain embodiments, the pharmaceutical composition used in the method of the present invention may contain antioxidants. 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.

[0126] In certain embodiments, the pharmaceutical composition used in the method of the present invention may include a chelating agent. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate, disodium calcium edetate, dipotassium edetate, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Examples of antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and thimerosal.

[0127] In certain embodiments, the pharmaceutical composition may include a buffer along with fasudil or a salt or hydrate thereof. Exemplary buffers include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dicalcium phosphate, phosphoric acid, tricalcium 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, water free of pyrogens, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0128] In certain embodiments, the pharmaceutical composition used in the method of the present invention may include a lubricant. Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0129] In other embodiments, pharmaceutical compositions comprising fasudil or a salt or hydrate thereof are administered as liquid dosage forms. Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may also contain, for example, water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof, which are commonly used in the art. In addition to the inert diluent, the oral composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0130] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient is at least one inert, pharmaceutically acceptable additive or carrier, e.g., sodium citrate or dicalcium phosphate, and / or (a) excipients or fillers (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), (b) binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), (c) humectants (e.g., glycerol), and (d) disintegrants (e.g., agar, calcium carbonate, jasper). (e) potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, etc., (e) dissolution retarders (such as paraffin), (f) absorption enhancers (such as quaternary ammonium compounds), (g) wetting agents (e.g., 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 glycol, sodium lauryl sulfate), as well as mixtures thereof, are mixed. In the case of capsules, tablets, and pills, the dosage form may include buffers.

[0131] Some compositions of the present invention relate to sustained-release or controlled-release formulations. These may be, for example, diffusion-controlled products, dissolution-controlled products, erosion products, osmotic pump systems, or ion resin systems. Diffusion-controlled products include water-insoluble polymers that control the flow of water and the subsequent release of the dissolved drug from the dose. Dissolution-controlled products control the dissolution rate of the drug by using polymers that solubilize slowly or by microencapsulating the drug, using varying thicknesses to control release. Erosion products control the release of the drug by the erosion rate of a carrier substrate. Osmotic pump systems release the drug based on a constant inflow of water across a semipermeable membrane into a storage compartment containing an osmotic agent. Ion exchange resins may be used to bind the drug so that, upon ingestion, the release of the drug is determined by the ionic environment in the gastrointestinal tract.

[0132] Combination therapy The method of the present invention may also employ fasudil in combination with one or more other vasoactive agents. These may be administered together with fasudil in a single fixed-dose combination composition, or fasudil and the other vasoactive agents may be administered in separate compositions, but as part of a common therapeutic regimen. Examples of other vasoactive agents include angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), calcium channel blockers (CCBs), phosphodiesterase (PDE) inhibitors, and nitrates.

[0133] ARBs include, but are not limited to, valsartan, losartan, azilsartan, irbesartan, olmesartan, telmisartan, and fimasartan.

[0134] ACE inhibitors include, but are not limited to, alacepril, captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril, cilazapril, and fosinopril.

[0135] CCBs include, but are not limited to, amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, crebidipine, efonidine, felodipine, isradipine, lasidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipidine, pranidipine, fendiline, garopamil, verapamil, and diltiazem.

[0136] PDEs include, but are not limited to, sildenafil, tadalafil, vardenafil, udenafil, avanafil, dipyridamole, amrinone, milrinone, enoximon, anagrelide, cilostazol, and pimobendan.

[0137] Examples of nitrates include, but are not limited to, amyl nitrite, glyceryl trinitrate, isosorbide mononitrate (with or without hydralazine), nitroglycerin, and pentaerythritol tetranitrate.

[0138] The method of the present invention may also employ fasudil in combination with one or more other cognitive agents such as cholinesterase inhibitors (e.g., donepezil, galantamine, rivastigmine) or memantine.

[0139] The method of the present invention may also employ fasudil in combination with one or more other anti-migraine agents, including beta-blockers, triptans including sumatriptan, naratriptan, zolimitriptan, and rizatriptan, acetaminophen having codeine or hydrocodone, morphine, tramadol, propranolol, amitriptyline, pizotifen, acetazoleamide, and simple analgesics such as aspirin, acetaminophen, and ibuprofen.

[0140] Evaluation of the results of fasudil treatment Patients treated with fasudil or M3 in accordance with this disclosure may be evaluated using the following methods. Patients typically have a defect in one or more of these evaluations prior to treatment. Treatment generally improves performance in at least one of these evaluations. Typical improvements are 5%, 10%, 15%, 20%, or 25% or greater. Other improvements may be more descriptive, such as severe becoming moderate, moderate becoming moderate, moderate becoming mild, or mild becoming asymptomatic. In some cases, evaluation scores or categories stabilize, or the rate of deterioration slows compared to expected or control patients.

[0141] Those skilled in the art will be familiar with these techniques and other methods useful for evaluating patient defects and treatment outcomes. The present invention aims to improve one or more of the following evaluations in fasudil or M3 treatment.

[0142] Image. In one embodiment, patients with NOTCH3 mutations can be monitored with the MRI and other imaging described above. White matter loading, as assessed by MRI, increases over time and is associated with cognitive and functional decline.

[0143] In one embodiment, patients treated according to the present invention may experience a reduced rate of white matter lesion accumulation. Treated patients may also experience a reduced frequency of lacunar stroke and other ischemic events compared to their own clinical history or an untreated cohort.

[0144] In another embodiment, patients with white lesions may be diagnosed or monitored using diffusion tensor imaging, which measures water diffusion inside and outside the brain lesion. Diffusion tensor imaging can detect suprastrumatic tissue damage even in areas that appear normal on conventional MRI. The mean diffusion rate has been found to be a major predictor of clinical progression in patients with CADASIL (Holtmannspotter 2005).

[0145] Other assessment methods commonly used by healthcare providers include CT angiography.

[0146] Neuropsychological and disability assessment. The modified Rankin scale is a disability scale often used in patients with small vessel diseases, including CADASIL. It is a 7-point scale (0-6) ranging from asymptomatic to death. 0 - No symptoms. 1. No serious impairment. Able to perform all normal activities despite some symptoms. 2. Mild disability. Able to perform their own requirements without assistance, but unable to perform all of their previous activities. 3 - Moderate disability. Requires some assistance, but can walk without support. 4. Moderately severe disability. Unable to meet one's own physical needs without assistance; unable to walk without assistance. 5. Severe disability. Requires constant care and attention, is bedridden, and experiences incontinence. 6-Death.

[0147] The Bartel Index can also be used to monitor disease progression. The Bartel Index is a 100-point scale that measures activities of daily living. A score of 100 is completely independent. The categories are: eating (0-10), bathing (0-5), grooming (0-5), dressing (0-10), bowel control (0-10), bladder control (0-10), toilet use (0-10), mobility (bed to chair and back) (0-15), movement on a flat surface (0-15), and stairs (0-10).

[0148] Useful working memory tests for evaluating results also include the Digit Span test, which involves immediate retrieval of a certain number of digits, both from the beginning and the end (WAIS-III 1997).

[0149] The Weschler Memory Scale III (WMS-III 1997) or WMS-IV may also be used. The memory indicators are as follows: auditory immediate memory, visual immediate memory, immediate memory, auditory delayed memory, visual delayed memory, auditory cognitive delayed memory, general memory, and working memory.

[0150] The WMS long-term memory test includes logical memory tests and visual recall tests. The logical memory test measures immediate and delayed recall of short stories. This can also be used to monitor disease progression. The visual recall test measures immediate and delayed recall of diagrams.

[0151] The Brief Memory and Executive Function Test (BMET) is a previously validated cognitive screening tool designed to be sensitive to the cognitive impairments seen in sporadic SVD and has been shown to be sensitive to the cognitive impairments of CADASIL (Brookes 2015). The test includes eight tasks (domains) that provide a scale that forms two subscales and an overall score. The first subscale, Executive Function and Processing Speed, is calculated using tasks of letter-number matching, motor sequencing, letter sequencing, and letter-number sequencing. The second subscale, Orientation and Memory, consists of Orientation, 5-item repetition, 5-item recall, and 5-item recognition tasks. Measures from each task are converted to a maximum of two scales, with a maximum of eight scales in each subscale and 16 scales overall. These scores are age-adjusted, and each measure is adjusted to the age of the participant.

[0152] Processing speed in patients with NOTCH3 mutations can be assessed using the BIRT memory and information processing battery, the digit-code test, and the grooved pegboard test. The BIRT involves canceling out the second highest value of five two-digit numbers within a time limit (Coughlin 2007). The digit-code test is a time-limited transcoding test (WAIS-III 1997). The grooved pegboard involves picking up, rotating, and placing small pegs within a time limit (Matthews 1964, Dawson 2010). The DSST is a paper-and-pencil cognitive test presented on a sheet of paper, requiring subjects to match symbols with numbers according to keys located at the top of the page. Subjects copy the symbols into the space below the rows of numbers. The score is determined by the number of correct symbols within a given time, typically 90-120 seconds.

[0153] The DSST measures the range of cognitive actions. The DSST can prove to be a practical and effective method for clinical use to detect declines in cognitive processing over time. Good performance on the DSST requires full motor speed, attention, and visual perception functions, including the ability to scan and write or draw lines (i.e., basic manual dexterity). Performance can also be influenced by collaborative learning. For example, if pairings are learned rapidly after the first few tests, performance speed improves because the subject does not need to refer to the keys to check the accuracy of each pairing. The decision to consciously engage in this learning strategy and seek to improve performance speed requires executive functions of planning and strategizing. Another executive function, working memory, is likely required to keep task rules in mind and continuously update the necessary symbol-digit pairs. CADASIL patients score approximately 1 SD below normal on the DSST.

[0154] Symbol code paradigms comparable to the DSST (with different symbols) are included as subtests in the Simplified Cognitive Function Test for Schizophrenia and the Repeatable Battery for the Assessment of Neuropsychological Status. The Symbol Digit Modalities Test uses essentially the same method, but in reverse: instead of drawing symbols that match numbers, subjects are required to fill in matching numbers in the blanks. This version of the 9-code paradigm has the advantage of being easily modifiable to allow verbal responses in cases of impairment affecting motor function. Computer-based tests offer another option for monitoring changes over time, such as the Cambridge Cognition developed by Cambridge Cognition, Cognigram, Cogstate Brief Battery, Cognigram (a version of the Cogstate Brief Battery), and THINC-IT (a freely available computer-based test). (Jaeger 2018)

[0155] Executive function in NOTCH3 mutation patients can be assessed using the Trailmaking Test (the entire test or individual parts A or B), the DSST, verbal fluidity tests, and the modified Wisconsin Card Sorting Test. The Trailmaking Test involves arranging letters and numbers alternately using pen and paper (Reitan 1955, Mitrushina 2005; eatspeakthink.com / wp-content / uploads / 2018 / 06 / IowaTrailMaking.pdf). Verbal fluidity tests assess a subject's ability to generate words beginning with specific letters within a set time (Delis 2001). The modified Wisconsin Card Sorting Test involves asking subjects to sort a set of cards without instruction, receiving feedback on their performance, and then adapting their strategy (Nagahama 2003). This tests flexibility and the ability to learn the rules of the game. The Stroop Test can also assess executive function. Similar to the word "red" written in blue letters, this evaluates responses to contradictory stimuli.

[0156] Cognitive assessments for dementia, including the Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MocA), may also be used. The maximum score for the MMSE is 30. Scores of 25 or higher are classified as normal. Scores of 24 or lower are considered normal and indicate a possible cognitive impairment. In one embodiment, a patient may be selected for treatment based on their MMSE, and / or their MMSE score will improve with treatment according to the present invention. The maximum score for the MoCA is 30. Scores of 26 or higher are classified as normal. Scores below 26 are considered normal and indicate a possible cognitive impairment. In one embodiment, a patient may be selected based on their MoCA, and / or their MoCA score will improve with treatment according to the present invention.

[0157] Migraine. Migraine in patients with NOTCH3 mutations can be assessed in several ways. One measure is frequency (i.e., how many times per day / week / month / year). Another is severity. Severity can be assessed using a simple visual analog scale (e.g., 0-10 or 0-100) or using a functional assessment such as the Disability Scale (FDS). The FDS asks subjects to assess their performance in daily activities using four response options, from 0 (no impairment, able to function normally) to 3 (severe impairment, unable to do all or most, may require bed rest). The impact on function can also be assessed using the Migraine-Specific Quality of Life Questionnaire or the Migraine-Related Activity Disability Diary (AIM-D).

[0158] In one embodiment, treatment of a patient with fasudil or M3 reduces the frequency and / or severity of migraines.

[0159] Vascular assessment. Furthermore, patients may show improvement in hemodynamic parameters such as cerebral vascular reactivity (the ability of blood vessels to dilate and constrict in response to stimuli) or cerebral hypoperfusion during treatment with fasudil or M3. Improvement may also be seen in vascular patency. In another embodiment, hypertensive patients may improve (blood pressure decreases or returns to normal).

[0160] In another embodiment, retinal vascular density (VD), measured using optical coherence tomography (OCTA), has been suggested as a potential marker of cerebral vascular changes. Specifically, a decrease in retinal VD in young people is associated with CADASIL patients. Treatment with fasudil or M3 may prevent the decrease in VD.

[0161] In further embodiments, revealed diffuse irregularities of the vascular wall, vascular thickening, and coronary symptoms including a mixture of non-calcified / calcified coronary artery lesions and intermediate stenosis will improve with treatment.

[0162] Caregiver assessment. Caregiver assessment may also be used to monitor the progression of NOTCH3 in patients. NPI-Q. In one embodiment, patients with NOTCH3-related small vessel disease treated with ROCK inhibitors such as fasudil are assessed using the Neuropsychological Assessment Questionnaire (NPI-Q).

[0163] The NPI-Q is designed to be a self-administration questionnaire completed by informants for the patients they care for. Each of the twelve NPI-Q domains contains survey questions that reflect the main symptoms of that domain. The first answer to a question in each domain is either "yes" (present) or "no" (not present). Thus, a total score of 36 points will represent the maximum severity across all parameters considered.

[0164] If the answer to a domain question is "no," the informant proceeds to the next question. If the answer is "yes," the informant assesses both the severity of the symptoms present since last month on a 3-point scale and the impact of the symptom on them (i.e., caregiver distress) using a 5-point scale. The NPI-Q provides either a table of symptom severity and distress for each reported symptom, as well as a severity and distress score that reflects the sum of the individual domain scores. The NPI-Q distress assessment has a maximum score of 60 (a score of 5 for all questions). [Examples]

[0165] Approximately 40 patients diagnosed with CADASIL who meet the following criteria will be mobilized.

[0166] Selection criteria: • Patients aged 30-65 • Diagnosis of CADASIL based on clinical symptoms, MRI, and genetic analysis. Specifically, patients with known or new mutations in the NOTCH3 gene involving odd-numbered cysteine ​​residues in at least one EGFR domain, or patients with positive skin biopsies showing typical granular osmiumophilic material (GOM) in the walls of small blood vessels on electron microscopy. Patients without a NOTCH3 sequence at the time of enrollment will undergo sequencing as part of the study. • An alternative to this criterion is as follows: The diagnosis of CADASIL is recorded as follows: (a) Genetic testing of the NOTCH3 gene indicating the presence of high-risk or medium-risk NOTCH3 mutations by Hack (2023) and Fazekas deep or surface white matter score ≥ 1 or (b) Genetic testing of the NOTCH3 gene indicating the presence of a low-risk NOTCH3 mutation by Hack (2023), Fazekas deep or surface white matter score ≥ 1, and at least one of the following clinical signs / symptoms (in the opinion of the principal investigator, and after review and approval by the sponsor): • Subcortical ischemic events, and / or • Cognitive impairment, and / or • Migraine with aura • MMSE > 18 Fazekas Grade 1-3 • Ability to follow clinical trial procedures 1 Unless a standard therapeutic MRI obtained within the past X months is available, confirmation will be made by screening MRI.

[0167] Exclusion criteria. Patients will be excluded if they meet any of the following criteria: • Stroke with symptoms within the past 90 days • Any serious neurological disorder / condition other than CADASIL • Localized lesions that may cause cognitive impairment (e.g., major artery stroke, infection, space-occupying lesion, history of normal pressure hydrocephalus) • Any other illness / condition that may affect adherence to the protocol, such as uncontrolled mental illness or alcohol or substance abuse. • Unable to withstand MRI scans

[0168] Physical examination, vital signs, and standard laboratory assessments will be performed during each site visit. Gluteal muscle biopsies will be performed at baseline and at 6 months.

[0169] Patients are randomized to receive either placebo or fasudil. On day one, patients come to the clinic and undergo a baseline MRI sequence. This includes structural sequences to assess white matter loading, examine brain structures such as Virchow-Robins space, and provide a baseline for assessing changes such as atrophy at later time points. In addition, the sequence is performed to assess local and total cerebral blood flow (arterial spin labeling), responsiveness to CO2 (blood oxygenation level-dependent MRI), and responsiveness to visual or motor stimuli (blood oxygenation level-dependent MRI). 2D phase contrast is used to examine pulsatile (carotides, CSF, and veins) and diffusive (diffusion-weighted imaging) tracts in white matter. Following the first MRI session, patients are orally administered 60 mg of fasudil or the corresponding placebo, and a functional MRI scan is repeated one hour after treatment.

[0170] Patients return to the clinic between one day and one month after their initial MRI visit. Pre-treatment functional MRI scans are performed again before and after treatment, but the treatment assignment is reversed from the initial MRI visit. Patients continue this treatment assignment for 12 months, taking fasudil 60 mg orally three times a day for a total daily dose of 180 mg. Patients in the placebo group receive the corresponding placebo.

[0171] Patients return to the clinic at one month, three months, and six months. Structural MRI is repeated at three and six months. Functional MRI is repeated at every visit. Physical and laboratory assessments are performed at each visit, along with standard cognitive tests, including the Trailmaking Test B and the Wisconsin Card Classification Test for cognition, including processing speed and / or executive function, as well as the Digit Sign Substitution Test.

[0172] The population showed improvements in gray matter perfusion, along with significant improvements in white matter perfusion and overall perfusion at one month visit (after 3 scans) in the drug treatment group compared to the placebo group. Enhanced responses to CO2 and visual or motor stimuli were observed in the drug treatment group. Improved processing speed was evidenced in the drug group after a single treatment and persisted up to six months visit. Executive function remained stable in the drug treatment group throughout six months, but worsened in the placebo group, and executive function in the treatment group was better than in the placebo group at six months. Biopsies showed decreased rho kinase activity in smooth muscle cells, along with activation of survival / anti-apoptotic markers in the drug treatment group compared to the placebo group, suggesting that fasudil reduces apoptotic stress on the vascular system. This is supported by MRI pulsatile measurements showing less vascular rigidity in the drug treatment group compared to the placebo group.

[0173] List of References This specification and all the following references are incorporated herein by reference in their entirety. 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. Cedres N et al.,Predicting Fazekas scores from automatic segmentations of white matter signal abnormalities.Aging.2020;12(1):894-901. 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 Coughlan AK,Oddy M,Crawford JR.The BIRT Memory and Information Processing Battery(B-MIPB).Wakefield,UK:The Brain Injury Rehabilitation Trust(BIRT);2007. 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 Delis DC,Kaplan E,Kramer JH.Delis-Kaplan Executive Function Scale(D-KEFS).San Antonio,TX:The Psychological Corporation;2001. Fischl B et al.,Whole brain segmentation:automated labeling of neuroanatomical structures in the human brain.Neuron.2002;33(3):341-55. 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. 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. 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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. 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. Neves KB et al.,ER stress and Rho kinase activation underlie the vasculopathy of CADASIL.JCI Insight.2019;4(23):e131344. 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):e191350. 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Claims

1. A method for treating a human patient having a mutation in the NOTCH3 gene, comprising administering a therapeutically effective amount of oral fasudil or hydroxyfasudil to the patient.

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

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

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

5. The method according to any one of claims 1 to 4, wherein the NOTCH3 mutation is a gain-of-function mutation that results in an odd number of cysteines in the extracellular domain.

6. The method according to any one of claims 1 to 5, wherein the mutation is not a gain-of-function mutation.

7. The method according to any one of claims 1 to 6, wherein the patient has at least one high-signal area in white matter.

8. The method according to any one of claims 1 to 7, wherein the patient has at least one Fazekas score in the deep or periventricular white matter.

9. The method according to any one of claims 1 to 8, wherein the patient has a defect in DSST or SDDT.

10. The method according to any one of claims 1 to 9, wherein the patient has granular osmiumophilic substances.

11. The method according to any one of claims 1 to 10, wherein the patient has had at least one stroke or transient ischemic attack.

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

13. The method according to claim 10, wherein the existing infarcts are of lacunar origin, and all previous cerebral hemorrhages were microhemorrhages.

14. The method according to claim 9, wherein the patient has had at least one stroke or transient ischemic attack at least 90 days prior to the start of treatment.

15. The method according to any one of claims 1 to 14, wherein the patient exhibits evidence of cerebrovascular reactivity, abnormally reduced neurovascular connectivity, or localized or global cerebral hypoperfusion.

16. The method according to any one of claims 1 to 15, wherein the patient shows evidence of an abnormal increase in vascular muscle tone.

17. The method according to any one of claims 1 to 16, wherein the patient has vascular mild cognitive impairment.

18. The method according to any one of claims 1 to 17, wherein the patient has vascular dementia.

19. The method according to any one of claims 1 to 18, wherein the NOTCH3 variant is an EGFr1 to 6 pathogenicity variant.

20. The method according to any one of claims 1 to 19, wherein the NOTCH3 variant is an EGFr7-34 pathogenic variant.

21. The method according to any one of claims 1 to 20, wherein the NOTCH3 variant is an EGFr18-34 pathogenic variant.

22. The method according to any one of claims 1 to 21, wherein the NOTCH3 variant is a high-risk variant.

23. The method according to any one of claims 1 to 22, wherein the NOTCH3 variant is a moderate-risk variant.

24. The method according to any one of claims 1 to 23, wherein the NOTCH3 variant is a low-risk variant.

25. The method according to any one of claims 1 to 24, wherein the NOTCH3 variant is an unknown risk variant.

26. The method according to any one of claims 1 to 25, wherein the patient is treated with a therapeutically effective dose of a second vasoactive drug.

27. The method according to claim 20, wherein the second vasoactive agent is selected from the group consisting of angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, phosphodiesterase inhibitors, and nitrates.

28. The method according to any one of claims 1 to 27, wherein the patient has migraines.

29. The method according to claim 22, wherein the migraine occurs at least once a month.

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

31. The method according to any one of claims 1 to 30, wherein the patient does not have the R169C NOTCH3 mutation.

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