Diaminophenothiazines for the treatment of microvascular cerebral disease.

Abstract

The present invention provides a method for the therapeutic or prophylactic treatment of microvascular cerebral disease in a subject, comprising orally administering to said subject a reduced form of a methylthioninium (MT)-containing compound, said administration providing a total daily dose of 8-200 mg of MT to the subject per day, optionally divided into two or more doses.

Description

[Technical Field]

[0001] Technical Field The present invention relates generally to methods and materials for use in the treatment of microvascular brain diseases. [Background technology]

[0002] Background of the Invention Microvascular encephalopathy results from alterations or loss of the integrity of small blood vessels in the brain and is a common finding in older adults. It can manifest as silent ischemic lesions identified as white matter hyperintensities (WMHs) or lacuneas on computed tomography and magnetic resonance imaging (MRI) scans (see Koton, Silvia, et al. “Microvascular brain disease progression and risk of stroke: the ARIC study.” Stroke 51.11 (2020):3264-3270).

[0003] However, if left untreated, microvascular disease can lead to cognitive decline, dementia, stroke, and other undesirable outcomes.

[0004] Treatment of microvascular encephalopathy generally depends on the suspected underlying cause, but may typically involve reducing or managing risk factors such as high blood pressure, cholesterol levels, diabetes, and smoking. For example, subjects suffering from or considered at high risk for microvascular encephalopathy may be encouraged to adopt a healthier lifestyle and may be prescribed medications to lower high blood pressure and control cholesterol and glucose levels.

[0005] WO 2022 / 029606 reports that known pharmacological treatments for microvascular damage include drugs for controlling narrowing of small blood vessels, such as nitroglycerin, beta-blockers, calcium channel blockers, statins, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), ranolazine (Ranexa), and aspirin. WO 2022 / 029606 reports that cannabinoid-based compositions can be used to prevent and / or treat damage to the integrity of microvessels, and thus to treat pathologies associated with damaged microvessels. Summary of the Invention [Problem to be solved by the invention]

[0006] It is recognized that providing novel treatments for microvascular brain diseases would provide a useful contribution to the art. [Means for solving the problem]

[0007] DISCLOSURE OF THE INVENTION The inventors have shown that diaminophenothiazines are effective in reducing the rate of progression of white matter hyperintensities and microvascular pathology in subjects, as demonstrated in a clinical trial over a one-year course of treatment.

[0008] The diaminophenothiazine compound used in this study was the compound leucomethylthioninium (LMT). Methylthioninium (MT) is a redox molecule that can form both a reduced form (LMT) and an oxidized form (MT) depending on environmental conditions (e.g., pH, oxygen, reducing agents). + ) exists in equilibrium between

[0009] MT (methylthioninium chloride, MTC, also known as methylene blue) has been reported to potentially slow the progression of perfusion-diffusion mismatch to infarct in persistent ischemic stroke (see Rodriguez, Pavel, et al. “Methylene blue treatment delays progression of perfusion-diffusion mismatch to infarct in permanent ischemic stroke.” Brain research 1588 (2014):144-149) and may also be neuroprotective in ischemic stroke (see Shen, Qiang, et al. “Neuroprotective efficacy of methylene blue in ischemic stroke: an MRI study.” PloS one 8.11 (2013):e79833).

[0010] Both of these studies modeled ischemic stroke using occlusion of the middle cerebral artery and reported a reduction in lesion volume after reperfusion. The exact mechanism of neuroprotection was unclear.

[0011] However, neither study reported nor suggested any effect on MTC on the progression of microvascular pathology or white matter intensity in human subjects, and effects on this entirely different pathology could not reasonably be predicted from those reported based on occlusive events in a single large vessel.

[0012] The treatment of white matter hyperintensities and microvascular pathology demonstrated by the present inventors was part of a clinical trial for the treatment of mild to moderate Alzheimer's disease (AD).

[0013] MT acts as a tau aggregation inhibitor in vitro (Wischik CM, Edwards PC, Lai RYK, Roth M, Harrington CR. Selective inhibition of Alzheimer's disease-like tau aggregation by phenothiazines. Proc Natl Acad Sci USA 1996; 93:11213-8; Harrington CR, Storey JMD, Clunas S, et al. Cellular models of aggregation-dependent template-directed proteolysis to characterize tau aggregation inhibitors for treatment of Alzheimer's disease. J Biol Chem 2015; 290:10862-75.), dissolves PHFs from Alzheimer's disease brain tissue (Wischik et al., 1996), and reduces tau pathology and associated behavioral deficits in transgenic mouse tau models at brain concentrations consistent with human oral administration (Melis V, Magbagbeolu M, Rickard JE, et al. Effects of oxidized and reduced forms of methylthioninium in two transgenic mouse tauopathy models. Behav Pharmacol 2015; 26:353-68;Baddeley T, C., McCaffrey J, Storey JMD, et al. Complex disposition of methylthioninium redox forms determines efficacy in tau aggregation inhibitor therapy for Alzheimer's disease. J Pharmacol Exptl Therapeutics 2015; 352:110-8.).MT has also been shown to inhibit other disease-associated protein aggregation (see, eg, WO 2007 / 110629 and references therein).

[0014] However, MT or LMT have not previously been described for the treatment of microvascular pathology. Indeed, the fact that the burden of microvascular pathology was a significant contributor to cognitive impairment in AD was quite unexpected, and even more surprising, this burden was seen in typical or "pure" AD but not in the mixed AD / vascular subtype.

[0015] Thus, in one aspect, there is provided a method for the therapeutic or prophylactic treatment of microvascular cerebral disease in a subject, comprising orally administering to said subject a methylthioninium (MT)-containing compound, wherein the MT-containing compound is [ka] or a hydrate or solvate thereof.

[0016] "Microvascular encephalopathy" is caused by changes or loss of the integrity of small blood vessels in the brain. Depending on the severity of these changes, complications ranging from difficulty focusing to stroke can occur. Therefore, this term encompasses microvascular dysfunction in the brain that causes microvascular ischemic disease, such as cerebral small vessel disease (CSVD), chronic microvascular ischemic (brain) disease, and microvascular dysfunction that causes small vessel ischemic disease. Cerebral microhemorrhage (MB) is a small chronic cerebral hemorrhage likely caused by structural abnormalities in the brain's small blood vessels (Anand Viswanathan and Hugues Chabriat (2006) Cerebral microhemorrhage. Stroke. 37:550-555).

[0017] Such changes or loss of integrity can occur due to injury, but are thought to occur gradually even in normal aging subjects. Microvascular ischemic disease therefore occurs in older adults, affecting both men and women equally. Approximately 5% of people aged 50 are affected. However, nearly 100% of people over the age of 90 are affected. Microvascular ischemic disease is a very common condition in older adults. It is estimated that microvascular ischemic disease accounts for 45% of dementia cases and 25% of strokes. Transient ischemic attacks (TIAs) or "mini-strokes" are caused by a temporary interruption in blood supply to part of the brain, which can also result from microvascular ischemic disease.

[0018] Suitable targets and indications for treatment or prevention according to the present invention include those mentioned above and those discussed in more detail below.

[0019] The methods of the present invention are intended to reduce the rate of progression of microvascular pathology in a subject, i.e., reduce the increase in total lesion volume over time that would otherwise be seen in the same subject or in a corresponding subject not receiving the treatment. The treatment period is typically at least six months but can be longer, e.g., one, two, three, four, or five years, or even longer. As shown in the Examples below, subjects progressed in microvascular pathology, as measured by total volume of white matter hyperintensities, over a one-year period, and this progression could be inhibited by the use of LMT compounds.

[0020] Thus, the methods of the present invention may be used with disease-modifying effects on microvascular pathology, for example, to prevent and / or treat damage to microvascular integrity and thus treat pathologies associated with damaged microvascular vessels.

[0021] The method may be used to reduce cognitive impairment in a subject that would otherwise result from progressive microvascular pathology.

[0022] The mechanisms underlying the effects of LMTM on microvascular pathology are unclear.

[0023] Without wishing to be bound by theory, it is possible that this LMTM effect may be exerted by enhancing mitochondrial metabolism. The reduction in the effectiveness of LMTM treatment with concurrent symptomatic therapy would be consistent with its effect on mitochondrial function (Atamna, H., Mackey, J. & Dhahbi, JM (2012) Mitochondrial pharmacology: electron transport chain bypass as strategies to treat mitochondrial dysfunction. Biofactors 38, 158-66; Riedel, G., Klein, J., Niewiadomska, G., Kondak, C., Schwab, K., Lauer, D., Magbagbeolu, M., Steczkowska, M., Zadrozny, M., Wydrych, M., Cranston, A., Melis, V., Santos, RX, Theuring, F., Harrington, CR & Wischik, CM (2020) Mechanisms of anticholinesterase interference with tau aggregation inhibitor activity in a tau-transgenic mouse model. Current Alzheimer Research 17, 285-296).

[0024] Microvascular brain disease and microvascular pathology can be measured as the total lesion volume (TLV) of hyperintense areas seen on MRI. As described herein, it has surprisingly been found that the burden of microvascular pathology is a significant contributor to cognitive impairment in AD, and this burden can be reduced by the use of the compounds described herein.

[0025] In particular, treatment with LMT has been shown to result in an exposure-dependent reduction in the progression of microvascular pathology, thereby providing a novel approach to the treatment of microvascular pathology in subjects. In one embodiment, the subject is a human.

[0026] In one embodiment, the subject is between 40 and 89 years old, for example, at least 60 years old or about 70 years old.

[0027] In one embodiment, a subject may be diagnosed and / or selected, or has been diagnosed and / or selected, as having said microvascular brain disease, for example, based on imaging such as TLV as described above.

[0028] A further unexpected finding described herein is that hypertension was more common in typical AD than in the mixed subtype (AD and CVD), and that this association is driven by the previously unquestioned contribution of microvascular pathology to cognitive impairment in typical AD.

[0029] Thus, in one embodiment, the subject may be a subject who has been diagnosed with pure AD (i.e., not mixed AD) and / or other indications associated with microvascular pathology as described herein.

[0030] Cerebrovascular disease (CVD) has risk factors such as hypertension, diabetes, atherosclerosis, hypercholesterolemia, and dyslipidemia.

[0031] As described in the Examples below, brain atrophy was the primary driver of cognitive impairment in AD patients, while microvascular pathology in the form of WMHs was significantly higher in hypertensive patients regardless of subtype (p≦0.0001).

[0032] As such, treatment or prevention in the present invention can be based on any of these risk factors, or on other predisposing familial or genetic data.

[0033] WO 2008 / 155533 describes the use of MT compounds for the treatment of mild cognitive impairment. WO 2021 / 001306 describes the use of MT compounds for general cognitive function. None of these publications discusses the role of MT in microvascular cerebral disease or any effect on microvascular pathology.

[0034] In some embodiments, the subject may not suffer from any type of AD. For example, the subject of the present invention may not suffer from or have not been diagnosed with, for example, vascular dementia, senile dementia, age-related memory impairment, Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease, or mild cognitive impairment. Therefore, such a subject may be diagnosed as not suffering from these diseases. In this regard, diagnosis may be according to the generally recognized criteria of The Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5, American Psychiatric Association, 2013). Similarly, such a subject may not suffer from PTSD or a defect in mitochondrial energy metabolism.

[0035] For purposes of the present invention, it is expected that a wide range of dosages (8 mg / day to 200 mg / day) will be usable.

[0036] Thus, administration may provide a total daily dose of 8-200 mg of MT to a subject per day, optionally divided into two or more doses, although it may be preferable to use a lower dose (e.g., 8-60 mg / day, e.g., 8 mg / day).

[0037] Based on the concentration-dependence described below, the total daily MT dose can advantageously be 12-60 mg. An exemplary dosage is 16-32 mg. A further exemplary dosage is 16-30 mg.

[0038] Thus, the total daily dose can be about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 mg.

[0039] The present invention relates to the administration of MT in its reduced (LMT) form.

[0040] The total daily dose of the compound may be administered in divided doses two or three times daily.

[0041] As explained below, when administering divided MT doses in more doses per day, it may be desirable to use a lower total amount within the recited ranges compared to single daily administration or fewer doses per day.

[0042] Currently approved treatments for Alzheimer's disease include acetylcholinesterase inhibitors (AChEIs) and the N-methyl-D-aspartate receptor antagonist memantine.

[0043] Analyses in the Examples below show that LMTM as monotherapy was associated with a lower increase in TLV compared to subjects receiving LMTM in combination with these symptomatic treatments.

[0044] Thus, in some embodiments, the treatment is monotherapy, meaning that the combination of an AChEI and memantine is excluded.

[0045] In some embodiments, subjects are selected who have not recently been treated with an AChEI or memantine or other symptomatic therapy.

[0046] In other embodiments, the treatment is a combination therapy, e.g., a combination with an AChEI and / or memantine, such that patients already receiving an AChEI and / or memantine may benefit from administration of these doses of an MT compound, and subjects receiving these doses of an MT compound may benefit from an AChEI and / or memantine.

[0047] The treatment may also be combined with other drugs or procedures intended to treat microvascular brain diseases, for example, drugs to lower high blood pressure and control cholesterol and glucose levels.

[0048] These aspects and embodiments will now be described in more detail.

[0049] Methylthioninium moiety

[0050] [Table 1]

[0051] The MT-containing compounds used in the present invention contain the MT moiety as the reduced active ingredient (referred to as "LMT"). The LMT moiety itself is not stable. Therefore, the LMT moiety is administered as an LMT compound (e.g., an LMT salt).

[0052] LMT-containing compounds are generally stabilized, for example, by the presence of one or more protic acids (eg, two protic acids).

[0053] The MT content of such salts can be readily calculated by one of skill in the art based on the molecular weight of the compound and the molecular weight of the MT moiety. Examples of such calculations are provided herein.

[0054] WO 2007 / 110627 disclosed certain 3,7-diamino-10H-phenothiazinium salts that are effective as drugs or prodrugs for the treatment of diseases, including Alzheimer's disease and other diseases such as frontotemporal dementia (FTD). This compound is also in the "reduced" or "leuco" form when considered in relation to MTC. This leucomethylthioninium compound was designated the "LMTX" salt.

[0055] WO 2012 / 107706 describes other LMTX salts with superior properties to those listed above, including leucomethylthioninium bis(hydromethanesulfonate), LMTM (USAN name hydromethylthionine mesylate, HMTM).

[0056] [Table 2]

[0057] Specifically, LMTM retains TAI activity in vitro and in vivo (Harrington et al., 2015; Melis et al., 2015), has excellent pharmaceutical properties in terms of solubility and pKa, and possesses the same activity as MT + It is not subject to the absorption limitations of the form ( Baddeley et al., 2015 ).

[0058] WO 2018 / 019823 describes a novel regimen for the treatment of neurodegenerative disorders utilizing methylthioninium (MT)-containing compounds. Briefly, this regimen identified two important factors: the first was related to the dosage of the MT compound, and the second was an interaction with symptomatic treatments based on modulating acetylcholinesterase levels.

[0059] In analyses described in WO 2018 / 019823, low doses of MT compounds (e.g., 4 mg bid) demonstrated therapeutic efficacy when compared with monotherapy and combination therapy. The efficacy profile was similar in mild and moderately affected subjects for most of the outcomes measured.

[0060] Furthermore, the therapeutic effect in AD (according to study criteria) was limited to patients receiving LMTM as monotherapy. In contrast, the declines seen in the majority of patients receiving LMTM in combination with AD-labeled treatments (acetylcholinesterase inhibitors [AChEIs] and / or memantine) at the corresponding doses were indistinguishable from those seen in the control arm for all parameters.

[0061] WO 2018 / 019823 did not provide any teaching regarding the effect of LMTM on microvascular encephalopathy, nor did it provide any teaching regarding its interaction with AD labeling treatment in the treatment of microvascular encephalopathy.

[0062] WO 2020 / 020751 describes a novel pharmacokinetic (PK) model designed for dosing MT compounds in patient populations. This versatile model was used to estimate the Cmax of parent MT in patients receiving LMTM in two Phase 3 AD trials described in WO 2018 / 019823 (Studies "005" and "015" for the treatment of patients with mild or mild-to-moderate AD, respectively, as discussed in Example 1 below). Estimating Cmax in each subject allowed for the derivation of a distribution of Cmax estimates for each treatment population. This novel analysis revealed the presence of a concentration response within the low-dose treatment population.

[0063] WO 2020 / 020751 did not provide any teaching regarding the effect of LMTM on microvascular cerebral disease, nor did it provide any teaching regarding exposure dependency in such treatment.

[0064] Preferred compounds for use in the present invention are "LMTX" compounds of the type described in WO 2007 / 110627 or WO 2012 / 107706.

[0065] Thus, the compound may be selected from compounds of the formula: or a hydrate or solvate thereof:

[0066] [Table 3]

[0067] H n A and H n Each B, if present, is a protic acid which may be the same or different.

[0068] "Protonic acid" refers to the acid that reacts with protons (H + ) donor. Therefore, in the protonic acid, A - or B - is the conjugate base. Therefore, protic acids have a pH in water less than 7 (i.e., the concentration of hydronium ions is less than 10 per liter). -7 (more than a mole).

[0069] In one embodiment, the salt is a mixed salt having the formula: where HA and HB are different monoprotic acids.

[0070] [Table 4]

[0071] However, preferably the salt is not a mixed salt but has the formula:

[0072] [Table 5] wherein H n Each X is a protic acid, such as a diprotic acid or a monoprotic acid.

[0073] In one embodiment, the salt has the formula: where H2A is a diprotic acid.

[0074] [Table 6]

[0075] Preferably, the salt has the formula:

[0076] [Table 7]

[0077] Examples of protic acids that may be present in the LMTX compounds used herein include: Inorganic acids: hydrohalide acids (e.g., HCl, HBr), nitric acid (HNO3), sulfuric acid (H2SO4) Organic acids: carbonic acid (H2CO3), acetic acid (CH3COOH), methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid.

[0078] Preferred acids are monoprotic acids and the salts are bis(monoprotic acid) salts.

[0079] A preferred MT compound is LMTM:

[0080] [Table 8]

[0081] Weighting Factor The anhydrous salt has a molecular weight of approximately 477.6. Based on the molecular weight of the LMT core of 285.1, the weighting factor for the use of this MT compound in the present invention is 1.67. "Weighting factor" refers to the relative weight of the pure MT-containing compound to the weight of the MT it contains.

[0082] Other weighting factors may be calculated, for example, for the MT compounds herein, from which corresponding dosage ranges may be calculated.

[0083] Other example LMTX compounds are as follows, with their molecular weights (anhydrous) and weighting factors also shown:

[0084] [Table 9]

[0085] [Table 10]

[0086] In some embodiments, the LMT compound is not Compound 8.

[0087] As such, the dosages set forth herein for MT apply mutatis mutandis to these MT-containing compounds as adjusted for molecular weight.

[0088] Methods for chemically synthesizing the MT-containing compounds described herein are known in the art, for example: The synthesis of compounds 1-7 can be carried out according to the method described in WO 2012 / 107706 or methods analogous thereto. The synthesis of compound 8 can be carried out according to the method described in WO 2007 / 110627 or methods analogous thereto.

[0089] Accumulation Factor As will be appreciated by those skilled in the art, for a given daily dosage, more frequent administration may result in greater accumulation of the drug.

[0090] Thus, in certain embodiments of the claimed invention, the total daily dosage of the MT compound may be relatively low when administered more frequently (e.g., twice daily "bid" or three times daily "tid"), or may be higher when administered once daily "qd."

[0091] Treatment and Prevention The term "treatment", as used herein in reference to treating a condition, generally relates to human or animal (e.g., veterinary) treatments and therapies to achieve some desired therapeutic effect, such as inhibition of progression of the condition, including slowing the rate of progression, halting the rate of progression, regression of the condition, amelioration of the condition, and cure of the condition.

[0092] The term "therapeutically effective amount," as used herein, relates to an amount of a compound of the present invention, or a material, composition, or dosage form comprising said compound, that, when administered in accordance with a desired treatment regimen, is effective to produce some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. The inventors have demonstrated that the therapeutically effective amount of an MT compound for the diseases of the present invention can be much lower than previously understood in the art.

[0093] The term "prophylactically effective amount," as used herein, relates to an amount of a compound of the invention, or a material, composition, or dosage form comprising said compound, that, when administered in accordance with a desired treatment regimen, is effective for producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio.

[0094] "Prophylaxis" in the context of this specification should not be understood to encompass complete success, i.e., complete protection or complete prevention. Rather, prophylaxis in this context refers to measures taken prior to the detection of a symptomatic condition, with the goal of maintaining health by helping to delay, alleviate, or avoid that particular condition.

[0095] Combination Treatments and Monotherapy The term "treatment" includes "combined" treatments and therapies in which two or more treatments or therapies for the same disorder are combined, for example sequentially or simultaneously. These may be symptomatic or disease-modifying simultaneous treatments.

[0096] The specific combination will be at the discretion of the physician.

[0097] In combination treatment, the agents (i.e., an MT compound described herein and one or more other agents) can be administered simultaneously or sequentially, and can be administered individually at different dose schedules and via different routes. For example, when administered sequentially, the agents can be administered closely spaced (e.g., over 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4, or more, or even longer, times apart, as needed), with the exact dosing regimen being commensurate with the properties of the therapeutic agents.

[0098] An example of a combination treatment of the present invention for the treatment of microvascular cerebral disease would be a drug that is an MT-containing compound at a specified dosage combined with a drug that acts to lower blood pressure, such as aspirin, candesartan, and simvastatin (Smith and Markus, 2020, Stroke 51:38-46).

[0099] In other embodiments, the treatment is a "monotherapy," i.e., the MT-containing compound is not used in combination (within the meaning discussed above) with another active agent for treating microvascular cerebral disease in a subject.

[0100] Subjects, patients, and patient populations The teachings of the present invention may be applied to a subject / patient that is an animal, mammal, placental mammal, rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), lagomorph (e.g., rabbit), avian (e.g., bird), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), porcine (e.g., pig), ovine (e.g., sheep), bovine (e.g., cow), primate, simian (e.g., monkey or ape), monkey (e.g., marmoset, baboon), monotreme (e.g., platypus), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human.

[0101] The patient may be an adult human, and the population-based dosages described herein are based on this assumption (typical body weight 50-70 kg). If necessary, corresponding dosages may be utilized for subjects outside this range by using a subject weight factor, which is the subject weight divided by 60 kg to obtain a multiplication factor for that individual subject.

[0102] Parts Labels, Instructions, and Kits Therapeutic or prophylactic compositions described herein may be provided in a labeled packet along with instructions for their use.

[0103] In one embodiment, the pack is a bottle as is well known in the pharmaceutical arts. A typical bottle may be made from pharmacopoeia-grade HDPE (high density polyethylene) with a child-resistant HDPE push-lock closure and contains a silica gel desiccant present in a sachet or canister. The bottle itself may include a label and may be packaged in a cardboard container along with instructions and, optionally, a further copy of the label.

[0104] In one embodiment, the pack or packet is a substantially moisture-tight blister pack (preferably one with an aluminum cavity and aluminum foil), in which case the pack may be packaged in a cardboard container with instructions and a label on the container.

[0105] The label or instructions may provide information about the disorder for which the drug is intended.

[0106] The label or instructions may provide information regarding the maximum allowable daily dosage of the compositions described herein, for example, on a once-daily, bid or tid basis.

[0107] The label or instructions may provide information regarding the proposed duration of treatment, as described herein.

[0108] Treatment method Another aspect of the present invention relates to a method of treatment, as described above, comprising administering to a patient in need of treatment a prophylactically or therapeutically effective amount of a compound described herein (preferably in the form of a pharmaceutical composition).

[0109] Use in therapeutic methods Another aspect of the present invention pertains to compounds or compositions described herein for use in a method of treatment of the human or animal body (e.g., of a disease state) by therapy.

[0110] Use in pharmaceutical manufacturing Another aspect of the present invention relates to the use of an MT compound or composition described herein in the manufacture of a medicament for use in the treatment (eg, of a disease state).

[0111] In order to more fully describe and disclose the present invention and the prior art to which it pertains, several patents and publications are cited herein. Each of these references is incorporated by reference in its entirety into the present disclosure herein to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0112] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not to the exclusion of any other integer or step or group of integers or steps.

[0113] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0114] Ranges are often expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

[0115] Any subheadings herein are included for convenience only and should not be construed as limiting the disclosure in any way.

[0116] The invention will now be further described with reference to the following non-limiting figures and examples, in light of which other embodiments of the invention will occur to those skilled in the art.

[0117] The disclosures of all references cited herein, insofar as they may be used by those skilled in the art to carry out the invention, are hereby specifically incorporated by cross-reference. [Brief explanation of the drawings]

[0118] Figures and Tables [Figure 1] Examples of altered FDG-PET patterns characteristic of metabolic activity in patients with AD (A and C) and mixed subtype (B and D) in the axial, sagittal, and coronal planes. These images also support temporoparietal hypometabolism in AD. FDG uptake in mixed AD confirmed reduced uptake in vascular regions of the brain, such as the frontal and temporal lobes and cerebellum. [Figure 2] Various signs of cerebral small vessel disease include small subcortical infarcts, white matter hyperintensities (WMH), lacunae (usually with a hyperintense rim), perivascular spaces (mostly linear without a hyperintense rim), and cerebral microbleeds (round or oval) (Wardlaw, JM, Smith, EE, Biessels, GJ, Dichgans, M. (2013) Lancet Neurol, 12, 822-38). [Figure 3] Illustration of WMHs from two subjects in three coronal planes. The upper panel shows an occasional small WMH, and the lower panel shows an example with numerous larger WMHs. [Figure 4] Frequency of WMH by various metrics in an ageing population (A, total Scheltens score; B, total lesion volume by LGA; and C, % WMH by LGA) (Aberdeen Birth Cohort ('Children of the Nineteen Fifties')). [Figure 5] History of diabetes as a risk factor for WMH in the elderly population. The figure shows raw WMH%. [Figure 6] Exposure-dependent differences in the rate of progression of microvascular pathology (total lesion volume (TLV) measured in cm3, change from baseline) for LMTM as monotherapy, modeled using a repeated measures mixed effects model. [Figure 7A] LMTM as monotherapy at two different doses (4 and 100 mg bid) was associated with a lower increase in TLV compared to subjects receiving LMTM in combination with AChEI / Mem. [Figure 7B] LMTM as monotherapy at two different doses (4 and 100 mg bid) was associated with a lower increase in TLV compared to subjects receiving LMTM in combination with AChEI / Mem. DETAILED DESCRIPTION OF THE INVENTION

[0119] Table 1: Summary of demographic and clinical characteristics at baseline of the population studied.

[0120] Table 2: Statistical analysis of demographic and clinical characteristics at baseline according to FDG-PET AD classification.

[0121] Table 3: Association between scan subtype and common vascular risk factors recorded in the patient's case report file.

[0122] Table 4: Frequency of cardiovascular risk factors.

[0123] Table 5: Association between microvascular pathology and gender in both AD subtypes.

[0124] Table 6: ADAS-cognitive scores in both AD subtypes 11 Association of scores with microvascular pathology, brain atrophy, age, and gender.

[0125] Table 7: Comparison of microvascular pathology and brain fractions in the presence or absence of hypertension. [Example]

[0126] Example 1 - Clinical Trial Background Dementia is a progressive, acquired condition of intellectual degeneration that significantly impairs the performance of personal, social, or occupational functions and prevents an individual from living independently. This condition persists for at least six months. [1] Dementia is a collective term for a range of symptoms that can be caused by several diseases affecting the brain. Alzheimer's disease (AD) is the most common brain pathology that causes dementia and frequently coexists with cerebrovascular disease (CVD). In the absence of definitive clinical diagnostic tests to distinguish dementia subtypes, brain imaging and body fluid biomarkers are used to aid in etiological diagnosis. Recent classification schemes based on the presence of positive markers for amyloid (Aβ), tau, and indicators of neurodegeneration have been proposed. [2,3] However, this presents a problem in that etiological, diagnostic, and ultimately therapeutic advantages must be attributed to amyloid. Given the dissociation between amyloid pathology and cognitive decline,[4,5] the failure of numerous therapeutic approaches targeting amyloid pathways,[6] and the uncertain clinical benefit in clinical trials showing an effect on amyloid burden,[7] doubt remains as to whether amyloid burden is a valid surrogate marker for predicting clinical benefit.[8] Even if that goes so far, the practical clinical relevance of defining AD in terms of amyloid pathology remains in question.

[0127] A more important practical distinction in the etiological subclassification of dementia is that between AD and CVD. These conditions share specific characteristics and can be distinguished by various imaging modalities, thus contributing to differential diagnosis and helping to better understand the pathological substrate of cognitive impairment across the lifespan.[9,10] Over the past few decades, a growing body of literature has supported the possible role of vascular risk factors (VRFs) in the pathogenesis of AD.

[11] The co-occurrence of AD and CVD pathology is more frequent than expected based on age alone.

[12] Reduced cerebral perfusion is generally thought to result from vascular changes leading to brain dysfunction and cognitive impairment.

[13] AD and CVD have been reported to share common risk factors, including hypertension, diabetes, atherosclerosis, hypercholesterolemia, and apolipoprotein E4 genotype.

[14] However, these relationships are not simple and depend on when and how risk factors and AD are defined, for example, on the stage of life when hypertension develops and whether AD diagnosis is based on amyloid biomarkers. Among the common VRFs that contribute to cognitive impairment, hypertension in midlife and diabetes in later life have relative risks of 1.6 and 1.5, respectively.

[15] These associations have led to the emergence of the vascular hypothesis of AD. VRFs have been proposed to play a causative role in the development of AD neuropathology.

[16] This has generated significant interest in the potential of lifestyle and dietary interventions in preventing AD based on reducing risk factors that overlap with VRFs.[15,17]

[0128] Structural and molecular imaging techniques are being used in the evaluation of patients with dementia to improve diagnostic accuracy. In comparative studies, 18F-fluoro-deoxy-glucose-positron emission tomography (FDG-PET) appears to be superior to other diagnostic methods, such as clinical diagnosis, magnetic resonance imaging (MRI), computed tomography (CT), and single-photon emission computed tomography (SPECT) using the technetium-99m hexamethylpropyleneamine oxime (HMPAO) tracer.[18-23] A key feature of FDG-PET is that it measures the functional impact of pathology on cerebral metabolism. It is important to note that AD and CVD commonly coexist, and patients with both conditions have poorer cognitive outcomes, worse prognosis, and less favorable responses to candidate disease-modifying treatments currently in development.

[24] Because both AD and CVD are independently associated with cognitive impairment, there are limitations to the extent to which the contribution of VRF to the etiology of AD pathology can be inferred based on clinical diagnostic criteria alone. If the combination of AD pathology and CVD lowers the threshold for detecting clinically significant cognitive impairment, risk factor attribution may be biased toward attributing causal significance to vascular factors that they may not have.

[0129] A way to avoid the confounding effects of biased case ascertainment is to use FDG-PET in patients who meet clinical diagnostic criteria for AD to distinguish between patients with only typical temporoparietal metabolic deficits of AD and those with mixed AD / CVD changes. We reasoned that if typical vascular risk factors (hypertension, diabetes, and dyslipidemia) indeed contribute to pure AD pathogenesis, this should be demonstrable in a subgroup of patients who meet both clinical and FDG-PET criteria for typical AD in the absence of evidence of mixed vascular pathology. On the other hand, if VRFs primarily contribute to the mixed AD / CVD phenotype, these factors should be overrepresented in the mixed population. To examine this suspicion, we used baseline data from a large cohort of 794 patients available from two completed phase 3 trials of mild to moderate AD [25, 26] who had FDG-PET scans available at baseline and met the National Institute of Aging and the Alzheimer's Association (NIA / AA) diagnostic criteria for probable AD

[27] with VRF documented in the trial case report forms.

[0130] Materials and methods for testing Patient population The design and results of two large, phase 3, double-blind, controlled, randomized clinical trials of hydromethylthionine in probable AD have been described elsewhere.[25,26] Study TRx-237-015 (clintrials.gov NCT01689246) included 890 patients with mild / moderate AD, and Study TRx-237-005 (clintrials.gov NCT01689233) had 800 patients with mild AD who met the NIA / AA diagnostic criteria for probable AD.

[27] Eligible patients were under 90 years of age. Patients were excluded if they had the following: uncontrolled hypertension, a recent history of clinically significant cardiovascular disease (e.g., hospitalization for symptoms consistent with acute coronary syndrome or angina pectoris), or a modified Hachinski ischemia score greater than 4. Patients were also excluded if they had significant vascular pathology on brain MRI, such as large confluent white matter hyperintensities that would have led to a diagnosis other than AD. A detailed list of all inclusion and exclusion criteria for both studies is available in the supplemental materials. FDG-PET scans were performed at baseline only in a subgroup of TRx-237-015 subjects (n=198) but were also required for all TRx-237-005 participants if appropriate imaging facilities were available at the study site. Of the 1,690 participants enrolled in both studies, 794 had FDG-PET scans available at baseline.

[0131] In both studies, medical history recorded on case report forms at baseline included any previous diagnosis of vascular disorders, as well as the presence of hypertension, diabetes, and dyslipidemia. In addition, information on concomitant medications related to the treatment of potential vascular risk factors, such as antihypertensives, insulin, or other hypoglycemic medications, was also recorded.

[0132] Classification of FDG-PET images FDG-PET images from 794 participants were visually classified into those with a typical AD imaging pattern and those with a mixed AD / CVD pattern. Subjects with typical temporoparietal hypometabolism combined with defects in one or more vascular territories were classified as having a mixed AD / CVD pattern, whereas subjects with only temporoparietal defects were classified as having only typical AD. Classification was based on visual review of scans displayed in three planes using a standard color scale representing FDG uptake using the PMOD Alzheimer's Discriminant Analysis Tool (PALZ). Classification was performed independently by two observers, an ADM and a SMT. Inter-rater reliability, measured using Cohen's kappa, was 0.55, indicating an acceptable level of agreement. In cases of disagreement, images were collaboratively reviewed and discussed until consensus was reached.

[0133] MRI brain acquisition and analysis This study used only GE, Siemens, or Philips (1.5-Tesla and 3.0-Tesla) machines to reduce variability in imaging data. The MRI sequences utilized in this protocol were based on the ADNI protocol. Recommended ranges of acquisition parameters for individual sequences on GE, Siemens, and Philips MRI machines were developed after technical assessment forms from all facilities. The following sequences were included: FLAIR, which allowed for the assessment of other clinically significant focal intracranial pathology and the detection and quantification of WMHs; and unenhanced T1-weighted 3D sequences (e.g., MP-RAGE or SPGR) were used to assess whole-brain, ventricular, and hippocampal volumes, as well as WMHs. MR images were also reviewed to ensure participants did not have mass lesions or new infarcts.

[0134] WMHs obtained from brain MRI were quantified using the Lesion Growth Algorithm (LGA) part of the Lesion Segmentation Toolbox (LST

[28] ), an automated method for extracting WMHs that has been validated on various datasets. [29-31] This algorithm classifies voxels using T1 and FLAIR MR images, segmenting tissue classes into white matter (WM), gray matter (GM), and cerebrospinal fluid (CSF) using T1 images, and fluid-attenuated inversion recovery (FLAIR) imaging to determine areas of hyperintense signal (i.e., WMHs). WMH data were analyzed in cm. 3 The total lesion volume (TLV) measured in the whole brain is provided as a function of time.

[0135] Brain MRI volumes were measured using Freesurfer, and white matter, gray matter, and cerebrospinal fluid volumes were extracted. Brain fraction (BF) was calculated as the ratio of brain size (gray matter volume + white matter volume) to total intracranial volume (TICV, gray matter volume + white matter volume + CSF).

[0136] statistics Statistical analysis was performed using R version 3.5.1. Paired sample t-tests were used to compare baseline characteristics across AD imaging subtypes (Table 2). Chi-square tests were used to compare gender distribution of AD subtypes (Tables 2 and 5). Associations between AD subtypes and vascular risk factors (hypertension, diabetes, and dyslipidemia) were examined by binary logistic regression analysis (Table 3). In addition, regression analysis using a generalized linear model was performed to determine the association between AD subtypes and vascular risk factors, controlling for age and gender (Table 4). Baseline cognitive function was assessed using the ADAS-cognitive score. 11 The significance level was set at p<0.05.

[0137] Regression analysis with linear models was used to evaluate ADAS-cognitive function. 11 The associations of scores with microvascular pathology, brain atrophy, age, and sex were determined. The relative contribution of covariates to the variance in ADAS-cog11 scores was calculated using partial R2 The total model contribution to this variance is evaluated using the coefficient of determination, R 2 This analysis was repeated for both AD subtypes (Table 6). In addition, unpaired t-tests were used to compare microvascular pathology and brain fractions in the presence or absence of hypertension (Table 7).

[0138] Example 2 - Test Results 2.1 Baseline Demographics Baseline demographic and clinical data for the 794 subjects are shown in Table 1. The mean age (±se) was 70.56±0.32 years, ranging from 40 to 89 years, and there was a female predominance (55.2%). The overall frequency of vascular risk factors is also shown below: approximately half had hypertension, approximately half had elevated cholesterol, and 14% had diabetes.

[0139] 2.2 FDG-PET FDG-PET images were classified into typical AD and mixed AD / CVD. Scans with a typical AD FDG-PET profile showed reduced glucose uptake limited to the temporoparietal region (Fig. 1A, C). Scans with a mixed AD / CVD profile showed reduced FDG uptake in specific vascular territories, such as the middle cerebral artery, and / or patchy uptake in addition to typical temporoparietal hypometabolism (Fig. 1B, D). Therefore, the mixed AD / CVD category includes patients with macrovascular disease.

[0140] Of the 794 participants, 533 (67.1%, 47.1% male) were classified as having a typical AD scan pattern, and 261 (32.8%, 41.0% male) were classified as having a mixed pattern (Table 2). Patients with a mixed AD / CVD scan profile were significantly younger (difference ± SE, p = 0.002) and more cognitively impaired (difference ± SE, p < 0.001) compared with patients with a typical AD scan profile.

[0141] 2.3. Vascular risk factors Next, we analyzed the association between scan subtype and common vascular risk factors (cardiovascular, diabetes, and dyslipidemia) recorded in patient case report forms. The distribution of these common vascular risk factors is shown in Table 3. Surprisingly, only cardiovascular factors differed in distribution across AD subtypes, being more frequent in subjects with typical AD scans (59%) compared with subjects with mixed scans (47%), a difference that was statistically significant (p = 0.002). Dyslipidemia was also more frequent in patients with typical AD scans (51% typical, 45% mixed), but the difference was not significant (p = 0.392). Similarly, the frequency of diabetes was similar in the two groups (14% typical, 13% mixed; p = 0.567).

[0142] Conditions reported in case report forms that were specifically classified as cardiovascular in nature are listed in Table 4. Hypertension was present in 52% (279 / 533) of the typical AD subgroup, whereas nonhypertensive cardiac factors were present in only 10% (54 / 533) overall. However, these distributions were uneven (p<0.0001), with nonhypertensive factors being more frequent in hypertensive patients. Hypertension was also more frequent in typical AD scan patients (52%) than in patients with a mixed AD profile (42%), and this difference was statistically significant (p=0.0087). We compared baseline blood pressure (sitting and standing) between subtypes to determine whether risk factors could be attributed to the presence of inadequately controlled blood pressure. There were no differences in baseline blood pressure between typical and mixed AD subtypes. Thus, a history of adequately treated hypertension is a major cardiovascular risk factor associated with typical AD and is more frequent in this group than in patients with mixed AD.

[0143] 2.4 White matter hyperintensities and brain segments To better understand why hypertension is more frequently present in typical AD subtypes compared with mixed AD subtypes, we examined the relationship between AD subtype and imaging metrics associated with cognitive impairment using available MRI scans from the same patients. These metrics were brain atrophy (measured by brain fraction, BF)

[32] and microvascular pathology (measured by total lesion volume of white matter hyperintensities, TLV). [33, 34] While there were no overall differences between AD subtypes with respect to either brain atrophy or microvascular pathology, subanalysis showed that lesion volume was significantly higher in typical compared with mixed AD in patients stratified as having moderate impairment at baseline (MMSE 14–19, inclusive; 5.64 ± 0.73 cm in typical AD). 3 and 3.54±0.56cm for mixed AD. 3 , p=0.0268). This suggests that microvascular pathology may contribute to the severity of cognitive impairment in patients with typical AD. To further explore this, we used linear regression models to compare the ADAS-cognitive score at baseline in both the typical AD / CVD and mixed AD / CVD groups. 11 The relative contributions of microvascular pathology, brain atrophy, age, and gender to the severity of dementia as measured by the score were determined. Each variable was standardized (mean = 0, standard deviation = 1) so that linear coefficients in the analysis could be used directly to compare the relative contributions of these factors. In the typical AD scan group, both brain atrophy and microvascular pathology contributed highly significantly to cognitive impairment, with brain atrophy having the greater effect of the two. Age and gender also contributed significantly to cognition in this group. This is in contrast to the mixed AD group, where brain atrophy was the primary factor determining the level of cognitive impairment, with age contributing even more. Microvascular pathology and gender were not significant (Table 5). This also supports the ADAS-cognitive score. 11This can also be illustrated by examining their relative contributions to the variance in the scores (Table 6). The overall model accounts for 16% of the variance in the typical AD subtype and 12% in the mixed AD subtype. Brain atrophy and microvascular pathology independently contribute 10% and 4% of the variance in the typical AD group, respectively. Brain atrophy is also the main contributor to the variance in the mixed AD / CVD group (9%). Only 1% of the variance is explained by microvascular pathology. Although these two forms of pathology differ in their contributions to cognitive impairment in the two AD subtypes, overall there was a high and significant correlation between the two (r = -0.35, p = <0.0001).

[0144] Finally, we investigated the impact of hypertension on the severity of microvascular pathology and whole-brain atrophy. As shown in Table 7, the burden of microvascular pathology was significantly higher in hypertensive patients regardless of subtype. Similarly, brain atrophy was slightly, but still significantly, greater in hypertensive patients. Thus, hypertension significantly contributes to both microvascular pathology and brain atrophy.

[0145] Example 3 - Discussion of Clinical Trial Findings The fundamental objective of this study was to investigate the role of vascular risk factors in a large, well-characterized cohort of patients with mild to moderate AD who met the NIA / AA clinical diagnostic criteria for probable AD, for whom FDG-PET and MRI scan data were also available. Using FDG-PET scans, patients were classified according to whether they had typical temporoparietal defects alone or in combination with known vascular perfusion defects, signifying a mixed AD / CVD phenotype. The expectation was that vascular risk factors (dyslipidemia, diabetes, and hypertension) would have a higher prevalence in patients with the mixed AD / CVD pattern. Instead, we found that only cardiovascular risk factors differed by subtype and were more frequent in typical AD compared with the mixed AD / CVD subtype. The primary cardiovascular factor was found to be a history of clinically controlled hypertension. Further analysis revealed that patients with hypertension had a 34% higher burden of white matter hyperintensities. In contrast, brain fraction decreased by less than 2%. Brain atrophy was the main contributor to cognitive impairment in both AD subtypes and the only pathology that made a significant contribution in the mixed AD / CVD subtype. Although a history of hypertension is known to be a risk factor for cerebral microvascular pathology

[35] , the main surprise from this study was that it contributed primarily to cognitive impairment in the typical AD subtype but not at all in the mixed AD / CVD subtype. These unexpected findings contribute to a better understanding of the role of vascular risk factors in AD. These findings suggest that the contribution of vascular risk factors to cognitive impairment in AD is primarily mediated through small vessel disease, as measured by white matter hyperintensity (TLV) burden. The effect of small vessel disease is not significant in the presence of large vessel areas with reduced FDG uptake, as in the mixed group. It is significant in the absence of large vessel areas with reduced FDG uptake. A history of hypertension is the major vascular risk factor in AD, which selectively contributes to the non-mixed AD subtype.

[0146] Both brain atrophy and microvascular lesion burden are strongly associated with a history of hypertension. Indeed, a history of hypertension, even if now clinically well controlled, has a profoundly detrimental effect on brain pathology and cognitive impairment, especially in the typical AD subtype. Both the association between hypertension and having a typical AD profile on FDG-PET and the lack of such an association in those with a mixed AD / CVD profile are surprising. An important motivation for conducting this analysis was to see whether the association between vascular risk factors and AD could be explained by the confounding effect of mixed AD / CVD pathology. Our results demonstrate a much closer association between microvascular lesions and pathologies underlying brain atrophy than we initially assumed. Available reports linking AD to vascular risk factors tend to include hypertension, diabetes, and dyslipidemia as a cluster.

[36] However, the association we identify between hypertension and the typical AD subtype does not apply to diabetes and dyslipidemia.

[0147] We demonstrate that microvascular pathology, measured by the total volume of hyperintensities seen on MRI, provides an important link, as it is known to be associated with both hypertension and cognitive impairment.

[37] While brain atrophy is a major driver of cognitive impairment in patients with any AD subtype, microvascular pathology burden is a significant contributor only in typical AD. This suggests that the presence of microvascular pathology may determine the reported association between vascular risk factors and AD.

[38] Comparing patients with and without hypertension, there is a 34% difference in brain volume, but a <2% difference in microvascular pathology burden. Therefore, a simple volumetric relationship between these two parameters is unlikely. An alternative explanation is that microvascular pathology burden is associated with more general impairments in perfusion and tissue oxygenation, which increase neuronal vulnerability to either neurofibrillary or amyloid pathology. Tau aggregation pathology burden is directly related to brain atrophy. 39 The presence of hypertension has also been shown to increase susceptibility to AD, 15, 40-42 and is associated with neurofibrillary tangle density. 43 Our data support a surprisingly high correlation of 0.35 between microvascular pathology and brain atrophy. Further studies are needed to more directly measure tau or amyloid pathology burden and their relationship to microvascular pathology.

[0148] Despite using MRI scans to exclude patients with significant CVD from study inclusion, only 67% of our study population had a typical AD profile on FDG-PET scans without defects characteristic of cerebrovascular disease. Furthermore, the combination of clinical criteria and MRI scans failed to exclude 33% of patients without a typical AD pattern on FDG-PET. This corresponds to the misdiagnosis rate when using amyloid-PET criteria.

[44] The primary underlying cause of mixed AD / CVD pathology is attributed to cerebral amyloid angiopathy (CAA).[24,45] If this is true, the recently proposed ATN classification system, which requires evidence of amyloid pathology for AD diagnosis, may increase rather than decrease the confounding effect of CVD on diagnosis and the development of disease-modifying treatments. The contribution of amyloid to microvascular pathology is unclear.

[46] However, if increased in amyloid-positive patients, this may also increase the risk of amyloid-related imaging abnormalities in clinical trials targeting amyloid pathology. The diagnostic accuracy of distinguishing between AD and healthy controls is approximately 94% for FDG-PET and amyloid-PET, although FDG-PET is superior in MCI.[47,48]

[0149] Typical AD and mixed AD differed in age and disease severity in our cohort. Mixed cases were younger and more severely affected. Previous studies have shown that the burden of comorbid AD pathology increases with age, while "pure AD" pathology decreases. [45, 49] However, our results from the study-derived population differed; those with a mixed AD / CVD phenotype were significantly younger than those with typical AD. The most likely explanation for this is that the coexistence of CVD and AD pathology lowered the threshold for a clinical diagnosis of AD [50, 51], increasing the likelihood of meeting study inclusion criteria. Nevertheless, we found that the severity of cognitive impairment was higher in patients with mixed pathology compared with those with a more typical AD pattern. This is consistent with previous findings that patients with "pure" AD pathology are older and have less severe disease compared with patients with cerebrovascular disease.

[52]

[0150] Dyslipidemia is considered a risk factor associated with AD. Elevated cholesterol levels and its derivatives in the brain have been shown to induce neuronal apoptosis and oxidative stress [53, 54], potentially increasing the risk of developing dementia due to neuronal loss and dysfunction. A study by Mielke et al. (2005) examined total serum cholesterol levels in AD patients and found an association between high cholesterol levels and a reduced risk of dementia in later life.

[55] In contrast, follow-up studies have found that high serum cholesterol levels in middle age are associated with an increased risk of developing AD in later life. [56, 57] Clinical trials have not found benefit in individuals with mild to moderate AD treated with statins (simvastatin), despite significant reductions in cholesterol levels.

[58] Despite the role of hypercholesterolemia in the pathogenesis of AD, we have not reported a difference in the frequency of hypercholesterolemia between typical and mixed AD subtypes.

[0151] The relationship between neurodegenerative disorders and metabolic dysfunction, such as diabetes, has attracted considerable attention.[15,59] Diabetes has been reported to have the strongest association with AD among various vascular risk factors.

[60] The Rotterdam study was one of the first to demonstrate an increased risk of dementia with type 2 diabetes.

[61] Similarly, the Kungsholmen Project, a longitudinal population-based study, found that type 2 diabetes and prediabetes accelerate the progression from MCI to dementia by 3.18 years.

[62] Other studies have also reported that diabetes shares similar mechanisms with AD, such as mitochondrial dysfunction, energy homeostasis, and abnormalities in neuroinflammation.

[63] This has motivated trials of antidiabetic drugs in AD.

[64] However, nothing promising has been found to date.

[65] Despite the role of diabetes in the pathogenesis of AD, we have not reported any difference in frequency between typical and mixed AD subtypes.

[0152] Although this study is from a large and well-characterized population, it is a clinical trial population. Therefore, it is unclear to what extent these findings are applicable to the more general AD population. In this study, we used hypometabolic patterns from FDG-PET images to classify patients according to typical AD and mixed AD / CVD subtypes. Therefore, this finding only addresses the question of whether vascular risk factors are equally represented in both subtypes. Therefore, the possibility remains that dyslipidemia and diabetes contribute equally to both subtypes. We show that AD patients of either subtype are more severely impaired if they have hypertension. Further possibilities explaining the relatively weak role of hypertension in the mixed AD / CVD group could be due to exclusion criteria that prevent the enrollment of participants with significant vascular pathology or poorly controlled hypertension. However, although this study had fewer mixed cases (261 cases) than typical AD cases (533 cases), this number is sufficient to be informative. The proportion of cases without hypertension in the mixed AD / CVD group (58%) was significantly higher than in the typical AD group (48%).

[0153] In summary, we attempted to resolve some confounding considerations in the discussion of the role of vascular risk factors in the etiology of AD by using a large population of patients who met clinical research criteria for the diagnosis of mild to moderate AD. The main difficulty in this discussion is the inclusion of individuals with mixed AD / CVD in the population diagnosed with AD in many studies. We used FDG-PET to distinguish between typical AD and mixed AD. Contrary to expectations, we show that hypertension is overrepresented in the typical AD group compared with the mixed group. This is not the case for either dyslipidemia or diabetes. This supports the idea that a history of hypertension (or drugs used to treat hypertension) may play a role in typical AD that is not explained by the presence of mixed vascular and AD lesions that can be seen on FDG-PET.

[0154] Despite attempts to define "pure AD" solely based on biomarker-based evidence of amyloid pathology, there is no evidence that this has helped improve treatment efficacy in trials targeting amyloid pathology.[6] Indeed, the ATN system may have the reversal effect of further mixing typical AD and mixed AD / CVD, with important implications for interpreting clinical trial results. Clinical classification of dementia remains controversial due to the complex nature of the diseases that cause cognitive impairment and the difficulty of defining "pure AD" as a specific category using any of the available diagnostic criteria.

[66] Recognizing the distinction between AD and mixed AD / CVD remains challenging in clinical practice, despite the availability of novel biomarkers aimed at the AD phenotype.

[67] Mixed brain pathology is the most common cause of cognitive impairment in older adults and the most common pathological finding in patients with dementia.[45,68] This means that regardless of the classification system adopted, the reality of underlying complexity may persist in everyday clinical practice and needs to be recognized in the development of disease-modifying treatments for dementia.

[0155] Example 4 - Microvascular cerebral disease in an aging population Microvascular encephalopathy (including cerebral small vessel disease) results from alterations or loss of the integrity of small blood vessels in the brain and is a common finding in older adults. As shown in Figure 2, signs of cerebral small vessel disease include subcortical infarctions, white matter hyperintensities (WMHs), lacunae, perivascular spaces, and cerebral microbleeds.

[0156] Figure 3 shows a representative image of a WMH. WMH have been reported to predict increased risk of stroke, gait disturbance, cognitive decline, and vascular / mixed dementia. Risk factors include aging, hypertension, smoking, and diabetes. Prevalence in the general population increases from approximately 11–21% (age 64) to 94% (age 82; see, e.g., Debette, Stephanie, and H.S. Markus. “The clinical importance of white matter hyperintensities on brain magnetic resonance imaging: a systematic review and meta-analysis.” Bmj 341 (2010)).

[0157] Figure 4 shows the frequency (Scheltens, TLV, and %) of WMH by various metrics in a population with a mean age of 62 years (see the Aberdeen Birth Cohort 'Children of the 1950s' ('ACONF') - Batty, G. David, et al. "The Aberdeen Children of the 1950s cohort study: background, methods, and follow-up information on a new resource for the study of life course and intergenerational influences on health." Paediatric and perinatal epidemiology 18.3 (2004):221-239).

[0158] A summary of prevalence data can be found below.

[0159] [Table 11]

[0160] Regarding risk factors for WMH, data from ACONF indicate that increased WMH burden* is significantly correlated with the following factors: Age (r=.129, p=.032) Mean systolic BP (r=0.163, p=0.007) Self-reported diabetes (r=0.149, p=0.013) (*WMH burden measured as WMH% (Log10 transformed)) WMH% - total lesion volume as a % of total intracranial volume Correlation - Pearson's r Group Differences - Independent-Samples t-Test

[0161] ACONF participants with diabetes had significantly greater WMH burden compared with participants without diabetes: (t(275) = -2.49, p = .013; see also Figure 5).

[0162] Further analysis of the ACONF cohort (normal population) found that increased WMH burden was significantly correlated with poorer performance on the following cognitive tests*: Digit symbol task (r=-.133, p=.028) Mill Hill Vocabulary Test (r=-.141, p=.019) Matrix Reasoning Total correct answers (r=-.195, p=.001) Total number of fraudulent transactions (r=.193, p=.001) Total time required (r=-.119, p=.048) *Transformed WMH% log 10 WMH burden measured as; correlation: Pearson's r

[0163] Example 5 - Treatment of Microvascular Pathology Due to its prevalence in aging populations and its association with poor cognitive outcomes (both in normal populations and in typical AD), as well as being a risk factor for other undesirable outcomes, it is recognized that treatments that slow the rate of progression of microvascular pathology would be a contribution to the art.

[0164] In the studies referenced in Examples 1-3, the inventors found that LMTM as monotherapy was associated with a lower increase in total lesion volume (TLV, measured in ml) compared to subjects receiving LMTM in combination with symptomatic treatment for AD, with statistically significant differences present in both the high-dose and low-dose groups:

[0165] [Table 12]

[0166] Figure 6 shows exposure-dependent differences in the rate of progression of microvascular pathology for LMTM as a low-dose monotherapy. Exposure-dependent groups were defined according to the methods described in more detail in WO 2020 / 020751, discussed above.

[0167] Figures 7A and B show that LMTM as monotherapy is associated with a lower increase in TLV compared to subjects receiving LMTM in combination with AD symptomatic treatment.

[0168] There was a statistically significant difference in both the high-dose group (mean high dose, 150-250 mg / day) and the low-dose group:

[0169] [Table 13]

[0170] In conclusion, microvascular pathology, measured as total lesion volume (TLV) of hyperintense areas, can be readily identified on MRI, and increased microvascular pathology is associated with measurable cognitive impairment in normal aging subjects. Furthermore, the burden of microvascular pathology appears to be a significant contributor to cognitive impairment in AD (surprisingly, this is observed in typical AD but not in the mixed AD / vascular subtype).

[0171] Example 6 - Literature review of microvascular pathology in neurodegenerative diseases Cerebral microvascular pathology precedes and accompanies age-related cognitive impairment and neurodegeneration (Bell and Zlokovic, 2009; Brown et al., 2009; Van Dijk et al., 2008). Progressive functional decline and accompanying morphological collapse are typical features of the aging central nervous system (Farkas and Luiten, 2001). Physiological neuronal changes are also accompanied by reduced oxygen and glucose metabolic rates, reduced cerebral blood flow, and impaired structural integrity of cerebral blood vessels (Farkas and Luiten, 2001). These are all typical degenerative features of the aging brain vasculature. In AD, there is extensive literature linking alterations in the vasculature to disease progression ( Agrawal and Schneider, 2022 ; Farkas and Luiten, 2001 ; Steinman et al., 2021 ), with one study reporting that up to one-third of AD patients have vascular pathology ( Knopman et al., 2003 ), suggesting a strong vascular component to cognitive impairment.

[0172] In addition, cerebrovascular lesions, such as white matter hyperintensities (WMHs), are associated with an increased risk of clinical AD (Prins and Scheltens, 2015). WMHs are one of the most prominent age-related changes observed on brain magnetic resonance imaging scans (Knopman et al., 2003). These WMHs and lesions represent morphological brain changes thought to indicate brain dysfunction and may result in specific symptoms depending on the affected brain region (Jimenez-Balado et al., 2022). Increased WMH burden has also been associated with cognitive decline, increased risk of stroke, and changes in gait.

[0173] Although the relationship between WMH burden and mild cognitive impairment (MCI) remains unclear, some evidence suggests that WMH may increase the risk of MCI (Brickman et al., 2012; Tosto et al., 2015; Van Straaten et al., 2008). A recent study of over 350 elderly participants found that the presence of WMH was associated with a significantly increased risk of MCI, independent of gray matter volume, vascular risk factors, and vascular disease (Boyle et al., 2016). Similarly, WMH was associated with increased rates of cognitive decline, working memory, episodic memory, and semantic memory (Boyle et al., 2016).

[0174] In AD, WMHs may increase underlying AD pathology (i.e., neurofibrillary tangles), thus lowering the threshold for cognitive impairment (Corriveau et al., 2017; Silbert et al., 2009) and may contribute to AD pathophysiology (Garnier-Crussard et al., 2022). In the Rotterdam study (de Leeuw et al., 2002), 87% of participants aged 60–70 years had subcortical WMHs, compared with 100% of participants aged 80–90 years (de Leeuw et al., 2002). Furthermore, WMH volume appears to be larger in AD compared with cognitively unimpaired older adults (Maniega et al., 2015). Additionally, important recent studies have demonstrated that increased cortical tau burden is associated with increased WMH severity (McAleese et al., 2015). The authors showed that cortical tau burden predicted WMH severity, independently of both cortical amyloid burden and small vessel disease severity, in frontal, temporal, and parietal regions ( McAleese et al., 2015 ).

[0175] LMTM and related compounds may be used to prevent and / or treat damage to microvascular integrity in both normal aging populations and subjects with AD, and thus to treat pathologies associated with damaged microvasculature, such as those described herein. In either case, use without ongoing prior treatment with AD symptomatic medications is preferred, although benefits in terms of reduced rate of progression (i.e., less increase in total lesion volume) may be seen regardless.

[0176] References for Example 6 Agrawal, S., Schneider, JA, 2022. Vascular pathology and pathogenesis of cognitive impairment and dementia in older adults. Cereb. Circ. - Cogn. Behav. 3, 100148. https: / / doi.org / 10.1016 / j.cccb.2022.100148 Bell, RD, Zlokovic, BV, 2009. Neurovascular mechanisms and blood-brain barrier disorder in Alzheimer's disease. Acta Neuropathol. 118, 103-113. https: / / doi.org / 10.1007 / s00401-009-0522-3 Boyle, P.A., Yu, L., Fleischman, D.A., Leurgans, S., Yang, J., Wilson, R.S., Schneider, J.A., Arvanitakis, Z., Arfanakis, K., Bennett, D.A., 2016. White matter hyperintensities, incident mild cognitive impairment, and cognitive decline in old age. Ann. Clin. Transl. Neurol. 3, 791-800. https: / / doi.org / 10.1002 / acn3.343 Brickman, A.M., Provenzano, F.A., Muraskin, J., Manly, J.J., Blum, S., Apa, Z., Stern, Y., Brown, T.R., Luchsinger, J.A., Mayeux, R., 2012. 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[0178] [Table 14]

[0179] [Table 15]

[0180] [Table 16]

[0181] [Table 17]

[0182] [Table 18]

[0183] [Table 19]

[0184] [Table 20]

Claims

1. 1. A method for the therapeutic or prophylactic treatment of microvascular cerebral disease in a subject, comprising: orally administering to said subject a methylthioninium (MT)-containing compound; said administering administers a total daily dose of 8-200 mg of MT to said subject per day, optionally divided into two or more doses; The MT-containing compound 【Chemistry 1】 or a hydrate or solvate thereof, method.

2. 10. The method of claim 1, wherein the treatment has a disease-modifying effect on the microvascular pathology.

3. 3. The method of claim 1 or 2, wherein said treatment reduces the rate of progression of microvascular pathology in said subject.

4. The method of any one of claims 1 to 3, wherein the subject is a human.

5. 5. The method of claim 4, wherein the human subject is at least 60 years old.

6. 6. The method of any one of claims 1 to 5, wherein the subject has been diagnosed and / or selected as having said microvascular cerebral disease, optionally by brain imaging.

7. The method of any one of claims 1 to 5, wherein the subject is assessed as being susceptible to or at risk for microvascular encephalopathy.

8. 8. The method of claim 7, wherein the subject has been assessed as susceptible or at risk based on familial or genetic data.

9. 9. The method of claim 7 or 8, wherein the subject has been assessed as susceptible or at risk based on age, history or presence of diabetes, hypertension.

10. 10. The method of any one of claims 1 to 9, wherein the total daily dose is from 12 to 60 mg, or optionally from 16 to 32 mg.

11. 11. The method of claim 10, wherein the total daily dose is about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 mg.

12. 12. The method of any one of claims 1 to 11, wherein the total daily dose of the MT-containing compound is administered as a single dose or as divided doses twice or three times daily.

13. The subject is (i) has not been historically treated with neurotransmission-modifying compounds that are modulators of acetylcholine or glutamate neurotransmitter activity, or (ii) has previously received treatment with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity, but this treatment was discontinued at least 1, 2, 3, 4, 5, 6, or 7 days prior to treatment with the MT-containing compound, or at least 2, 3, 4, 5, 6, 7, or 8 weeks prior to treatment with the MT-containing compound; or (iii) has been selected as having undergone treatment with the neurotransmission-modifying compound, which is a modulator of acetylcholine or glutamate neurotransmitter activity, and the treatment has been discontinued prior to treatment with the MT-containing compound; The method according to any one of claims 1 to 12.

14. The method according to any one of claims 1 to 11, wherein the therapeutic treatment is not combined with a neurotransmission-modifying compound that is a modulator of the activity of acetylcholine or glutamate neurotransmitters.

15. 15. The method of claim 13 or 14, wherein the neurotransmission modifying compound is an acetylcholinesterase inhibitor, and the acetylcholinesterase inhibitor is optionally selected from donepezil; rivastigmine; and galantamine.

16. 15. The method of claim 13 or 14, wherein the neurotransmission modifying compound is an N-methyl-D-aspartate receptor (NMDA) antagonist, and the N-methyl-D-aspartate receptor (NMDA) antagonist is optionally memantine.

17. The MT-containing compound has the following formula: 【Chemistry 2】 is a compound of In the formula, H ｎ A and H ｎ each B, if present, is a protic acid which may be the same or different; wherein p=1 or 2; q=0 or 1; n=1 or 2; (p+q)×n=2. The method according to any one of claims 1 to 16.

18. The MT-containing compound has the following formula: 【Transformation 3】 18. The method of claim 17, having the formula:

19. The MT-containing compound has the following formula: 【Chemistry 4】 wherein H ｎ Each X is a protic acid; 18. The method of claim 17.

20. The MT-containing compound has the formula: ２ A is a diprotic acid: 【Transformation 5】 18. The method of claim 17.

21. The MT-containing compound has the following formula and is a bis-monoprotic acid: 【Transformation 6】 20. The method of claim 19.

22. A method according to any one of claims 17 to 21, wherein the or each protic acid is an inorganic acid.

23. 23. The method of claim 22, wherein each protonic acid is a hydrohalic acid.

24. The or each protonic acid is HCl; HBr; HNO ３ ;H ２ SO ４ 23. The method of claim 22, wherein the

25. A method according to any one of claims 17 to 21, wherein the or each protonic acid is an organic acid.

26. The or each protonic acid may be H ２ CO ３ ;CH ３ 26. The method of claim 25, wherein COOH is selected from methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid.

27. The MT-containing compound is LMTM: 【Transformation 7】 The method according to any one of claims 1 to 16 or claim 26, wherein

28. The MT-containing compound is 【Transformation 8】 【Chemistry 9】 43. The method of any one of claims 1 to 42, selected from the list consisting of:

29. 29. An MT-containing compound according to any one of claims 1 to 28 for use in a method of treatment according to any one of claims 1 to 28.

30. 29. Use of an MT-containing compound or composition according to any one of claims 1 to 28 in the manufacture of a medicament for use in a method of treatment according to any one of claims 1 to 28.

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