Methylthioninium as a drug for improving cognitive function

Abstract

The present invention relates generally to nootropic compositions comprising leucomethylthioninium acid salts and their use for cognitive enhancement in normal (non-demented) individuals.

Description

[Technical Field]

[0001] Technical field The present invention generally relates to nootropic compositions and their use for cognitive enhancement in normal individuals. [Background technology]

[0002] Background technology The use of psychoactive substances to enhance the abilities of normal, healthy (unimpaired) individuals in work or learning has become an increasingly important topic of interest in recent years (Maier et al. 2018). Such substances are sometimes referred to as pharmacological cognitive enhancers (PCEs), nootropes, or smart drugs.

[0003] Such substances may be sought after by students striving for better grades, military personnel who need to remain alert for extended periods of service, and individuals generally keen to enhance their cognitive abilities.

[0004] Examples of drugs used for this purpose include methylphenidate and modafinil. Other drugs commonly used for cognitive enhancement in healthy individuals include caffeine, nicotine, amphetamines, and drugs that modulate acetylcholine breakdown or NMDA receptor activity. Various traditional herbs, vitamins, and supplements have also been suggested. These drugs function through various mechanisms and affect cognition in different ways (Husain and Mehta, 2011).

[0005] Due to the high level of interest in providing novel nootropic compositions, a wide range of patent documents exist for them. For example, International Publication No. 2014 / 037412 relates to a composition comprising at least two drugs selected from cinacalcet, baclofen, acamprosate, mexiletine, sulfisoxazole, and torasemide, which are useful for enhancing memory and mental functions in a subject, such as arousal, attention, logical thinking, concentration, learning, or language processing.

[0006] However, characterizing novel PCEs or nootropic substances that enhance cognition in healthy subjects can be seen as a contribution to this field. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Disclosure of the invention The inventors unexpectedly found that leucomethylthioninium acid salt (hereinafter referred to as “LMTX” salt) can activate neuronal function at therapeutically relevant doses in normal (wild-type) animals. This is evidenced by increased basal acetylcholine (“ACh”) levels in the hippocampus and, furthermore, by increased mean synaptophysin levels in various brain regions. [Means for solving the problem]

[0008] It is well known that ACh is important for cognitive function. Similarly, increased synaptophysins may enhance the release of neurotransmitters needed to support cognitive and other mental functions.

[0009] These findings demonstrate novel utility of LMTX salts at therapeutically relevant doses for use as nootropic agents in healthy, unimpaired control groups.

[0010] Bis(hydromethanesulfonate) (LMTM; USAN name, hydromethylthionine mesylate) is being developed as a treatment targeting pathological aggregation of tau protein in Alzheimer's disease (AD) (Wischik et al., 2018). The methylthioninium (MT) moiety is in its oxidized form (MT + ) and can exist in reduced form (LMT). LMTM is an oxidized form of MT. + This is a stabilized salt of LMT that has significantly better pharmaceutical properties than its morphological counterpart (Baddeley et al., 2015; Harrington et al., 2015). + Rather, we recently reported that LMT is an active species that blocks tau aggregation in vitro (Al-Hilaly et al., 2018). LMT blocks tau aggregation in vitro in cell-free and cell-based assays (Harrington et al., 2015; Al-Hilaly et al., 2018) and reduces tau aggregation pathology and associated behavioral disorders in vivo at clinically relevant doses in a tau transgenic mouse model (Melis et al., 2015a). LMT also deaggregates tau protein in paired helical fibrils (PHFs) isolated from AD brain tissue, converting tau into a form more susceptible to protease (Wischik et al., 1996; Harrington et al., 2015).

[0011] Orally administered LMTM produced brain levels sufficient for in vitro and in vivo activity (Baddeley et al., 2015), but it had minimal apparent efficacy when administered as an add-on to previously received symptomatic treatments in two major Phase 3 clinical trials (Gauthier et al., 2016; Wilcock et al., 2018). However, in subjects receiving LMTM as monotherapy, the treatment resulted in a significant slowing of cognitive and functional decline, a reduction in the rate of progression of brain atrophy as measured by MRI, and a reduction in the decrease in glucose uptake as measured by FDG-PET (Gauthier et al., 2016; Wilcock et al., 2018). When these results were analyzed in combination with population pharmacokinetic data available from the subjects participating in the trials, LMTM was found to produce concentration-dependent effects, whether administered alone or in combination with symptomatic treatments, such as acetylcholinesterase inhibitors. However, the therapeutic effect in monotherapy was substantially greater in those taking LMTM after prior chronic treatment with symptomatic treatments approved for AD (acetylcholinesterase inhibitors and / or memantine).

[0012] LMTM and other leucomethylthioninium bis-protonates have been suggested for the treatment of various diseases and pathologies in several publications, such as International Publication Nos. 2007 / 110627, 2009 / 044127, 2012 / 107706, 2018019823, and 2018041739.

[0013] However, the inventors' findings have been conducted in wild-type animals that do not exhibit tau pathology or other diseases or functional impairments.

[0014] International Publication 2008 / 155533 instructs on the use of various diaminophenothiazines in the treatment of mild cognitive impairment (MCI). MCI is being discussed by the FDA in the context of being an effective disease target. It is defined as having a slight degree of cognitive impairment but not yet meeting the clinical criteria for a diagnosis of dementia. Therefore, patients are neither normal nor dementia-prone. One group of patients featured in International Publication 2008 / 155533 had a Short-Term Mental State Examination (MMSE) score of 24–29.

[0015] MT + Methylene blue (MB, methylthioninium chloride, or MTC), a salt of mitochondrial enzymes, undergoes redox cycling catalyzed by complex I using NADH as a cofactor. This cycling involves methylene blue accepting electrons, which then transfer to complex IV. Thus, methylene blue has been suggested to prevent or delay mitochondrial-mediated damage (Atamna et al., 2012).

[0016] Several published documents have suggested that MB may be used to improve memory in a variety of situations, as described in those documents, which are typically impairment models. These include Martinez et al. (1978); Callaway et al. (2002); Gonzalez-Lima and Bruchey. (2004); Callaway et al. (2004); Riha et al. (2005); and Wrubel et al. (2007).

[0017] The implications that can be drawn from this technical field are extensively discussed in International Publication No. 2008 / 155533.

[0018] However, these published materials do not teach or suggest the use of the compounds described herein in the claimed context.

[0019] More recent publications also suggest that MB can be used to enhance learning (Zoellner, et al., 2017).

[0020] In that study, there was no clear advantage of MB over placebo during and immediately after treatment, although it was suggested that performance improved several months after treatment. Nevertheless, the model used in that paper was one of posttraumatic stress disorder (PTSD).

[0021] Therefore, this publication also does not teach or suggest the use of the compounds described herein in the claimed context.

[0022] For a drug that acts as a nootropic in healthy subjects, it must have a mechanism that allows its beneficial effects to occur in the absence of a disease or a biochemical or physiological target associated with that disease.

[0023] This study was conducted with the aim of understanding the mechanisms involved in the reduced efficacy of LMTM as an add-on to previous symptomatic treatments discussed above. In these studies, well characterized tau transgenic mouse models (line 1, "L1"; (Melis et al., 2015b)) were compared to wild-type mice.

[0024] One conclusion from these studies is that homeostatic mechanisms downregulate multiple nervous systems at different levels of brain function, compensating for chronic pharmacological activation induced by previous symptomatic treatments. When LMTM is given against the background of previous chronic exposure to acetylcholinesterase inhibitors, the effect of this downregulation is to reduce neurotransmitter release, levels of synaptic proteins, mitochondrial function and behavioral advantages compared to LMTM given alone. The behavioral advantages of LMTM are also reduced by previous chronic treatment with memantine.

[0025] Unexpectedly, however, these studies also revealed that LMTX salts can activate neuronal function even in unimpaired mice. As described below, these activating effects on baseline acetylcholine levels and synaptophysin release do not appear to be mediated by enhanced mitochondrial function or low levels of acetylcholinesterase inhibition.

[0026] These findings suggest novel utility for LMTX salts at therapeutically relevant doses for non-therapeutic use as nootropics in healthy, unimpaired control groups.

[0027] Thus, in one embodiment, a non-therapeutic use of methylthioninium (MT)-containing compounds for stimulating cognitive function in healthy human subjects is provided. The aforementioned use involves orally administering 2 to 100 mg of MT per day to the subject, which may be optionally divided into two or more doses. The MT compound is the LMTX compound shown in the following formula. [ka] (In the formula, H n A and H n Each of B (if present) is a protonate, which may be the same or different. p = 1 or 2; q = 0 or 1; n = 1 or 2; (p + q) × n = 2.

[0028] In a further embodiment, the LMTX compound provides non-therapeutic uses for stimulating baseline acetylcholine levels or increased levels of synaptophysins, which are synaptic vesicle proteins, in healthy subjects. The latter exhibits more or larger vesicles required for the release of several neurotransmitters (e.g., acetylcholine, norepinephrine, dopamine, glutamate, serotonin) in healthy subjects. This may be for nootropic purposes as described herein.

[0029] Another aspect of the present invention relates to a non-therapeutic method for treating healthy human subjects to stimulate their cognitive functions, the treatment comprising administering to the subjects an effective amount of a methylthioninium (MT)-containing LTMX compound as described herein in a dose as described herein.

[0030] Another aspect of the present invention relates to methylthioninium (MT)-containing LTMX compounds, as described herein, for use in non-therapeutic methods of treating healthy human subjects to stimulate the cognitive functions described above.

[0031] Another aspect of the present invention relates to the use of methylthioninium (MT)-containing LTMX compounds as described herein in the preparation of nootropic compositions for stimulating cognitive function in healthy human subjects as described above.

[0032] Non-therapeutic stimulation of cognitive function may be for the purpose of stimulating (e.g., improving, enhancing, or increasing) one or more memory and mental functions in a healthy subject, such as arousal, attention, logical thinking, concentration, learning, or language processing.

[0033] This may be for a more specific purpose, for example, to support the ability of the subject to cope with specific socio-occupational burdens.

[0034] The present invention is suitable for non-therapeutic use in normal, non-demented ("healthy") subjects, meaning subjects who do not have known clinical signs of amnesia, cognitive impairment, or disease. Subjects may have other (physical or mental) impairments that are completely unrelated to amnesia, cognitive decline, or disease.

[0035] The treatment is not intended to alleviate or relieve clinical amnesia or other cognitive impairments, nor is it intended to treat depression.

[0036] The subjects of this invention are, for example, individuals who do not suffer from and have not been diagnosed with vascular dementia, senile dementia, age-related memory impairment, Alzheimer's disease, Lewy body dementia, Parkinson's disease, or mild cognitive impairment. Such subjects can thus be diagnosed as not suffering from these diseases. In this context, the diagnosis can be made according to the generally accepted criteria of The Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5, American Psychiatric Association, 2013).

[0037] Similarly, these subjects do not suffer from PTSD or defects in mitochondrial energy metabolism.

[0038] Such subjects may have 30 MMSEs.

[0039] The subjects may be those who have not received, or have not previously received, treatment with an acetylcholinesterase inhibitor (AChEI) or the N-methyl-D-aspartate receptor antagonist memantine. Examples of acetylcholinesterase inhibitors include donepezil (Aricept®), rivastigmine (Exelon®), or galantamine (Reminyl®). An example of an NMDA receptor antagonist is memantine (Ebixa®, Namenda®).

[0040] Nevertheless, such individuals may have a desire for improved or stimulated cognitive abilities, whether temporary or over a longer period.

[0041] For example, the control group may be completely untreated in relation to these other treatments, and may have never received one or both of them before.

[0042] However, the subjects may have previously received one or both of these treatments, but prior to treatment with the MT compound according to the present invention, they have discontinued the drug for at least 1, 2, 3, 4, 5, 6, 7 days, or 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 12 weeks, or 16 weeks, or more preferably, at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0043] Any aspect of the present invention may include an active step of selecting a target group based on these criteria.

[0044] Positive results were achieved in wild-type NMRI mice at a dose of 5 mg / kg / day, as described in the examples below in this specification.

[0045] Based on the results described herein, as well as previous and concurrent results of using LMTM in the treatment of diseases, it can be concluded that MT doses in the range of 2 to 80 or 100 mg / day may be beneficial for the nootropic effects described herein.

[0046] More specifically, further analysis of the concentration response of LMTM in relation to disease treatment supports the assertion that a preferred dose is at least 2 mg / day, and doses in the range of 20–40 mg / day, or 20–60 mg / day, are expected to maximize cognitive benefits while nevertheless maintaining a desirable profile with minimal side effects and good tolerability. Since nootropics are indicated for healthy individuals, it is important to minimize rare adverse events or side effects, and therefore lower doses may be preferred.

[0047] Thus, in one embodiment, the total MT dose may range from approximately 2, 2.5, 3, 3.5, or 4 mg to approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mg.

[0048] The example dosage is 2 to 60 mg, for example, 20, 30, 40, 50, or 60 mg.

[0049] The typical dosage is 20-40 mg.

[0050] Further dosages include 8, 16, or 24 mg / day.

[0051] The subjects of this invention may be adult humans, and the dosages described herein are based on their baseline weight (typical body weight 50-70 kg). If necessary, the corresponding dosages may be used for subjects outside this range by using a weight factor for the subject, thereby dividing the subject's body weight by 60 kg and providing a multiplier factor for each individual subject.

[0052] As described herein, in some embodiments, the treatment is monotherapy or excludes at least prior administration of AChEI or memantine.

[0053] Some of these aspects and embodiments will now be described in more detail. [Modes for carrying out the invention]

[0054] LMTX compound Preferably, the MT compound is an "LMTX" compound of the type described in International Publication No. 2007 / 110627 or International Publication No. 2012 / 107706.

[0055] Thus, the compound can be selected from compounds of the following formula, or hydrates or solvates thereof.

[0056] [Table 1]

[0057] H n A and H n Each of B (if present) is a protonic acid which may be the same or different.

[0058] "Protonic acid" means a proton (H + ) donor in an aqueous solution. Within the protonic acid, A - or B - is thus the conjugate base. Thus, the protonic acid has a pH of less than 7 in water (i.e., the concentration of hydronium ions is more than 10 -7 moles per liter).

[0059] In one embodiment, the salt is a mixed salt having the following formula

[0060] [Table 2] wherein HA and HB are different monoprotic acids.

[0061] However, preferably, the salt is not a mixed salt but has the following formula

[0062] [Table 3] wherein each of H n X is a protonic acid, for example, a diprotic acid or a monoprotic acid.

[0063] In one embodiment, the salt is of the following formula

[0064] [Table 4] The formula contains, where H2A is a diprotonic acid.

[0065] Preferably, the salt is a bismonoprotonic acid with the following formula:

[0066] [Table 5] It holds.

[0067] Examples of protonates that may be present in LMTX compounds used herein are: 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 Includes.

[0068] The preferred acid is a monoprotonic acid, and the preferred salt is a bis(monoprotonic acid) salt.

[0069] The preferred MT compound is LMTM.

[0070] [Table 6]

[0071] The anhydrous salt has a molecular weight of approximately 477.6. Based on the molecular weight of 285.1 for the LMT core, the weight factor for using this MT compound in the present invention is 1.67. "Weight factor" means the relative weight of the pure MT-containing compound to the weight of MT contained in the pure MT-containing compound.

[0072] Other weight factors can be calculated for the example MT compounds described herein, and the corresponding dosage ranges can be calculated from them.

[0073] Therefore, the present invention encompasses a total daily dose of LMTM of approximately 0.8 to 33 mg / day.

[0074] More preferably, a total dose of LMTM of approximately 6-12 mg / day is used, which corresponds to approximately 3.5-7 mg of MT.

[0075] Other examples of LMTX compounds are listed below. Their molecular weights (anhydrous) and weight coefficients are also shown.

[0076] [Table 7]

[0077] [Table 8]

[0078] In the various embodiments of the present invention described herein (as these relate to MT-containing compounds), this may optionally be any of the above-mentioned compounds.

[0079] In one embodiment, it is compound 1.

[0080] In one embodiment, it is compound 2.

[0081] In one embodiment, it is compound 3.

[0082] In one embodiment, it is compound 4.

[0083] In one embodiment, it is compound 5.

[0084] In one embodiment, it is compound 6.

[0085] In one embodiment, it is compound 7.

[0086] In one embodiment, it is compound 8.

[0087] Alternatively, the compound may be a hydrate, a solvate, or a mixture of either of these.

[0088] Accumulation coefficient As those skilled in the art will recognize, more frequent dosing of a given daily dose leads to greater accumulation of the drug.

[0089] The inventors derived the following estimated accumulation coefficient for MT.

[0090] [Table 9]

[0091] For example, considering a total daily dose of 3.5-7 mg of MT: When given as a single daily dose, this may be equivalent to an accumulation of 4.5–8 MT in plasma. When splitting by bid, this may be equal to the accumulation of MT in plasma from 5.1 to 10.3. When splitting by tid, this may be equal to the accumulation of MT in plasma between 5.8 and 11.6.

[0092] Therefore, in certain embodiments of the present invention, the total daily dose of the MT compound may be lower when administered more frequently (e.g., twice a day [bid] or three times a day [tid]).

[0093] In one embodiment, LMTM is administered at approximately 9 mg once daily; 4 mg bid; 2.3 mg tid (based on the weight of LMTM).

[0094] In one embodiment, LMTM is administered at approximately 34 mg once daily; 15 mg bid; 8.7 mg tid (based on the weight of LMTM).

[0095] Combined treatment The term “treatment” includes non-therapeutic treatments in “combination,” where two or more treatments that stimulate cognitive function in a healthy subject (and / or stimulate baseline acetylcholine levels in a healthy subject and / or increase levels of synaptophysins, which are synaptic vesicle proteins (representing more or larger vesicles required for the release of certain neurotransmitters in a healthy subject)) are combined, for example, sequentially or simultaneously.

[0096] In combination therapy, the drugs (i.e., MT compounds as described herein, plus one or more other drugs) may be administered simultaneously or sequentially, in individually varying dose schedules and via different routes. For example, when administered sequentially, the drugs may be administered at close intervals (e.g., over periods of 5 to 10 minutes) or at longer intervals (e.g., depending on the circumstances, 1 hour, 2 hours, 3 hours, 4 hours or more, or even longer intervals), and the precise dosage regimen is in balance with the properties of the therapeutic agents.

[0097] One example of a combined treatment according to the present invention is the use of a nootropic MT compound that is known in the art.

[0098] Known nootropics belong to many different categories, including traditional herbs, vitamins and supplements, recreational drugs, racetams, dopamine agonists, serotonin agonists, antidepressants, adaptogenics (anti-stress agents) and mood stabilizers, vasodilators, antioxidants, neuroprotective agents, hormones and other stimulants, as well as concentration and memory enhancers.

[0099] The use of MT compounds in the methods or uses described herein, in combination with any of these or other nootropic agents, constitutes one aspect of the present invention.

[0100] In other embodiments, the treatment is a "monotherapy," meaning that the MT-containing compound is not used in combination with another activator (within the sense discussed above), whether it be a nootropic agent or something else.

[0101] As mentioned above, it is particularly anticipated that administration of MT compounds may be initiated in subjects who have not previously received (and are not currently receiving) AChEI or memantine.

[0102] However, such AChEI or memantine treatment may be optionally initiated or resumed after the start of treatment with the MT compound, for example, after at least 3 months or about 3 months of treatment with the MT compound.

[0103] Oral dosage form The MT compound of the present invention, or a composition containing the same, is administered orally to the subject.

[0104] In some embodiments, the MT compound is administered as a nootropic composition comprising an LMTX compound as described herein and a pharmaceutically acceptable carrier, excipient, or additive.

[0105] The term "pharmaceutically acceptable," as used herein, refers to compounds, components, materials, compositions, dosage forms, etc., that are suitable for use in contact with the target tissue, without excessive toxicity, irritation, allergic response, or other problems or complications, in proportion to a reasonable benefit / risk ratio. Each carrier, excipient, additive, etc., must also be "acceptable" in the sense that it is compatible with the other components of the formulation.

[0106] Compositions containing LMTX salts are described in several publications, for example, International Publication Nos. 2007 / 110627, 2009 / 044127, 2012 / 107706, 2018019823, and 2018041739.

[0107] In some embodiments, the composition is a nootropic composition comprising at least one LMTX compound as described herein, together with one or more other pharmaceutically acceptable components well known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, excipients, additives, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners.

[0108] In some embodiments, the composition further comprises other nootropic activators.

[0109] Appropriate carriers, excipients, and additives can be found in standard pharmaceutical textbooks. For example, see Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0110] In some embodiments, the composition is a tablet, or a dosage unit.

[0111] In some embodiments, the composition is a capsule, or a dosage unit.

[0112] In some embodiments, the capsule is a gelatin capsule.

[0113] In some embodiments, the capsule is an HPMC (hydroxypropyl methylcellulose) capsule.

[0114] In some embodiments, the amount of MT in a unit is 2 to 60 mg.

[0115] In some embodiments, the amount of MT in a unit is 10 to 40 or 10 to 60 mg.

[0116] In some embodiments, the amount of MT in a unit is 20-40 or 20-60 mg.

[0117] The example dosage unit may contain 2 to 10 mg of MT.

[0118] Further example dose units may contain 2–9 mg of MT.

[0119] Further example dose units may contain 3–8 mg of MT.

[0120] Further preferred dosage units may contain 3.5–7 mg of MT.

[0121] A further preferred dosage unit may contain 4–6 mg of MT.

[0122] In some embodiments, the amount is MT of approximately 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg.

[0123] Using the weight coefficients described or explained herein, those skilled in the art can select an appropriate amount of MT-containing compound for use in an oral formulation.

[0124] As explained above, the MT weight factor for LMTM is 1.67. Since it is convenient to use a unified or simple fraction of the active ingredient, non-limiting examples of LMTM dosage units may include approximately 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 34, 50, 63 mg, etc.

[0125] The nootropic compositions described herein (e.g., low-dose MT-containing compounds plus optionally other components) may be provided in labeled packets along with instructions for the use of their nootropic agents.

[0126] In one embodiment, the pack is a bottle as is well known in pharmaceutical technology. A typical bottle may be made from pharmacopoeia-grade HDPE (high-density polyethylene) with an HDPE push-lock closure that cannot be opened by children and may contain silica gel desiccant present in a sachet or canister. The bottle itself may be packaged in a corrugated container with a label, instructions for use, and optionally, additional copies of the label.

[0127] In one embodiment, the pack or packet is a blister pack (preferably having an aluminum cavity and aluminum foil) that is substantially moisture-impervious. In this case, the pack may be packaged in a corrugated container along with instructions for use and a label on the container.

[0128] The label or instructions may provide information, for example, on the maximum permissible daily dose of a composition as described herein, based on once daily, bid, or tid.

[0129] The aforementioned label or instructions may provide information regarding the recommended duration of treatment.

[0130] Salts and solvates The LMTX-containing compounds described herein are salts, but they may also be provided in the form of mixed salts (i.e., the compounds of the present invention combined with another salt). Such mixed salts are intended to be encompassed by the term “and its pharmaceutically acceptable salts.” Unless otherwise specified, references to specific compounds also include their salts.

[0131] The compounds of the present invention may also be provided in the form of solvates or hydrates. The term “solvate” is used herein in its ordinary sense to refer to a complex of a solute (e.g., a compound, a salt of a compound) and a solvent. When the solvent is water, the solvate may conveniently be referred to as a hydrate, e.g., monohydrate, dihydrate, trihydrate, pentahydrate, etc. Unless otherwise specified, references to a compound also include the solvate and any hydrate form thereof.

[0132] Naturally, solvates or hydrates of salts of compounds are also included in the present invention.

[0133] As used herein, the term “improvement” means an increase in ability in memory, selective attention, and / or related mental functions compared to a previous scale or reference data. Such abilities in memory and / or related mental functions can be measured using several memory and cognitive tests well known in the art.

[0134] Several patents and published materials are referenced herein to more fully describe and disclose the present invention and the existing art to which the invention relates. Each of these references is incorporated in whole by reference to the same extent as each individual reference is specifically and individually indicated as being incorporated by reference.

[0135] Throughout this specification, including the claims below, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” are understood to mean including the integer or step, or group of integers or steps, described, but not to mean excluding any other integer or step, or group of integers or steps.

[0136] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to “pharmaceutical carriers” includes two or more such carriers or mixtures thereof.

[0137] In this specification, the range is often expressed as from one specific value of “approximately” to and / or another specific value of “approximately.” When such a range is expressed, an alternative embodiment includes from one specific value to and / or another specific value. Similarly, when a value is expressed as an approximation by the use of the antecedent “approximately,” it is understood that a specific value forms an alternative embodiment.

[0138] Any subtitles in this specification are included for convenience only and shall not be construed as limiting the disclosure in any way.

[0139] The present invention will now be described further with reference to the following non-limiting drawings and embodiments. Other embodiments of the present invention will be apparent to those skilled in the art in consideration thereof.

[0140] All references cited herein are incorporated herein by cross-reference in particular, to the extent that they can be used by those skilled in the art to carry out the invention. [Brief explanation of the drawing]

[0141] [Figure 1]Treatment effects of LMTM alone or LMTM following chronic pretreatment with rivastigmine on hippocampal acetylcholine levels (A) or synaptophysin levels (B), measured immunohistochemically as the mean of the hippocampus, visual cortex, diagonal zone, and septal cortex, in wild-type mice. (**, p<0.01; ***, p<0.001). [Examples]

[0142] Examples Example 1 - Provision of MT-containing compounds The methods for the chemical synthesis of MT-containing compounds described herein are known in the art. For example: Compounds 1 to 7 can be synthesized by the methods described in International Publication No. 2012 / 107706, or by similar methods. Compound 8 can be synthesized by the method described in International Publication No. 2007 / 110627, or by a similar method.

[0143] Example 2 - Characteristics of a tau transgenic mouse model used in interference studies In the L1 mouse model used in some of this study, overexpression of a 3-repeat tau fragment containing residues 296-390 of the 2N4R tau isoform exists under the control of the Thy1 promoter in the NMRI mouse line (International Publication No. 2002 / 059150). This fragment corresponds to the tau segment first identified within the core stable to proteolysis of PHF (Wischik et al., 1988a; Wischik et al., 1988b), and has recently been confirmed by cryo-electron microscopy observations of AD and PHF in tau filaments in Pick's disease (Fitzpatrick et al., 2017; Falcon et al., 2018).

[0144] Further features of the L1 mouse model include a marked loss of neuronal immunoreactivity to choline acetyltransferase in the basal forebrain, as well as a corresponding reduction in acetylcholinesterase in the neocortex and hippocampus, indicating a reduction in acetylcholine. There is also a roughly 50% reduction in glutamate release from brain synaptosome preparations from L1 mice compared to those from wild-type mice. In this regard, L1 mice thus also mimic the neurochemical impairments in cholinergic (Mesulam, 2013; Pepeu and Grazia Giovannini, 2017) and glutamatergic (Revett et al., 2013) functions that characterize AD.

[0145] These impairments in neurotransmitter function underlie the L1 mouse model, which exhibits impaired synaptic protein integration. Quantitative immunohistochemistry of multiple synaptic proteins in the forebrain basal zone (vertical diagonal) shows that in wild-type mice, there is normally a high degree of correlation at the levels of proteins including the SNARE complex (e.g., SNAP-25, syntaxin, VAMP2; outlined in Li and Kavalali, 2017), as well as the vesicular glycoproteins synaptophysin and α-synuclein. These correlations are greatly lost in L1 mice (Table 1). The only remaining correlation is between synaptophysin, syntaxin, and VAMP2. Therefore, synaptic vesicular protein levels are no longer quantitatively linked to the SNARE complex or α-synuclein proteins. This suggests that the tau oligomer pathology in L1 mice interferes with the functional integration between vesicular and membrane-docking proteins at the synapse.

[0146] [Table 10]

[0147] Example 3 - Experimental Paradigm, Results, and Discussion Experimental paradigms The treatment schedule used to study the negative interaction between symptomatic treatment and LMTM was designed to mimic clinical symptoms, in which subjects were first treated chronically with a cholinesterase inhibitor or memantine, followed by LMTM. Below, we summarize some of the key results obtained for AChEI and rivastigmine.

[0148] Wild-type and L1 mice (n=7–16 for each group) were pre-treated for 5 weeks by force-feeding with rivastigmine (0.1 or 0.5 mg / kg / day) or memantine (2 or 20 mg / kg / day) or vehicle. For the following 6 weeks, LMTM (5 and 15 mg / kg) or vehicle was added to this daily treatment regimen via gastric tube feeding. The animals were tested for behavior using a problem-solving task in an open-field water maze during weeks 10 and 11, and then sacrificed for immunohistochemistry and other histological analysis.

[0149] Converting doses from mouse to human requires considering several factors. 5 mg / kg / day in mice corresponds to the C2 of parental MT in plasma. max The level roughly corresponds to 8 mg / day in humans, but this dose is at the threshold for its effects on disease state and behavior. Higher doses of 15 mg / kg / day are generally required for LMTM to be sufficiently effective in the L1 mouse model (Melis et al., 2015a). This may be related to the much shorter half-life of MT in mice (4 hours) compared to humans (37 hours in aged humans). Tissues dissected for immunohistochemistry were labeled with antibodies, processed using Image J, and protein expression was determined by concentration measurements. Data are presented as unitless Z-score conversions.

[0150] To measure acetylcholine (ACh) levels in the hippocampus, animals (wild-type or L1) were treated with LMTM (5 mg / kg / day for 2 weeks) after a 2-week prior treatment with or without rivastigmine (0.5 mg / kg / day). Rivastigmine was administered subcutaneously using an Alzet minipump, while LMTM was administered via oral gastric tube feeding. ACh levels in the hippocampus were measured using an implanted microdialysis probe and HPLC analysis of extracellular fluid.

[0151] The data are presented as group mean and standard error of the mean, and analyzed using parametric statistics with alpha set to 0.05.

[0152] Experiments on animals were conducted in accordance with the European Communities Council Directive (63 / 2010 / EU), with local ethical approvals and project licenses under the UK Scientific Procedures Act (1986), as well as the German Law for Animal Protection (Tierschutzgesetz) and the Polish Law on the Protection of Animals.

[0153] result Effects of LMTM and rivastigmine treatment in wild-type mice The effects of LMTM treatment alone or in the background of chronic rivastigmine are summarized in Table 2.

[0154] In wild-type mice, LMTM treatment was followed by a significant twofold increase in baseline ACh levels in the hippocampus, and a 30% reduction was observed when mice received LMTM after prior treatment with rivastigmine (Figure 1A).

[0155] A three-fold increase in mean synaptophysin levels, measured in the hippocampus, visual cortex, diagonal zone, and septal area, was observed following LMTM treatment alone, as well as a statistically significant reduction of the same magnitude when LMTM was administered against a background of prior rivastigmine treatment (Figure 1B).

[0156] [Table 11]

[0157] Efficacy of LMTM and rivastigmine treatment in tau transgenic L1 mice The activating effect of LMTM alone and the inhibitory effect of its combination with rivastigmine were greater and more general in tau transgenic L1 mice than in wild-type mice (results not shown).

[0158] Discussion of Example 3 The results presented here demonstrate that the reduction in the efficacy of LMTM can be reproduced in both wild-type and tau transgenic mouse models when it is given as an add-on to symptomatic treatment in humans.

[0159] The results we report hereby demonstrate the existence of two classes of effects induced by LMTM treatment in wild-type mice and tau transgenic mice: those subject to dynamic modulation by prior exposure to cholinesterase inhibitors and those not subject to such modulation. In tau transgenic mice, modulable treatment effects include increased ACh release in the hippocampus, alterations in synaptic proteins, increased mitochondrial complex IV activity, and reversal of impaired behavioral function. The only treatment effects not subject to pharmacological modulation are the primary effects on tau aggregation pathology and its immediate effects on neuronal function, such as those measured by the restoration of choline acetyltransferase expression in the forebrain basum.

[0160] The effects of pharmacological modulation are of two types: those enhanced by the effect on tau aggregation pathology, and those also observed in wild-type mice. Among the results measured by the inventors, the positive treatment effect of LMTM administered alone in wild-type mice included an increase in ACh levels in the hippocampus and an increase in synaptophysin levels in multiple brain regions. Therefore, LMTM treatment can activate neuronal function at therapeutically relevant doses in wild-type mice lacking tau aggregation pathology.

[0161] In experimental models, cholinergic function is primarily associated with selective attention (Botly and De Rosa, 2007; 2008; Sarter et al., 2016), and the cognitive function improvements resulting from cholinesterase inhibitors in AD are thought to be a consequence of elevated levels of acetylcholine in the synaptic cleft. However, these drugs are thought not to increase acetylcholine levels in wild-type mice due to efficient homeostatic adaptations that mitigate the inhibition of acetylcholinesterase inhibitors (e.g., by reducing the level of synaptic vesicles in the presynaptic region).

[0162] In contrast, LMTM results in a significant increase in acetylcholine levels in the hippocampus, which is known to be important for cognitive function.

[0163] Similarly, an increase in synaptophysins indicates an increase in the number or size of synaptic vesicles required for the release of neurotransmitters from the presynapse following activation via action potentials. Therefore, an increase in synaptophysin levels appears to be associated with an increase in several neurotransmitters needed to support cognitive and other mental functions.

[0164] The MT portion has been reported to be a weak cholinesterase inhibitor (Pfaffendorf et al., 1997; Deiana et al., 2009), but it is unlikely that this is the mechanism involved in the increase of ACh levels.

[0165] Specifically, further experiments using scopolamine, which increases ACh levels (by blocking M2 / M4 negative feedback receptors), showed that the increase induced by LMTM was less than that seen with rivastigmine alone, indicating that the combination was again inhibitory in wild-type mice. Under the cholinesterase inhibitory conditions used in these experiments (very small amounts of cholinesterase inhibitor added to the perfusion fluid, 100 nanomoles of rivastigmine), ACh levels in the hippocampus increased, and when they increased sufficiently, they limited further ACh release by activating muscarinic receptors (so-called negative feedback receptors) at the presynaptic sites of M2 / M4 subtypes.

[0166] In this situation, adding scopolamine (1 μM) to the perfusion fluid blocks these presynaptic receptors, resulting in a 3- to 5-fold increase in ACh levels. The fact that LMTM is not additive with rivastigmine in these experiments suggests that LMTM has a different mechanism of action than rivastigmine. In other words, although LMTM has been described as a weak cholinesterase inhibitor at high concentrations, the absence of an additive effect with small amounts of rivastigmine suggests that this effect is unrelated to cholinesterase inhibition.

[0167] The MT moiety is known to enhance mitochondrial complex IV activity (Atamna et al., 2012), and mitochondria play a crucial role in regulating presynaptic homeostasis (Devine and Kittler, 2018). Therefore, increases in ACh and synaptophysin levels can be theoretically explained by increases in presynaptic mitochondrial activity. In particular, the MT moiety is thought to enhance oxidative phosphorylation by acting as an electron shuttle between complex I and complex IV (Atamna et al., 2012). The MT moiety has a redox potential of approximately 0 mV, which is intermediate between the redox potentials of complex I (-0.4 mV) and complex IV (+0.4 mV).

[0168] However, direct measurement of complex IV activity in wild-type mice did not show an increase following LMTM treatment. Furthermore, the activating effect of LMTM was not associated with improvements in spatial recognition memory in wild-type mice.

[0169] Chronic pretreatment with rivastigmine suppressed cholinergic activation in the hippocampus and more generally reduced synaptophysin levels in the brains of wild-type mice. Since the pathogenesis is absent in wild-type mice, this effect is clearly independent of the effect of LMTM on tau aggregation pathogenesis. Rather, these point to a general downregulation of homeostasis, offsetting the effects of combining two drugs, each having an activating effect on neuronal function. Perhaps the primary mechanism that normally protects against excessive levels of ACh in the synaptic cleft is increased AChE activity. Because rivastigmine causes chronic impairment of this regulatory system, pathways separately activated by LMTM are suppressed to preserve cholinergic and other neuronal homeostasis. Thus, when the brain is already subjected to chronic stimulation with a cholinesterase inhibitor, the effects induced by LMTM are subject to dynamic downregulation.

[0170] A further consideration is whether the homeostatic downregulation demonstrated by the inventors would function similarly if LMTM treatment were the primary treatment followed by symptomatic treatment. While the inventors' experiments to date have been designed to mimic clinical symptoms, here LMTM is administered to patients who have already received symptomatic treatment. If homeostatic downregulation is determined by the initial treatment, the treatment effect of LMTM is inevitably dominant, even though the response to add-on symptomatic treatment may be reduced to some extent.

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Claims

1. 1. A non-therapeutic use of a methylthioninium (MT)-containing compound to stimulate cognitive function in a healthy subject, comprising: said use comprising administering to said subject 2 to 100 mg of MT per day, optionally divided into two or more doses; The MT compound is an LMTX compound of the following formula: 【Chemistry 1】 (In the formula, H ｎ A and H ｎ each B (if present) is a protic acid, which may be the same or different; p = 1 or 2; q = 0 or 1; n = 1 or 2; (p + q) x n = 2 or a hydrate or solvate thereof.

2. 2. The non-therapeutic use of claim 1, wherein the LMTX compound stimulates basal choline levels and synaptophysin in the subject.

3. 3. The non-therapeutic use of claim 1 or claim 2, wherein the stimulation of cognitive function is for the purpose of stimulating alertness, attention, reasoning, concentration, learning, or language processing in the subject.

4. 4. The non-therapeutic use according to any one of claims 1 to 3, wherein the subject has an MMSE of 30.

5. 5. The non-therapeutic use of any one of claims 1 to 4, wherein the subject has not previously received treatment with an acetylcholinesterase inhibitor or an N-methyl-D-aspartate receptor antagonist, or has discontinued such treatment prior to administration of the LMTX compound.

6. The non-therapeutic use according to any one of claims 1 to 5, wherein the total daily dose of MT is 10 to 60 mg.

7. The non-therapeutic use according to any one of claims 1 to 6, wherein the total daily dose of MT is 20 to 60 mg.

8. The non-therapeutic use according to any one of claims 1 to 6, wherein the total daily dose of MT is 10 to 40 mg.

9. The non-therapeutic use according to any one of claims 1 to 8, wherein the total daily dose of MT is 20 to 40 mg.

10. 6. The non-therapeutic use of any one of claims 1 to 5, wherein the total daily dose of MT is from about any of 2, 2.5, 3, 3.5, or 4 mg to about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, or 60 mg.

11. 11. The non-therapeutic use according to any one of claims 1 to 10, wherein the total daily dose of the compound is administered as divided doses twice a day or three times a day.

12. The compound has the formula: 【Chemistry 2】 12. The non-therapeutic use according to any one of claims 1 to 11, having the formula: wherein HA and HB are different monoprotic acids.

13. The compound has the formula: 【Transformation 3】 wherein H ｎ 12. The non-therapeutic use of any one of claims 1 to 11, wherein each X is a protic acid.

14. The compound has the formula: 【Chemistry 4】 and H ２ 12. The non-therapeutic use according to any one of claims 1 to 11, wherein A is a diprotic acid.

15. The compound has the formula: 【Transformation 5】 and is a bis-monoprotic acid.

16. 16. The non-therapeutic use according to any one of claims 1 to 15, wherein the or each protic acid is an inorganic acid.

17. 17. The non-therapeutic use of claim 16, wherein each protic acid is a hydrohalic acid.

18. The or each protonic acid is HCl; HBr; HNO ３ ;H ２ SO ４ 17. The non-therapeutic use according to claim 16, wherein the non-therapeutic use is selected from the group consisting of:

19. 16. The non-therapeutic use according to any one of claims 1 to 15, wherein the or each protic acid is an organic acid.

20. The or each protonic acid is H ２ CO ３ ;CH ３ 20. The non-therapeutic use according to claim 19, wherein the COOH is selected from methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid.

21. 21. The non-therapeutic use of claim 20, wherein the compound is LMTM. 【Transformation 6】

22. 22. The non-therapeutic use of claim 21, wherein the total daily dose of LMTM is approximately 3.4 to 100 mg / day, more preferably 34 to 100 mg / day of total LMTM.

23. 23. The non-therapeutic use of claim 22, wherein the dose of LMTM is approximately 34 mg once daily; 15 mg b.i.d.; 8.7 mg t.i.d.

24. The compound is 【Transformation 7】 【Transformation 8】 21. The non-therapeutic use of claim 20, wherein the compound is selected from the list consisting of:

25. 25. The non-therapeutic use according to any one of claims 1 to 24, wherein the MT compound is provided as a nootropic composition comprising the MT compound and a pharmaceutically acceptable carrier or excipient, optionally in the form of a dosage unit.

26. 26. The non-therapeutic use of claim 25, wherein the amount of MT in the unit is from about 4, 5, 6, 7, 8, 9, 10, 20, or 30 to about 40, 50, or 60 mg.

27. 27. The non-therapeutic use according to any one of claims 1 to 26, wherein the MT compound is provided as a nootropic composition comprising the MT compound and a further nootropic agent, optionally in the form of a dosage unit.

28. 27. The non-therapeutic use of claim 25 or claim 26, which is a tablet or capsule.

29. (i) a plurality of dosage units according to any one of claims 25 to 28; (ii) Labels and / or instructions for their non-therapeutic use according to any one of claims 1 to 24. a container comprising:

30. 30. The container of claim 29, wherein the dosage units are present in a blister pack that is substantially moisture impermeable.

31. 31. The container of claim 29 or claim 30, wherein the label or instructions provide information regarding the cognitive stimulation that the composition is intended to provide.

32. 32. The container of any one of claims 29 to 31, wherein the label or instructions provide information regarding the maximum allowable daily dosage of the dosage unit.

33. 1. A non-therapeutic method of treating healthy human subjects to stimulate their cognitive function, comprising:

29. The method, wherein the treatment comprises orally administering to the subject a nootropically effective amount of a methylthioninium (MT)-containing LTMX compound according to the use of any one of claims 1 to 28.

34. 29. A methylthioninium (MT)-containing LTMX compound according to any one of claims 1 to 28 for use in a non-therapeutic method of treating healthy human subjects to stimulate their cognitive function by the use according to any one of claims 1 to 28.

35. 29. Use of a methylthioninium (MT)-containing LTMX compound according to any one of claims 1 to 28 in the manufacture of a nootropic composition for stimulating cognitive function in a healthy human subject by the use according to any one of claims 1 to 28.

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