Treatment of neurodegenerative disorders using methylthioninium (MT)-containing compounds

Abstract

The present invention provides a method for treating a neurodegenerative disease in a subject, comprising the step of orally administering a methylthioninium (MT)-containing compound to the subject, wherein the administration is at a frequency of less than once a day, and the administration provides the subject with an amount of MT corresponding to an average between 0.05 mg and 30 mg of MT per day, preferably between 0.1 mg and 20 mg of MT per day. The present invention further provides related compositions and kits of parts for use in the method.

Description

Technical Field

[0001] Cross - reference to related applications This patent application claims the benefit of priority of GB2208038.6 filed on May 31, 2022 and GB2214646.8 filed on October 5, 2022, which are incorporated herein by reference in their entirety.

[0002] The present invention generally relates to methods and materials for use in the treatment or prevention of diseases of protein aggregation, such as cognitive impairment, using diaminophenothiazines.

Background Art

[0003] Abnormal protein aggregation is thought to be the proximate cause of a number of disease states that can manifest as neurodegeneration, clinical dementia, and other pathological symptoms.

[0004] Generally, abnormal protein aggregation results from induced conformational polymerization interactions, i.e., conformational changes in a protein or its fragment that self - propagatively give rise to templated binding and aggregation of additional (precursor) protein molecules.

[0005] Once nucleation is initiated, an aggregation cascade ensues involving induced conformational polymerization of additional protein molecules, which can lead to the formation of toxic product fragments in aggregates that are substantially resistant to further proteolysis.

[0006] For example, certain cognitive conditions can be characterized by the progressive accumulation of intracellular and / or extracellular deposits of proteinaceous structures, such as β - amyloid plaques and neurofibrillary tangles (NFTs) in the brains of affected patients. The appearance of these lesions strongly correlates with pathological neurodegeneration and brain atrophy, as well as cognitive impairment (see, for example, Mukaetova - Ladinska, E.B. et al., 2000, Am. J. Pathol., Vol. 157, No. 2, pp. 623 - 636).

[0007] Currently approved treatments for Alzheimer's disease include acetylcholinesterase inhibitors (AChEIs) and the N-methyl-D-aspartic acid receptor antagonist memantine. These are symptomatic and do not address the underlying disease pathology. Treatments targeting amyloid pathology have so far been found to be unsuccessful in late-stage clinical trials (Geerts et al., 2013; Mullane and Williams, 2013). According to a recent Lancet Neurology Commission, in particular, the global economic cost of dementia is estimated to be $818 billion, or 0.65% of the world's gross domestic product (Alzheimer's Disease International, 2015), so "effective treatment for AD is perhaps the greatest unmet medical need facing modern medicine" (Winblad et al., 2016).

[0008] NFT (a pathology discovered by Alois Alzheimer, (Alzheimer, 1907)) is composed of paired helical filaments (PHFs) mainly consisting of 12 kDa repeat domain fragments of the microtubule-binding protein tau (Wischik et al., 1985; Wischik et al., 1988a, b). Numerous studies have confirmed the quantitative relationship between the extent of neurofibrillary change pathology and the amount of aggregated tau, both with the degree of clinical dementia and functional molecular imaging defects in Alzheimer's disease (Arriagada et al., 1992; Brier et al., 2016; Giannakopoulos et al., 2003; Josephs et al., 2003; Maruyama et al., 2013). Since the pathological aggregation of tau protein begins at least 20 years before any clinical symptoms (Braak and del Tredici, 2013), targeting this pathology provides a rational approach to both the treatment and prevention of AD and related tau aggregation disorders (Huang and Mucke, 2012; Wischik et al., 2014; Wischik et al., 2010).

[0009] The tau fragments first identified as an intrinsic structural component of PHF cores capture normal tau protein with very high affinity (Lai et al., 2016) and have prion-like properties in vitro in that they convert it into its own proteolytically stable replicas in a self-propagating and autocatalytic process (Wischik et al., 1996; Harrington et al., 2015). Phosphorylation inhibits aggregation (Lai et al., 2016) and is unlikely to drive the cascade (Mukaetova-Ladinska et al., 2000; Schneider et al., 1999; Wischik et al., 1995). The direct inhibition of tau aggregation represents a valid point of therapeutic intervention.

[0010] Methylthioninium (MT) acts as a tau aggregation inhibitor (TAI) in vitro (Wischik et al., 1996; Harrington et al., 2015), dissolves PHF from Alzheimer's diseased brain tissue (Wischik et al., 1996), and reduces tau pathology and associated behavioral impairments in a transgenic mouse model of tau at brain concentrations consistent with human oral administration (Melis et al., 2015; Baddeley et al., 2015). MT has also been shown to inhibit other disease-related protein aggregations (see, e.g., WO 2007 / 110629).

[0011] MT is a redox molecule and exists in an equilibrium state between the reduced form [leucomethylthioninium (LMT)] and the oxidized form (MT + ) depending on environmental conditions (e.g., pH, oxygen, reducing agents).

[0012] Leucomethylthioninium (LMT) is the active moiety in compounds such as leucomethylthioninium mesylate (LMTM). Leucomethylthioninium (LMT) may also be referred to as hydroxymethylthionine (HMT). To avoid ambiguity, these two terms are synonymous and may be used interchangeably herein. Similarly, the terms LMTM (leucomethylthioninium mesylate) and HMTM (hydroxymethylthionine mesylate) may be used interchangeably herein.

[0013] International Publication No. 96 / 30766 describes such MT-containing compounds for use in the treatment and prevention of various diseases including AD and Lewy body diseases. One exemplary compound is methylthioninium chloride (「MTC」), which is commonly known as methylene blue, and this is the oxidized form of methylthioninium (MT), i.e., MT + chloride salt.

[0014]

Table 1

[0015] International Publication No. 96 / 30766 describes a daily dosage of about 50 mg to about 700 mg, preferably about 150 mg to about 300 mg, preferably divided into 1 to 3 unit doses for oral administration.

[0016] International Publication No. 2007 / 110630 discloses certain specific diaminophenothiazine compounds related to MTC, such as (so-called) ETC, DEMTC, DMETC, DEETC, MTZ, ETZ, MTI, MTILHI, ETI, ETLHI, MTN, and ETN, which are useful as drugs in the treatment of Alzheimer's disease, for example.

[0017] International Publication No. 2007 / 110630 describes dosage units containing 20 to 300 mg of the 3,7-diaminophenothiazine (DAPTZ) compounds described therein, such as 30 to 200 mg, such as 30 mg, 60 mg, 100 mg, 150 mg, 200 mg. Suitable dosages of the DAPTZ compounds are proposed in the range of about 100 ng to about 25 mg (more typically about 1 μg to about 10 mg) per kilogram of the subject's body weight per day, for example, 100 mg three times a day, 150 mg twice a day, 200 mg twice a day. Administration of 50 mg three or four times a day has also been discussed.

[0018] A preliminary pharmacokinetic model of methylene blue based on studies of urinary excretion datasets in humans, dogs, and rats was proposed by DiSanto and Wagner, J Pharm Sci 1972, 61:1086-1090 and 1972, 61:1090-1094 and Moody et al., Biol Psych 1989, 26:847-858.

[0019] Peter et al. (2000) Eur J Clin Pharmacol 56:247-250 provided a model integrating blood level data that was inconsistent with previous data from DiSanto and Wagner regarding the terminal phase elimination half-life.

[0020] May et al. (Am J Physiol Cell Physiol, 2004, Vol. 286, pp. C1390-C1398) showed that human erythrocytes continuously reduce and take up MTC, i.e., it is not MTC itself that is taken up by the cells, but rather the reduced form of MT that crosses the cell membrane. They also showed that the rate of uptake is enzyme-dependent and that both oxidized and reduced MT are concentrated intracellularly (the reduced form of MT re-equilibrates once intracellularly to form oxidized MT).

[0021] Based on these and other disclosures, orally administered MTC and similar drugs are thought to be taken up in the intestine, enter the bloodstream, and unabsorbed drug penetrates to the distal intestine through the digestive tract. One important undesirable side effect is the effect of unabsorbed drug in the distal intestine, e.g., sensitization of the distal intestine and / or the antimicrobial effect of unabsorbed drug on the bacterial flora in the distal intestine, both leading to diarrhea.

[0022] MTC has been clinically tested in a Phase 2 trial (Wischik et al., 2015). The minimum safe and effective dose was identified as 138 mg / day, but higher doses of 218 mg / day are most likely to enable efficient absorption by passive diffusion for MT +It was limited in effectiveness due to absorption limitations resulting from the need to reduce it to the leuco-MT (LMT) form.

[0023] WO 2009 / 044127 discloses the results of a Phase II clinical trial, which showed that MTC has two systemic pharmacological actions, a cognitive effect and a hematological effect, and that these actions are separable. Specifically, the cognitive effect did not show a monotonic dose-response relationship, while the hematological effect did. It has been proposed that two different species are responsible for the two types of pharmacological activity: MTC absorbed as the uncharged leuco-MT form is responsible for beneficial cognitive activity, and MTC absorbed as the oxidized dimer species is responsible for the oxidation of hemoglobin. WO 2009 / 044127 describes a method for maximizing the bioavailability of the therapeutically active (cognitively effective) species using a dosage form, regardless of whether an oxidized DAPTZ compound or a leuco-DAPTZ compound is administered.

[0024] Since it is the reduced form of MT that is taken up by cells, it has been proposed to administer the reduced form to patients. This can also reduce the dependence on the rate-limiting step of enzymatic reduction.

[0025] MTC, a phenothiazine-5-ium salt, can be considered "oxidized" relative to the corresponding 10H-phenothiazine compound, N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine, which can be considered "reduced":

[0026]

Chemical formula

[0027] The "reduced form" (or "leuco form") is known to be unstable and can be easily and rapidly oxidized to give the corresponding "oxidized" form.

[0028] WO 02 / 055720 discloses the use of the reduced form of certain diaminophenothiazines for treating protein aggregation diseases, mainly tauopathies. Based on the in vitro activity of the reduced form of the diaminophenothiazines therein, the recommended daily dosage is 3.2 - 3.5 mg / kg, and dosages of 20 mg t.d.s., 50 mg t.d.s. or 100 mg t.d.s. in combination with ascorbic acid at a 2-fold mg ratio to achieve more than 90% reduction before ingestion were also described.

[0029] WO 2007 / 110627 discloses certain 3,7-diamino-10H-phenothiazinium salts that are effective as drugs or prodrugs for treating diseases including Alzheimer's disease. These compounds are also in the'reduced' or 'leuco' form in view of the MTC. These leuco methylthioninium compounds are referred to as 'LMTX' salts and included the following salts:

[0030] [Table 2]

[0031] WO 2012 / 107706 describes other LMTX salts having properties superior to those of the LMTX salts listed above, including leuco-methylthioninium bis(hydromethanesulfonate) (LMTM; also known as HMTM, HMT, hydromethylthionine):

[0032] [Table 3]

[0033] Specifically, LMTM retains TAI activity in vitro and in vivo (Wischik et al., 1996; Harrington et al., 2015; Melis et al., 2015), has excellent pharmaceutical properties in terms of solubility and pKa, and is not subject to the absorption limitations of the MT + type (Baddeley et al., 2015).

[0034] International Publication Nos. WO 2007 / 110627 and WO 2012 / 107706 describe dosage units containing 20 to 300 mg, such as 30 to 200 mg, such as 30 mg, 60 mg, 100 mg, 150 mg, 200 mg of the DAPTZ compound described therein. Appropriate dosages of the DAPTZ compound are proposed in the range of about 100 ng to about 25 mg (more typically about 1 μg to about 10 mg) per kilogram of the subject's body weight per day, for example, 100 mg three times a day, 150 mg twice a day, 200 mg twice a day.

[0035] International Publication No. WO 2008 / 155533 describes the use of the MT compound for treating mild cognitive impairment (MCI). A total daily dosage of 10 mg to 400 mg, preferably administered twice a day (b.i.d.) or three times a day (t.i.d.), is disclosed.

[0036] International Publication No. WO 2018 / 019823 describes a novel regimen for treating neurodegenerative disorders utilizing methylthioninium (MT)-containing compounds. Briefly, these regimens identified two important factors. The first was related to the dosage of the MT compound, and the second was the interaction with symptomatic treatment based on the regulation of acetylcholinesterase levels.

[0037] In the analysis described in International Publication No. WO 2018 / 019823, low doses of the MT compound (e.g., 4 mg b.i.d.) showed a therapeutic benefit when monotherapy was compared to add-on. The efficacy profile was similar in mild and moderate subjects for most of the measured outcomes.

[0038] Furthermore, the treatment benefit (by the test criteria) in AD was restricted to patients taking LMTM as monotherapy. In contrast, in the majority of patients taking LMTM in combination with AD-labeled treatments (acetylcholinesterase inhibitors [AChEI] and / or memantine), the decreases seen at corresponding doses were indistinguishable from those seen in the control group for all parameters.

[0039] The possibility that the LMT compound is active at low doses and the apparent lack of a dose response have been discussed in WO 2018 / 019823, and it is hypothesized that there may be a critical threshold of activity at the tau aggregation inhibitor target and that the effect of high doses may reach a plateau or even become negative at brain concentrations above 1 μM (Melis et al., 2015). The absorption and distribution of MT into the brain are complex and are likely mediated via erythrocytes rather than plasma (Baddele et al., 2015), and it has previously been shown that a pathway is provided that protects MT from first-pass metabolism. In the same study, erythrocyte uptake of MT was approximately 20-fold higher in vivo when administered intravenously as LMTM compared to MTC, most likely due to direct erythrocyte uptake of LMT by passive diffusion without the need for prior reduction of MT as in the case of MTC (Baddeley et al., 2015; May et al., 2004). + Based on the analysis and considering that the low dose (4 mg twice daily) has a better overall clinical profile than the high dose (100 mg twice daily), WO 2018 / 019823 teaches a method of treating neurodegenerative disorders of protein aggregation that comprises oral administration of an MT-containing compound, said administration being provided, optionally as a single administration or divided into two or more administrations, for a total of 0.5 to 20 mg of MT per day. For a given daily dose, WO 2018 / 019823 teaches that more frequent administration leads to more accumulation of the drug.

[0040]

[0041] Other publications that use "low dose" or "low dosage" in relation to the MT-containing compound are described in International Publication No. WO 2018 / 019823. For example: Telch, Michael J. et al., "Effects of post-session administration of methylene blue on fear extinction and contextual memory in adults with claustrophobia." American Journal of Psychiatry 171.10 (2014): 1091-1098: This publication refers to the use of "low-dose methylene blue" in the retention of fear extinction and contextual memory after fear extinction training. This paper reports that "methylene blue is a diaminophenothiazine drug with neuro-metabolism promoting properties at low doses (0.5-4 mg / kg)". The dosage used in the publication corresponded to a 4 mg / kg dose, which was 260 mg / day for adult participants.

[0042] Gonzalez-Lima F and Auchter A (2015), "Protection against neurodegeneration with low-dose methylene blue and near-infrared light." Front. Cell. Neurosci. 9:179. doi:10.3389 / fncel.2015.00179: This publication discusses the cellular mechanisms mediating the neuroprotective effects of low-dose methylene blue and near-infrared light. It refers to previous studies that cite 0.5-4 mg / kg of methylene blue as being safe and effective.

[0043] Alda, Martin et al., "Methylene blue treatment for residual symptoms of bipolar disorder: randomised crossover study." The British Journal of Psychiatry (2016): doi:10.1192 / bjp.bp.115.173930: This publication described the use of 15 mg of "low-dose" methylene blue as a placebo in a 6-month trial. The "active dose" was 195 mg. In each case, the dose was divided into three daily administrations.

[0044] Rodriguez, Pavel et al., "Multimodal Randomized Functional MR Imaging of the Effects of Methylene Blue in the Human Brain." Radiology (2016): 152893: This publication also referred to the "known" pharmacokinetics and side effects of "low-dose" (0.5 - 4.0 mg / kg) methylene blue, which is in contrast to the effects of doses above 10 mg / kg. The dose used in this publication was 280 mg / day for adult participants, which approximated a dose of 4 mg / kg.

[0045] Naylor et al. (1986), "A two-year double-blind crossover trial of the prophylactic effect of methylene blue in manic-depressive psychosis." Biol. Psychiatry 21:915 - 920 and Naylor et al. (1987), "A controlled trial of methylene blue in severe depressive psychosis." Biol. Psychiatry 22:657 - 659: These studies used a treatment of methylene blue at 15 mg / day versus 300 mg / day nominally as a placebo. However, in the latter paper, the authors proposed that placebo administration might act as an antidepressant.

[0046] As discussed above, due to their activity regarding tau aggregation and TDP-43 aggregation, MT-based compounds have also been proposed for the treatment of FTD (see all of the above WO 2007 / 110630; WO 2007 / 110627; WO 2009 / 044127; WO 2012 / 107706).

[0047] WO 2018 / 041739 describes the results of a Phase 3 clinical trial investigating the treatment of frontotemporal dementia (FTD) disease using LMTM.

[0048] The results showed that even relatively low doses of the MT compound (used in the trial as a control) can show benefit in FTD compared to the cognitive decline seen in historical controls.

[0049] Furthermore, unexpectedly, the results showed a strong interaction effect when MT was co-administered with AD treatments that modify synaptic neurotransmission in the brain. Significant cognitive benefits were seen in FTD patients taking MT in combination with such AD treatments (e.g., acetylcholinesterase inhibitors and / or memantine) compared to MT alone. WO 2018 / 041739 further describes ways in which MT compounds can be combined with acetylcholinesterase inhibitors and / or memantine without apparent incompatibility.

[0050] More recently, WO 2020 / 020751 described a novel pharmacokinetic (PK) model for administering LMT compounds in a patient population. As expected, for a given low dose, MT C across the population maxThere were substantial variations in the values. Analysis of the distribution confirmed the findings of WO 2018 / 019823 that low doses (4 mg MT b.i.d.) are effective (measured, for example, by the reduced decline in the Alzheimer's Disease Assessment Scale - Cognitive Subscale (ADAS-cog)). It was further confirmed that monotherapy provides a substantial benefit according to this criterion compared to add-on therapy with AChEI and / or memantine (the average benefit between monotherapy and add-on is approximately 4 ADAS-cog units over 65 weeks).

[0051] However, unexpectedly, considering the lack of any recognizable dose-response as described above, the analysis in WO 2020 / 020751 revealed the existence of a concentration-response within the low-dose treatment population. These insights show that, despite maintaining relatively low doses to maintain a desirable clinical profile in relation to good tolerability with minimal side effects, the MT concentration is C max or C ave It is advantageous to adopt a dosing regimen that maximizes the proportion of subjects with MT concentrations exceeding the threshold and maximizes the expected therapeutic efficacy of LMTM, whether taken alone or in combination with (or at least prior to) symptomatic treatment. WO 2020 / 020751 indicates that the minimum dose to achieve all these objectives is at least 20 mg / day, and doses in the range of 20 - 40 mg / day, or 20 - 60 mg / day, are expected to maximize the therapeutic benefit, although good efficacy is also seen at doses of 100 mg or more, particularly in AD patients who have not been pretreated with symptomatic treatment. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0052] The inventors conducted a randomized, double-blind, placebo-controlled, three-group, 12-month safety and efficacy study of L-methylthioninium mesylate (LMTM) monotherapy in subjects with Alzheimer's disease.

[0053] The daily doses of 8 mg and 16 mg of LMTM (i.e., 8 mg and 16 mg of MT delivered as LMTM) were compared twice weekly to a control dose containing 4 mg of MT as MTC. The control dosing was intended to maintain blinding with respect to fecal discoloration.

[0054] The study results show that administration of 16 mg / day of LMTM as monotherapy is effective and that patients in the treatment group of the study experience zero or minimal decline on cognitive and functional scales. Thus, the results confirm previous clinical trial results (described above) and show that patients receiving 16 mg / day of LMTM decline at a rate substantially lower than the typical rate in Alzheimer's disease. This was seen across a wide range of severities from mild cognitive impairment (MCI) to moderate disease in cognitive and functional assessment items and up to measures of brain atrophy.

[0055] However, unexpectedly, the twice-weekly 4 mg "control" dose of MTC also showed a therapeutic benefit. Steady-state plasma MT levels in the control group were on average low in the majority of patients (significantly lower than the 16 mg LMTM / day group), yet there appears to be an equivalent therapeutic benefit even at very low concentrations in patients receiving twice-weekly dosing of 4 mg of MTC.

[0056] The inventors have previously shown that LMTM administered twice daily provides exposure-dependent benefits for ADAS, ADL, and WBV as monotherapy and as an add-on for ADAS and WBV (Schelter et al., 2019; WO 2018 / 019283, supra). Thus, this drug has pharmacological activity with respect to dementia-related outcomes. However, it is extremely surprising that such very low dosing levels and low dosing frequencies can result in equivalent benefits.

[0057] For a given daily dose, WO 2018 / 019823 teaches that more frequent dosing results in greater drug accumulation, which should be noted means that more frequent dosing is preferred for therapeutic efficacy. Thus, this result is highly unexpected.

[0058] Without wishing to be bound by theory, this surprising result may at least in part be due to a substantial portion of patients being highly sensitive to quite low levels of the drug, and / or a relatively selective accumulation of the drug administered at low levels in these patients. PK analysis revealed that the control dose achieved therapeutic blood levels as the blood levels of the active moiety increased over time. The blood levels of the drug clarify the exposure-response such that a clear response threshold can be identified.

[0059] Without wishing to be bound by theory, this result may also be at least in part explained by the timing of administration always being in the evening when the brain is undergoing endogenous repair (Alhola et al., 2007; Eugene et al., 2015). The inventors have previously shown in vitro that HMT prevents aggregation at a stoichiometric ratio of tau:HMT of 1:0.1 (Al-Hilaly et al., 2018). Thus, in some patients, even very low levels of MT overnight may be sufficient to help the brain remove aggregated tau.

[0060] Regardless of the mechanism, the disclosure herein surprisingly shows that low dose and / or intermittent dosing of an MT compound can provide substantial clinical benefit. Since it is desirable to give the patient the minimum amount of drug necessary to treat the disease, the ability to achieve clinical benefit with relatively low frequency, low doses represents a contribution to the art.

[0061] Thus, in one aspect, a method of treating a neurodegenerative disease in a subject, the method comprising orally administering to the patient a methylthioninium (MT)-containing compound, wherein the administration is at a dosing frequency of less than once a day, is disclosed.

[0062] Preferably, the dosage is selected such that the low-frequency dosing provides MT in an amount corresponding to an average between 0.05 and 30 mg of MT per day. In some embodiments, the dosage is selected such that the low-frequency dosing provides MT in an amount corresponding to an average between 0.1 and 20 mg of MT per day.

[0063] In a related aspect, a method of treating a neurodegenerative disease in a subject, comprising orally administering to the patient a methylthioninium (MT)-containing compound, wherein the administration provides MT in an amount corresponding to an average of less than 0.5 mg of MT per day, is disclosed.

[0064] In some embodiments, the neurodegenerative disorder can be AD.

[0065] In other embodiments, the neurodegenerative disorder can be a neurodegenerative disorder other than AD.

[0066] In some embodiments, the neurodegenerative disorder can be mild cognitive impairment (MCI). Also provided herein is a method of prophylactically treating a neurodegenerative disorder of protein aggregation.

[0067] Also provided are methylthioninium (MT)-containing compounds and compositions thereof for use in the methods described herein.

[0068] These aspects and embodiments are described in more detail herein:

[0069] Dosing frequency / timing / amount Without wishing to be bound by theory, it has been observed that there is a threshold plasma level of the active drug moiety (i.e., MT) necessary to provide a clinical effect. The inventors have surprisingly discovered that this threshold is significantly lower than previously considered.

[0070] While not wishing to be bound by theory, in the methods disclosed herein, prevention of cognitive decline (e.g., in patients with mild to moderate AD) appears to result from a threshold plasma MT level of 0.10 ng / ml. Thus, in some embodiments, administration of the MT compounds described herein provides a subject with an amount of MT that results in a steady-state plasma MT level of at least 0.10 ng / ml.

[0071] Furthermore, improvement of cognitive function (e.g., in patients with MCI) may be associated with a threshold plasma MT level of 0.23 ng / ml. Thus, in some embodiments, administration of the MT compounds provides a subject with an amount of MT that results in a steady-state plasma MT level of at least 0.23 ng / ml.

[0072] In the methods of the invention, the dosing frequency, timing, and amount are appropriately selected to result in a plasma level of the active pharmaceutical moiety (i.e., MT) that exceeds the threshold necessary to provide the associated clinical effect.

[0073] In some embodiments, the above-described threshold plasma MT levels are measured 12 months after treatment (i.e., at the 12-month time point from the first administration of the MT compound). In some embodiments, the above-described threshold plasma MT levels can be achieved at an earlier time point, i.e., prior to 12 months of treatment, e.g., during 4 weeks to 12 months of continuous treatment.

[0074] The methods of the invention are based in part on the surprising discovery that infrequent or intermittent administration (i.e., less than once daily) of a methylthioninium (MT)-containing compound can provide a therapeutic benefit in patients with neurodegenerative disorders.

[0075] In the methods of the invention, treatment of a subject with a methylthioninium (MT)-containing compound is carried out under a dosing regimen in which the compound can be administered to the subject less than once daily.

[0076] In other embodiments, the method of the present invention is based on the surprising discovery that an even lesser amount of MT than previously considered can provide a clinical benefit. Thus, in some embodiments, regardless of the dosing frequency, the administration provides an amount of MT corresponding to an average of less than 0.5 mg of MT per day.

[0077] As used herein, "dosing frequency" can be defined as the average frequency over a treatment period or duration (e.g., at least 8 weeks). That is, a frequency of less than once a day means that over an 8-week treatment period, the total number of administrations given is less than the number of days in that period (56). Preferably, the total number of administrations given is substantially less than the number of days in the treatment period.

[0078] The treatment period or duration can be, for example, equal to or at least 8 weeks, 3 months, 6 months, or 12 months. The treatment duration will be discussed in more detail below.

[0079] In a preferred embodiment, the dosing frequency is such that the total number of administrations given over a defined treatment period is less than or equal to half the number of days in the treatment period. For example, over an 8-week treatment period (56 days), the total number of administrations given is 28 or less. This dosing frequency can be referred to herein as a ≤ 1 / 2 dosing frequency.

[0080] In a preferred embodiment, the dosing frequency is such that the total number of administrations given over a defined treatment period is less than or equal to one-third the number of days in the treatment period. For example, over an 8-week treatment period (56 days), the total number of administrations given is 18 or less. This dosing frequency can be referred to herein as a ≤ 1 / 3 dosing frequency.

[0081] In a preferred embodiment, the dosing frequency is such that the total number of administrations given over a defined treatment period is at most one quarter of the number of days of the treatment period. For example, over an 8-week treatment period (56 days), the total number of administrations is 14 or less. This dosing frequency may be referred to herein as a ≤1 / 4 dosing frequency.

[0082] In some embodiments, the administration of the MT compound is not given daily. In other words, there is at least a 1-day gap between each administration of the MT compound.

[0083] In an alternative embodiment, some administrations of the MT compound may be given daily, as long as the overall dosing frequency defined herein is not exceeded.

[0084] In some embodiments, the administration of the MT compound is given for no more than 2 consecutive days. In some embodiments, the administration of the MT compound is given for no more than 3 consecutive days.

[0085] In some embodiments, the administrations can be at regular intervals. For example, the administrations can be given every other day (once every 2 days), once every 3 days, or once or twice a week on a fixed day of the week.

[0086] In other embodiments, the administrations can be irregular or intermittent. For example, the administrations can be given once, twice, or three times a week on a changing or random schedule, i.e., on a different day or a random day each week.

[0087] The dosing frequency can be, for example, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, twice a week, or three times a week (3×).

[0088] In some embodiments, the dosing frequency is selected from every other day; once a week; or twice a week.

[0089] In some embodiments, the MT compound is administered to the patient every other day. In some embodiments, the MT compound is administered to the patient twice a week on a changing schedule. In some embodiments, the MT compound is administered to the patient twice a week on a fixed schedule (i.e., on a fixed day of each week). In some embodiments, the MT compound is administered to the patient once a week on a variable schedule. In some embodiments, the MT compound is administered to the patient once a week on a fixed schedule.

[0090] Without wishing to be bound by theory, evening administration may be more effective in at least some patients. In some embodiments, the MT compound is administered in the evening. In some embodiments, at least some administrations of the MT compound are administered in the evening. As defined herein, for a given dosing frequency, the average amount of MT provided by a given dose is calculated by dividing the amount of MT provided in each administration of the MT compound by the number of days in each dosing period. For example, as shown in Table 1:

[0091] [Table 4]

[0092] In some embodiments, the administration provides an amount of MT corresponding to an average amount per day of from any of about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, and 2 mg to any of about 2.5, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, and 30 mg.

[0093] In some embodiments, the average amount of MT per day is from 0.05 to 30 mg. In some embodiments, the average amount of MT per day is from 0.05 to 25 mg. In some embodiments, the average amount of MT per day is from 0.05 to 20 mg. In some embodiments, the average amount of MT per day is from 0.1 to 30 mg. In some embodiments, the average amount of MT per day is 0.1 to 25 mg. In some embodiments, the average amount of MT per day is 0.1 to 20 mg. In some embodiments, the average amount of MT per day is 0.2 to 30 mg. In some embodiments, the average amount of MT per day is 0.2 to 25 mg. In some embodiments, the average amount of MT per day is 0.2 to 20 mg. In some embodiments, the average amount of MT per day is 0.2 to 10 mg. In some embodiments, the average amount of MT per day is 0.2 to 5 mg. In some embodiments, the average amount of MT per day is 0.2 to 3 mg. In some embodiments, the average amount of MT per day is 0.3 to 10 mg. In some embodiments, the average amount of MT per day is 0.3 to 5 mg. In some embodiments, the average amount of MT per day is 0.3 to 3 mg. In some embodiments, the average amount of MT per day is 0.4 to 10 mg. In some embodiments, the average amount of MT per day is 0.4 to 5 mg. In some embodiments, the average amount of MT per day is 0.4 to 3 mg. In some embodiments, the average amount of MT per day is 0.5 to 10 mg. In some embodiments, the average amount of MT per day is 0.5 to 5 mg. In some embodiments, the average amount of MT per day is 0.5 to 3 mg.

[0094] In some embodiments, the average amount of MT per day is less than 0.5 mg. In some embodiments, the average amount of MT per day is 0.05 to less than 0.5 mg. In some embodiments, the average amount of MT per day is 0.1 to less than 0.5 mg. In some embodiments, the average amount of MT per day is 0.05 to 0.49 mg. In some embodiments, the average amount of MT per day is 0.10 to 0.49 mg.

[0095] In one embodiment, the average amount of MT per day, delivered, for example, as MTC, is 0.75 to 1.25 mg / day, or about 1 mg / day.

[0096] As will be appreciated, the appropriate dosage depends on the dosing frequency. The dosage is preferably selected such that the amount of MT provided to the subject corresponds to the desired average amount of MT per day as defined above.

[0097] In some embodiments, the amount of MT provided by each administration of the methylthioninium (MT)-containing compound is from any of about 0.05, 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2, and 1.4 mg of MT to any of about 5, 10, 20, 40, 50, 60, 70, 80, 100, 120, and 140 mg of MT.

[0098] In some embodiments, the amount of MT per administration is 0.1 to 100 mg. In some embodiments, the amount of MT per administration is 0.1 to 50 mg. In some embodiments, the amount of MT per administration is 0.2 to 100 mg. In some embodiments, the amount of MT per administration is 0.2 to 50 mg. In some embodiments, the amount of MT per administration is 0.2 to 20 mg. In some embodiments, the amount of MT per administration is 0.5 to 50 mg. In some embodiments, the amount of MT per administration is 0.5 to 20 mg. In some embodiments, the amount of MT per administration is 0.5 to 10 mg. In some embodiments, the amount of MT per administration is 0.5 to 5 mg.

[0099] In some embodiments, the dosing frequency is ≤ 1 / 2, and the amount of MT provided per dose is between 0.4 and 40 mg. In some embodiments, the dosing frequency is every other day, and the amount of MT provided per dose is between 0.4 and 40 mg. In some embodiments, the dosing frequency is ≤ 1 / 2, and the amount of MT provided per dose is between 0.1 and 60 mg. In some embodiments, the dosing frequency is every other day, and the amount of MT provided per dose is between 0.1 and 60 mg. In some embodiments, the dosing frequency is ≤ 1 / 3, and the amount of MT provided per dose is between 0.6 and 60 mg. In some embodiments, the dosing frequency is every three days, and the amount of MT provided per dose is between 0.6 and 60 mg. In some embodiments, the dosing frequency is ≤ 1 / 3, and the amount of MT provided per dose is between 0.15 and 90 mg. In some embodiments, the dosing frequency is every three days, and the amount of MT provided per dose is between 0.15 and 90 mg. In some embodiments, the dosing frequency is ≤ 1 / 4, and the amount of MT provided per dose is between 0.8 and 80 mg. In some embodiments, the dosing frequency is every four days, and the amount of MT provided per dose is between 0.8 and 80 mg. In some embodiments, the dosing frequency is ≤ 1 / 4, and the amount of MT provided per dose is between 0.2 and 120 mg. In some embodiments, the dosing frequency is every four days, and the amount of MT provided per dose is between 0.2 and 120 mg. In some embodiments, the dosing frequency is every five days, and the amount of MT provided per dose is between 1 and 100 mg. In some embodiments, the dosing frequency is every six days, and the amount of MT provided per dose is between 1.2 and 120 mg. In some embodiments, the dosing frequency is once a week, and the amount of MT provided per dose is between 1.4 and 140 mg. In some embodiments, the dosing frequency is every 5 days, and the amount of MT provided in each dose is between 0.25 and 150 mg. In some embodiments, the dosing frequency is every 6 days, and the amount of MT provided in each dose is between 0.3 and 180 mg. In some embodiments, the dosing frequency is once a week, and the amount of MT provided in each dose is between 0.35 and 210 mg. In some embodiments, the dosing frequency is twice a week, and the amount of MT provided in each dose is between 0.7 and 70 mg. In some embodiments, the dosing frequency is three times a week (3 times), and the amount of MT provided in each dose is between 0.5 and 50 mg. In some embodiments, the dosing frequency is twice a week, and the amount of MT provided in each dose is between 0.2 and 100 mg. In some embodiments, the dosing frequency is three times a week (3 times), and the amount of MT provided in each dose is between 0.15 and 70 mg.

[0100] Methylthioninium moiety

[0101] [Table 5]

[0102] The MT-containing compounds used in the present invention can contain either reduced or oxidized MT. "MT" is the active ingredient, i.e., it is present to provide the listed therapeutic effects. Specifically, the compound can include any of the above MT moieties. The above MT moieties themselves are not stable. Therefore, these are administered as MT compounds, for example, LMT or MT + salts.

[0103] MT + salts generally contain one or more anionic counterions (X - ) to achieve electrical neutrality. The compound can be a hydrate, solvate, or mixed salt of the MT + salt.

[0104] The LMT-containing compound is generally stabilized, for example, by the presence of one or more protonic acids, such as two protonic acids.

[0105] The MT content of such salts can be readily calculated by those skilled in the art based on the molecular weight of the compound and the molecular weight of the MT moiety. Examples of such calculations are shown herein.

[0106] LMT compound In some embodiments, the MT compound is preferably an LMT compound.

[0107] In some embodiments, the MT compound is a "LMTX" compound of the type described in International Publication No. WO 2007 / 110627 or International Publication No. WO 2012 / 107706.

[0108] Accordingly, the compound can be selected from compounds of the following formula, or hydrates or solvates thereof:

[0109] [Table 6] H n A and H n Each of A and H (when present), and B (when present) is a protonic acid which may be the same or different.

[0110] "Protonic acid" means a proton (H + ) donor in aqueous solution. Thus, protonic acid A - or B - contains a conjugate base. Thus, the protonic acid has a pH of less than 7 in water (i.e., the concentration of hydronium ions is greater than 10 -7 moles per liter).

[0111] In one embodiment, the salt is a mixed salt having the following formula (wherein HA and HB are different monoprotic acids):

[0112]

Table 7

[0113] However, preferably, the salt is not a mixed salt and has the following formula:

[0114]

Table 8

[0115] In one embodiment, the salt has the following formula (wherein H 2 A is a diprotic acid):

[0116]

Table 9

[0117] Preferably, the salt has the following formula which is a bis-monoprotic acid:

[0118]

Table 10

[0119] Examples of protonic acids that may be present in the LMTX compound used herein include inorganic acids: hydrohalic acids (e.g., HCl, HBr), nitric acid (HNO 3 ), sulfuric acid (H 2 SO 4 ) organic acids: carbonic acid (H 2 CO 3 ), acetic acid (CH 3 COOH), methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid and the like can be mentioned.

[0120] Preferred acids are monoprotic acids and the salts are bis(monoprotic acid) salts.

[0121] A preferred MT compound is LMTM:

[0122] [Table 11]

[0123] The anhydrous salt has a molecular weight of about 477.6. Based on a 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 with respect to the weight of MT it contains.

[0124] Other weight factors can be calculated for the exemplary MT compounds herein, and the corresponding dosage ranges can then be calculated therefrom.

[0125] Accordingly, the present invention encompasses dosing regimens that provide an average amount of LMTM of about 0.08 - 50 mg / day, including dosing regimens that provide an average amount of LMTM of about 0.17 - 33 mg / day.

[0126] More preferably, the average amount of LMTM provided to the subject is any one of 0.1, 0.2, 0.3, 0.4, 0.5 per day to any one of 2, 3, 5, 10, 20, 25, or 50 mg.

[0127] In some embodiments, the method includes administering about 1 mg of LMTM every other day. In some embodiments, the method includes administering about 3.5 mg of LMTM every other day. In some embodiments, the method includes administering about 7 mg of LMTM every other day. In some embodiments, the method includes administering about 14 mg of LMTM every other day. In some embodiments, the method includes administering about 27 mg of LMTM every other day.

[0128] In some embodiments, the method comprises administering about 2 mg of LMTM twice a week. In some embodiments, the method comprises administering about 7 mg of LMTM twice a week. In some embodiments, the method comprises administering about 14 mg of LMTM twice a week. In some embodiments, the method comprises administering about 27 mg of LMTM twice a week.

[0129] In some embodiments, the method comprises administering about 2 mg of LMTM once a week. In some embodiments, the method comprises administering about 7 mg of LMTM once a week. In some embodiments, the method comprises administering about 14 mg of LMTM once a week. In some embodiments, the method comprises administering about 27 mg of LMTM once a week.

[0130] Other exemplary LMTX compounds are as follows. Their molecular weights (anhydrous) and weight coefficients are also shown:

[0131] [Table 12-1]

[0132] [Table 12-2]

[0133] Therefore, the dosages described herein for MT are adjusted for molecular weight and applied mutatis mutandis to these MT-containing compounds.

[0134] Oxidized MT compound In another embodiment, the MT compound is MT + compound.

[0135] Preferably, the MT compound is a type of MT + compound described in WO 96 / 30766 or WO 2007 / 110630.

[0136] Accordingly, the compound can be selected from compounds of the following formula, or hydrates, solvates, or mixed salts thereof:

[0137]

Table 13

[0138] In some embodiments of the present invention, the MT + compound is MTC, for example, the following "high-purity" MTC.

[0139] In some embodiments of the present invention, the MT + compound is not MTC.

[0140]

Table 14

[0141] As described in International Publication No. 2011 / 036561 and International Publication No. 2011 / 036558, MTC occurs in several polymorphic forms with different hydration levels.

[0142] In some embodiments of the present invention, the MT + compound is high-purity MTC. In this context, "high-purity" is defined by one or more of the criteria shown below.

[0143] In some embodiments, MTC has a purity of greater than 97%. In some embodiments, MTC has a purity of greater than 98%. In some embodiments, MTC has a purity of greater than 99%.

[0144] In some embodiments, MTC has less than 2% of azur B as an impurity. In some embodiments, MTC has less than 1% of azur B as an impurity. In some embodiments, MTC has less than 0.5% of azur B as an impurity. In some embodiments, MTC has less than 0.1% Azure B as an impurity.

[0145] In some embodiments, MTC has less than 0.15% Azure A as an impurity. In some embodiments, MTC has less than 0.10% Azure A as an impurity. In some embodiments, MTC has less than 0.05% Azure A as an impurity.

[0146] In some embodiments, MTC has less than 0.15% Azure C as an impurity. In some embodiments, MTC has less than 0.10% Azure C as an impurity. In some embodiments, MTC has less than 0.05% Azure C as an impurity.

[0147] In some embodiments, MTC has less than 0.13% MVB (Methylene Violet Bernthsen) as an impurity. In some embodiments, MTC has less than 0.05% MVB as an impurity. In some embodiments, MTC has less than 0.02% MVB as an impurity.

[0148] Unless otherwise specified, all percentage purities recited herein are by weight.

[0149] In some embodiments, MTC has elemental purity superior to that specified by the European Pharmacopoeia (EP).

[0150] As used herein, the term "elemental purity" relates to the amounts of twelve (12) metals specified by the European Pharmacopoeia: Al, Cd, Cr, Cu, Sn, Fe, Mn, Hg, Mo, Ni, Pb, and Zn. The current version of the European Pharmacopoeia (8th edition, Supplement 8.8) specifies the following limits for these metals:

[0151]

Table 15

[0152] In one embodiment, the MTC has an elemental purity that is equal to or better than (i.e., lower than) the value of EP8.8 shown in the above table (e.g., for each of Al, Cd, Cr, Cu, Sn, Fe, Mn, Hg, Mo, Ni, Pb, and Zn).

[0153] In one embodiment, the MTC has an elemental purity that is equal to or better than 0.9 times the value of EP8.8 shown in the above table.

[0154] In one embodiment, the MTC has an elemental purity that is equal to or better than 0.8 times the value of EP8.8 shown in the above table.

[0155] In one embodiment, the MTC has an elemental purity that is equal to or better than 0.7 times the value of EP8.8 shown in the above table.

[0156] In one embodiment, the MTC has an elemental purity that is equal to or better than 0.5 times the value of EP8.8 shown in the above table. (For example, 0.5 times the value of EP8.8 shown above is 50 μg / g Al, 0.5 μg / g Cd, 50 μg / g Cr, etc.)

[0157] In one embodiment, the MTC has a chromium level that is equal to or better than (i.e., lower than) 100 μg / g.

[0158] In one embodiment, the MTC has a chromium level that is equal to or better than (i.e., lower than) 10 μg / g.

[0159] In one embodiment, the MTC has a copper level that is equal to or better than (i.e., lower than) 300 μg / g.

[0160] In one embodiment, the MTC has a copper level that is equivalent to or better than (i.e., lower than) 100 μg / g.

[0161] In one embodiment, the MTC has a copper level that is equivalent to or better than (i.e., lower than) 10 μg / g.

[0162] In one embodiment, the MTC has an iron level that is equivalent to or better than (i.e., lower than) 200 μg / g.

[0163] In one embodiment, the MTC has an iron level that is equivalent to or better than (i.e., lower than) 100 μg / g.

[0164] All valid and compliant combinations of the above purity grades are disclosed herein as if each individual combination was specifically and explicitly listed.

[0165] In certain embodiments, the MTC is high-purity MTC, where "high-purity" is a purity greater than 98% and the following: (i) less than 2% azurite B as an impurity; (ii) less than 0.13% MVB (methylene violet burns stain) as an impurity; or (iii) elemental purity better than the European Pharmacopoeia limits of less than 100 μg / g aluminum (Al); less than 1 μg / g cadmium (Cd); less than 100 μg / g chromium (Cr); less than 300 μg / g copper (Cu); less than 10 μg / g tin (Sn); less than 200 μg / g iron (Fe); less than 10 μg / g manganese (Mn); less than 1 μg / g mercury (Hg); less than 10 μg / g molybdenum (Mo); less than 10 μg / g nickel (Ni); less than 10 μg / g lead (Pb); and less than 100 μg / g zinc (Zn) characterized by one or more of the above.

[0166] In certain embodiments, the MTC is high-purity MTC, and the high purity is greater than 98% purity and the following: (i) less than 1% azur B as an impurity; (ii) less than 0.15% azur A as an impurity; (iii) less than 0.15% azur C as an impurity; (iv) less than 0.13% methylene violet burns stain (MVB) as an impurity; (v) element purity superior to the European Pharmacopoeia limits of less than 100 μg / g aluminum (Al); less than 1 μg / g cadmium (Cd); less than 100 μg / g chromium (Cr); less than 300 μg / g copper (Cu); less than 10 μg / g tin (Sn); less than 200 μg / g iron (Fe); less than 10 μg / g manganese (Mn); less than 1 μg / g mercury (Hg); less than 10 μg / g molybdenum (Mo); less than 10 μg / g nickel (Ni); less than 10 μg / g lead (Pb); and less than 100 μg / g zinc (Zn) characterized by one or more of the above.

[0167] In certain embodiments, the MTC is high-purity MTC, and the high purity is greater than 98% purity and the following: (i) less than 1% azur B as an impurity; (ii) less than 0.15% azur A as an impurity; (iii) less than 0.15% azur C as an impurity; (iv) less than 0.05% methylene violet burns stain (MVB) as an impurity; or (v) element purity superior to the European Pharmacopoeia limits of less than 100 μg / g aluminum (Al); less than 1 μg / g cadmium (Cd); less than 100 μg / g chromium (Cr); less than 300 μg / g copper (Cu); less than 10 μg / g tin (Sn); less than 200 μg / g iron (Fe); less than 10 μg / g manganese (Mn); less than 1 μg / g mercury (Hg); less than 10 μg / g molybdenum (Mo); less than 10 μg / g nickel (Ni); less than 10 μg / g lead (Pb); and less than 100 μg / g zinc (Zn) characterized by one or more of the above.

[0168] In certain embodiments, the MTC is high-purity MTC, and the high purity is characterized by a purity of at least 98% and less than 1% azurite B as an impurity.

[0169] In certain embodiments, the MTC is high-purity MTC, and the high purity is (i) a purity of at least 98%; (i) less than 1% azurite B as an impurity; and (ii) elemental purity superior to the European Pharmacopoeia limits of less than 100 μg / g aluminum (Al); less than 1 μg / g cadmium (Cd); less than 100 μg / g chromium (Cr); less than 300 μg / g copper (Cu); less than 10 μg / g tin (Sn); less than 200 μg / g iron (Fe); less than 10 μg / g manganese (Mn); less than 1 μg / g mercury (Hg); less than 10 μg / g molybdenum (Mo); less than 10 μg / g nickel (Ni); less than 10 μg / g lead (Pb); and less than 100 μg / g zinc (Zn) characterized thereby.

[0170] In certain embodiments, the MTC is high-purity MTC, and the high purity is characterized by a purity of at least 98% and elemental purity superior to the European Pharmacopoeia limits of less than 100 μg / g aluminum (Al); less than 1 μg / g cadmium (Cd); less than 100 μg / g chromium (Cr); less than 300 μg / g copper (Cu); less than 10 μg / g tin (Sn); less than 200 μg / g iron (Fe); less than 10 μg / g manganese (Mn); less than 1 μg / g mercury (Hg); less than 10 μg / g molybdenum (Mo); less than 10 μg / g nickel (Ni); less than 10 μg / g lead (Pb); and less than 100 μg / g zinc (Zn).

[0171] Methods for the production of "high purity" diaminophenothiazinium compounds, including MTC, are described, for example, in WO 2006 / 032879 and WO 2008 / 007074 (WisTa Laboratories Ltd) and WO 2008 / 006979 (Provence Technologies).

[0172] The preferred MTC polymorph for use in the methods and compositions described herein is the "morphology A" described in WO 2011 / 036561, which is the above-mentioned "high purity", pentahydrate form. It has a molecular weight of about 409.9. Based on the molecular weight of the MT core of 284.1, the weight coefficient for using this MT compound in the present invention is 1.44. + Based on the molecular weight of the core of 284.1, the weight coefficient for using this MT compound in the present invention is 1.44.

[0173] Other weight coefficients can be calculated for the exemplary MT compounds herein, and the corresponding dosage ranges can be calculated therefrom.

[0174] Accordingly, the present invention includes dosing regimens that provide an average amount of MTC·5H₂O of about 0.14 - 29 mg / day, and encompasses dosing regimens that provide an average amount of MTC·5H₂O of about 0.07 - 43 mg / day. 2 In some embodiments, the average amount of MTC·5H₂O provided to the subject is any one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 to any one of 2, 3, 5, 6, 10, 12, 20, 24, 35, or 43 mg per day. 2 In some embodiments, the method includes administration of about 2 mg of MTC (e.g., MTC·5H₂O) every other day. In some embodiments, the method includes administration of about 6 mg of MTC (e.g., MTC·5H₂O) every other day. In some embodiments, the method includes administration of about 12 mg of MTC every other day. In some embodiments, the method includes administration of about 24 mg of MTC every other day.

[0175] In some embodiments, the average amount of MTC·5H₂O provided to the subject is any one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 to any one of 2, 3, 5, 6, 10, 12, 20, 24, 35, or 43 mg per day. 2 In some embodiments, the average amount of MTC·5H₂O provided to the subject is any one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 to any one of 2, 3, 5, 6, 10, 12, 20, 24, 35, or 43 mg per day.

[0176] In some embodiments, the method includes administration of about 2 mg of MTC (e.g., MTC·5H₂O) every other day. In some embodiments, the method includes administration of about 6 mg of MTC (e.g., MTC·5H₂O) every other day. In some embodiments, the method includes administration of about 12 mg of MTC every other day. In some embodiments, the method includes administration of about 24 mg of MTC every other day. 2 In some embodiments, the method includes administration of about 2 mg of MTC (e.g., MTC·5H₂O) every other day. In some embodiments, the method includes administration of about 6 mg of MTC (e.g., MTC·5H₂O) every other day. In some embodiments, the method includes administration of about 12 mg of MTC every other day. In some embodiments, the method includes administration of about 24 mg of MTC every other day. 2 In some embodiments, the method includes administration of about 2 mg of MTC (e.g., MTC·5H₂O) every other day. In some embodiments, the method includes administration of about 6 mg of MTC (e.g., MTC·5H₂O) every other day. In some embodiments, the method includes administration of about 12 mg of MTC every other day. In some embodiments, the method includes administration of about 24 mg of MTC every other day.

[0177] In some embodiments, the method comprises administering about 2 mg of MTC (e.g., MTC.5H 2 O) twice a week. In some embodiments, the method comprises administering about 6 mg of MTC (e.g., MTC.5H 2 O) twice a week. In some embodiments, the method comprises administering about 12 mg of MTC twice a week. In some embodiments, the method comprises administering about 24 mg of MTC twice a week.

[0178] In some embodiments, the method comprises administering about 2 mg of MTC (e.g., MTC.5H 2 O) once a week. In some embodiments, the method comprises administering about 6 mg of MTC (e.g., MTC.5H 2 O) once a week. In some embodiments, the method comprises administering about 12 mg of MTC once a week. In some embodiments, the method comprises administering about 24 mg of MTC once a week.

[0179] Other exemplary MT compounds are described in International Publication No. WO 2007 / 110630. Their molecular weights (anhydrous) and weight coefficients are also shown:

[0180] [Table 16]

[0181] Accordingly, the dosages described herein for MT are adjusted for molecular weight and for the choice of hydrate when used, and are applied mutatis mutandis to these MT-containing compounds. For example, MTC.0.5ZnCl 2 (also referred to as "methylene blue zinc chloride double salt"; CI52015) can be commercially obtained as a monohydrate by several suppliers, having an 18-fold higher molecular weight and a correspondingly changed weight coefficient. MTI is reportedly available as a hemihydrate.

[0182] Adsorption coefficient The inventors have found that administering the LMTX salt is MT +It has been previously determined to enable efficient adsorption compared to salts. Typically, MT + In contrast to salts, MT adsorption can be approximately 1.5 times greater when delivered as the LMTX salt. This factor of 1.5 can be referred to herein as the "adsorption coefficient".

[0183] Thus, in certain embodiments of the present invention, MT + The amount of salt administered can be higher than when using the LMTX salt to achieve a similar plasma concentration.

[0184] However, without wishing to be bound by theory, at the very low doses and reduced dosing frequencies envisioned by the present invention, at very low levels, substantially all of the MT + is converted to HMT in vivo, so the adsorption coefficient may become less important.

[0185] Any of the MT compounds described herein can be formulated with a reducing agent. In particular, MT + salts such as MTC can be formulated with a reducing agent such as ascorbic acid and then lyophilized (as described in WO 02 / 055720). This can improve the adsorption of MT delivered by the compound.

[0186] In various aspects of the invention described herein (when they relate to MT-containing compounds), the MT-containing compound can optionally be any of the above compounds: In one embodiment, it is Compound 1. In one embodiment, it is Compound 2. In one embodiment, it is Compound 3. In one embodiment, it is Compound 4. In one embodiment, it is Compound 5. In one embodiment, it is Compound 6. In one embodiment, it is Compound 7. In one embodiment, it is Compound 8. In one embodiment, it is Compound 9. In one embodiment, it is Compound 10. In one embodiment, it is Compound 11. In one embodiment, it is Compound 12. In one embodiment, it is Compound 13.

[0187] Alternatively, the compound can be a hydrate, solvate, or mixed salt of any of these.

[0188] Treatment and Prevention As used herein in the context of treating a condition, the term "treatment" generally relates to treatment and therapy in humans or animals (e.g., in veterinary use) that achieve a desired therapeutic effect, such as inhibition of the progression of a condition, including a decrease in the rate of progression, a halt in the rate of progression, regression of the condition, improvement of the condition, and cure of the condition.

[0189] As used herein, the term "therapeutically effective amount" relates to an amount of a compound of the present invention, or a material, composition, or dosage form comprising said compound, that is effective to produce a desired therapeutic effect when administered according to a desired treatment regimen and that provides 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 the amounts previously understood in the art.

[0190] The present invention also encompasses treatment such that prophylactic measures are included.

[0191] Accordingly, the present invention also provides a method for preventing a neurodegenerative disorder of protein aggregation in a subject, comprising the step of orally administering a methylthioninium (MT)-containing compound to said patient, wherein said administration is at a dosing frequency of less than once a day, and said administration provides to the subject an amount of MT corresponding to an average between 0.05 mg and 30 mg of MT per day, preferably between 0.1 mg and 20 mg of MT per day.

[0192] The present invention also provides a method for preventing neurodegenerative disorders of protein aggregation in a subject, comprising orally administering to the subject a methylthioninium (MT)-containing compound, wherein the administration provides to the subject an amount of MT corresponding to less than 0.5 mg of MT per day.

[0193] As used herein, the term "prophylactically effective amount" refers to an amount of a compound of the invention, or a material, composition or dosage form containing said compound, effective to produce a desired prophylactic effect commensurate with a reasonable benefit / risk ratio when administered in accordance with a desired treatment regimen.

[0194] "Prophylaxis" in the context of this specification should not be understood to define complete success, i.e., complete protection or complete prevention. Rather, prophylaxis in this context refers to a measure administered prior to detection of a particular condition, for the purpose of maintaining health by helping to delay, reduce or avoid symptomatic states.

[0195] Combination Treatments and Monotherapy The term "treatment" includes "combination" treatments and therapies in which two or more treatments or therapies for the same neurodegenerative disorder of protein aggregation are combined, e.g., sequentially or simultaneously. These can be symptomatic treatments or disease-modifying treatments.

[0196] A particular combination is made at the discretion of the physician.

[0197] In combination treatments, the agents (i.e., the MT compounds described herein + one or more other agents) can be administered simultaneously or sequentially, at individually varying dosing schedules, and via different routes. For example, when administered sequentially, the agents can be administered at short intervals (e.g., over 5 - 10 minutes) or at longer intervals (e.g., separated by 1, 2, 3, 4 hours or more, or even longer intervals as needed), and the exact dosing regimen will be commensurate with the properties of the therapeutic agent.

[0198] An example of a combination treatment of the invention is an agent that is a specific dosage of an MT-containing compound in combination with an agent that is an inhibitor of amyloid precursor protein that inhibits β-amyloid (e.g., an inhibitor of amyloid precursor protein processing that results in enhanced production of β-amyloid).

[0199] In other embodiments, the treatment is a "monotherapy", i.e., the MT-containing compound is not used in combination (within the meaning described above) with another active agent to treat the same neurodegenerative disorder of protein aggregation in a subject.

[0200] As described below, in the present invention, when treating at least AD, it is preferred that the treatment does not include administration of either or both of an acetylcholinesterase inhibitor or an N-methyl-D-aspartic acid receptor antagonist. The MT compound-based treatment of AD can optionally be monotherapy.

[0201] Duration of treatment To treat the neurodegenerative disorders of protein aggregation described herein, a treatment regimen based on a low-dose MT compound preferably extends over a prolonged period. The specific duration is determined at the discretion of the physician.

[0202] For example, the duration of treatment is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more; at least 2, 3, 4, 5 years or more; for a period of 6 to 12 months; for a period of 1 to 5 years may be.

[0203] If the disorder is AD, the duration is an improvement of 3, 4 or 5 points on the 11-item Alzheimer's Disease Assessment Scale - Cognitive Subscale (ADAS-cog) over 52 weeks; an improvement of 4, 5, or 6 points on the 23-item Alzheimer's Disease Cooperative Study - Activities of Daily Living (ADCS-ADL) over 52 weeks; a decrease in the increase in lateral ventricular volume (LVV) measured by ventricular boundary shift integral (VBSI) of 1 or 2 cm over 52 weeks 3 ; a decrease in the annual rate of global brain atrophy on brain MRI using BSI may be such that any one or more of the above are achieved.

[0204] In the case of prevention, treatment may be ongoing.

[0205] In all cases, the treatment duration is generally subject to medical advice and review.

[0206] Pharmaceutical Form Preferably, the MT compound of the present invention is administered in the form of a pharmaceutical composition. Preferably, such a composition comprises a compound described herein and a pharmaceutically acceptable carrier or diluent.

[0207] In some embodiments, the composition is a pharmaceutical composition (e.g., a formulation, preparation, medicament) comprising a compound described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0208] ​As used herein, the term "pharmaceutically acceptable" pertains to compounds, ingredients, materials, compositions, dosage forms, etc. that are suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic reaction, or other problems or complications, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0209] In some embodiments, the composition is a pharmaceutical composition comprising at least one compound described herein in combination with one or more other pharmaceutically acceptable ingredients known to those of skill in the art, including but not limited to pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavorants, and sweeteners.

[0210] In some embodiments, the composition further comprises other active agents, such as other therapeutic or prophylactic agents.

[0211] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical textbooks. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (editors M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, NY, USA), Remington’s Pharmaceutical Sciences, 20th Edition, publisher Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd Edition, 1994.

[0212] In some embodiments, the composition is in the form of a dosage unit (e.g., a pharmaceutical tablet or capsule) comprising an MT compound as described herein (e.g., obtainable or obtained by the methods described herein; having the purity described herein; etc.) and a pharmaceutically acceptable carrier, diluent, or excipient.

[0213] The "MT compound" is present in relatively small amounts but is the active agent in the dosage unit, i.e., is intended to have a therapeutic or prophylactic effect with respect to neurodegenerative disorders of protein aggregation. Rather, other components in the dosage unit, such as carriers, diluents, or excipients, are therapeutically inert. Thus, preferably, there are no other active ingredients in the dosage unit and no other agents intended to have a therapeutic or prophylactic effect with respect to the disorder for which the dosage unit is intended to be used.

[0214] In some embodiments, the dosage unit is a tablet. In some embodiments, the dosage unit is a capsule.

[0215] In some embodiments, the capsule is a gelatin capsule. In some embodiments, the capsule is a HPMC (hydroxypropylmethylcellulose) capsule.

[0216] In some embodiments, the amount of MT in the unit is 0.1 - 10 mg. In some embodiments, the amount of MT in the unit is 0.05 - 10 mg. In some embodiments, the amount of MT in the unit is 0.05 - 5 mg.

[0217] Exemplary dosage units can contain 1 - 10 mg of MT. Further exemplary dosage units can contain 2 - 9 mg of MT. Further exemplary dosage units can contain 3 - 8 mg of MT. Even more preferred dosage units can contain 3.5 - 7 mg of MT. More preferred dosage units may contain 4 - 6 mg of MT.

[0218] In some embodiments, the amount is about 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10 mg of MT.

[0219] Using the weight coefficients described or illustrated herein, one of ordinary skill in the art can select an appropriate amount of the MT - containing compound for use in an oral formulation.

[0220] As described above, the MT weight coefficient of LMTM is 1.67. Because it is convenient to use units or fractional amounts of the active ingredient, exemplary non - limiting LMTM dosage units can include 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18 mg, etc.

[0221] As described above, MTC·5H 2 O has an MT weight coefficient of 1.44. Because it is convenient to use units or fractional amounts of the active ingredient, exemplary non - limiting MTC·5H 2 O dosage units can include 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 18, 20 mg, etc.

[0222] The surprising results leading to the present invention suggest that lower doses of MT compounds than previously thought may be useful in the treatment of neurodegenerative disorders. Thus, dosage units containing very small amounts of MT compounds may be beneficial. Accordingly, in another aspect of the present invention, there is provided a pharmaceutical composition comprising, optionally in the form of a dosage unit, an MT compound as defined herein and a pharmaceutically acceptable carrier or diluent, wherein the amount of MT in the composition or unit is less than 0.5 mg.

[0223] In some embodiments, the amount of MT in the composition or unit is any one of 0.05 mg, 0.1 mg, 0.2 mg, and 0.3 mg to any one of 0.45, 0.46, 0.47, 0.48, 0.49, and about 0.5 mg.

[0224] Oral dosage form The MT compound of the present invention, or a pharmaceutical composition containing the same, is preferably orally administered to a subject / patient.

[0225] Dietary supplement composition The "dietary supplement composition" of the present invention contains a low dose of the MT compound described herein in combination with one or more nutrients in an edible form (e.g., an oral dosage form).

[0226] The novel dietary supplement composition of the present invention can be used as a supplement to food and beverages, as well as a pharmaceutical composition.

[0227] "Nutrient", as used herein, refers to a component of a dietary supplement composition that plays a biochemical and / or physiological role in the body of a human or animal. "Nutrients" include substances such as vitamins, minerals, trace elements, micronutrients, antioxidants, and other bioactive substances such as enzymes, or compounds biosynthetically produced by human or animal enzymes; as well as herbs and herb extracts; fatty acids, amino acids, and derivatives thereof.

[0228] "Edible form" refers to a composition that can be ingested directly or can be converted into an ingestible form such as by dissolving in water.

[0229] Alternatively, the dietary supplement composition can be in the form of a food or beverage such as a defined proportion of food products (this term includes both food and beverages) supplemented with a defined dose of the MT compound. These food products typically contain one or more of fat, protein, or carbohydrates.

[0230] The term "dietary supplement", as used herein, indicates utility in both the nutritional and pharmaceutical fields of application, and the disclosure of this specification regarding pharmaceutical dosage forms is made applicable mutatis mutandis to dietary supplement compositions.

[0231] Oral dosage forms particularly suitable for dietary supplement compositions are well known in the art and are described in more detail elsewhere in this specification. These include powders, capsules, pills, tablets, caplets, gel caps, and defined proportions of edible foods. Liquid forms include solutions or suspensions. General examples of dosage forms and dietary supplement forms are shown, for example, in WO 2010 / 078659.

[0232] Some examples of nutrients useful in the compositions of the present invention are as follows. Any combination of these nutrients is contemplated by the present invention:

[0233] Vitamins B vitamin supplementation (folic acid [folate, vitamin B 9 , vitamin B 12 , vitamin B 6) is an important component of the AD process and has been reported to be able to delay the atrophy of specific brain regions associated with cognitive decline. This is particularly true for elderly subjects with high homocysteine levels (see also Douaud, Gwenaelle et al. "Preventing Alzheimer’s disease-related gray matter atrophy by B-vitamin treatment." Proceedings of the National Academy of Sciences 110.23 (2013): 9523~9528; Quadri, Pierluigi et al. "Homocysteine, folate, and vitamin B12 in mild cognitive impairment, Alzheimer disease, and vascular dementia." The American journal of clinical nutrition 80.1 (2004): 114~122; Rosenberg IH, Miller JW. Nutritional factors in physical and cognitive functions of elderly people. The American journal of clinical nutrition. June 1, 1992; 55(6): 1237S~1243S).

[0234] Vitamin C, along with other antioxidants (see below), can be useful in protecting neural tissue and potentially reducing β-amyloid production and acetylcholinesterase activity, and has been suggested to prevent endothelial dysfunction by regulating nitric oxide (see, for example, Heo JH, Hyon-Lee, Lee KM. The possible role of antioxidant vitamin C in Alzheimer’s disease treatment and prevention. American Journal of Alzheimer’s Disease & Other Dementias. March 2013; 28(2): 120-5).

[0235] It has also been suggested that vitamin E supplementation may play a role in AD treatment (see, for example, Mangialasche, Francesca et al., "Serum levels of vitamin E forms and risk of cognitive impairment in a Finnish cohort of older adults.", Experimental Gerontology 48.12 (2013): 1428-1435).

[0236] Micronutrients, antioxidants Micronutrients or antioxidants such as polyphenols have been reported to be beneficial in relation to the protection or treatment of neurodegenerative diseases, particularly age-related diseases including cognitive impairment and AD.

[0237] The micronutrients and / or antioxidants that can be used in the nutraceutical compositions described herein include the flavonoids shown in the following table (reproduced from Mecocci, Patrizia et al., "Nutraceuticals in cognitive impairment and Alzheimer’s disease.", Frontiers in Pharmacology 5:147 (2014)): Flavonoid chemical subgroups and related food sources:

[0238] [Table 17]

[0239] Other micronutrients that have potential utility in relation to the protection or treatment of age-related diseases and are described by Mecocci et al. include the following: · Non-flavonoid polyphenols: resveratrol and curcumin, · Carotenoids: lycopene, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, and the most prominent carotenoid, β-carotene, · Crocin (the main compound identified in saffron), · Diterpenes: For example, carnosic acid and rosmarinic acid are two of the most important antioxidant compounds in rosemary.

[0240] Herbs and plant extracts In addition to the plants described or cross-referenced above in relation to micronutrients and antioxidants, other plant extracts and herbs have been reported to be beneficial for CNS disorders - see Kumar, Vikas. "Potential medicinal plants for CNS disorders: an overview." Phytotherapy Research 20.12 (2006): 1023 - 1035. These include Ginkgo biloba, Hypericum perforatum (St. John's wort), Piper methysticum Forst. (also known as kava kava, Piperaceae), Valeriana officinalis L. (valerian), Bacopa monniera (locally known in India as Brahmi or Jalanimba), Convolvulus pluricaulis (also known as Shankhpushpi or shankapushpi).

[0241] Oils and fats For example, ω-3 polyunsaturated fatty acids (PUFAs) have been reported to be a promising tool for preventing age-related brain deterioration. Sources of PUFAs such as docosahexaenoic acid (DHA, 22:6) and eicosapentaenoic acid (EPA, 20:5) include fish oil (Denis, I. et al. "Omega-3 fatty acids and brain resistance to ageing and stress: body of evidence and possible mechanisms." Ageing Research Reviews 12.2 (2013): 579-594).

[0242] Immediate release dosage unit Formulations and compositions (especially pharmaceutical compositions) can be prepared to provide rapid or delayed release; immediate, delayed, timed, or sustained release; or combinations thereof.

[0243] Immediate release products dissolve the components or active moieties in the gastrointestinal tract without causing delay or prolongation of drug dissolution or absorption. The requirements for dissolution testing of immediate release products are shown in Guidance for Industry (CDER 1997) "Dissolution testing for immediate release solid oral dosage forms", (CDER 1997) "Immediate release solid oral dosage forms - Scale up and Post approval Changes", ICH Guidance Q6A, Specifications: Test Procedures and Acceptance Criteria For New Drug Substances And New Drug Products. The most commonly used dissolution test methods described in USP and the European Pharmacopoeia (6th Edition) are the basket method (USP1) and the paddle method (USP2). The methods described are simple, robust, well standardized and are used worldwide. They have sufficient flexibility to enable dissolution testing of various pharmaceuticals. The following parameters that affect dissolution behavior may be relevant, for example, to the selection of appropriate in vitro dissolution test conditions for immediate release solid oral products: apparatus, stirring speed, dissolution medium, and temperature. Due to the biopharmaceutical characteristics of MTC and its expected desirable absorption characteristics in the upper gastrointestinal tract, it was preferred to manufacture rapidly dissolving tablets of MTC.

[0244] The composition according to the present invention can be dissolution tested in a USP-2 apparatus in 900 ml of 0.1 N HCl using a paddle rotating at 50 to 75 rpm. The composition according to the present invention shows at least the acceptance criteria cited for the Stage 1 (S1) test in USP32 (United States Pharmacopeia, edited by the United States Pharmacopeial Convention, 12601 Twinbrook Parkway, Rockville, MD 20852; published by Rand McNally, Inc., 32nd Edition, 2008): Acceptance criteria: For each tablet, after inserting the coated tablet into 0.1N HCl, 85% dissolution of MTC was achieved within 30 minutes.

[0245] Thus, in some embodiments, when the MTC-based formulation of the present invention is evaluated using this method, it is at least: 75% dissolution of MTC within 45 minutes after inserting the coated tablet into 0.1N HCl; or 85% dissolution of MTC within 30 minutes after inserting the coated tablet into 0.1N HCl; 85% dissolution of MTC within 15 minutes after inserting the coated tablet into 0.1N HCl is provided.

[0246] Another aspect of the present invention is a method for preparing a low-dose MT compound pharmaceutical composition, comprising mixing at least one MT compound as defined herein with one or more other pharmaceutically acceptable components well-known to those skilled in the art, such as carriers, diluents, excipients, etc. When formulated as separate units (e.g., tablets, etc.), each unit contains a predetermined amount (dose) of the compound.

[0247] The formulation can be prepared by any method well-known in the pharmaceutical art. Such methods include associating the compound with a carrier that constitutes one or more auxiliary components. Generally, the formulation is prepared by uniformly and intimately associating the compound with a carrier (e.g., a liquid carrier, a fine solid carrier, etc.), and then shaping the product as necessary.

[0248] In some embodiments, the pharmaceutically acceptable carrier, diluent, or excipient is one or both of glycerides (e.g., Gelucire 44 / 14 (registered trademark); Lauroyl Macrogol-32 Glyceride PhEur, USP) and colloidal silicon dioxide (e.g., 2% Aerosil 200 (registered trademark); Colliodal Silicon Dioxide PhEur, USP), or includes one or both of them.

[0249] Preferably, the pharmaceutical composition containing the compound of the present invention is in a solid dosage form. The composition preferably further comprises at least one diluent suitable for dry compression. The pharmaceutical composition is characterized in that the compound is present in a substantially stable form.

[0250] The pharmaceutical composition generally also contains a lubricant. Examples of lubricants include magnesium stearate, calcium stearate, sodium stearyl fumarate, stearic acid, glyceryl behenate, polyethylene glycol, ethylene oxide polymers (e.g., those available under the registered trademark Carbowax from Union Carbide, Inc., Danbury, Connecticut), sodium lauryl sulfate, magnesium lauryl stearate, a mixture of magnesium stearate and sodium lauryl sulfate, and hydrogenated vegetable oil. Preferred lubricants include calcium stearate, magnesium stearate, and sodium stearyl fumarate. Most preferred as the lubricant is magnesium stearate. The lubricant generally constitutes about 0.5 to about 5.0% of the total (uncoated) tablet weight. The amount of lubricant employed is generally about 1.0 to about 2.0%, preferably 0.5 to 2.0% w / w.

[0251] In addition to diluents and lubricants, other conventional excipients may also be present in the pharmaceutical composition of the present invention. Such additional excipients include disintegrants, binders, flavoring agents, coloring agents, and glidants. Some excipients can perform multiple functions, for example, as both a binder and a tablet disintegrant.

[0252] Disintegrants may be present in the amounts necessary to achieve rapid dissolution. Disintegrants are excipients that oppose the physical forces of particle binding in tablets or capsules when the dosage form is placed in an aqueous environment. Examples of disintegrants include cross-linked polyvinylpyrrolidone (crospovidone), sodium starch glycolate, cross-linked sodium carboxymethylcellulose (crosscarmellose sodium), and pregelatinized starch. Generally, the amount of disintegrant can be from 0 to about 25% w / w of the composition, more commonly from about 1% to about 15% w / w, and usually less than 10% or less than 5% w / w.

[0253] Binders are excipients that contribute to particle adhesion in solid formulations. Examples of binders include cellulose derivatives (carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, ethylcellulose, microcrystalline cellulose), as well as sugars such as lactose, sucrose, dextrose, glucose, maltodextrin, and mannitol, xylitol, polymethacrylate, polyvinylpyrrolidone, sorbitol, pregelatinized starch, alginic acid, and its salts such as sodium alginate, magnesium aluminum silicate, polyethylene glycol, carrageenan, etc. Generally, the amount of binder can vary widely, for example, from 0% to 95% w / w of the composition. As noted above, excipients can perform multiple functions. For example, a tablet diluent can also function as a binder.

[0254] Glidants are substances added to powders to improve flowability. Examples of glidants include magnesium stearate, colloidal silicon dioxide (such as grades sold as Aerosil), starch, and talc. Glidants can be present in pharmaceutical compositions at levels of 0 to about 5% w / w. However, it should also be noted that excipients can perform multiple functions. Lubricants, such as magnesium stearate, can also function as glidants.

[0255] Examples of colorants that can be incorporated into the pharmaceutical compositions of the present invention include titanium dioxide and / or those known as FD&C dyes and dyes suitable for food such as natural colorants. The colorants are unlikely to be used in the powder mixture that is compressed according to the aspects of the present invention described above, but as described below, can form part of the coating applied to the composition, in which case the colorant can be present in the film coating in an amount of up to about 2.0% w / w.

[0256] Tablets are desirably coated with a conventional film coating that imparts toughness, ease of swallowing, and an elegant appearance to the final product. Many polymeric film coating materials are known in the art. Preferred film coating materials are hydroxypropylmethylcellulose (HPMC) or partially hydrolyzed polyvinyl alcohol (PVA). HPMC and PVA are commercially available from Colorcon, for example, in coating formulations containing excipients that function as coating aids under the registered trademark Opadry. Opadry formulations can also contain talc, polydextrose, triacetin, polyethylene glycol, polysorbate 80, titanium dioxide, and one or more dyes or lakes. Other suitable film-forming polymers can also be used, including vinyl copolymers such as hydroxypropylcellulose, polyvinylpyrrolidone, and polyvinyl acetate, and acrylate-methacrylate copolymers. The use of a film coating is beneficial for ease of handling, as the uncoated core with a blue color can stain the inside of the mouth during swallowing. The coating also improves the light stability of the dosage form.

[0257] The coating of tablets can be easily carried out using a conventional coating pan. In a preferred embodiment of the process, the coating pan is preheated using inlet air heated until the exhaust temperature reaches 35°C to 55°C, more preferably 40 to 50°C. This may typically require the application of inlet air heated at an inlet temperature of 45 to 75°C, preferably 50 to 65°C for 10 to 15 minutes. Subsequently, a tablet core containing an active ingredient (e.g., LMTM) is added to the coating pan and an aqueous film coat is applied. The spraying rate is controlled such that the floor temperature is maintained at 38 to 48°C, more preferably 42 to 44°C until the desired weight gain (coating weight) is achieved.

[0258] Subjects, patients, and patient groups Subjects / patients can be animals, mammals, placental mammals, rodents (e.g., guinea pigs, hamsters, rats, mice), mice (e.g., mice), rabbits (e.g., rabbits), birds (e.g., birds), dogs (e.g., dogs), cats (e.g., cats), horses (e.g., horses), pigs (e.g., pigs), sheep (e.g., sheep), cows (e.g., cows), primates, euprimates (e.g., monkeys or anthropoid apes), monkeys (e.g., marmosets, baboons), monotremes (e.g., platypuses), anthropoid apes (e.g., gorillas, chimpanzees, orangutans, gibbons), or humans.

[0259] In a preferred embodiment, the subject / patient is diagnosed as having one of the cognitive or CNS disorders described herein, or (in the case of prophylactic treatment) is evaluated as being susceptible to one of the neurodegenerative disorders of protein aggregation (e.g., cognitive or CNS disorders) described herein, based on, for example, family data or genetic data or other data.

[0260] The patient can be an adult human, and the dosages described herein are premised on that standard (typical weight of 50 - 70 kg). If desired, the dosage for a subject outside this range can be utilized by dividing the subject's weight by 60 kg to obtain an individual subject multiplication factor and using the subject's weight coefficient.

[0261] The reduction in the concentration of the active ingredient necessarily reduces the risk of adverse side effects (and increases the safety profile), and thus the low-dose treatment of the invention increases the feasibility of a purely prophylactic treatment in order to increase the risk / benefit ratio of such prophylactic treatment.

[0262] Thus, for example, for the initial selection of patients for the diagnosis of AD and assessment of severity, it can involve any one or more of: a rigorous assessment by an experienced clinician; exclusion of possible non-AD diagnoses by supplementary laboratory and other investigations; an objective assessment of the cognitive function level using a neuropathologically validated battery.

[0263] The diagnosis of AD and other disorders described herein can be performed by a physician by methods well known to those skilled in the art.

[0264] In some embodiments, when a subject or group of patients is being treated for AD, it is preferred that they have not been treated with either an acetylcholinesterase inhibitor or an N-methyl-D-aspartic acid receptor antagonist. Examples of acetylcholinesterase inhibitors include donepezil (Aricept (trademark)), rivastigmine (Exelon (trademark)), or galantamine (Reminyl (trademark)). An example of an NMDA receptor antagonist is memantine (Ebixa (trademark), Namenda (trademark)).

[0265] For example, the AD subject or patient group may be completely naive to these other treatments and may not have historically received one or both of them. For example, the AD subject or patient group may have historically received one or both of them, but prior to treatment with the MT compound according to the present invention, the dosing thereof was discontinued for at least 1, 2, 3, 4, 5, 6, 7 days, or 2, 3, 4, 5, 6, 7, 8, 12, or 16 weeks, or more preferably for at least 1, 2, 3, 4, 5, or 6 months, etc.

[0266] The inventors have found that certain patients appear to be particularly responsive to intermittent dosing of low-dose MT compounds.

[0267] As described in the examples herein, a subgroup of patients receiving 4 mg of MTC twice weekly was found to have much higher steady-state HMT plasma levels (measured 1 hour after dosing) than average.

[0268] Without wishing to be bound by theory, these patients may be particularly good at absorbing and / or accumulating HMT for reasons yet unknown. Demographic and genetic factors do not appear to be important. Furthermore, patients in this subgroup, i.e., those with higher steady-state HMT plasma levels, appear to be responsive to the therapeutic effects of MT. For example, as described in the following examples, patients in the "high HMT plasma" subgroup of the control group did not experience a decrease or experienced an improvement above baseline in the ADAS-cog11 assessment over 12 months.

[0269] Thus, without wishing to be bound by theory, patients showing steady-state HMT plasma levels above the average may be particularly suitable for treatment using the methods of the present invention.

[0270] Thus, in some embodiments, the present invention provides the methods described herein where the subject has a steady-state plasma level of MT of at least 0.1 ng / ml.

[0271] Thus, in some embodiments, the present invention provides a method as described herein, wherein the subject has a steady-state plasma level of MT of at least 0.23 ng / ml.

[0272] Thus, in some embodiments, the present invention provides a method as described herein, wherein the subject has a steady-state plasma level of MT of at least 0.25 ng / ml.

[0273] Thus, in some embodiments, the present invention provides a method as described herein, wherein the subject has a steady-state plasma level of MT of at least 0.6 ng / ml.

[0274] In some embodiments, the steady-state plasma level is measured 1 hour after administration of the MT compound. In some embodiments, the steady-state plasma level is measured 1 hour after administration of between 0.2 - 4 mg of MT.

[0275] Any aspect of the present invention may include a positive step of selecting an AD subject or patient group according to these criteria.

[0276] For example, the method of the present invention may - administering to the subject a low dose, e.g., an MT-containing compound at a dose as described herein; - collecting a blood sample after administration of the MT-containing compound at said dose; - measuring or estimating the MT plasma concentration of said subject; - using said measured or estimated value to determine whether to treat said patient with the treatment method of the present invention and may involve a patient selection step including.

[0277] For example, if the MT plasma concentration is higher than a threshold value after a certain period, this would indicate a high likelihood of a therapeutic benefit from low-frequency, low-dose treatment.

[0278] For example, the method of the present invention may - Administering to a subject a MT-containing compound at a dosage provided for low dosages, e.g., about 4 mg of MT; - Preferably, collecting a blood sample about 1 hour after administering the MT-containing compound at said dosage; - Determining the steady-state MT plasma concentration of said subject can involve a patient selection step including, A steady-state MT plasma concentration higher than the threshold defined herein indicates a high likelihood of a therapeutic benefit from treatment.

[0279] In some embodiments, the invention provides a method as described herein for treating, e.g., Alzheimer's disease, wherein the subject has a steady-state plasma level of MT of at least 0.1 ng / ml.

[0280] In some embodiments, the invention provides a method as described herein for treating, e.g., mild cognitive impairment (MCI), wherein the subject has a steady-state plasma level of MT of at least 0.23 ng / ml.

[0281] Labels, Instructions, and Kits of Parts The unit dosage compositions described herein (e.g., low-dose MT-containing compounds + optionally other ingredients, or more generally MT compositions for treatment in AD) can be provided in labeled packets together with their instructions for use.

[0282] In one embodiment, the pack is a bottle as is well known in the pharmaceutical art. A typical bottle can be made of USP-grade HDPE (high density polyethylene) with a child-resistant HDPE push-lock closure and can contain a silica gel desiccant present in a sachet or canister. The bottle itself can include a label and can be packaged in a cardboard container with instructions for the user and optionally an additional copy of the label.

[0283] In one embodiment, the pack or packet is a blister pack (preferably one having an aluminum cavity and an aluminum foil), and thus is substantially impermeable to moisture. In this case, the pack may be packaged in a cardboard container provided with instructions and labels for the user on the container.

[0284] The label or instructions may provide information regarding neurodegenerative disorders of protein aggregation for which the drug is intended (e.g., cognitive or CNS disorders).

[0285] When the drug is indicated for AD, the label or instructions may provide information instructing the user that the composition should not be used in combination with either an acetylcholinesterase inhibitor or an N-methyl-D-aspartic acid receptor antagonist.

[0286] The label or instructions may provide information regarding the maximum allowable daily dose of the composition described herein.

[0287] The label or instructions may provide information regarding the recommended dosing regimen for treatment described herein. For example, recommended dosing frequencies such as every other day, twice a week, once a week, etc. Accordingly, the disclosure of the above dosing frequencies is incorporated herein by reference in this regard.

[0288] The label or instructions may provide information regarding the recommended duration of treatment described herein. Accordingly, the disclosure of the above treatment duration is incorporated herein by reference in this regard.

[0289] Reversal and / or inhibition of protein aggregation One aspect of the invention is the use of the MT compounds or compositions described herein for modulating (e.g., reversing and / or inhibiting) protein aggregation, such as protein aggregation associated with neurodegenerative diseases and / or clinical dementia. Aggregation is associated with the disease states discussed below.

[0290] Similarly, one aspect of the invention is a method of modulating (e.g., reversing and / or inhibiting) protein aggregation in the brain of a mammal, wherein the aggregation is associated with a disease state described herein, and the treatment comprises administering to the mammal in need of the treatment a prophylactically effective amount or a therapeutically effective amount of an MT compound or composition described herein that is an inhibitor of said aggregation.

[0291] Disease states treatable via the present invention are discussed in more detail below.

[0292] Methods of Treatment Another aspect of the invention relates to a method of treatment comprising administering to a patient in need of treatment a prophylactically effective amount or a therapeutically effective amount of a compound described herein, preferably in the form of a pharmaceutical composition, as described above.

[0293] Use in Methods of Treatment Another aspect of the invention relates to a compound or composition described herein for use in a method of treating (e.g., a disease state of) the body of a human or animal by therapy.

[0294] Use in the Manufacture of a Medicament Another aspect of the invention relates to the use of an MT compound or composition described herein in the manufacture of a medicament for use in treating (e.g., a disease state of).

[0295] In some embodiments, the medicament is a composition, e.g., a low-dose unit-dose composition described herein.

[0296] Diseases of Protein Aggregation The compounds and compositions of the invention are useful for the treatment or prevention of diseases of protein aggregation.

[0297] Thus, in some embodiments, the disease state is a disease of protein aggregation and, for example, the treatment is by a compound or composition described herein in an amount sufficient to inhibit the aggregation of a protein associated with said disease state.

[0298] The following table lists various disease-related aggregation proteins and the corresponding neurodegenerative diseases of protein aggregation. The use of the compounds and compositions of the present invention related to these proteins or diseases is encompassed by the present invention.

[0299]

Table 18-1

[0300]

Table 18-2

[0301]

Table 18-3

[0302] As described in International Publication No. 02 / 055720, International Publication No. 2007 / 110630, and International Publication No. 2007 / 110627, diaminophenothiazine is useful in the inhibition of such protein aggregation diseases.

[0303] Accordingly, unless context requires otherwise, descriptions of embodiments regarding tau protein or tau-like proteins (e.g., MAP2; see below) are equally applicable to other proteins discussed herein (e.g., β-amyloid, synuclein, prions, etc.), or other proteins that may initiate or be susceptible to similar pathological aggregation by conformational changes in domains important for aggregation growth, or other proteins that confer proteolytic stability to aggregates thus formed (e.g., see the paper by Wischik et al. in “Neurobiology of Alzheimer’s Disease”, 2nd Edition, 2000, editors Dawbarn, D. and Allen, S.J., The Molecular and Cellular Neurobiology Series, Bios Scientific Publishers, Oxford). All such proteins may be referred to herein as “aggregation disease proteins”.

[0304] Similarly, when referred to herein as in “tau-tau aggregation”, this may also be considered applicable to other “aggregation protein aggregations” such as β-amyloid aggregation, prion aggregation, synuclein aggregation, etc. The same applies to “tau proteolysis” and the like.

[0305] Preferred aggregation disease target proteins Preferred embodiments of the present invention are based on tau protein. As used herein, the term "tau protein" generally refers to any protein of the tau protein family. Tau proteins are characterized as one of a number of protein families that co-purify with microtubules during repeated cycles of assembly and disassembly (see, e.g., Shelanski et al., 1973, Proc. Natl. Acad. Sci. USA, Vol. 70, pp. 765-768), and are known as microtubule-associated proteins (MAPs). Members of the tau family share common features including a characteristic N-terminal segment, an approximately 50 amino acid sequence inserted into the developmentally regulated N-terminal segment in the brain, a characteristic tandem repeat region consisting of three or four tandem repeats of 31-32 amino acids, and a C-terminal tail.

[0306] MAP2 is the dominant microtubule-binding protein in the somatodendritic compartment (see, e.g., Matus, A., "Microtubules" [ed. Hyams and Lloyd] pp. 155-166, John Wiley and Sons, New York, USA). MAP2 isoforms are nearly identical to tau proteins in the tandem repeat region, but are substantially different in both the sequence and extent of the N-terminal domain (see, e.g., Kindler and Garner, 1994, Mol. Brain Res., Vol. 26, pp. 218-224). Nevertheless, aggregation in the tandem repeat region is not selective for the tau repeat domain. Thus, it will be recognized that any discussion herein related to tau protein or tau-tau aggregation should also be considered relevant to tau-MAP2 aggregation, MAP2-MAP2 aggregation, etc.

[0307] In some embodiments, the protein is a tau protein.

[0308] In some embodiments, the protein is synuclein, such as α- or β-synuclein.

[0309] In some embodiments, the protein is TDP-43.

[0310] TAR DNA-binding protein 43 (TDP-43) is a 414-amino acid protein encoded by TARDBP on chromosome 1p36.2. This protein is highly conserved, widely expressed, and mainly localized in the nucleus, but can shuttle between the nucleus and the cytoplasm (Mackenzie et al. 2010). It is involved in transcriptional and splicing regulation and may have roles in other processes such as microRNA processing, apoptosis, cell division, messenger RNA stabilization, regulation of neuronal plasticity, and maintenance of dendritic integrity. Furthermore, since 2006, considerable evidence has accumulated to support the toxic gain-of-function hypothesis of TDP-43 in amyotrophic lateral sclerosis (ALS). TDP-43 is a protein that is intrinsically prone to aggregation, and the aggregates formed in vitro are ultrastructurally similar to the TDP-43 deposits found in the degenerating neurons of ALS patients (Johnson et al. 2009). Johnson et al. (2008) showed that when TDP-43 is overexpressed in a yeast model, only the aggregated form is toxic. Several in vitro studies have also shown that the C-terminal fragment of TDP-43 is more ubiquitinated and is likely to form insoluble cytoplasmic aggregates that are toxic to cells than full-length TDP-43 (Arai et al. 2010; Igaz et al. 2009; Nonaka et al. 2009; Zhang et al. 2009). Nonaka et al. (2009) suggested that these cytoplasmic aggregates bind to the endogenous full-length protein that depletes it from the nucleus, while Zhang et al. (2009) found normal nuclear expression and suggested a pure toxic effect regarding the aggregates. Yang et al. (2010) described the sequestration of full-length TDP-43 within aggregates of the C-terminal and N-terminal fragments of TDP-43 in NSC34 motor neurons in culture. The neurite outgrowth impaired as a result of the presence of such cleaved fragments can be rescued by overexpression of the full-length protein. Although the role of neurite outgrowth in vivo has not been established, this model would support the suggestion made by Nonaka and colleagues regarding the role of TDP-43 aggregation in ALS pathogenesis.

[0311] Mutant TDP-43 expression in cell cultures has been repeatedly reported to result in increased production of C-terminal fragments with even greater cytoplasmic aggregation and toxic effects than the wild-type protein (Kabashi et al. 2008; Sreedharan et al. 2008; Johnson et al. 2009; Nonaka et al. 2009; Arai et al. 2010; Barmarda et al. 2010; Kabashi et al. 2010).

[0312] When the protein is the tau protein, in some embodiments of the invention, a method of inhibiting the production of protein aggregates (e.g., optionally, in the form of paired helical filaments (PHF) in neurofibrillary tangles (NFT) in the mammalian brain) is provided, and the treatment is as described above.

[0313] Preferred indications - diseases of protein aggregation In one embodiment, the invention is used for the treatment of Alzheimer's disease (AD), e.g., mild, moderate, or severe AD.

[0314] In particular, tau protein (and its abnormal function or processing) can play a role not only in Alzheimer's disease (AD). The onset of neurodegenerative diseases such as Pick's disease and progressive supranuclear palsy (PSP) each appears to correlate with the accumulation of pathological truncated tau aggregates in the dentate gyrus and stellate pyramidal cells of the neocortex. Other dementias include frontotemporal dementia (FTD); FTD associated with Parkinsonism linked to chromosome 17 (FTDP-17); disinhibition-dementia-Parkinsonism-amyotrophy complex (DDPAC); pallido-ponto-nigral degeneration (PPND); Guam ALS syndrome; pallido-nigral Lewy body degeneration (PNLD); corticobasal ganglionic degeneration (CBD), etc. (see Wischik et al., "Neurobiology of Alzheimer’s Disease", 2nd Edition, 2000, edited by Dawbarn, D. and Allen, S.J., The Molecular and Cellular Neurobiology Series, Bios Scientific Publishers, Oxford; see especially Table 5.1). All of these diseases characterized mainly or in part by abnormal tau aggregation are referred to herein as "tauopathies".

[0315] Accordingly, in some embodiments, the disease state is a tauopathy. In some embodiments, the disease state is a neurodegenerative tauopathy.

[0316] In some embodiments, the disease state is selected from Alzheimer's disease (AD), Pick's disease, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), FTD with parkinsonism linked to chromosome 17 (FTDP17), frontotemporal lobar degeneration (FTLD), disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC), pallido-ponto-nigral degeneration (PPND), Guam ALS syndrome, pallidonigral Lewy body degeneration (PNLD), corticobasal ganglionic degeneration (CBD), argyrophilic grain dementia (AgD), boxer dementia (DP) or chronic traumatic encephalopathy (CTE), Down syndrome (DS), dementia with Lewy bodies (DLB), subacute sclerosing panencephalitis (SSPE), MCI, Niemann-Pick disease type C (NPC), Sanfilippo syndrome type B (mucopolysaccharidosis IIIB), or myotonic dystrophy (DM), DM1 or DM2, or chronic traumatic encephalopathy (CTE).

[0317] In some embodiments, the disease state is a lysosomal storage disorder having tau pathology. NPC is caused by a mutation in the gene NPC1 that affects cholesterol metabolism (Love et al. 1995), Sanfilippo syndrome type B is caused by a mutation in the gene NAGLU, and there is lysosomal accumulation of heparan sulfate (Ohmi et al. 2009). Tau pathology is observed in these lysosomal storage disorders, and treatment thereof may reduce disease progression. Other lysosomal storage disorders may also be characterized by tau accumulation.

[0318] The use of phenothiazine diaminium salts in the treatment of Parkinson's disease and MCI is described in detail by PCT / GB2007 / 001105 and PCT / GB2008 / 002066.

[0319] In some embodiments, the disease state is Parkinson's disease, MCI, or Alzheimer's disease.

[0320] In some embodiments, the disease state is MCI or Alzheimer's disease. In some embodiments, the disease state is MCI.

[0321] In a further aspect of the invention, there is provided a method of treating MCI in a subject, the method comprising the step of orally administering to the subject a methylthioninium (MT)-containing compound, wherein the administration provides a daily dosage of from about 21 mg to about 29 mg of MT. For example, the daily dosage of MT is about 21, 22, 23, 24, 25, 26, 27, 28, or 29 mg. The MT compound in this aspect is preferably MT + salt, most preferably methylthioninium chloride (MTC). In certain embodiments, the method comprises the administration of MTC at a total daily dosage of from about 21, 22, 23, 24, 25, 26, 27, 28, or 29 mg.

[0322] In another aspect of the invention, there is provided a method of treating MCI in a subject, the method comprising the step of orally administering to the subject a methylthioninium (MT)-containing compound, wherein the administration is at a frequency of less than once a day; and the administration provides to the subject an amount of MT corresponding to an average of 21 to 29 mg of MT per day. In certain embodiments, the method comprises the administration of MT at a total daily dosage of from about 21, 22, 23, 24, 25, 26, 27, 28, or 29 mg. The MT compound in this aspect is preferably MT + salt, most preferably methylthioninium chloride (MTC). In certain embodiments, the method comprises the administration of MTC at a total daily dosage between 21 and 29 mg. In certain embodiments, the method comprises the administration of MTC at a total daily dosage of from about 21, 22, 23, 24, 25, 26, 27, 28, or 29 mg.

[0323] Optionally, treating MCI according to the methods of the invention includes inhibiting a decline, preventing an expected decline, or improving a condition. For example, in some embodiments, treating MCI can include improving cognitive ability or function in a subject.

[0324] In some embodiments, the disease state is Huntington's disease, or spinal bulbar muscular atrophy (or Kennedy disease), and other polyglutamine disorders such as dentatorubral-pallidoluysian atrophy and various spinocerebellar ataxias.

[0325] In some embodiments, the disease state is an FTLD syndrome (see below, which can be, for example, tauopathy or TDP-43 proteinopathy).

[0326] In some embodiments, the disease state is PSP or ALS.

[0327] TDP-43 proteinopathy includes amyotrophic lateral sclerosis (ALS; ALS-TDP) and frontotemporal lobar degeneration (FTLD-TDP).

[0328] The role of TDP-43 in the neurodegeneration of ALS and other neurodegenerative disorders has been reviewed in several recent publications (Chen-Plotkin et al. 2010; Gendron et al. 2010; Geser et al. 2010; Mackenzie et al. 2010).

[0329] ALS is a neurodegenerative disease characterized by progressive paralysis and muscle wasting due to the degeneration of both upper and lower motor neurons in the primary motor cortex, brainstem, and spinal cord. It is sometimes referred to as a motor neuron disease (MND), but there are diseases other than ALS that affect either upper or lower motor neurons. A definitive diagnosis requires signs of both upper and lower motor neurons in the muscle tissue of the bulbar, arm, and leg, with clear evidence of clinical progression that cannot be explained by other disease processes (Wijesekera and Leigh 2009).

[0330] Most cases are ALS-TDP, but there are other cases in which the pathological protein is different from TDP-43. Mutant misfolded SOD1 is the pathological protein of ubiquitin-positive inclusions in ALS with SOD1 mutations (Seetharaman et al. 2009), and in a very small subset (approximately 3 - 4%) of familial ALS due to mutations in FUS (fused in sarcoma protein), the ubiquitinated pathological protein is FUS (Vance et al. 2009; Blair et al. 2010). FUS, like TDP-43, appears to be important in the nucleo-cytoplasmic shuttle, but the way in which nuclear import impairment of FUS remains unclear. The new molecular classification of ALS, modified from Mackenzie et al. (2010), reflects different underlying pathological mechanisms in different subtypes (see the table below).

[0331] New molecular classification of ALS (modified from Mackenzie et al. 2010). In most cases, TDP-43 is the pathological ubiquitinated protein found in ALS.

[0332]

Table 19

[0333] Amyotrophic lateral sclerosis has been recognized as a nosological entity for almost a century and a half, is recognized in ICD-10, and is classified as a subtype of MND in ICD10 (G12.2). Reliable clinical diagnostic criteria, which are little different from Charcot's original description, are available for ALS, and neuropathological criteria reflecting the underlying molecular pathology are also agreed upon.

[0334] ALS is pathologically classified into three subgroups: ALS-TDP, ALS-SOD1, and ALS-FUS, with the latter two conditions being rare. In the largest study to date, all sporadic ALS cases have been shown to have TDP-43 pathology (Mackenzie et al. 2007). Only about 5% of ALS cases are familial (Byrne et al. 2010), and mutations in SOD1, the most common mutation seen in FALS, account for between 12 and 23% of cases (Andersen et al. 2006). SOD1 can also be involved in 2 to 7% of SALS cases. Mutations in FUS appear to be much less common, accounting for only about 3 to 4% of FALS cases (Blair et al. 2010). Therefore, clinical cases of SALS can be reliably predicted to have TDP-43-based pathology. Similarly, this can be reliably predicted in FALS due to mutations in TDP-43, which account for about 4% of cases (Mackenzie et al. 2010). ALS cases with mutations in VCP (Johnson et al. 2010), ANG (Seilhean et al. 2009), and CHMP2B (Cox et al. 2010), which account for 1 to 2% of FALS cases, have also been reported to be associated with TDP-43 positive pathology. Mutations in SOD1, FUS, and ATXN2 have not been found to be associated with TDP-43 positive aggregates, but TDP-43 has been reported to be involved in the pathological processes presumably arising from these mutations (Higashi et al. 2010; Ling et al. 2010; Elden et al. 2010).

[0335] Therefore, it has been established that TDP-43 has an important and potentially central role in the pathogenesis of the majority of SALS cases and may be involved in the pathogenesis of a significant proportion of FALS cases. ALS is currently widely considered to be a TDP-43 proteinopathy (Neumann et al. 2009), and numerous in vitro and in vivo studies support the hypothesis that a toxic gain of function due to TDP-43 aggregation underlies at least part of the neurotoxicity in the disease.

[0336] FTLD syndrome is a neurodegenerative condition with insidious onset and relentless progression, with peak onset in the late middle age. In many cases, there is a positive family history of a similar disorder in first-degree relatives.

[0337] Behavioral variant FTD is characterized by early prominent changes in social and interpersonal functioning, often accompanied by repetitive behaviors and changes in eating patterns. In semantic dementia, there are prominent word-finding problems with a decline in object knowledge and an impairment in word comprehension in cognitive assessment, despite otherwise fluent speech. Progressive non-fluent aphasia presents a combination of problems with motor speech and grammatical deficits. The main clinical diagnostic features of these three FTLD syndromes are shown in the following table and the full criteria of Neary et al. (1998).

[0338] Clinical profile and main diagnostic features of FTLD syndrome

[0339]

Table 20

[0340] The discovery that TDP-43 positive inclusions characterize ALS and FTLD-TDP (Neumann et al., 2006) was soon followed by the identification of missense mutations in the TARDBP gene in both familial and sporadic cases of ALS (Gitcho et al. 2008; Seedharan et al., 2008). To date, 38 different TARDBP mutations have been reported in 79 phylogenetically unrelated families worldwide (Mackenzie et al 2010). TARDBP mutations account for approximately 4% of all familial ALS cases and about 1.5% of sporadic ALS cases.

[0341] As of December 2010, mutations in 13 genes associated with familial and sporadic ALS have been identified. Linkage of ALS to five other chromosomal loci has been demonstrated, but to date, no specific mutations have been identified.

[0342] TDP-43 proteinopathy MT targets intracellular TDP-43 protein aggregates, which are a major pathological feature of both familial and sporadic ALS and also a feature of FTLD-P, and has a mode of action that can reduce them.

[0343] In addition, laboratory data have shown that methylthioninium inhibits the formation of TDP-43 aggregates in SH-SY5Y cells. After treatment with 0.05 μM MT, the number of TDP-43 aggregates decreased by 50%. These findings were confirmed by immunoblot analysis (Yamashita et al. 2009).

[0344] Therefore, the compounds and compositions of the present invention may be useful for the treatment of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).

[0345] Huntington's disease and polyglutamine disorders MT can reduce intracellular polyglutamine protein aggregates, which are a pathological feature of Huntington's disease. Huntington's disease is caused by an expansion of the translated CAG repeat located at the N-terminus of huntingtin. Wild-type chromosomes contain 6 - 34 repeats, while in Huntington's disease, the chromosomes contain 36 - 121 repeats. The age of onset of the disease is inversely correlated with the length of the CAG tract encoding the polyglutamine repeat within the protein.

[0346] Laboratory data have shown that methylthioninium inhibits the formation of aggregates of a huntingtin derivative containing a 102-residue polyglutamine extension in zebrafish (van Bebber et al. 2010). MT dose-dependently prevented the formation of such aggregates in zebrafish when tested at 0, 10, and 100 μM.

[0347] Accordingly, the compounds and compositions of the present invention may be useful for the treatment of Huntington's disease, as well as other polyglutamine disorders such as spinal bulbar muscular atrophy (or Kennedy's disease), and dentatorubral-pallidoluysian atrophy and various spinocerebellar ataxias (Orr & Zoghbi, 2007).

[0348] Mitochondrial diseases and Lafora disease In addition to skeletal muscle, the organ most frequently affected by mitochondrial disorders, particularly respiratory chain diseases (RCDs), is the central nervous system (CNS). CNS symptoms of RCDs include stroke-like episodes, epilepsy, migraine, ataxia, spasticity, movement disorders, mental disorders, cognitive decline, or even dementia (mitochondrial dementia). To date, mitochondrial dementia has been reported in MELAS, MERRF, LHON, CPEO, KSS, MNGIE, NARP, Leigh syndrome, and Alpers-Huttenlocher disease (Finsterer, 2009). The mitochondrial respiratory chain consists of four complexes that involve a series of electron transfers. Abnormal function of any of these complexes can lead to mitochondrial diseases that result from an abnormal electron transport chain and subsequent abnormal mitochondrial respiration. Complex III of the mitochondrial respiratory chain acts to transfer electrons to cytochrome c.

[0349] The compounds and compositions of the present invention may also be used to treat mitochondrial diseases associated with deficiencies and / or impairments in the function of complex III of the respiratory chain. Since the thioninium moiety has a low redox potential that converts between an oxidized and a reduced form, the compounds have the ability to act as effective electron carriers and / or mediators. When a disorder and / or deficiency in the function of complex III results in a mitochondrial disease, the compounds of the present invention can also play the role of electron transport and transfer in complex III due to the ability of the thioninium moiety to shuttle between the oxidized and reduced forms, and thus act as an electron carrier instead of a complex III that functions sub-optimally to transfer electrons to cytochrome c.

[0350] The compounds and compositions of the present invention also have the ability to generate active thioninium moieties that have the ability to divert misfolded proteins / amino acid monomers / oligomers away from the Hsp70 ADP-related protein accumulation and / or refolding pathways and instead direct these abnormally folded protein monomers / oligomers into a pathway that directly leads to the Hsp70 ATP-dependent ubiquitin-proteasome system (UPS), a pathway that removes these misfolded proteins / amino acid monomers / oligomers (Jinwal et al. 2009).

[0351] Lafora disease (LD) is an autosomal recessive, fatal epilepsy that develops in the teens and is associated with the slow accumulation of a poorly branched and insoluble glycogen called polyglucosan in many tissues. In the brain, polyglucosan bodies, or Lafora bodies, form within neurons. Inhibition of the Hsp70 ATPase by MT (Jinwal et al. 2009) can upregulate the removal of misfolded proteins. Lafora disease is caused primarily by a deficiency in the lysosomal ubiquitin-proteasome system (UPS) due to mutations in either the laforin gene or the malin gene, both of which are located on chromosome 6 and can lead to inclusion bodies that can accelerate the aggregation of misfolded tau proteins. Secondary mitochondrial damage from UPS impairment further results in the suppression of mitochondrial activity and disruption of the electron transport chain, leading to further lipofuscin and triggering seizures that are characteristic of Lafora disease.

[0352] The MT moiety can deaggregate existing tau aggregates, reduce further tau accumulation, and enhance lysosomal efficiency by inhibiting the Hsp70 ATPase. MT can lead to a reduction in tau tangles by enhancing the ubiquitin proteasome system removal of tau monomers / oligomers through its inhibitory action on the Hsp70 ATPase.

[0353] Therefore, the compounds and compositions of the present invention may be useful for the treatment of Lafora disease.

[0354] Mixture of oxidized MT compound and reduced MT compound The MT compounds for use in the present invention may include a mixture of oxidized and reduced forms.

[0355] In particular, LMT-containing compounds may contain oxidized (MT + ) compounds as "impurities" during synthesis and may be oxidized (e.g., auto-oxidized) after synthesis to yield the corresponding oxidized form. Thus, unless otherwise inevitable, compositions containing the compounds of the present invention will contain, as impurities, at least some of the corresponding oxidized compounds. For example, "LMT" salts may contain 10-15% MT + salts.

[0356] When using mixed MT compounds, the MT dosage can be easily calculated using the molecular weight coefficients of the compounds present.

[0357] Salts and solvates The MT-containing compounds described herein are salts themselves, 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, reference to a particular compound includes its salts as well.

[0358] 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 conventional meaning 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., a monohydrate, dihydrate, trihydrate, pentahydrate, etc. Unless otherwise specified, any reference to a compound includes its solvate and any hydrate form.

[0359] Naturally, solvates or hydrates of salts of the compounds are also encompassed by the present invention.

[0360] To more fully describe and disclose the present invention and the prior art to which the present invention pertains, several patents and publications are cited herein. Each of these references is incorporated by reference in its entirety into this disclosure to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0361] Throughout this specification, including the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be interpreted as meaning that the recited integer or step, or group of integers or steps, is included, but not to the exclusion of any other integer or step, or group of integers or steps.

[0362] It should 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.

[0363] Ranges are expressed herein as from "about" a particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the particular value and / or to the other particular value. Similarly, it will be understood that when values are expressed using the antecedent "about", the particular value forms another embodiment.

[0364] Any subheadings in this specification are included for convenience only and should not be construed as limiting the disclosure in any way.

[0365] Here, the present invention will be further described with reference to the following non-limiting drawings and examples. In light of these, other embodiments of the present invention will occur to those skilled in the art.

[0366] To the extent that it can be used by those skilled in the art to practice the present invention, the disclosures of all references cited herein are specifically incorporated herein by cross-reference.

Brief Description of the Drawings

[0367]

Figure 1

Figure 2A

Figure 2B

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7A

Figure 7B

Figure 8A

Figure 8B

Figure 9A

Figure 9B

Figure 10A

Figure 10B

Figure 11A

Figure 11B

Figure 12A

Figure 12B

Figure 12C

Figure 13

Mode for Carrying Out the Invention

Examples

[0368] Example 1 - Provision of MT-containing Compounds The chemical synthesis methods of the MT-containing compounds described in this specification are known in the art. For example: The synthesis of Compounds 1 to 7 can be carried out according to the methods described in International Publication No. WO 2012 / 107706, or methods similar thereto. The synthesis of Compound 8 can be carried out according to the methods described in International Publication No. WO 2007 / 110627, or methods similar thereto. The synthesis of Compound 9 (MTC) is well-known in the art. Exemplary syntheses of high-purity MTC are provided in International Publication No. WO 2006 / 032879 and International Publication No. WO 2008 / 007074. The synthesis of Compounds 10 to 13 can be carried out according to the methods described in International Publication No. WO 2007 / 110630, or methods similar thereto.

[0369] Example 2 - Formulation of MT-containing Compounds The chemical synthesis methods of the MT-containing compounds described in this specification are known in the art. For example, an exemplary method using dry compression is provided in International Publication No. WO 2012 / 072977.

[0370] Example 3 - Phase 3 Clinical Trials in Mild to Moderate AD Protocol

[0371] [Table 21]

[0372] [Table 22]

[0373] [Table 23-1]

[0374]

Table 23-2

[0375]

Table 23-3

[0376]

Table 24

[0377]

Table 25-1

[0378]

Table 25-2

[0379]

Table 25-3

[0380]

Table 25-4

[0381]

Table 25-5

[0382]

Table 25-6

[0383]

Table 25-7

[0384]

Table 25-8

[0385]

Table 25-9

[0386]

Table 25-10

[0387]

Table 25-11

[0388] Test Design The LUCIDITY trial (NCT03446001; EudraCT: 2017-003558-17) is a Phase 3, randomized, double-blind, placebo-controlled, outpatient trial to evaluate the safety, efficacy, and tolerability of hydroxymethylthionine mesylate monotherapy in participants with severity ranging from mild cognitive impairment (MCI) to moderate AD. Following an initial 12-month blinded period, a 12-month open-label extension period follows, providing relatively late-start data. There are 76 trial sites located in Canada, France, Italy, Poland, Spain, the United Kingdom, and the United States. The protocol was approved by the institutional review board or independent ethics committee at each site.

[0389] The trial design is summarized in Figure 1. After screening, eligible participants are randomized at baseline in a 4:1:4 ratio to receive 16 mg / day of hydroxymethylthionine mesylate, 8 mg / day of hydroxymethylthionine mesylate, or placebo. After completion of the 52-week double-blind treatment period, all participants continue with open-label treatment with 16 mg / day of hydroxymethylthionine mesylate for an additional 52 weeks. Participants randomized to initiate HMTM are considered early starters; patients who initiate placebo and switch to HMTM at week 52 are considered late starters. Unlike conventional delayed-start designs where participants remain blinded to treatment throughout the trial, treatment between weeks 52 and 104 is open-label, but participants and site study staff remain blinded to the prior treatment assignment. Randomization is stratified by severity (3 levels: MMSE 16–19, 20–25, or 26–27, approximate target ratio of 2:3:1), prior use of symptomatic treatment (AChEI and / or memantine - 2 levels: prior use or none), and region (2 levels: Canada / USA or UK / Europe). To achieve this goal, enrollment was monitored and controlled at the site level of high-recruitment sites and upper limits were set at the trial level as needed. Patients who dropped out after randomization were not replaced, but participants were encouraged to continue with out-of-treatment study visits until the scheduled completion of the double-blind treatment period (visit 7). Only participants who continued the trial without adding concomitant AChEI and / or memantine and received hydroxymethylthionine mesylate treatment until the last visit (visit 10) were eligible to continue treatment in the subsequent expanded access program.

[0390] Test drug and placebo formulations The active and placebo treatment formulations are visually identical tablets. Hydroxymethylthionine mesylate can cause variable urine discoloration. Therefore, to maintain blinding, the placebo group is administered tablets containing a urine discolorant (MTC, 4 mg) included between blank tablets containing only excipients on a schedule that varies with an average frequency of twice / week.

[0391] The dosing frequency of the 4 mg MTC tablets was a variable schedule such that each patient received the 4 mg tablets on average twice a week during the blanks. All subjects (all groups) received 4 tablets twice a day. This allowed for 2 x 4 mg, followed by 2 x 4 mg, giving a daily dose of 16 mg. Four tablets (2 x placebo, then 2 x placebo) were also administered daily for the placebo, with 2 of the 28 tablets per week being MTC tablets inserted twice a week, and the rest being true placebo.

[0392] Inclusion and exclusion criteria Participants had to be under 90 years of age, meet the diagnostic criteria for possible AD or MCI-AD, and not have taken either an AChEI or memantine for at least 60 days at baseline. Participants had to be community residents, have a Mini-Mental State Examination (MMSE) score of 16 - 27 at screening, and have no functional impairment as demonstrated by a Clinical Dementia Rating (CDR) stage of 0.5 - 2. Patients also had to have a negative amyloid PET scan. All patients also had to have at least one study partner who consented to their participation; the study partner could be changed as long as there was sufficient contact to meaningfully complete the outcome and safety assessments and verify compliance with the study treatment. Patients were excluded from the study if they had MCI other than AD or a substantial CNS cause of dementia including significant vascular pathology seen on brain MRI. Other exclusions included severe, unstable, or poorly controlled medical or psychiatric conditions; pregnancy or lactation; contraindications or prior adverse reactions to MT or excipients; and involvement in another clinical trial or potential lack of compliance as determined by the study investigator. Stable doses of antipsychotics and antidepressants were permitted. Patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency or who were taking drugs with warnings or precautions regarding methemoglobinemia were also excluded. Other pharmacological agents that could affect cognition or pose an excessive risk were also excluded.

[0393] Recruitment and consent procedures Patients were recruited from memory clinics, outpatient clinics, or other component facilities of specialists in neurology, psychiatry, or geriatric services. Whenever possible, written informed consent was obtained from the patients. If the patient's decision-making capacity was impaired, consent for participation in the trial was obtained at the highest level of understanding of the patient, by a legal representative or the like consenting on behalf of the patient.

[0394] Assessment For participants not receiving AChEI and / or memantine, the screening period is up to 9 weeks. For participants receiving AChEI and / or memantine who consent to a washout, the screening period can be extended by up to an additional 6 weeks to allow for washout. Five visits are scheduled during the double-blind treatment period (3 visits for safety assessment; and 4, 5, 6, and 7 visits at 3-month intervals for efficacy, safety, and MRI assessments), and 3 of the 8 post-baseline visits are scheduled during the open-label phase. Visit 8, 4 weeks after the start of the open-label phase, is for safety assessment; visits 9 and 10 at 6-month intervals are for efficacy and safety assessments, and brain imaging is only at visit 10 (Table 1).

[0395] At visits 2, 3, 7, and 10, timed morning blood samples are collected to determine the plasma levels of the drug. Samples are collected before dosing and then at 1, 2, and 4 hours after dosing. A single blood sample for apolipoprotein E (ApoE) is obtained from participants who provide informed consent at any time after eligibility determination and before visit 7. Blood may also be analyzed for other biomarkers for future research that may be relevant to the determination of potential biomarker predictors or surrogates for treatment response as described in a separate protocol.

[0396] Safety and tolerability All safety evaluations are conducted during screening by independent qualified medical evaluators not involved in the efficacy evaluation to assess subject eligibility. For enrolled participants, safety evaluations are conducted at baseline and at each clinic visit at 4, 13, 26, 39, and 52 weeks during the double-blind treatment period, and at 56 (telephone evaluation, or on-site in the UK), 78, and 104 weeks during the open-label, delayed start phase; when follow-up of adverse events (AEs) occurring during treatment is required; and at early termination (Table 1). Patients are followed as needed for resolution or stabilization of any AE, in accordance with the medical judgment of the study investigator.

[0397] Primary Efficacy Endpoints The co-primary endpoints of the LUCIDITY trial are evaluated in participants taking 16 mg / day of hydromethylthionine mesylate and compared to participants taking placebo. The co-primary endpoints are the change from baseline to week 52 in cognitive function as measured by the ADAS-Cog 11 and in functional ability as measured by the ADCS-ADL 23

[0398] Secondary Efficacy Endpoints Secondary endpoints evaluated in participants taking placebo compared to those taking 8 mg / day of hydromethylthionine mesylate include the following: 1. The change from baseline to week 52 in cognitive function as measured by the ADAS-Cog 11 and in functional ability as measured by the ADCS-ADL 23 Secondary endpoints evaluated in participants taking placebo compared separately to those taking 8 mg / day or 16 mg / day of hydromethylthionine mesylate include the change from baseline to week 52 in the following: 1. Cognitive and functional ability as measured by the MMSE and CDR ​​2. Volumes of the whole brain, parietal lobe, and temporal lobe measured by MRI; the annualized rate of atrophy from baseline to week 52 is quantified using the boundary shift integral (BSI). 3. In the standardized uptake value ratio (SUVR) of the temporal lobe (normalized to the pons) 18 F-fluorodeoxyglucose positron emission tomography( 18 F-FDG-PET) changes in cerebral metabolic function; this analysis is limited to participants with a CDR score of 0.5 at screening if a sufficient number of participants providing data reach a predefined threshold. Secondary evaluation items evaluated over the open-label delayed start period (week 52 to week 104) compare participants originally randomized to placebo (delayed starters) with participants originally randomized to any dose of hydroxymethylthionine mesylate (early starters). 4. Cognitive function measured by changes in ADAS-Cog 11 from week 52 to week 104.

[0399] Exploratory evaluation items 1. The TauRx composite scale is a new composite scale constructed based on data available from the completed TauRx Phase 3 trial and Alzheimer’s Disease Neuroimaging Initiative data, and is designed to be sensitive to decline in early AD. This scale consists of the cognitive subdomains of the standard ADAS-Cog 13 (orientation, constructional praxis, word recall, examiner ratings of subject speech, and examiner ratings of subject comprehension), and the functional items of the ADCS-ADL 23 (use of the telephone, keeping appointments, cooking and meal preparation, and dishwashing). Scores range from 0 to 48, with lower scores indicating greater impairment. 2. Changes from week 52 to week 104 in the ADCS-ADL 23 , cerebral atrophy (MRI), and cerebral metabolic function( 18 F-FDG-PET) comparing delayed starters with early starters. 3. Influence of APOE genotype on primary and secondary outcomes. 4. Perform population pharmacokinetic (PK) analysis to estimate the exposure of each subject for use in the evaluation of the exposure-response relationship.

[0400] Statistical analysis Sample size estimation to achieve 90% power (two-sided α = 0.05) to detect the difference between 16 mg / day of hydroxymethylthionine mesylate and placebo, which is the primary treatment group comparison during the double-blind treatment period, was performed assuming a dropout rate of 20% - 25% for the two co-primary clinical evaluation items. An approximate sample size of 450 test samples was based on the ADCS-ADL 11 with a larger standard deviation (SD) than the ADAS-Cog 23 Based on the estimated decline of 7.7 units in the ADCS-ADL 23 over 52 weeks in the control group and an estimated SD of 8.5 units, the study has more than 90% power to detect a reduction in decline of more than 3.4 units. The effect size of 3.4 units was derived from the estimated treatment effect of 5.0 ± 1.6 (mean ± standard error) units in the completed hydroxymethylthionine mesylate trials. Based on the estimated decline of 6.5 units in the ADAS-Cog 11 over 52 weeks and an estimated SD of 5.9 units, 200 participants per treatment group provide more than 90% power to detect a reduction in decline of more than 2.6 units, based on a conservative value of the pooled estimated treatment effect of -5.2 ± 1.3 (mean ± standard error) units in the completed phase 3 trials in the completed hydroxymethylthionine mesylate trials. For the primary comparison during the double-blind treatment period, assuming a dropout rate of 20% - 25% as above, 160 - 170 participants per group enter the open-label, delayed-start treatment phase with 200 participants per group randomized. Assuming an additional 10% dropout during the delayed-start phase, an important secondary analysis to demonstrate disease modification by comparing early starters and late starters using a non-inferiority margin of -2 ADAS-Cog 11 units has approximately 80% power.

[0401] The primary analysis is conducted using the intention-to-treat (ITT) population and the efficacy-modified intention-to-treat (E-MITT) population. The ITT population includes all randomized participants. The E-MITT population includes all randomized participants who took the investigational drug at least once and had a baseline and post-baseline efficacy assessment. The comprehensive null hypothesis is as follows: · H01: From baseline to week 52, there is no difference in the change in ADAS-Cog 11 between the 16 mg / day hydroxymethylthionine mesylate group and the placebo group. · H02: From baseline to week 52, there is no difference in the change in ADCS-ADL 23 between the 16 mg / day hydroxymethylthionine mesylate group and the placebo group. The comprehensive null versus alternative primary efficacy hypothesis is the Union-Intersection test, which requires that both co-primary endpoints show statistical significance at the 5% two-sided significance level for the comprehensive null hypothesis to be rejected.

[0402] Evolution of the Protocol Design The LUCIDITY trial protocol had three major revisions motivated by regulatory expectations for AD treatment and changes in new data. As initially devised in 2017 (version 1.0, August 2017), a limited trial was intended to confirm the pharmacological activity of an 8 mg / day dose over 6 months in a population of 180 participants meeting the diagnostic criteria for early AD, using changes in FDG-PET as the primary outcome. At that time, it was assumed that clinical endpoints would be evaluated in a larger subsequent trial in mild to moderate AD. The first revision corresponded to draft guidance issued by the FDA in February 2018 and by the European Medicines Agency (EMA) in March 2018 (28, 29), showing that a single trial could form the basis for regulatory approval in early AD based on a statistically significant benefit over placebo in a single composite clinical outcome measure that includes cognitive and functional components. Therefore, the trial uses data from completed trials to ADAS-Cog 11and ADCS-ADL 23 Intended to use a composite scale developed by TauRx based on items that were found to be the most sensitive and discriminatory from the scales, it was expanded to 375 participants and extended to 9 months. In light of the new exposure-response data summarized above, a dose of 16 mg / day was added to the design. Scientific advice from the EMA in May 2019 indicated that if the trial was successful, the scope of approval would be limited to early AD. Since hydromethylthionine mesylate has clinically relevant pharmacological activity across the range of AD severity from early to moderate disease (21), this trial was changed to a more conventional design with co-primary cognitive (ADAS-Cog 11 ) and functional (ADCS-ADL 23 ) assessment items. This required further expansion to 450 participants in a revised version of the protocol and an extension of the double-blind, placebo-controlled treatment phase to 12 months to ensure sufficient power. The main focus of this trial was also changed to the 16 mg / day dose. The basic structure of the final amended design (version 5.0) agreed upon by the EMA consisted of a 12-month double-blind, placebo-controlled treatment period followed by a 12-month modified delayed start open-label extension period in which participants initially randomized to placebo were switched to 16 mg / day to approximate a delayed start design to investigate disease modification potential. Further amendments were introduced in October 2020 in consideration of the impact of COVID-19. The statistical analysis plan was adjusted to accommodate these changes and the current version is briefly described above. This trial was conducted based on the main comparison of 16 mg / day and placebo. The small 8 mg / day group was maintained at a 1:4 ratio with 16 mg / day and placebo to provide a bridge to previously completed trials and to include participants randomized to an earlier version of the protocol receiving this low dose.

[0403] Patient selection criteria for the completed trial included an MMSE score of 16 - 17, age less than 90 years, meeting the diagnostic criteria for mild cognitive impairment (MCI) due to AD, or mild to moderate AD with a positive amyloid-PET scan. Further details are provided in Wischik et al., 2022.

[0404] This trial recruited over 20% of the target in April 2021. The target MMSE ratio was achieved. All participants are expected to complete the blinded phase by the end of March 2022, and the topline results are scheduled for mid-2022. Currently, 20% of the participants have prematurely terminated from the double-blind phase. Less than 3% of the clinic attendees were affected by COVID-19, which does not appear to have affected the validity of the trial. A significant number of participants with CDR 0.5 have 18 F-FDG-PET data available to enable analysis of this assessment item.

[0405] Results / Discussion Due to the slight urine discoloration caused by LMTM, participants in the control group were administered a 4 mg dose of the active drug at varying schedules twice a week. This was necessary to maintain the blindness of the trial in this placebo-controlled study. Most of the participants in this group declined as expected over 52 weeks (i.e., the decline predicted without active treatment, based on a meta-analysis of data from previously published studies). However, this group of participants benefited even from such a low dose of the active drug (see Figures 12B and 12C).

[0406] While not wishing to be bound by theory, this may be due to the accumulation of low blood levels of the drug in these participants, suggesting that some people are extremely sensitive to LMTM. As further discussed below, twice-weekly MTC 4 mg appears to result in the accumulation of MT in these patients.

[0407] As shown in Figure 2, for the primary efficacy assessment items (change in ADAS-cog 11 and ADCS-ADL 23 ), the results of the control group were, on average, equivalent to those of the treatment group, with minimal or no decline during the study duration. The change in total brain volume (WBV) was also equivalent (Figure 3).

[0408] From the available results, a subset of MCI patients appears to be particularly responsive to low-dose MTC.

[0409] Example 4 - Analysis of Plasma HMT Levels The distribution of HMT concentrations in the control group at steady state (blood levels 1 hour after dosing) at the first administration (Visit 2), 4 weeks (Visit 3), and 12 months (Visit 7) is shown in Figure 4. Concentrations of 8 mg LMTM / day and 16 mg LMTM / day are also shown. All patients were administered 16 mg / day (i.e., 8 mg twice a day) after Visit 7.

[0410] Figure 5 shows the correlation between pre-dose and post-dose plasma levels at the first administration (Visit 2) and at 4 weeks of treatment (Visit 3) across all three groups of the study.

[0411] Due to the dosing frequency of the control group (4 mg MTC-containing capsules administered only twice a week), the timing of plasma measurements is a factor: in particular, for the study design, the placebo administration given 1 hour before the measurement may or may not actually contain MTC. Therefore, evidence of MT accumulation in some patients cannot be completely conclusive. However, this fact alone cannot explain the distribution of plasma levels across the entire control group. Furthermore, the evening dose the previous day may have randomly improved the cognitive score the next day in 2 / 7 of the visits (since MTC is known to have nootropic properties at very low doses), but this alone cannot explain the treatment effect observed in the control group.

[0412] The steady-state plasma concentrations (Visit 4, 1 hour after dosing) in each dosing group were further investigated. Patients were grouped based on plasma concentration. The mean values for each dosing group and subgroup are shown in Table 2:

[0413]

Table 26

[0414]

Table 27

[0415] Further analysis of plasma HMT levels within the control group is shown in Figure 6, which shows the distribution of different steady-state plasma concentrations within that group (4 hours after hospital visit, 1 hour after administration).

[0416] Generally, as expected, it can be seen that the HMT plasma levels in the 16 mg LMTM / day group are higher than those in the control group. At week 4, the overall mean plasma concentration in the control group (1 hour after administration) is 0.12 ng / ml compared to 0.50 ng / ml in the 16 mg LMTM / day group. However, some patients in the control group appear to have higher levels than the other groups.

[0417] For example, as shown in Figure 5, MTC 4 mg twice a week appears to result in the accumulation of MT in these patients. This results in a non-linear relationship between dose and steady-state plasma levels at these very low doses. The half-life of MT in this elderly population is approximately 22 hours, and thus accumulation should be negligible at an average dosing interval of 84 hours (i.e., twice a week), which is unexpected. As shown in Figure 6, the majority of control patients have measurable levels of HMT in plasma. The overall mean HMT concentration in this group was approximately 0.012 ng / ml. All MT+ is likely to be converted to HMT at this very low dose. Absorption limits that are dependent on food only appear at much higher MTC doses (Baddeley et al., 2015).

[0418] There are 111 control group patients with plasma levels between 0.005 - 0.025 ng / ml (very low), 47 cases between 0.025 - 0.06 ng / ml (medium), and 16 cases above 0.06 ng / ml (high). There are also 68 cases with plasma levels below 0.005 ng / ml (undetectable).

[0419] In the control group, as expected, there was a low number of cases and a decline in ADAS-cog was observed (3.75±0.79). The expected decline, i.e., the decline that occurs without active treatment, can be modeled based on a meta-analysis of the expected decline data from previously published studies (see, for example, Figure 9).

[0420] Compared to this expected decline, the treatment effect in the high group is -4.73±2.01, so it is zero or above the baseline. Including all patients, the overall treatment effect at 16 mg / day compared to the control group with ultra-low plasma levels is -1.79 ADAS-cog units (p0.0400). The exposure-response as a function of the mean plasma HMT levels in the three groups is shown in Figure 7 (for the control group). For comparison, the exposure-response across the entire study (for all three treatment groups) is shown in Figure 8.

[0421] Example 5 - Clinical Trial Results - Summary The Phase 3 LUCIDITY trial compared a dose of 16 mg / day of methylthioninium chloride (MTC) given at a dose of 4 mg twice a week, which is the minimum amount necessary to prevent bias from potential urine discoloration, with a dose of 16 mg / day. This trial was conducted in 598 patients with AD severity ranging from mild cognitive impairment (MCI) to moderate stages.

[0422] TauRx has currently completed the first 12-month double-blind phase of the trial. This was followed by a further 12-month period that is still ongoing, during which all patients were administered HMTM at 16 mg / day. All patients in this trial were required to have a positive amyloid-PET scan and not to have received standard symptomatic treatment for AD.

[0423] Unexpectedly, it was found that the majority (85%) of patients administered 4 mg of MTC twice a week had blood levels of the active drug above the threshold required to produce a clinical effect (see Figure 10).

[0424] The PK of MTC / LMTM appears to show further accumulation from visit 3 (4 weeks) to visit 7 (12 months), regardless of dose (a two-fold increase is observed) (see Figure 13). The trough level was found to increase nine-fold more than the predicted steady state over time. Interestingly, no corresponding increase was seen in the inactive glucuronide metabolite (which accounts for approximately 99% of total plasma HMT / MT). Thus, there was no overall accumulation of HMT / MT, but there may have been a time-dependent inhibition of the conversion of HMT to its glucuronide metabolite. Overall, the HMT accumulation from the MTC 8 mg / week dose is sufficient to produce a treatment effect.

[0425] In the absence of a true placebo, the designed trial was unable to determine the outcome on the primary clinical evaluation items compared to a pre-specified therapeutically inactive placebo. In light of currently available evidence, TauRx does not consider a blinded placebo-controlled trial using clinical evaluation items to be technically feasible. Thus, TauRx analyzed the data from the perspective of the relationship between blood concentration and treatment effect, the change from the pre-treatment baseline, and comparison with historical controls available from closely matching data from the Alzheimer’s Neuroimaging Initiative (ADNI).

[0426] The overall baseline MMSE score was 21 for the test population ranging from MCI to moderate disease. In patients administered the 16 mg / day dose, over the first 12 months, a minimal decline was seen in both co-primary cognitive and functional evaluation items (1.3 ADAS-cog 11 units and -1.0 ADCS-ADL 23 units). The expected decline in the untreated population would be approximately 5 units on both scales.

[0427] In 105 patients with MCI (baseline MMSE score 23) administered the 16 mg / day dose, ADAS-cog 13At the scales, statistically significant cognitive improvements of 2 units were seen at 6 months (p = 0.0002), 12 months (p = 0.0391) and 18 months (p = 0.0473) compared to baseline. The plasma level thresholds required to bring about this improvement were determined (Figure 11). The mean change in the ADCS-ADL instrumental activities of daily living subscale also remained above the pre-treatment baseline at 6, 12 and 18 months.

[0428] In 147 patients with mild / moderate AD (baseline MMSE 20) administered 16 mg / day, there was a cognitive decline of 2.5 units in the first 9 months, and no further decline over the subsequent 9 months. The functional decline on the ADCS-ADL scale was -2 units at 12 months and -3 units at 18 months, representing a reduction in decline of approximately 75% compared to the published meta-analysis of publicly available placebo decline data from historical trials in mild to moderate AD.

[0429] A statistically significant decrease in disease progression, measured by change in cognitive function (p = 0.0008) and brain atrophy (p < 0.0001), was confirmed by comparison of participants administered the 16 mg / day dose with ADNI subjects who were closest to the study population by age and clinical severity (see Figure 12A). These differences were statistically significant in both the MCI and AD subgroups. As expected, LUCIDITY trial participants with MCI had greater brain atrophy than healthy elderly ADNI subjects and entered the trial in line with ADNI MCI subjects. Participants treated with HMTM 16 mg / day had significantly lower (p < 0.0001) and a rate of brain atrophy progression equivalent to that seen in healthy elderly ADNI subjects.

[0430] Recent trials testing treatments targeting amyloid have been conducted in populations that are equal to or less severely affected than the MCI group in the LUCIDITY trial. When comparing HMTM to placebo reduction data publicly available from these trials as benchmarks, the treatment effect on cognitive and functional decline is approximately three times greater over 18 months. The benefits seen with HMTM are clinically meaningful for Alzheimer's patients.

[0431] The safety profile seen with LUCIDITY remains strong and is consistent with previously published HMTM trial data. There were also no treatment-related serious adverse events or evidence of amyloid-related imaging abnormalities (ARIA).

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[0434] References related to the disclosure and examples of the invention Alhola P, Polo-Kantola P. Sleep deprivation: Impact on cognitive performance. Neuropsychiatr Dis Treat. 2007;3:553-67. Eugene, A.R., and Masiak, J. (2015). The Neuroprotective Aspects of Sleep. MEDtube Sci 3, 35-40. Wischik CM, Bentham P, Gauthier S, Miller S, Kook K, Schelter BO; “Oral Tau aggregation inhibitor for Alzheimer’s disease: design, progress and basis for selection of the 16 mg / day dose in a Phase 3, randomized, placebo-controlled trial of hydromethylthionine mesylate”; Journal of Prevention of Alzheimer’s Disease; 2022; 9:780-790.

Claims

1. A methylthioninium (MT)-containing compound for use in methods of treating neurodegenerative diseases in the subject, The method includes the step of orally administering a methylthioninium (MT)-containing compound to the subject over a treatment period. The aforementioned administration is administered less than once a day. The aforementioned administration provides the subject with an amount of MT corresponding to an average of 0.05 mg to 30 mg per day, preferably between 0.1 mg and 20 mg per day. The aforementioned treatment period is at least seven months for the compound for use.

2. The aforementioned administration frequency is ≤1 / 2, ≤1 / 3, or ≤1 / 4, The compound for use according to claim 1, wherein the administration frequency is defined as the total number of doses during a given treatment period divided by the number of days in that treatment period.

3. The compound for use according to claim 1, wherein the MT compound is not administered on consecutive days.

4. The administration is at regular intervals. (i) The administration frequency is every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or (ii) The compound for use according to claim 1, wherein the administration frequency is once, twice, or three times a week on a fixed day each week.

5. The administration is irregular or intermittent. The compound for use according to claim 1, wherein the administration frequency is once, twice, or three times a week on a variable or random day each week, and more preferably twice a week on a weekly changing schedule.

6. The compound for use according to claim 1, wherein the average daily amount of MT is 0.5 to 3 mg.

7. The aforementioned compound, 【Chemistry 1】 The compound for use according to claim 1, which is a salt of any of the, or a hydrate or solvate thereof.

8. The aforementioned compound is an LMTX compound of the following formula: 【Chemistry 2】 (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) The compound for use according to claim 7.

9. The compound has the following formula and is a bis-monoprotonic acid: 【Transformation 3】 The compound for use according to claim 8.

10. The aforementioned compound is LMTM: 【Chemistry 4】 The compound for use according to claim 9.

11. The compound for use according to claim 7, wherein the compound is MTC.

12. Each dose is administered at MTC 5H ２ The compound for use according to claim 11, comprising approximately 16 mg, approximately 8 mg, approximately 4 mg, or approximately 2 mg.

13. The compound for use according to claim 10, wherein each dose contains approximately 4 mg of MT, and the frequency of administration is twice a week.

14. The compound for use according to claim 1, wherein the disorder is a tauopathy and is optionally selected from the list consisting of Pick's disease, progressive supranuclear palsy, frontotemporal dementia, FTD with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, disinhibition-dementia-parkinsonism-muscle atrophy complex, globus pallidus-pontine-nigroniospheric degeneration, Guam ALS syndrome, globus pallidus-nigroluys's body degeneration, corticobasal degeneration, argyrophilic grain dementia, Boxer dementia or chronic traumatic encephalopathy, Down syndrome, subacute sclerosing panencephalitis, mild cognitive impairment, Niemann-Pick disease type C, Sanfilippo syndrome type B, or myotonic dystrophy DM1 or DM2.

15. The compound for use according to claim 1, wherein the impairment is Alzheimer's disease or mild cognitive impairment.

16. The compound for use according to claim 1, wherein the disorder is a polyglutamine disorder such as Huntington's disease, spinal and bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, or spinocerebellar ataxia; the disorder is a TDP-43 proteinopathy such as FTLD-TDP; the disorder is a synucleinopathy such as Parkinson's disease, Lewy body dementia, or multiple system atrophy; the disorder is a hereditary cerebral angiopathy; the disorder is amyotrophic lateral sclerosis; or the disorder is familial encephalopathy with intranuclear inclusions.

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