Long term dosing of neurodegenerative disorders utilising methylthioninium (MT)-containing compounds

Abstract

The present invention is provides a method of treatment of a neurodegenerative disease mild cognitive impairment due to AD, as in a subject, which method comprises orally administering to said subject a methylthioninium (MT) compound, at a first dosage level for a first dosage period and then at a second dosage level for a second dosage period wherein the second dosage level is higher than the first dosage level, and wherein the first dosage period is between 6 and 18 months, and wherein the second dosage period is at least 12 months, and wherein the first dosage level of MT is 12mg / day or more, and wherein the second dosage level of MT is 16mg / day or more. The invention is based on analysis of long term dosing of subjects suffering from AD and MCI-AD with MT-containing compounds.

Description

[0001] Long term dosing of neurodegenerative disorders utilising methylthioninium (MT)-containing compounds

[0002] Cross-reference to related applications

[0003] This patent application claims the benefit of priority of GB2500018.3 filed on 2ndJanuary 2025 which is herein incorporated in its entirety.

[0004] Field of the Invention

[0005] The present invention relates generally to methods and materials for use in the treatment or prophylaxis of diseases of protein aggregation, for example cognitive disorders, using diaminophenothiazines.

[0006] Background

[0007] Aberrant protein aggregation is believed to be a proximal cause of numerous disease states, which may be manifested as neurodegeneration, clinical dementia, and other pathological symptoms.

[0008] In general, the aberrant protein aggregation is that which arises from an induced conformational polymerisation interaction, i.e., one in which a conformational change of the protein, or in a fragment thereof, gives rise to templated binding and aggregation of further (precursor) protein molecules in a self-propagating manner.

[0009] Once nucleation is initiated, an aggregation cascade may ensue which involves the induced conformational polymerisation of further protein molecules, leading to the formation of toxic product fragments in aggregates which are substantially resistant to further proteolysis. For example certain conditions of dementia may be characterised by a progressive accumulation of intracellular and / or extracellular deposits of proteinaceous structures such as -amyloid plaques and neurofibrillary tangles (NFTs) in the brains of affected patients. The appearance of these lesions largely correlates with pathological neurofibrillary degeneration and brain atrophy, as well as with cognitive impairment (see, e.g., Mukaetova-Ladinska, E.B. et al., 2000, Am. J. Pathol., Vol. 157, No. 2, pp. 623-636).

[0010] Current approved treatments for Alzheimer’s disease include acetylcholinesterase inhibitors (AChEls) and the N-methyl-D-aspartate receptor antagonist memantine. These are symptomatic and do not address the underlying disease pathology. Therapies targeting the amyloid pathology have so far proved unsuccessful in late-stage clinical trials (Geerts et al., 2013; Mullane and Williams, 2013). According to a recent Lancet Neurology Commission, “an effective treatment for AD is perhaps the greatest unmet medical need facing modern medicine”, (Winblad et al., 2016) not least because the global economic cost of dementia is estimated to be $818 billion, or 0.65% of global gross domestic product (Alzheimer’s Disease International, 2015).NFTs (the pathology discovered by Alois Alzheimer, (Alzheimer, 1907)) are made up of paired helical filaments (PHFs), composed predominantly of a 12-kDa repeat-domain fragment of the microtubule-associated protein tau (Wischik et al., 1985; Wischik et al., 1988a,b). Numerous studies have confirmed a quantitative link for the spread of neurofibrillary tangle pathology and the quantity of aggregated tau with both the extent of clinical dementia and functional molecular imaging deficits 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 pathological aggregation of tau protein begins at least 20 years prior to any of the clinical manifestations, (Braak and del Tredici, 2013) targeting this pathology offers a rational approach to both treatment and prevention of AD and related tau aggregation disorders (Huang and Mucke, 2012; Wischik et al., 2014; Wischik et al., 2010). The tau fragment originally identified as an intrinsic structural constituent of the PHF core has prion-like properties in vitro in that it captures normal tau protein with very high affinity (Lai et al., 2016) and converts it to a proteolytically stable replicate of itself (Wischik et al., 1996; Harrington et al., 2015) in a process which is self-propagating and autocatalytic.

[0011] Phosphorylation is inhibitory to 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). Direct inhibition of tau aggregation represents a plausible point for therapeutic intervention. Methylthioninium (MT) acts as a tau aggregation inhibitor (TAI) in vitro, (Wischik et al., 1996; Harrington et al., 2015), dissolves PHFs from Alzheimer’s disease brain tissue, (Wischik et al., 1996) and reduces tau pathology and associated behavioural deficits in transgenic mouse tau models at brain concentrations consistent with human oral dosing. (Melis et al., 2015; Baddeley et al., 2015) MT has also been shown to inhibit other disease-associated protein aggregation (see e.g. W02007 / 110629).

[0012] MT is a redox molecule and, depending on environmental conditions (e.g., pH, oxygen, reducing agents), exists in equilibrium between a reduced [leucomethylthioninium (LMT)] and oxidized form (MT+).

[0013] Leucomethylthioninium (LMT) is the active moiety in compounds such as leucomethylthioninium mesylate (LMTM). Leucomethylthioninium (LMT) may also be referred to as hydromethylthionine (HMT). For the avoidance of doubt, these two terms are synonymous and may be used interchangeably herein. Similarly, the terms LMTM (leucomethylthioninium mesylate) and HMTM (hydromethylthionine mesylate) may be used interchangeably herein.

[0014] WO96 / 30766 describes such MT-containing compounds for use in the treatment and prophylaxis of various diseases, including AD and Lewy Body Disease. One example compound was methylthioninium chloride (“MTC”) commonly known as methylene blue, which is the chloride salt of the oxidized form of methylthioninium (MT) i.e. MT+.

[0015]

[0016] WO96 / 30766 describes, in the case of oral administration, a daily dosage of about 50 mg to about 700 mg, preferably about 150 mg to about 300 mg, divided in preferably 1 -3 unit doses.

[0017] W02007 / 110630 discloses certain specific diaminophenothiazine compounds related to MTC, including (so-called) ETC, DEMTC, DMETC, DEETC, MTZ, ETZ, MTI, MTILHI, ETI, ETLHI, MTN, and ETN, which are useful as drugs, for example in the treatment of Alzheimer’s disease.

[0018] W02007 / 110630 describes dosage units comprising 20 to 300 mg of 3,7-diaminophenothiazine (DAPTZ) compounds described therein e.g. 30 to 200 mg, for example 30 mg, 60 mg, 100 mg, 150 mg, 200 mg. A suitable dose of the DAPTZ compound is suggested in the range of about 100 ng to about 25 mg (more typically about 1 pg to about 10 mg) per kilogram body weight of the subject per day e.g. 100 mg, 3 times daily, 150 mg, 2 times daily, 200 mg, 2 times daily. A dosage of 50mg 3 or 4 times daily is also discussed. A preliminary pharmacokinetic model for methylene blue, based on studies of urinary excretion data sets 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 eta / . (2000) Eur J Clin Pharmacol 56: 247-250 provided a model which integrated blood level data, which contradicted the earlier data from DiSanto and Wagner as regards terminal elimination half-life.

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

[0021] Based on these and other disclosures, it is believed that orally administered MTC and similar drugs are taken up in the gut and enter the bloodstream, with unabsorbed drug percolates down the alimentary canal, to the distal gut. One important undesired side-effect is the effect of the unabsorbed drug in the distal gut, for example, sensitisation of the distal gut and / or antimicrobial effects of the unabsorbed drug on flora in the distal gut, both leading to diarrhoea.

[0022] MTC was tested clinically in a phase 2 study (Wischik et al., 2015). Although the minimum safe and effective dose was identified as 138 mg / day, a higher dose of 218 mg / day hadlimited efficacy due to absorption limitations, most likely due to the need for the MT+to be reduced to the leuco-MT (LMT) form to permit efficient absorption by passive diffusion.

[0023] W02009 / 044127 disclosed the results of a phase 2 clinical trial, which indicated that MTC had two systemic pharmacological actions: cognitive effects and haematological effects, but that these actions were separable. Specifically the cognitive effects did not show a monotonic dose-response relationship, whereas the haematological effects did. It was proposed that two distinct species were responsible for the two types of pharmacological activity: MTC absorbed as the uncharged Leuco-MT form being responsible for the beneficial cognitive activity, and MTC absorbed as an oxidised dimeric species being responsible for the oxidation of haemoglobin. W02009 / 044127 described how dosage forms could be used to maximise the bioavailability of the therapeutically active (cognitively effective) species whether dosing with oxidised or leuco-DAPTZ compounds.

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

[0025] MTC, a phenothiazin-5-ium salt, may be considered to be an “oxidized form” in relation to the corresponding 10H-phenothiazine compound, N,N,N’,N’-tetramethyl-10H-phenothiazine- 3,7-diamine, which may be considered to be a “reduced form”:

[0026]

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

[0028] WO 02 / 055720 discloses the use of reduced forms of certain diaminophenothiazines for the treatment of protein aggregating diseases, primarily tauopathies. Based on in vitro activity for the reduced forms of diaminophenothiazines therein, a suggested daily dosage was 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., combined with 2x mg ratio of ascorbic acid in such a manner as to achieve more than 90% reduction prior to ingestion were also described.

[0029] W02007 / 110627 disclosed certain 3,7-diamino-10H-phenothiazinium salts, effective as drugs or pro-drugs for the treatment of diseases including Alzheimer’s disease. These compounds are also in the “reduced” or “leuco” form when considered in respect of MTC.These leucomethylthioninium compounds were referred to as “LMTX” salts, and included the following salts:

[0030] > <

[0031] >

[0032]

[0033] WO2012 / 107706 described other LMTX salts having superior properties to the LMTX salts listed above, including leuco-methylthioninium bis(hydromethanesulfonate) (LMTM; also known as HMTM, HMT, hydromethylthionine):

[0034]

[0035] Specifically, LMTM retains TAI activity in vitro and in vivo (Wischik et al, 1996; Harrington et al., 2015; Melis et al., 2015), has superior pharmaceutic properties in terms of solubility and pKa, and is not subject to the absorption limitations of the MT+form (Baddeley et al., 2015) W02007 / 110627 and WO2012 / 107706 describe dosage units comprising 20 to 300 mg of the DAPTZ compounds described therein e.g. 30 to 200 mg, for example 30 mg, 60 mg, 100 mg, 150 mg, 200 mg. A suitable dose of the DAPTZ compound is suggested in the range of about 100 ng to about 25 mg (more typically about 1 pg to about 10 mg) per kilogram bodyweight of the subject per day e.g. 100 mg, 3 times daily, 150 mg, 2 times daily, 200 mg, 2 times daily.

[0036] W02008 / 155533 describes the use of MT compounds for the treatment of mild cognitive impairment (MCI). Total daily doses of from 10 mg to 400 mg are disclosed, preferably administered as twice daily (b.i.d.) or three times daily (t.i.d.) dosage.

[0037] WO2018 / 019823 describes novel regimens for treatment of neurodegenerative disorders utilising methylthioninium (MT)-containing compounds. Briefly, these regimens identified two key factors. The first was in relation to the dosage of MT compounds, and the second was their interaction with symptomatic treatments based on modulation of acetylcholinesterase levels.

[0038] In the analysis described in WO2018 / 019823, low doses of MT compounds (for example 4 mg b.i.d) showed therapeutic benefits when monotherapy was compared against add-on. The efficacy profiles were similar in mild and moderate subjects for most of the measured outcomes.

[0039] Furthermore, treatment benefit in AD (according to the trial criteria) was restricted to patients taking LMTM as monotherapy. By contrast, the decline seen at corresponding doses in patients taking LMTM in combination with AD-labelled treatments (acetylcholinesterase inhibitors [AChEls] and\or memantine), who were the majority, was indistinguishable on all parameters from that seen in the control arm.

[0040] The potential for LMT compounds to be active at the low dose, and the apparent lack of a dose-response, are discussed in WO2018 / 019823 and it is hypothesised that there may be a critical threshold for activity at the tau aggregation inhibitor target, and that the effect of higher doses may plateau or may even become negative at brain concentrations above 1 pM (Melis et al., 2015). It had previously been shown that the absorption and distribution of MT to the brain is complex, and likely to be mediated via red cells rather than plasma (Baddeley et al., 2015) providing a route which protects MT from first-pass metabolism. In the same study, MT uptake into red cells was approximately 20-fold higher in vivo when as administered intravenously as LMTM compared with MTC, most likely due to direct red cell uptake of LMT by passive diffusion without need for prior reduction of MT+as is the case for MTC (Baddeley et al., 2015; May et al., 2004).

[0041] Based on analyses, and given that lower doses (4 mg twice a day) had a better overall clinical profile than the high dose (100 mg twice a day), WO2018 / 019823 teaches methods of treatment of neurodegenerative disorders of protein aggregation which comprise oral administration of MT-containing compounds, wherein said administration provides a total of between 0.5 and 20mg of MT to the subject per day, optionally as a single dose or split into 2 or more doses. For a given daily dosage, WO2018 / 019823 teaches that more frequent dosing will lead to greater accumulation of a drug.

[0042] Other publications using “low dose” or “low dosage” in relation to MT-containing compounds are described in WO2018 / 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” on retention of fear extinction and contextual memory following fear extinction training. The paper reports that “Methylene blue is a diamino phenothiazine drug that at low doses (0.5-4 mg / kg) has neurometabolic-enhancing properties. The dosages used in the publication were 260 mg / day for adult participants, corresponding to a 4 mg / kg dose.

[0043] 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:

[0044] 10.3389 / fncel.2015.00179: this publication discusses the cellular mechanisms mediating the neuroprotective effects of low doses of methylene blue and near-infrared light. It refers to earlier work citing 0.5-4 mg / kg of methylene blue as safe and effective.

[0045] Alda, Martin, et al. "Methylene blue treatment for residual symptoms of bipolar disorder: randomised crossover study." The British Journal of Psychiatry (2016): doi:

[0046] 10.1192 / bjp.bp.115.173930: this publication described the use of a 15 mg “low dose” of methylene blue as a placebo in a 6-month trial. The “active dose” was 195 mg. In each case the dose was split three times daily.

[0047] 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 refers to the ‘known’ pharmacokinetic and side effects of “low-dose” (0.5-4.0 mg / kg) methylene blue, which are contrasted with the effects of dosages greater than 10 mg / kg. The dosages used in the publication were 280mg / day for adult participants, approximating to a 4mg / kg dose.

[0048] 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.

[0049] Psychiatry 22:657-659: these studies used 15mg / day methylene blue, nominally as a placebo vs. a treatment of 300 mg / day methylene blue. However, in the latter paper the authors proposed that the placebo dosage may act as an antidepressant.

[0050] As discussed above, because of their activity in respect of tau aggregation and TDP-43 aggregation, MT-based compounds have also been suggested for the treatment of FTD (see W02007 / 110630 ; W02007 / 110627; W02009 / 044127; WO2012 / 107706, all described supra).

[0051] WO2018 / 041739 describes the results of a phase 3 clinical trial investigating the treatment of Frontotemporal dementia (FTD) disease using LMTM.The results indicated that even a relatively low dose of the MT compound (which was used in the trial as a control) may show benefit in FTD, as compared to the cognitive decline seen in historical controls.

[0052] Furthermore, unexpectedly, the results indicated strong interaction effects when MT is comedicated with AD treatments which modify synaptic neurotransmission in the brain. There appeared significant cognitive benefits in FTD patients taking MT in combination with such AD treatments (e.g. acetylcholinesterase inhibitors and / or memantine) compared to MT alone. WO2018 / 041739 further describes how MT compounds can be combined with acetylcholinesterase inhibitors and / or memantine without apparent incompatibility.

[0053] W02020 / 020751 described a novel pharmacokinetic (PK) model for dosing LMT compounds in patient populations. As expected, there was substantial variability in the MT Cmax values across the population for the given low dosage. Analysis of the distribution confirmed the findings in WO2018 / 019823 that low dosages (4 mg MT b.i.d.) were efficacious (as measured, for example, by reduced decline on the Alzheimer’s Disease Assessment Scale -cognitive subscale (ADAS-cog). It further confirmed that monotherapy gave a substantial benefit by this criterion compared to add-on therapy with AChEls and\or memantine (with the mean benefit of between monotherapy and add-on being ~ 4 ADAS-cog units over 65 weeks).

[0054] However, unexpectedly in view of the previously described lack of any recognisable dose response, the analysis in W02020 / 020751 revealed that there exists a concentration response within the low dose treated population. These insights suggested that it was advantageous to adopt a dosing regimen which both maximises the proportion of subjects in which the MT concentration will exceed the Cmax or Cavethreshold, and also maximises the expected therapeutic efficacy of LMTM whether it is taken alone or in combination with (or at least preceded by) symptomatic treatments, while nevertheless maintaining a relatively low dose so as to maintain a desirable clinical profile in relation to being well tolerated with minimal side-effects. W02020 / 020751 suggests that the minimum dose which achieves all these objectives is at least 20 mg / day, and doses in the range 20 - 40 mg / day, or 20 - 60 mg / day would be expected to maximise the therapeutic benefit, although good efficacy, particularly in AD patients not pre-treated with symptomatic treatments, can still be seen at dosages of 100mg or more.

[0055] W02020 / 020751 discloses regimens which combine the ‘low’ dosages and ‘higher’ ones -for example treatment regimens which comprise: (i) orally administering to said subject the MT-containing compound fora first period of time, wherein said administration provides a total daily dose of between 1 and 10 mg of MT to the subject per day, optionally 8 mg per day, optionally split into 2 or more doses; (ii) orally administering to said subject the MT-containing compound for an immediately subsequent period of time, wherein said administration provides a total daily dose of between 20.5 and 40 mg, 20.5 and 60, 20.5 and 80 or 20.5 and 100 mg of MT to the subject per day, optionally about 21 to 40, 50, or 60 mg per day, optionally split into 2 or more doses.WO2023 / 232764 described the results of a randomized, double-blind, placebo-controlled, three-arm, 12-month, safety and efficacy study of hydromethylthionine mesylate (LMTM) monotherapy in subjects with Alzheimer's disease. Daily doses of LMTM at 8 mg and 16 mg (i.e. 8mg and 16mg MT, delivered as LMTM) were compared with a control dose, which comprised dosages of 4 mg MT as MTC, twice weekly on a varying schedule every 2-4 days. The control dosage was intended to maintain blinding with respect to discolouration of excreta. Unexpectedly, however, the 4 mg twice weekly ‘control’ dose of MTC also showed therapeutic benefits. This revealed, surprisingly, low doses and / or intermittent administration of low doses of an MT compound can produce substantial clinical benefits. WO2023 / 232764 relates to methods of treatment of neurodegenerative diseases utilising in a subject, which method comprises orally administering to said patient a methylthioninium (MT) containing compound, wherein said administration is at a dosage frequency of less than once daily.

[0056] Disclosure of the invention

[0057] The present invention is based on analysis of long term dosing of subjects suffering from mild cognitive impairment due to AD (MCI-AD) and mild to moderate dementia caused by AD with MT-containing compounds.

[0058] In particular it is based on insights gained following a 12 month open label extension to the randomized, double-blind, placebo-controlled, three-arm, 12-month, study discussed in WO2023 / 232764 (see Figure 5).

[0059] The 12 month extension to the trial revealed unexpected divergence between subjects switching dosages after the first 12 months of treatment, based on cognitive and imaging criteria. Further analysis in turn revealed that the Cmax (dose normalized trough concentration of MT) at 24 months in subjects was profoundly affected by the Cmax attained at 12 months, and in particular the higher ne Cmax at 12 months, the greater the reduction in Cmax at 24 months (see Figures 8, 17, 18).

[0060] Without wishing to be bound by theory it appears that the initial inhibition of the body’s MT clearance mechanisms may saturate at higher doses, for example a gradually improved metabolism may lead to more efficient metabolite formation or improvement in renal function or both.

[0061] Irrespective of this, these novel findings imply a benefit to increasing MT dosages after an initial dosing period, to counteract the increasingly effective clearance that results from that initial dosing period.

[0062] Some of the results from the clinical trial were disclosed at the International Conference on Alzheimer's and Parkinson's Diseases (AD / PD™) between 5-9 March 2024 in Lisbon (Portugal). However the present insights of the inventors regarding the benefits in increasing dosage after a first period of time in order to address potential clearance or inhibition mechanisms were not disclosed.Thus in one aspect, there is disclosed a method of treatment of a neurodegenerative disease in a subject, which method comprises orally administering to said subject a methylthioninium (MT) containing compound,

[0063] at a first dosage level for a first dosage period and then

[0064] at a second dosage level for a second dosage period

[0065] wherein the second dosage level is higher than the first dosage level,

[0066] wherein the first dosage period is between 6 and 18 months,

[0067] wherein the second dosage period is at least 12 months,

[0068] wherein the first dosage level is at least 12 mg / day,

[0069] wherein the second dosage level is at least 16mg / day.

[0070] In some embodiments the first dosage level is 12 to 35 mg / day total.

[0071] In some embodiments the first dosage level may be about 12, 13, 14, 15, 16, 17, 18, 19, 20 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 mg.

[0072] In some embodiments the first dosage level is 23 mg / day or less.

[0073] In some embodiments the first dosage level is about 12, 13, 14, 15, 16, 17, 18, 19, 2021, 22, or 23 mg / day.

[0074] In some embodiments the second daily dosage may be about 16, 17, 18, 19, 2021 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 mg.

[0075] In some embodiments the second dosage level is 17 to 70 mg / day total.

[0076] In some embodiments the first dosage level is 32 mg / day or more.

[0077] In some embodiments the second dosage level is about 32, 33, 34, 35, 36, 37, 38, 39, 40 mg / day.

[0078] As explained above, it is believed that the increase in dosage is advantageous to address the clearance and recovery mechanisms which are induced in the first period of e.g. around 12 months, and which may profoundly reduce the effectiveness of the dosage. Preferably the initial dose is increased by at least 25%, more preferably at least 30%, more preferably at least 33% of the first dosage amount when progressing from the first to second dosage periods.

[0079] Also provided herein are related methods of prophylactic treatment of neurodegenerative disorders.Also provided are methylthioninium (MT) containing compounds and compositions thereof, for use in the methods described herein.

[0080] Also provided are use of methylthioninium (MT) containing compounds and compositions in the preparation of medicaments for use in the methods described herein.

[0081] These aspects and embodiments will now be described in more detail:

[0082] In some embodiments the neurodegenerative disorder may be AD, for example mild or moderate AD.

[0083] Where the disease is AD the first dosage level may be between 15 and 30 mg / day, for example 16mg per day. The second dosage level may be at least 22 mg / day - for example between 22 mg / day and 40mg / day. Example second dosage levels are 24, 28 or 32mg / day.

[0084] As explained below, the dosage periods and levels may be selected to achieve certain Cmax or Cavg MT thresholds, as appropriate to the pathology being addressed.

[0085] In some embodiments the first and second periods and dosages are selected to provide an amount of MT to the subject which results in a plasma MT level of at least 0.74 ng / ml throughout the entire first and second treatment periods.

[0086] In some embodiments the MT compound is administered twice daily and the second dosage level is 40 mg or more. Such a dosage should ensure >95% patients with Cmax > about 0.74 ng / ml_ at 24m, where the switch is around 9 months. Such a Cmax is believed to be an effective dosage for treating mild to moderate AD.

[0087] In some embodiments the MT compound is administered once daily and the second dosage level is 32 mg or more. Such a dosage should ensure >95% patients with Cmax > 0.74 ng / ml_ at 24m, where the switch is around 9 months.

[0088] In some embodiments the second dosage level is 56 mg / day or more. Switching to such a dosage should ensure >95% patients with Cavg > 0.693 ng / mL at 24m]

[0089] In some embodiments the neurodegenerative disorder may be mild cognitive impairment (MCI).

[0090] In the context of the present invention “MCI” is used specifically to relate to MCI as a predementia stage of AD (see e.g. Jack Jr, Clifford R., et al. "Revised criteria for diagnosis and staging of Alzheimer's disease: Alzheimer's Association Workgroup." Alzheimer's & Dementia 20.8 (2024): 5143-5169).

[0091] The terms “MCI due to AD” (Marilyn S. Albert et al. "The diagnosis of mild cognitive impairment due to Alzheimer's disease: Recommendations from the National Institute on Aging - Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease." Alzheimer's & Dementia: The Journal of the Alzheimer's Association 2011 ;7(3):270- 279), “AD with MCI", and “MCl-Early Alzheimer’s disease” are therefore used interchangeably herein to refer to this pre-dementia stage.

[0092] Where the disease is MCI the first dosage level may be between 12 and 20 mg / day, for example 12mg per day. The second dosage level may be at least 16 mg / day - for example between 16 mg / day and 20 mg / day. Example second dosage levels are 16 and 20mg / day. As explained below, the dosage periods and levels may be selected to achieve certain Cmax MT threshold.

[0093] For example in treating MCI the first and second periods and dosages may be selected to ensure that the Cmax is equal to or above about 0.40 ng / ml (for example more than 0.38 ng / ml) throughout the entire treatment period.

[0094] In other embodiments the neurodegenerative disorder may be a neurodegenerative disorder other than AD or MCI.

[0095] Neurodegenerative disorders of protein aggregation are discussed in more detail hereinafter. For any of the neurodegenerative disorders discussed herein, including AD and MCI, the dosage periods and levels may be selected based on a biomarker such as appropriate to the pathology being addressed. For example in treating AD the first and second periods and dosages may be selected to ensure that NfL does not increase throughout the entire treatment period.

[0096] In some embodiments the second dosage level is selected based on measurement of the subject’s parent HMT trough level Cmax after the first period.

[0097] HMT parent Cmax measurements may be made in subjects as described in the Examples below (see Example 4, 2.2).

[0098] In some embodiments the methods of the invention may involve steps comprising:

[0099] - treating a neurodegenerative disease in a subject by orally administering to a subject a methylthioninium (MT) containing compound at a first dosage level for a first dosage period (as described herein) and then

[0100] - taking a blood sample after said first dosage period;

[0101] - measuring or estimating the MT plasma concentration for said subject;

[0102] - if said measurement is below a threshold value proceeding to the second dosage level for the second dosage period;

[0103] - if said measurement is above a threshold value postponing the second dosage level for the second dosage period.

[0104] The plasma concentration measured may be a Cmax measurement (dose normalised trough) based on Tmax (time at which CLZF reach the minimum - see Example 4) which istypically around 2hr after administration, but those skilled in the art will appreciate that for convenience Tmax could be modelled based on other times after administration (typically 1 to 4h).

[0105] The threshold figure will be appropriate to the pathology being addressed e.g. Cmax is equal to or above 0.74 ng / ml for AD, equal to or above about 0.4 ng / ml for MCI.

[0106] The MT moiety targets pathological protein aggregation by blocking capture and stacking of aggregation-prone proteins such as tau. Additionally there is an aggregation independent symptomatic effect believed to be driven by increasing acetylcholine level in hippocampus. Without wishing to be bound by theory, it has been observed that there are threshold blood plasma levels of active drug moiety (i.e. MT) which are needed to produce a disease modifying effect, as measured by Neurofilament Light Chain (NfL).

[0107] NfL is a well-studied and broadly accepted marker of neurodegeneration in brain. For example brain tau pathology in temporal and frontal lobes correlates with NfL concentration in plasma.

[0108] Several independent studies have recently reported that NfL levels in blood corelate with NfL levels in CSF and with tau pathology. Additionally, changes in plasma NfL levels are also associated with AD severity and progression rate. Plasma NfL levels are elevated in the prodromal (Sanchez-Valle R, Heslegrave A, Foiani MS, etal. Serum neurofilament light levels correlate with severity measures and neurodegeneration markers in autosomal dominant Alzheimer’s disease. Alzheimer’s Res T / ?er2018; 10: 1-7) and dementia stages (Mattsson N, Cullen NC, Andreasson U, Zetterberg H, Blennow K. Association between Longitudinal Plasma Neurofilament Light and Neurodegeneration in Patients with Alzheimer Disease. JAMA Neurol 2019; 76: 791-9) of sporadic AD as well as in autosomal dominant AD.

[0109] Using the ADNI database, Ou et al. demonstrated that the rate of change in plasma NfL concentration is markedly increased in all groups including MCI (amyloid positive and negative) and AD (all p<0.001), with the greatest annual rate of change in the AD amyloid positive group (Ou Y-N, Hu H, Wang Z-T, Xu W, Tan L, Yu J-T. Plasma neurofilament light as a longitudinal biomarker of neurodegeneration in Alzheimer's disease. Brain Sci Adv 2019; 5: 94-105).

[0110] Importantly, NfL is not only able to distinguish between AD and healthy ageing, but pharmacodynamic reductions have been observed in AD clinical trials. In a phase 2 clinical trial of donanemab, plasma NfL was the only blood-based biomarker that significantly correlated with change in whole brain volume following 18-months of treatment (r=-0.1710; p=0.03) (Pontecorvo MJ, Lu M, Burnham SC, et al. Association of Donanemab Treatment With Exploratory Plasma Biomarkers in Early Symptomatic Alzheimer Disease: A Secondary Analysis of the TRAILBLAZER-ALZ Randomized Clinical Trial. JAMA Neurol 2022; 79: 1250-9). Therefore, plasma NfL is an effective, non-invasive diagnostic tool for assessing neurodegeneration and evaluating the efficacy of disease-modifying therapies. Therefore in one embodiment the present invention is aimed at ensuring appropriate levels of blood plasma MT are achieved and maintained to provide a protective effect against neurodegeneration e.g. as inferred by plasma NfL levels, measurement of which isdescribed in Example 4, 2.2. In the light of the present disclosure, the first and second dosage periods and levels may be selected to achieve Cmax MT thresholds to meet these aims.

[0111] In some embodiments the methods of the invention may involve steps comprising:

[0112] - treating a neurodegenerative disease in a subject by orally administering to a subject a methylthioninium (MT) containing compound at a first dosage level for a first dosage period (as described herein) and then

[0113] - taking a blood sample after said first dosage period;

[0114] - measuring or estimating the NfL plasma concentration for said subject;

[0115] - if said measurement is above a threshold value proceeding to the second dosage level for the second dosage period;

[0116] - if said measurement is below a threshold value postponing the second dosage level for the second dosage period.

[0117] As used herein, the term ‘dosage period’ (or “treatment duration”) is a period of time over which the subject is treated with the MT containing compound at a specified dosage. The compound will typically be intended to be administered regularly over this period (e.g. once or twice per day, or even three times each day).

[0118] In the practice of the invention it is intended that a minimum dosage period is 6 months. The first dosage period or duration may, for example, be equal to or at least 6 months, 9 months, 12 months, 15 months, or 18 months.

[0119] The second dosage period or duration may, for example, be:

[0120] At least 12 months or 18 months, or longer.

[0121] At least 2, 3, 4, 5 years, or longer.

[0122] Between 6 and 12 months.

[0123] Between 1 and 5 years.

[0124] For prophylaxis, the treatment may be ongoing.

[0125] In preferred embodiments the second dosage period will immediately follow from the first dosage period.

[0126] However it is envisaged within the present invention that the first and second dosage periods may be separated by an intermediate dosage period where the dosage is set at one or more intermediate levels between the first and second dosages (e.g. a ‘mid-way’ dosage) to permit dose ramp up. It is envisaged that such an intermediate dosage period will be substantially shorter than the first or second dosage periods. Examples intermediate dosage periods are 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, or 3 months.Methylthioninium moiety

[0127]

[0128] The MT-containing compounds used in the present invention can contain MT in either reduced or oxidised form. The “MT” is the active ingredient, which is to say that it is present to provide the recited therapeutic effect. Specifically, the compounds may comprise either of the MT moieties described above. The MT moieties perse described above are not stable. They will therefore be administered as MT compounds - for example LMT or MT+salts. MT+salts will generally include one or more anionic counter ions (X ) to achieve electrical neutrality. The compounds may be hydrates, solvates, or mixed salts of the MT+salt.

[0129] LMT containing compounds will generally be stabilised, for example by the presence of one or more protic acids e.g. two protic acids.

[0130] 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.

[0131] Examples of such calculations are given herein.

[0132] LMT compounds

[0133] In some embodiments, the MT compound is preferably an LMT compound.

[0134] In some embodiments, the MT compound is an “LMTX” compound of the type described in W02007 / 110627 or WO2012 / 107706.

[0135] Thus, the compound may be selected from compounds of the following formula, or hydrates or solvates thereof:

[0136]

[0137]

[0138] By “protic acid” is meant a proton (H+) donor in aqueous solution. Within the protic acid A-or B" is therefore a conjugate base. Protic acids therefore have a pH of less than 7 in water (that is the concentration of hydronium ions is greater than 10-7moles per litre).

[0139] In one embodiment the salt is a mixed salt that has the following formula, where HA and HB are different mono-protic acids:

[0140]

[0141] However preferably the salt is not a mixed salt, and has the following formula:

[0142]

[0143]

[0144] In one embodiment the salt has the following formula, where H2A is a di-protic acid:

[0145]

[0146] Preferably the salt has the following formula which is a bis monoprotic acid:

[0147]

[0148] Examples of protic acids which may be present in the LMTX compounds used herein include:

[0149] Inorganic acids: hydrohalide acids (e.g., HCI, HBr), nitric acid (HNO3), sulphuric acid (H2SO4)

[0150] Organic acids: carbonic acid (H2CO3), acetic acid (CH3COOH), methanesulfonic acid, 1,2-Ethanedisulfonic acid, ethansulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid, Preferred acids are monoprotic acid, and the salt is a bis(monoprotic acid) salt.

[0151] A preferred MT compound is LMTM:

[0152]

[0153] The anhydrous salt has a molecular weight of around 477.6. Based on a molecular weight of 285.1 for the LMT core, the weight factor for using this MT compound in the invention is 1.67. By “weight factor” is meant the relative weight of the pure MT containing compound vs. the weight of MT which it contains.

[0154] Other weight factors can be calculated for example MT compounds herein, and the corresponding dosage ranges can be calculated therefrom.

[0155] Other example LMTX compounds are as follows. Their molecular weight (anhydrous) and weight factor is also shown:

[0156] <

[0157] <

[0158] <

[0159]

[0160] <

[0161]

[0162]

[0163] The dosages described herein with respect to MT thus apply mutatis mutandis for these MT containing compounds, as adjusted for their molecular weight.

[0164] Oxidised MT compounds

[0165] In another embodiment the MT compound is an MT+compound.

[0166] Preferably the MT compound is an MT+compound of the type described in WO96 / 30766 or W02007 / 110630.

[0167] Thus, the compound may be selected from compounds of the following formula, or hydrates, solvates, or mixed salts thereof:

[0168]

[0169] Where X-is an anionic counter ion.

[0170] In some embodiments of the present invention the MT+compound is MTC, for example a “high purity” MTC as described below.In some embodiments of the present invention the MT+compound is not MTC.

[0171]

[0172] As explained in WO2011 / 036561 and WO2011 / 036558, MTC occurs in a number of polymorphic forms having different levels of hydration.

[0173] In some embodiments of the present invention, the MT+compound is a high purity MTC. In this context ‘high purity’ is defined by one or more of the criteria set out below.

[0174] In some embodiments, the MTC has a purity of greater than 97%.

[0175] In some embodiments, the MTC has a purity of greater than 98%.

[0176] In some embodiments, the MTC has a purity of greater than 99%.

[0177] In some embodiments, the MTC has less than 2% Azure B as impurity.

[0178] In some embodiments, the MTC has less than 1% Azure B as impurity.

[0179] In some embodiments, the MTC has less than 0.5% Azure B as impurity.

[0180] In some embodiments, the MTC has less than 0.1% Azure B as impurity.

[0181] In some embodiments, the MTC has less than 0.15% Azure A as impurity.

[0182] In some embodiments, the MTC has less than 0.10% Azure A as impurity.

[0183] In some embodiments, the MTC has less than 0.05% Azure A as impurity.

[0184] In some embodiments, the MTC has less than 0.15% Azure C as impurity.

[0185] In some embodiments, the MTC has less than 0.10% Azure C as impurity.

[0186] In some embodiments, the MTC has less than 0.05% Azure C as impurity.

[0187] In some embodiments, the MTC has less than 0.13% MVB (Methylene Violet Bernstein) as impurity.

[0188] In some embodiments, the MTC has less than 0.05% MVB as impurity.

[0189] In some embodiments, the MTC has less than 0.02% MVB as impurity.

[0190] All percentage purities recited herein are by weight unless otherwise specified.

[0191] In some embodiments, the MTC has an elementals purity that is better than that specified by the European Pharmacopeia (EP).

[0192] As used herein, the term ‘elementals purity’ pertains to the amounts of the twelve (12) metals specified by the European Pharmacopeia: Al, Cd, Cr, Cu, Sn, Fe, Mn, Hg, Mo, Ni, Pb,and Zn. The current edition of the European Pharmacopeia (8thEdition, suppiementum 8.8) specifies the following limits for these metals:

[0193]

[0194] In one embodiment, the MTC has an elementals purity (e.g. for each of Al, Cd, Cr, Cu, Sn, Fe, Mn, Hg, Mo, Ni, Pb, and Zn) which is equal to or better than (i.e. lower than) the EP8.8 values set out in the table above.

[0195] In one embodiment, the MTC has an elementals purity which is equal to or better than 0.9 times the EP8.8 values set out in the table above.

[0196] In one embodiment, the MTC has an elementals purity which is equal to or better than 0.8 times the EP8.8 values set out in the table above.

[0197] In one embodiment, the MTC has an elementals purity which is equal to or better than 0.7 times the EP8.8 values set out in the table above.

[0198] In one embodiment, the MTC has an elementals purity which is equal to or better than 0.5 times the EP8.8 values set out in the table above.

[0199] (For example, 0.5 times the EP8.8 values as set out above are 50 pg / g Al, 0.5 pg / g Cd, 50 pg / g Cr, etc.)

[0200] In one embodiment the MTC has a chromium level that is equal to or better than (i.e. lower than) 100 pg / g.

[0201] In one embodiment the MTC has a chromium level that is equal to or better than (i.e. lower than) 10 pg / g.

[0202] In one embodiment the MTC has a copper level that is equal to or better than (i.e. lower than) 300 pg / g.In one embodiment the MTC has a copper level that is equal to or better than (i.e. lower than) 100 pg / g.

[0203] In one embodiment the MTC has a copper level that is equal to or better than (i.e. lower than) 10 pg / g.

[0204] In one embodiment the MTC has an iron level that is equal to or better than (i.e. lower than) 200 pg / g.

[0205] In one embodiment the MTC has an iron level that is equal to or better than (i.e. lower than) 100 pg / g.

[0206] All plausible and compatible combinations of the above purity grades are disclosed herein as if each individual combination was specifically and explicitly recited.

[0207] In particular embodiments, the MTC is a high purity MTC wherein ‘high purity’ is characterised by a purity of greater than 98% and one or more of the following:

[0208] (i) less than 2% Azure B as impurity;

[0209] (II) less than 0.13% MVB (Methylene Violet Bernstein) as impurity; or

[0210] (ill) an elementals purity better than the European Pharmacopeia limits of less than 100 pg / g Aluminium (Al); less than 1 pg / g Cadmium (Cd); less than 100 pg / g Chromium (Cr); less than 300 pg / g Copper (Cu); less than 10 pg / g Tin (Sn); less than 200 pg / g Iron (Fe); less than 10 pg / g Manganese (Mn); less than 1 pg / g Mercury (Hg); less than 10 pg / g Molybdenum (Mo); less than 10 pg / g Nickel (Ni); less than 10 pg / g Lead (Pb); and less than 100 pg / g Zinc (Zn).

[0211] In particular embodiments, the MTC is a high purity MTC wherein high-purity is characterised by a purity of greater than 98% and one or more of the following:

[0212] (I) less than 1% Azure B as impurity;

[0213] (ii) less than 0.15% Azure A as impurity;

[0214] (iii) less than 0.15% Azure C as impurity;

[0215] (iv) less than 0.13% Methylene Violet Bernthsen (MVB) as impurity;

[0216] (v) an elementals purity better than the European Pharmacopeia limits of less than 100 pg / g Aluminium (Al); less than 1 pg / g Cadmium (Cd); less than 100 pg / g Chromium (Cr); less than 300 pg / g Copper (Cu); less than 10 pg / g Tin (Sn); less than 200 pg / g Iron (Fe); less than 10 pg / g Manganese (Mn); less than 1 pg / g Mercury (Hg); less than 10 pg / g Molybdenum (Mo); less than 10 pg / g Nickel (Ni); less than 10 pg / g Lead (Pb); and less than 100 pg / g Zinc (Zn).

[0217] In particular embodiments, the MTC is a high purity MTC wherein high-purity is characterised by a purity of greater than 98% and one or more of the following:

[0218] (i) less than 1% Azure B as impurity;

[0219] (ii) less than 0.15% Azure A as impurity;

[0220] (iii) less than 0.15% Azure C as impurity;

[0221] (iv) less than 0.05% Methylene Violet Bernthsen (MVB) as impurity; or

[0222] (v) an elementals purity better than the European Pharmacopeia limits of less than 100 pg / g Aluminium (Al); less than 1 pg / g Cadmium (Cd); less than 100 pg / g Chromium (Cr); less than 300 pg / g Copper (Cu); less than 10 pg / g Tin (Sn); less than 200 pg / g Iron (Fe); lessthan 10 pg / g Manganese (Mn); less than 1 pg / g Mercury (Hg); less than 10 pg / g Molybdenum (Mo); less than 10 pg / g Nickel (Ni); less than 10 pg / g Lead (Pb); and less than 100 pg / g Zinc (Zn).

[0223] In particular embodiments, the MTC is a high purity MTC wherein high-purity is characterised by at least 98% purity and less than 1 % Azure B as impurity.

[0224] In particular embodiments, the MTC is a high purity MTC wherein high-purity is characterised by:

[0225] (i) at least 98% purity

[0226] (i) less than 1 % Azure B as impurity; and

[0227] (ii) an elementals purity better than the European Pharmacopeia limits of less than 100 pg / g Aluminium (Al); less than 1 pg / g Cadmium (Cd); less than 100 pg / g Chromium (Cr); less than 300 pg / g Copper (Cu); less than 10 pg / g Tin (Sn); less than 200 pg / g Iron (Fe); less than 10 pg / g Manganese (Mn); less than 1 pg / g Mercury (Hg); less than 10 pg / g Molybdenum (Mo); less than 10 pg / g Nickel (Ni); less than 10 pg / g Lead (Pb); and less than 100 pg / g Zinc (Zn).

[0228] In particular embodiments, the MTC is a high purity MTC wherein high-purity is characterised by at least 98% purity and an elementals purity better than the European Pharmacopeia limits of less than 100 pg / g Aluminium (Al); less than 1 pg / g Cadmium (Cd); less than 100 pg / g Chromium (Cr); less than 300 pg / g Copper (Cu); less than 10 pg / g Tin (Sn); less than 200 pg / g Iron (Fe); less than 10 pg / g Manganese (Mn); less than 1 pg / g Mercury (Hg); less than 10 pg / g Molybdenum (Mo); less than 10 pg / g Nickel (Ni); less than 10 pg / g Lead (Pb); and less than 100 pg / g Zinc (Zn).

[0229] Methods for the production of ‘high purity’ diaminophenothiazinium compounds, including MTC, are described, for example, in W02006 / 032879 and W02008 / 007074 (WisTa Laboratories Ltd) and in W02008 / 006979 (Provence Technologies).

[0230] A preferred MTC polymorph for use in the methods and compositions described herein is ‘form A’ described in WO2011 / 036561 which is a pentahydrate, at a “high purity” described above. That has a molecular weight of around 409.9. Based on a molecular weight of 284.1 for the MT+core, the weight factor for using this MT compound in the invention is 1.44. Other weight factors can be calculated for example MT compounds herein, and the corresponding dosage ranges can be calculated therefrom.

[0231] Other example MT compounds are described in W02007 / 110630. Their molecular weight (anhydrous) and weight factor is also shown:

[0232]

[0233] The dosages described herein with respect to MT thus apply mutatis mutandis for these MT containing compounds, as adjusted for their molecular weight, and for choice of hydrate if used. For example MTC.O.SZnCI? (also referred to as 'METHYLENE BLUE ZINC CHLORIDE DOUBLE SALT; Cl 52015) may be obtained commercially as a monohydrate by several suppliers, which would have a molecular weight higher by 18, and correspondingly altered weight factor. MTI is reportedly available as a hemihydrate.

[0234] Any of the MT compounds described herein, may be formulated with a reducing agent. In particular, MT+salts such as MTC may be formulated with a reducing agent such as ascorbate, and then lyophilized (as described in W002 / 055720). This may improve adsorption of the MT delivered by the compound.

[0235] In the various aspects of the invention described herein (as they relate to an MT-containing compound) the MT-containing compound may optionally be any of those compounds described above:

[0236] In one embodiment, it is compound 1.

[0237] In one embodiment, it is compound 2.

[0238] In one embodiment, it is compound 3.

[0239] In one embodiment, it is compound 4.

[0240] In one embodiment, it is compound 5.

[0241] In one embodiment, it is compound 6.

[0242] In one embodiment, it is compound 7.

[0243] In one embodiment, it is compound 8.

[0244] In one embodiment, it is compound 9.

[0245] In one embodiment, it is compound 10.

[0246] In one embodiment, it is compound 11.

[0247] In one embodiment, it is compound 12.

[0248] In one embodiment, it is compound 13.

[0249] Or the compound may be a hydrate, solvate, or mixed salt of any of these.

[0250] Treatment and prophylaxis

[0251] The term “treatment,” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition.

[0252] The term “therapeutically-effective amount,” as used herein, pertains to that amount of a compound of the invention, or a material, composition or dosage from comprising said compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio, when administered in accordance with a desired treatment regimen.The invention also embraces treatment as a prophylactic measure is also included.

[0253] Thus the invention also provides a method of prophylaxis of a neurodegenerative disorder in a subject, which method comprises orally administering to said patient a methylthioninium (MT) containing compound,

[0254] at a first dosage level for a first dosage period and then

[0255] at a second dosage level for a second dosage period

[0256] wherein the second dosage level is higher than the first dosage level. The comments on dosage periods and levels herein in respect of treatment generally apply mutatis mutandis to such methods of prophylaxis.

[0257] The term “prophylactically effective amount,” as used herein, pertains to that amount of a compound of the invention, or a material, composition or dosage from comprising said compound, which is effective for producing some desired prophylactic effect, commensurate with a reasonable benefit / risk ratio, when administered in accordance with a desired treatment regimen.

[0258] “Prophylaxis” in the context of the present specification should not be understood to circumscribe complete success i.e. complete protection or complete prevention. Rather prophylaxis in the present context refers to a measure which is administered in advance of detection of a symptomatic condition with the aim of preserving health by helping to delay, mitigate or avoid that particular condition.

[0259] Combination treatments and monotherapy

[0260] 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, for example, sequentially or simultaneously. These may be symptomatic or disease modifying treatments.

[0261] The particular combination would be at the discretion of the physician.

[0262] In combination treatments, the agents (i.e., an MT compound as described herein, plus one or more other agents) may be administered simultaneously or sequentially, and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1 , 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).

[0263] An example of a combination treatment of the invention would be an agent which is MT-containing compound at the specified dosage in combination with an agent which is an inhibitor of amyloid precursor protein to beta-amyloid (e.g., an inhibitor of amyloid precursor protein processing that leads to enhanced generation of beta-amyloid).

[0264] In other embodiments the treatment is a “monotherapy”, which is to say that the MT-containing compound is not used in combination (within the meaning discussed above) withanother active agent for treating the same neurodegenerative disorder of protein aggregation in the subject.

[0265] As explained below, in the present invention, when treating AD at least, it is preferred that the treatment does not include administration of either or both of: an acetylcholinesterase inhibitor or an N-methyl-D-aspartate receptor antagonist. The MT-compound based treatment of AD may optionally be a monotherapy.

[0266] Pharmaceutical dosage forms

[0267] Preferably the MT compound of the invention is administered in the form of a pharmaceutical composition. Preferably such a composition comprises a compound as described herein, and a pharmaceutically acceptable carrier or diluent.

[0268] In some embodiments, the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a compound as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0269] The term “pharmaceutically acceptable,” as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, 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.

[0270] In some embodiments, the composition is a pharmaceutical composition comprising at least one compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.

[0271] In some embodiments, the composition further comprises other active agents, for example, other therapeutic or prophylactic agents.

[0272] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0273] 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., obtained by, or obtainable by, a method as described herein; having a purity as described herein; etc.), and a pharmaceutically acceptable carrier, diluent, or excipient.The “MT compound”, although present in relatively low amount, is the active agent of the dosage unit, which is to say is intended to have the therapeutic or prophylactic effect in respect of a neurodegenerative disorder of protein aggregation. Rather, the other ingredients in the dosage unit will be therapeutically inactive e.g. carriers, diluents, or excipients. Thus, preferably, there will be no other active ingredient in the dosage unit, no other agent intended to have a therapeutic or prophylactic effect in respect of a disorder for which the dosage unit is intended to be used.

[0274] In some embodiments, the dosage unit is a tablet.

[0275] In some embodiments, the dosage unit is a capsule.

[0276] In some embodiments, said capsules are gelatine capsules.

[0277] In some embodiments, said capsules are HPMC (hydroxypropylmethylcellulose) capsules. Oral dosage forms

[0278] The MT compound of the present invention, or pharmaceutical composition comprising it, is preferably administered to a subject / patient orally.

[0279] Immediate release dosage units

[0280] The formulations and compositions (especially pharmaceutical compositions) may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.

[0281] An immediate release product allows the ingredient or active moiety to dissolve in the gastrointestinal tract, without causing any delay or prolongation of the dissolution or absorption of the drug. Requirements for dissolution testing of immediate release products are set out in the Guidance for Industry (ODER 1997) "Dissolution testing for immediate release solid oral dosage forms", (ODER 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 employed dissolution test methods as described in the USP and European Pharmacopeia (6th edition) are the basket method (USP 1) and the paddle method (USP 2). The described methods are simple, robust, well standardized, and used worldwide. They are flexible enough to allow dissolution testing for a variety of drug products. The following parameters influencing the dissolution behaviour may for example be relevant for selecting the appropriate in vitro dissolution test conditions for an immediate release solid oral product: apparatus, stirring speed, dissolution medium and temperature. Because of the biopharmaceutical properties of MTC and its expected desirable absorption characteristics in the upper gastrointestinal tract, it was preferable to produce rapidly dissolving tablets of MTC.

[0282] Compositions according to the invention can be dissolution tested in a USP-2 apparatus in 900ml of 0.1 N HCI, with paddles rotating at 50-75 rpm. Compositions according to the invention exhibit at least the acceptance criteria cited for Stage 1 (S1) testing in the USP 32 (The United States Pharmacopeia, edited by the United States Pharmacopeial Convention,Inc., 12601 Twinbrook Parkway, Rockville, MD 20852; Published by Rand McNally, Inc., 32nd Edition, 2008):

[0283] Acceptance Criteria: Each tablet achieved 85% dissolution of MTC within 30 minutes after insertion of the coated tablet into the 0.1 N HCI.

[0284] Thus in some embodiments, the MTC based formulations of the invention, when evaluated using this method, provide at least:

[0285] 75% dissolution of MTC within 45 minutes after insertion of the coated tablet into the 0.1N HCI; or

[0286] 85% dissolution of MTC within 30 minutes after insertion of the coated tablet into the 0.1N HCI;

[0287] 85% dissolution of MTC within 15 minutes after insertion of the coated tablet into the 0.1N HCI.

[0288] Another aspect of the present invention pertains to methods of making a low dosage MT compound pharmaceutical composition comprising admixing at least one MT compound, as defined herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.

[0289] The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

[0290] In some embodiments, the pharmaceutically acceptable carrier, diluent, or excipient is or comprises one or both of a glyceride (e.g., Gelucire 44 / 14 ®; lauroyl macrogol-32 glycerides PhEur, USP) and colloidal silicon dioxide (e.g., 2% Aerosil 200 ®; Colliodal Silicon Dioxide PhEur, USP).

[0291] Preferably the pharmaceutical compositions comprising a compound of the invention, in solid dosage form. The composition preferably further comprises at least one diluent suitable for dry compression. The pharmaceutical composition is characterised in that the compound exists in a substantially stable form.

[0292] The pharmaceutical composition will generally also include a lubricant. Examples of lubricants include magnesium stearate, calcium stearate, sodium stearyl fumarate, stearic acid, glycerylbehaptate, polyethylene glycol, ethylene oxide polymers (for example, those available under the registered trademark Carbowax from Union Carbide, Inc., Danbury, CT), sodium lauryl sulphate, magnesium lauryl stearate, mixtures of magnesium stearate with sodium lauryl sulphate, and hydrogenated vegetable oil. Preferred lubricants include calcium stearate, magnesium stearate and sodium stearyl fumarate. Most preferred as the lubricant is magnesium stearate. Lubricants generally comprise from about 0.5 to about 5.0% of thetotal (uncoated) tablet weight. The amount of lubricant employed is generally from about 1.0 to about 2.0%, preferably 0.5 to 2.0% w / w.

[0293] In addition to the diluent(s) and lubricant(s), other conventional excipients may also be present in the pharmaceutical compositions of the invention. Such additional excipients include disintegrants, binders, flavouring agents, colours and glidants. Some excipients can serve multiple functions, for example as both binder and tablet disintegrant.

[0294] A tablet disintegrant may be present in an amount necessary to achieve rapid dissolution. Disintegrants are excipients which oppose the physical forces of particle bonding in a tablet or capsule when the dosage form is placed in an aqueous environment. Examples of disintegrants include crosslinked polyvinylpyrrolidone (crospovidone), sodium starch glycolate, crosslinked sodium carboxymethyl cellulose (sodium croscarmellose), and pregelatinized starch. Generally the amount of disintegrant can be from 0 to about 25% w / w, more commonly from about 1% to about 15% w / w, and usually less than 10% or less than 5% w / w, of the composition.

[0295] Binders are excipients which contribute to particle adhesion in a solid formulation. Examples of binders include cellulose derivatives (carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethylcellulose, ethylcellulose, microcrystalline cellulose) and sugars such as lactose, sucrose, dextrose, glucose, maltodextrin, and mannitol, xylitol, polymethacrylates, polyvinylpyrrolidone, sorbitol, pregelatinized starch, alginic acids, and salts thereof such as sodium alginate, magnesium aluminum silicate, polyethylene glycol, carrageenan and the like. Generally, the amount of binder can vary widely, e.g. from 0% to 95% w / w of the composition. As noted above, excipients may serve multiple functions. For instance, the tabletting diluent may also serve as a binder.

[0296] Glidants are substances added to a powder to improve its flowability. Examples of glidants include magnesium stearate, colloidal silicon dioxide (such as the grades sold as Aerosil), starch and talc. Glidants may be present in the pharmaceutical composition at a level of from 0 to about 5% w / w. Again, however, it should be noted that excipients may serve multiple functions. The lubricant, for example magnesium stearate, may also function as a glidant.

[0297] Examples of colours that may be incorporated into the pharmaceutical compositions of the invention include titanium dioxide and / or dyes suitable for food such as those known as FD&C dyes and natural colouring agents. A colouring agent is unlikely to be used in the powder mixture that is compressed in accordance with the aspects of the invention discussed above, but may form part of a coating applied to the composition, as described below, in which case the colouring agent may be present in the film coat in an amount up to about 2.0% w / w.

[0298] The tablet is desirably coated with a conventional film coating which imparts toughness, ease of swallowing, and an elegant appearance to the final product. Many polymeric filmcoating materials are known in the art. A preferred film-coating material is hydroxypropylmethylcellulose (HPMC) or polyvinyl alcohol-part hydrolysed (PVA). HPMC and PVA may be obtained commercially, for example from Colorcon, in coating formulationscontaining excipients which serve as coating aids, under the registered trademark Opadry. Opadry formulations may also contain talc, polydextrose, triacetin, polyethyleneglycol, polysorbate 80, titanium dioxide, and one or more dyes or lakes. Other suitable film-forming polymers may also be used, including hydroxypropylcellulose, vinyl copolymers such as polyvinyl pyrollidone and polyvinyl acetate, and acrylate-methacrylate copolymers. Use of a film coating is beneficial for ease of handling and because a blue coloured uncoated core may stain the inside of the mouth during swallowing. Coating also improves light stability of the dosage form.

[0299] Coating of the tablets may conveniently be carried out using a conventional coating pan. In preferred embodiments of the process, the coating pan is pre-heated using heated inlet air until the exhaust temperature reaches 35°-55°C, more preferably 40-50°C. This may typically require application of heated inlet air at an inlet temperature of 45-75°C, preferably 50-65°C, for 10-15 minutes. The tablet cores containing the active ingredient (e.g. LMTM) are then added to the coating pan and the aqueous film coat applied. The spray rate is controlled such that the bed temperature is maintained at 38-48°C, more preferably 42-44°C, until the desired weight gain (coating weight) has been achieved.

[0300] Subjects, patients and patient groups

[0301] The subject / patient may be an animal, a mammal, a placental mammal, a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), a monotreme (e.g. platypus), an ape (e.g., gorilla, chimpanzee, orangutan, gibbon), ora human.

[0302] In preferred embodiments, the subject / patient is a human who has been diagnosed as having one of the cognitive or CNS disorders described herein, or (for prophylactic treatment) assessed as being susceptible to one of the neurodegenerative disorders of protein aggregation (e.g. cognitive or CNS disorder) described herein - for example based on familial or genetic or other data.

[0303] The patient may be an adult human, and the dosages described herein are premised on that basis (typical weight 50 to 70kg). If desired, corresponding dosages may be utilised for subjects outside of this range by using a subject weight factor whereby the subject weight is divided by 60 kg to provide the multiplicative factor for that individual subject.

[0304] The low dosage treatments of the present invention increase the feasibility of purely prophylactic treatments, since the reduced concentration of active ingredients inevitably reduces risk of any adverse side effects (and increases the safety profile) and hence increases the risk / benefit ratio for such prophylactic treatments.

[0305] Thus, for example, for diagnosis of AD, and assessment of severity, the initial selection of a patient may involve any one or more of: rigorous evaluation by experienced clinician; exclusion of non-AD diagnosis as far as possible by supplementary laboratory and other investigations; objective evaluation of level of cognitive function using neuropathologically validated battery.Diagnosis of AD and other disorders described herein can be performed by physicians by methods well known to those skilled in the art.

[0306] In some embodiments it is preferred that the subject or patient group, if they are being treated in respect of AD, is one who is not receiving treatment with any of: an acetylcholinesterase inhibitor or an N-methyl-D-aspartate receptor antagonist. Examples of acetylcholinesterase inhibitors include Donepezil (Aricept™), Rivastigmine (Exelon™) or Galantamine (Reminyl™). An examples of an NMDA receptor antagonist is Memantine (Ebixa™, Namenda™).

[0307] For example, the AD subject or patient group may be entirely naive to these other treatments, and have not 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 ceased that medication 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 at least 1 , 2, 3, 4, 5 or 6 months etc. prior to treatment with an MT compound according to the present invention.

[0308] Labels, instructions and kits of parts

[0309] The unit dosage compositions described herein (e.g. a low dose MT containing compound plus optionally other ingredients, or MT composition more generally for treatment in AD) may be provided in a labelled packet along with instructions for their use.

[0310] In one embodiment, the pack is a bottle, such as are well known in the pharmaceutical art. Atypical bottle may be made from pharmacopoeial grade HDPE (High-Density Polyethylene) with a childproof, HDPE pushlock closure and contain silica gel desiccant, which is present in sachets or canisters. The bottle itself may comprise a label and be packaged in a cardboard container with instructions for us and optionally a further copy of the label.

[0311] In one embodiment, the pack or packet is a blister pack (preferably one having aluminium cavity and aluminium foil) which is thus substantially moisture-impervious. In this case the pack may be packaged in a cardboard container with instructions for us and label on the container.

[0312] Said label or instructions may provide information regarding the neurodegenerative disorders of protein aggregation (e.g. cognitive or CNS disorder) for which the medication is intended.

[0313] Where the medication is indicated for AD, said label or instructions may provide information instructing the user that the compositions should not be used in conjunction with any of: an acetylcholinesterase inhibitor or an N-methyl-D-aspartate receptor antagonist.

[0314] Said label or instructions may provide information regarding the maximum permitted daily dosage of the compositions as described herein.

[0315] Said label or instructions may provide information regarding the suggested dosage regimen for the treatment, as described herein. For example, a suggested dosage frequency of everyother day, twice weekly, once weekly, etc. The disclosure of dosage frequencies above thus applies mutatis mutandis to this aspect.

[0316] Said label or instructions may provide information regarding the suggested duration of treatment, as described herein. The disclosure of treatment duration above thus applies mutatis mutandis to this aspect.

[0317] Reversing and / or Inhibiting the Aggregation of a Protein

[0318] One aspect of the invention is the use of an MT compound or composition as described herein, to regulate (e.g., to reverse and / or inhibit) the aggregation of a protein, for example, aggregation of a protein associated with a neurodegenerative disease and / or clinical dementia. The aggregation will be associated with a disease state as discussed below. Similarly, one aspect of the invention pertains to a method of regulating (e.g., reversing and / or inhibiting) the aggregation of a protein in the brain of a mammal, which aggregation is associated with a disease state as described herein, the treatment comprising the step of administering to said mammal in need of said treatment, a prophylactically or therapeutically effective amount of an MT compound or composition as described herein, that is an inhibitor of said aggregation.

[0319] Disease conditions treatable via the present invention are discussed in more detail below. Methods of Treatment

[0320] Another aspect of the present invention, as explained above, pertains to a method of treatment comprising administering to a patient in need of treatment a prophylactically or therapeutically effective amount of a compound as described herein, preferably in the form of a pharmaceutical composition.

[0321] Use in Methods of Therapy

[0322] Another aspect of the present invention pertains to a compound or composition as described herein, for use in a method of treatment (e.g., of a disease condition) of the human or animal body by therapy.

[0323] Use in the Manufacture of Medicaments

[0324] Another aspect of the present invention pertains to use of an MT compound or composition as described herein, in the manufacture of a medicament for use in treatment (e.g., of a disease condition).

[0325] In some embodiments, the medicament is a composition e.g. a low-dose unit dose composition as described herein.

[0326] Diseases of Protein AggregationThe compounds and compositions of the present invention are useful in the treatment or prophylaxis of diseases of protein aggregation.

[0327] Thus, in some embodiments, the disease condition is a disease of protein aggregation, and, for example, the treatment is with an amount of a compound or composition as described herein, sufficient to inhibit the aggregation of the protein associated with said disease condition.

[0328] The following Table lists various disease-associated aggregating proteins and the corresponding neurodegenerative disease of protein aggregation. The use of the compounds and compositions of the invention in respect of these proteins or diseases is encompassed by the present invention:

[0329] &

[0330] &

[0331]

[0332]

[0333]

[0334] As described in WO 02 / 055720, W02007 / 110630, and W02007 / 110627, diaminophenothiazines have utility in the inhibition of such protein aggregating diseases. Thus it will be appreciated that, except where context requires otherwise, description of embodiments with respect to tau protein ortau-like proteins (e.g., MAP2; see below), should be taken as applying equally to the other proteins discussed herein (e.g., [3-amyloid, synuclein, prion, etc.) or other proteins which may initiate or undergo a similar pathological aggregation by virtue of conformational change in a domain critical for propagation of the aggregation, or which imparts proteolytic stability to the aggregate thus formed (see, e.g., the article by Wischik et al. in “Neurobiology of Alzheimer’s Disease”, 2nd Edition, 2000, Eds. 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 “aggregating disease proteins.”

[0335] Likewise, where mention is made herein of “tau-tau aggregation”, or the like, this may also be taken to be applicable to other “aggregating-protein aggregation”, such as p-amyloid aggregation, prion aggregation, synuclein aggregation, etc. The same applies for “tau proteolytic degradation” etc.

[0336] Preferred Aggregating Disease Target Proteins

[0337] Preferred embodiments of the invention are based on tau protein. The term “tau protein,” as used herein, refers generally to any protein of the tau protein family. Tau proteins are characterised as being one among a larger number of protein families which 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 the common features of having a characteristic N-terminal segment, sequences of approximately 50 amino acids inserted in the N-terminal segment, which are developmentally regulated in the brain, acharacteristic tandem repeat region consisting of 3 or 4 tandem repeats of 31 -32 amino acids, and a C-terminal tail.

[0338] MAP2 is the predominant microtubule-associated protein in the somatodendritic compartment (see, e.g., Matus, A., in “Microtubules” [Hyams and Lloyd, Eds.] pp. 155-166, John Wiley and Sons, New York, USA). MAP2 isoforms are almost identical to tau protein in the tandem repeat region, but differ substantially both in 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 appreciated that any discussion herein in relation to tau protein or tau-tau aggregation should be taken as relating also to tau-MAP2 aggregation, MAP2-MAP2 aggregation, and so on.

[0339] In some embodiments, the protein is tau protein.

[0340] In some embodiments, the protein is a synuclein, e.g., a- or |3-synuclein.

[0341] In some embodiments, the protein is TDP-43.

[0342] TAR DNA-Binding Protein 43 (TDP-43) is a 414 amino acid protein encoded by TARDBP on chromosome 1p36.2. The protein is highly conserved, widely expressed, and predominantly localised to the nucleus but can shuttle between the nucleus and cytoplasm (Mackenzie etal 2010). It is involved in transcription and splicing regulation and may have roles in other processes, such as: microRNA processing, apoptosis, cell division, stabilisation of messenger RNA, regulation of neuronal plasticity and maintenance of dendritic integrity. Furthermore, since 2006 a substantial body of evidence has accumulated in support of the TDP-43 toxic gain of function hypothesis in amyotrophic lateral sclerosis (ALS). TDP-43 is an inherently aggregation-prone protein and aggregates formed in vitro are ultrastructurally similar to the TDP-43 deposits seen in degenerating neurones in 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 C-terminal fragments of TDP-43 are more likely than full-length TDP-43 to form insoluble cytoplasmic aggregates that become ubiquitinated, and toxic to cells (Arai et al 2010; Igaz et al 2009; Nonaka et al 2009; Zhang et al 2009). Though Nonaka et al (2009) suggested that these cytoplasmic aggregates bind the endogenous full-length protein depleting it from the nucleus, Zhang et al (2009) found retention of normal nuclear expression, suggesting a purely toxic effect for the aggregates. Yang et al (2010) have described the capture of full-length TDP-43 within aggregates of C- and N-terminal fragments of TDP-43 in NSC34 motor neurons in culture. Neurite outgrowth, impaired as a result of the presence of such truncated fragments, could 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 for a role of TDP-43 aggregation in ALS pathogenesis.

[0343] Mutant TDP-43 expression in cell cultures has repeatedly been reported to result in increased generation 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; Aral et al 2010; Barmarda et al 2010; Kabashi et al 2010).

[0344] Where the protein is tau protein, in some embodiments of the present invention, there is provided a method of inhibiting production of protein aggregates (e.g. in the form of paired helical filaments (PHFs), optionally in neurofibrillary tangles (NFTs) in the brain of a mammal, the treatment being as described above.

[0345] Preferred Indications - Diseases of Protein Aggregation

[0346] In one embodiment the present invention is used for the treatment of Alzheimer’s disease (AD) - for example mild, moderate or severe AD.

[0347] Notably it is not only Alzheimer’s disease (AD) in which tau protein (and aberrant function or processing thereof) may play a role. The pathogenesis of neurodegenerative disorders such as Pick’s disease and progressive supranuclear palsy (PSP) appears to correlate with an accumulation of pathological truncated tau aggregates in the dentate gyrus and stellate pyramidal cells of the neocortex, respectively. Other dementias include fronto-temporal dementia (FTD); FTD with parkinsonism linked to chromosome 17 (FTDP-17); disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC); pallido-ponto-nigral degeneration (PPND); Guam-ALS syndrome; pallido-nigro-luysian degeneration (PNLD); cortico-basal degeneration (CBD) and others (see, e.g., the article by Wischik et al. in “Neurobiology of Alzheimer’s Disease”, 2nd Edition, 2000, Eds. Dawbarn, D. and Allen, S.J., The Molecular and Cellular Neurobiology Series, Bios Scientific Publishers, Oxford; especially Table 5.1). All of these diseases, which are characterized primarily or partially by abnormal tau aggregation, are referred to herein as “tauopathies”.

[0348] Thus, in some embodiments, the disease condition is a tauopathy.

[0349] In some embodiments, the disease condition is a neurodegenerative tauopathy.

[0350] In some embodiments, the disease condition is selected from Alzheimer's disease (AD), Pick’s disease, progressive supranuclear palsy (PSP), fronto temporal dementia (FTD), FTD with parkinsonism linked to chromosome 17 (FTDP 17), frontotemporal lobar degeneration (FTLD) syndromes; disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC), pallido-ponto-nigral degeneration (PPND), Guam-ALS syndrome, pallido nigro luysian degeneration (PNLD), cortico-basal degeneration (CBD), dementia with argyrophilic grains (AgD), dementia pugilistica (DP) or chronic traumatic encephalopathy (CTE), Down’s syndrome (DS), dementia with Lewy bodies (DLB), subacute sclerosing panencephalitis (SSPE), MCI, Niemann-Pick disease, type C (NPC), Sanfilippo syndrome type B (mucopolysaccharidosis III B), or myotonic dystrophies (DM), DM1 or DM2, or chronic traumatic encephalopathy (CTE).

[0351] In some embodiments, the disease condition is a lysosomal storage disorder with tau pathology. NPC is caused by mutations in the gene NPC1, which affects cholesterol metabolism (Love et al 1995) and Sanfilippo syndrome type B is caused by a mutation in the gene NAGLU, in which there is lysosomal accumulation of heparin sulphate (Ohmi et al. 2009). In these lysosomal storage disorders, tau pathology is observed and its treatmentmay decrease the progression of the disease. Other lysosomal storage disorders may also be characterised by accumulation of tau.

[0352] Use of phenothiazine diaminium salts in the treatment of Parkinson’s disease and MCI is described in more detail in PCT / GB2007 / 001105 and PCT / GB2008 / 002066.

[0353] In some embodiments, the disease condition is Parkinson’s disease, MCI, or Alzheimer’s disease.

[0354] In some embodiments, the disease condition is MCI or Alzheimer’s disease.

[0355] In some embodiments, the disease condition is MCI.

[0356] In a further aspect of the present invention, a method of treating MCI in a subject is provided, which method comprises orally administering to said subject a methylthioninium (MT) containing compound, wherein said administration provides a daily dosage of about 21 mg to about 29 mg MT. For example, a daily dosage of about 21 , 22, 23, 24, 25, 26, 27, 28, or 29 mg MT. The MT compound in this aspect is preferably an MT+ salt, most preferably methylthioninium chloride (MTC). In an embodiment, the method comprises administration of MTC at a total daily dosage of about 21 , 22, 23, 24, 25, 26, 27, 28, or 29 mg.

[0357] In another aspect of the present invention, a method of treating MCI in a subject is provided, which method comprises orally administering to said subject a methylthioninium (MT) containing compound, wherein said administration is at a dosage frequency of less than once daily and wherein said administration provides an amount of MT to the subject that corresponds to an average of 21 to 29 mg MT per day. In an embodiment, the method comprises administration of a total daily dosage of about 21 , 22, 23, 24, 25, 26, 27, 28, or 29 mg of MT. The MT compound in this aspect is preferably an MT+ salt, most preferably methylthioninium chloride (MTC). In an embodiment, the method comprises administration of MTC at a total daily dosage between 21 and 29 mg. In an embodiment, the method comprises administration of MTC at a total daily dosage of about 21 , 22, 23, 24, 25, 26, 27, 28, or 29 mg.

[0358] Optionally, treating MCI according to methods of the invention comprises inhibiting decline, preventing an expected decline, or improving the condition. For example, in some embodiments, treatment of MCI may comprise improving cognitive ability or function in a subject.

[0359] In some embodiments, the disease condition is Huntington’s disease or other polyglutamine disorder such as spinal bulbar muscular atrophy (or Kennedy disease), and dentatorubropallidoluysian atrophy and various spinocerebellar ataxias.

[0360] In some embodiments, the disease condition is an FTLD syndrome (which may for example be a tauopathy or TDP-43 proteinopathy, see below).

[0361] In some embodiments, the disease condition is PSP or ALS.

[0362] TDP-43 proteinopathies include amyotrophic lateral sclerosis (ALS; ALS-TDP) and frontotemporal lobar degeneration (FTLD-TDP).The role of TDP-43 in neurodegeneration in ALS and other neurodegenerative disorders has been reviewed in several recent publications (Chen-Plotkin eta / 2010; Gendron eta / 2010; Geser etal 2010; Mackenzie et al 2010).

[0363] ALS is a neurodegenerative disease, characterised by progressive paralysis and muscle wasting, consequent on the degeneration of both upper and lower motor neurones in the primary motor cortex, brainstem and spinal cord. It is sometimes referred to as motor neuron disease (MND) but there are diseases other than ALS which affect either upper or lower motor neurons. A definite diagnosis requires both upper and lower motor neurone signs in the bulbar, arm and leg musculature with clear evidence of clinical progression that cannot be explained by any other disease process (Wijesekera and Leigh 2009).

[0364] Although the majority of cases are ALS-TDP, there are other cases where the pathological protein differs from TDP-43. Misfolded SOD1 is the pathological protein in 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 etal 2009; Blair etal 2010). FUS, like TDP-43, appears to be important in nuclear-cytoplasmic shuttling although the ways in which impaired nuclear import of FUS remains unclear. A new molecular classification of ALS, adapted from Mackenzie etal (2010), reflects the distinct underlying pathological mechanisms in the different subtypes (see Table below).

[0365] New Molecular Classification of ALS (modified from Mackenzie et al 2010). In the majority of cases, TDP-43 is the pathological ubiquitinated protein found in ALS.

[0366]

[0367] Amyotrophic lateral sclerosis has been recognised as a nosological entity for almost a century and a half and it is recognised in ICD-10 is classified as a subtype of MND in ICD 10 (G12.2). Reliable clinical diagnostic criteria are available for ALS, which differ little from Charcot’s original description, and neuropathological criteria, reflecting the underlying molecular pathology, have also been agreed.

[0368] While ALS is classified pathologically into three subgroups, ALS-TDP, ALS-SOD1 and ALS-FUS, both latter conditions are rare. The largest study to date showed all sporadic ALScases to have TDP-43 pathology (Mackenzie et al 2007). Only around 5% of ALS is familial (Byrne et al 2010) and mutations in SOD1, the commonest mutations found in FALS, account for between 12-23% of cases (Andersen et al 2006). SOD1 may also be implicated in 2-7% of SALS. Mutations in FUS appear to be far less common, accounting for only around 3-4% of FALS (Blair et al 2010). So it can be reliably predicted that a clinical case of SALS will have TDP-43 based pathology. Similarly this can be reliably predicted in FALS due to mutations in TDP-43, which account for around 4% of cases (Mackenzie et al 2010). ALS with mutations in: VCP, accounting for 1-2% of FALS (Johnson et al 2010), ANG (Seilhean et al 2009), and CHMP2B (Cox et al 2010) have also been reported to be associated with TDP-43 positive pathology. Although SOD1, FUS and ATXN2 mutations have not been found to be associated with TDP-43 positive aggregates, it has however been reported that TDP-43 is implicated in the pathological processes putatively arising from these mutations (Higashi et al 2010; Ling et al 2010; Elden et al 2010).

[0369] It is therefore established that TDP-43 has an important, and potentially central role, in the pathogenesis of the vast majority of SALS cases and may be implicated in the pathogenesis of a significant proportion of FALS. ALS is now widely considered to be a TDP-43 proteinopathy (Neumann et al 2009) and numerous in vitro, and in vivo studies provide support to the hypothesis that toxic gain of function, due to TDP-43 aggregation is responsible for at least some of the neurotoxicity in the disease.

[0370] FTLD syndromes are insidious onset, inexorably progressive, neurodegenerative conditions, with peak onset in late middle age. There is often a positive family history of similar disorders in a first degree relative.

[0371] Behavioural variant FTD is characterised by early prominent change in social and interpersonal function, often accompanied by repetitive behaviours and changes in eating pattern. In semantic dementia there are prominent word finding problems, despite otherwise fluent speech, with degraded object knowledge and impaired single word comprehension on cognitive assessment. Progressive non-fluent aphasia presents with a combination of motor speech problems and grammatical deficits. The core clinical diagnostic features for these three FTLD syndromes are shown in the Table below and the full criteria in Neary etal (1998).

[0372] Clinical Profile and Core Diagnostic Features of FTLD Syndromes

[0373]

[0374]

[0375] The discovery that TDP-43-positive inclusions characterize ALS and FTLD-TDP (Neumann et al 2006) was quickly followed by the identification of missense mutations in the TARDBP gene in both familial and sporadic cases of ALS (Gitcho et al 2008; Sreedharan et al., 2008). So far, 38 different TARDBP mutations have been reported in 79 genealogically unrelated families worldwide (Mackenzie et al 2010). TARDBP mutations account for approximately 4% of all familial and around 1.5% of sporadic ALS cases.

[0376] As of December 2010, mutations in thirteen genes which are associated with familial and sporadic ALS have been identified. Linkage of ALS to five other chromosome loci has been demonstrated but thus far specific mutations have not been identified.

[0377] TDP-43 proteinopathies

[0378] MT has a mode of action which targets and can reduce TDP-43 protein aggregation in cells, which is a pathological feature of the vast majority of both familial and sporadic ALS and is also characteristic of FTLD-P.

[0379] In addition laboratory data shows that methylthioninium inhibits the formation of TDP-43 aggregates in SH-SY5Y cells. Following treatment with 0.05 pM MT, the number of TDP-43aggregates was reduced by 50%. These findings were confirmed by immunoblot analysis (Yamashita et al 2009).

[0380] The compounds and compositions of the invention may therefore be useful for the treatment of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).

[0381] Huntington’s disease and polyglutamine disorders

[0382] MT can reduce polyglutamine protein aggregation in cells, which is a pathological feature of Huntington’s disease. Huntington’s disease is caused by expansion of a translated CAG repeat located in the N-terminus of huntingtin. Wild-type chromosomes contain 6-34 repeats whereas, in Huntington’s disease, chromosomes contain 36-121 repeats. The age of onset of disease correlates inversely with the length of the CAG tracts that code for polyglutamine repeats within the protein.

[0383] Laboratory data shows that methylthioninium inhibits the formation of aggregates of a huntingtin derivative containing a polyglutamine stretch of 102 residues in zebrafish (van Bebber et al. 2010). MT, when tested at 0, 10 and 100 pM, prevented the formation of such aggregates in zebrafish in a dose dependent manner.

[0384] The compounds and compositions of the invention may therefore be useful for the treatment of Huntington’s disease and other polyglutamine disorders such as spinal bulbar muscular atrophy (or Kennedy disease), and dentatorubropallidoluysian atrophy and various spinocerebellar ataxias (Orr & Zoghbi, 2007).

[0385] Mitochondrial Diseases and Lafora Disease

[0386] The organ most frequently affected in mitochondrial disorders, particularly respiratory chain diseases (RCDs), in addition to the skeletal muscle, is the central nervous system (CNS). CNS manifestations of RCDs comprise stroke-like episodes, epilepsy, migraine, ataxia, spasticity, movement disorders, psychiatric disorders, cognitive decline, or even dementia (mitochondrial dementia). So far mitochondrial dementia has been reported in MELAS, MERRF, LHON, CPEO, KSS, MNGIE, NARP, Leigh syndrome, and Alpers-Huttenlocher disease (Finsterer, 2009). There are four complexes in the mitochondrial respiration chain, involving a series of electron transfers. Abnormal function of any of these complexes can result in mitochondrial diseases secondary to an abnormal electron transport chain and subsequent abnormal mitochondrial respiration. Complex III of the mitochondrial respiration chain acts to transfer electrons to cytochrome c.

[0387] Compounds and compositions of the invention may also be used to treat mitochondrial diseases which are associated with a deficient and / or impaired complex III function of the respiration chain. The compounds have the ability to act as effective electron carrier and / or transfer, as the thioninium moiety has a low redox potential converting between the oxidised and reduced form. In the event of an impaired and / or deficient function of Complex III leading to mitochondrial diseases, compounds of the invention are also able to perform the electron transportation and transfer role of complex III because of the ability of the thioninium moiety to shuttle between the oxidised and reduced form, thus acting as an electron carrier in place of sub-optimally functioning complex III, transferring electrons to cytochrome c.Compounds and compositions of the invention also have the ability to generate an active thioninium moiety that has the ability to divert misfolded protein / amino acid monomers / oligomers away from the Hsp70 ADP-associated protein accumulation and / or refolding pathways, and instead re-channel these abnormal folded protein monomers / oligomers to the pathway that leads directly to the Hsp70 ATP-dependent ubiquitin-proteasome system (UPS), a pathway which removes these misfolded proteins / amino acid monomers / oligomers via the direct route (Jinwal et al. 2009).

[0388] Lafora disease (LD) is an autosomal recessive teenage-onset fatal epilepsy associated with a gradual accumulation of poorly branched and insoluble glycogen, termed polyglucosan, in many tissues. In the brain, polyglucosan bodies, or Lafora bodies, form in neurons. Inhibition of Hsp70 ATPase by MT (Jinwal et al. 2009) may upregulate the removal of misfolded proteins. Lafora disease is primarily due to a lysosomal ubiquitin-proteasomal system (UPS) defect because of a mutation in either the Laforin or Malin genes, both located on Chromosome 6, which result in inclusions that may accelerate the aggregation of misfolded tau protein. Secondary mitochondrial damage from the impaired UPS may further result in a suppressed mitochondrial activity and impaired electron transport chain leading to further lipofuscin and initiating the seizures that are characteristic of Lafora disease.

[0389] The MT moiety may disaggregate existing tau aggregates, reduce more tau accumulating and enhance lysosomal efficiency by inhibiting Hsp70 ATPase. MT may lead to a reduction in tau tangles by enhancing the ubiquitin proteasomal system removal of tau monomers / oligomers, through its inhibitory action on Hsp70 ATPase.

[0390] Thus compounds and compositions of the present invention may have utility in the treatment of Lafora disease.

[0391] Mixtures of oxidised and reduced MT compounds

[0392] MT compounds for use in the present invention may include mixtures of the oxidised and reduced form.

[0393] In particular, the LMT-containing compounds may include oxidised (MT+) compounds as ‘impurities’ during synthesis, and may also oxidize (e.g., autoxidize) after synthesis to give the corresponding oxidized forms. Thus, it is likely, if not inevitable, that compositions comprising the compounds of the present invention will contain, as an impurity, at least some of the corresponding oxidized compound. For example an “LMT” salt may include 10 to 15% of MT+salt.

[0394] When using mixed MT compounds the MT dose can be readily calculated using the molecular weight factors of the compounds present.

[0395] Salts and solvates

[0396] Although the MT containing compounds described herein are themselves salts, they may also be provided in the form of a mixed salt (i.e., the compound of the invention in combination with another salt). Such mixed salts are intended to be encompassed by theterm “and pharmaceutically acceptable salts thereof”. Unless otherwise specified, a reference to a particular compound also includes salts thereof.

[0397] The compounds of the invention may also be provided in the form of a solvate or hydrate. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g., compound, salt of compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, a penta-hydrate etc. Unless otherwise specified, any reference to a compound also includes solvate and any hydrate forms thereof.

[0398] Naturally, solvates or hydrates of salts of the compounds are also encompassed by the present invention.

[0399] A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0400] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0401] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0402] Ranges are often expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

[0403] Any sub-titles herein are included for convenience only and are not to be construed as limiting the disclosure in anyway.

[0404] The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

[0405] The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by crossreference.Figures

[0406] Figure 1 : Prespecified efficacy results for the co-primary clinical endpoints for all participants at 12 months: (A) ADAS-cogn (B) ADCS-ADL23and (C) whole brain volume (WBV). Error bars denote SEM.

[0407] Figure 2: Twenty-four month change from baseline in (A) ADAS-cog , (B) ADCS-ADL23and (C) whole brain volume (WBV) in MCI-AD sub-population, p-values indicate statistical significance of differences between arms as randomised. Asterisks indicate statistically significant improvement over baseline; *P<0.05. Error bars denote SEM. (D) change from baseline in ADAS-cogn over 24 months for treatment of AD population. Closed square is HMTM 16mg / day; Open circle is MTC 8mg / wk for 12 months then HMTM 16mg / day. (E) change in whole brain volume (WBV; cm3) over 24 months for treatment of AD population. Closed square is HMTM 16mg / day; Open circle is MTC 8mg / wk for 12 months then HMTM 16mg / day.

[0408] Figure 3: Change in plasma NfL concentration over 12 months by randomized treatment group and categorized by diagnosis: (A) All Participants (MCI-AD & AD) (B) MCI-AD only and (C) Mild / Moderate AD only

[0409] Figure 4: (A) Change from baseline in ADAS-cogn and WBV relative to matched ADNI population over 24 months (closed squares, HMTM 16mg / day; open circles, matched ADNI subjects). (B) Change from baseline in ADAS-cogn, ADCS-ADL23and WBV relative to meta-analytic controls over 24 Months (closed squares, HMTM 16mg / day; open circles, meta-analytic controls). Error bars denote SEM.

[0410] Figure 5: LUCIDITY™ study design. For the first 12 months a ‘control’ treatment consisted of 4mg MTC tablets administered on a varying schedule with an average 2 tablets per week at least 2 days apart, to maintain the blind. MTC tablets were always given with the evening dose. MTC at low dose and frequency was chosen for control dosage, rather than LMTM, based on Phase 2 data showing that the minimal effective dose of MTC was 138 mg / day, with minimal efficacy at 69 mg / day. During the 12 month open-label extension all subjects received HMTM 16 mg / day.

[0411] Figure 6: Dose normalized parent HMT observed concentrations versus time after dose in the first twenty-four hours in single dose arms (A) and versus nominal time after dose in the first twenty-four hours in multiple dose arms (B). In the panels, the vertical bars represent mean ± SEM.

[0412] Figure 7: Parent HMT non-BLQ concentration versus nominal time after dose in Study TRx-237-039. Data presented as Median and 90% Cl. The MTC 8mg / day, HMTM 8 mg / day, HMTM 16 mg / day are respectively treatment arms of the control group receiving MTC tablet 4 mg twice weekly, 4 mg BID, and 8 mg BID up to Visit 7 (12 months). Patients in all treatment arms received 8 mg BID during the open label phase up to Visit 10 (24 months). Abbreviation: BLQ=below limit of quantitation.Figure 8: Visualization of parabolic function on clearance. Typical value of the time-varying parabolic clearance. Vertical dashed reference lines at time 0 and Visit 3 (week 4; 1 month), Visit 7 (week 52; 12 months) and Visit 10 (week 104; 24 months)

[0413] Figure 9: Overview of the simulation strategy. The simulation was based on N=1000 virtual patients randomly drawn from Study 39.

[0414] Figure 10: Line plots of median Cmax vs. time (months) for each simulated scenario. The dashed line is set at 0.5 ng / ml.

[0415] Figure 11 : Box plots of Cmax vs. time (months) for each simulated scenario. For Box-plots, the gray box the interquartile interval, the thick black line within the box corresponds to the median. The whiskers represent the 2.5% and 97.5% percentile (95% distribution interval). The dashed line is set at 0.5 ng / ml.

[0416] Figure 12: Line plots of the proportion of patients with Cmax > 0.74 ng / ml vs. time (months) for each simulated scenario. The dashed line represents the 95% limit.

[0417] Figure 13: Box plots of Cmax vs. Time (months) for each simulated scenario. The dashed is set at 0.693 ng / ml.

[0418] Figure 14: Line plots of the proportion of patients with Cavg > 0.693 ng / mL vs. time (months) for each simulated scenario. The dashed line represents the 95% limit.

[0419] Figure 15: Plots of median Cmax at 12 months and 24 months vs. second daily dose (mg / day). Note: exposures at 12 m where dose switch occurs (“Dose Switch at 12m”) are prior to the dose increase, leading to an opposite trend in the bottom two panels. The dashed line is 0.74 ng / ml.

[0420] Figure 16: Plots of median Cmax at 15 months and 24 months vs. second daily dose (mg / day). The dashed line is 0.74 ng / ml.

[0421] Figure 17: Median MT Cmax (ng / ml) at 12 months and 24 months achieved by three different initial (0-12 month) dosage regimens which are (i) MTC 8mg / wk (shown as initial 1.1 mg / day); (ii) HMTM 8mg / day; (iii) HMTM 16mg / day. In each case the subsequent regime (12-24 months) was HMTM 16mg / day. The upper line is the Cmax level (0.74 ng / ml) postulated to be required for disease-modifying effect (as measured by plasma NfL levels) in AD. The lower line is the Cmax level (0.38 ng / ml) postulated to be required for diseasemodifying effect (as measured by plasma NfL levels) in MCI.

[0422] Figure 18: Relationship between Cmax at 12 months (resulting from 3 different dosages: MTC 8mg / wk, HMTM 8mg / day, and HMTM 16mg / day) and Cmax at 24 months following subsequent dosage of HMTM 16mg / day between 12-24 months. The higher the Cmax at 12m, the greater the reduction in Cmax at 24m relative to Cmax for 16mg / day at 12m. The reduction can be fitted as follows: Cmax reduction = -0.30 + 0.57exp(-Cmax12*1 / 0.125) where: Lower limit (c) = -0.300; Upper limit (d) = 0.570; Decay rate (e) = 0.125.Examples

[0423] Example 1 - provision of MT-containinq compounds

[0424] Methods for the chemical synthesis of the MT-containing compounds described herein are known in the art. For example:

[0425] Synthesis of compounds 1 to 7 can be performed according to the methods described in WO2012 / 107706, or methods analogous to those.

[0426] Synthesis of compound 8 can be performed according to the methods described in W02007 / 110627, or a method analogous to those.

[0427] Synthesis of compound 9 (MTC) is well known in the art. Example syntheses of highly pure MTC are provided in W02006 / 032879 and W02008 / 007074.

[0428] Synthesis of compounds 10 to 13 can be performed according to the methods described in W02007 / 110630, or methods analogous to those.

[0429] Example 2 - formulation of MT-containing compounds

[0430] Methods for the chemical synthesis of the MT-containing compounds described herein are known in the art. Example methods using dry compression, for example, are provided in WO2012 / 072977.

[0431] Example 3 - structure of phase 3 clinical trial in MCI or AD

[0432] METHODS

[0433] Study design and participants

[0434] A description of the rationale and design of Study TRx -237-039 (LUCIDITY, TRx-237-039; NCT03446001; EudraCT: 2017-003558-17) along with amendments, their timing and justification have been published.20In its final form in May 2019, LUCIDITY was a two-phase 24-month study, with an initial 12-month randomised double-blind phase comparing HMTM as monotherapy at a dose 16 mg / day with MTC 4 mg twice weekly as control to ensure blinding. This was followed by a further 12-month modified delayed-start open label phase in which all participants received HMTM 16mg / day. The study also included a small HMTM 8 mg / day treatment arm to bridge to earlier studies (Figure 5.13,14,16

[0435] The study was conducted at 82 study sites located in Canada, European Union, United Kingdom and United States. Participants had to be less than 90 years of age and have a clinical diagnosis of probable AD or MCI-AD.18,19All were required to have been communitydwelling with a mini mental state examination (MMSE) score of 16 to 27, and a CDR stage of 0'5 to 2 at screening (with at least one non-zero functional domain score for CDR 0.5).

[0436] Concomitant use of acetylcholinesterase inhibitors (AChEls) or memantine was not permitted.

[0437] Inclusion and EthicsAll patients provided written informed consent prior to enrolling in the study; legal representatives provided consent on behalf of patients with reduced decision-making capacity. Informants for the participants also provided consent for involvement. The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonisation Guidelines for Good Clinical Practice, and approval of the study protocol and all related documents was obtained from the appropriate Independent Ethics Committees and Institutional Review Boards for all study sites. An independent Data and Safety Monitoring Board (DSMB) was established for oversight of accruing safety information.

[0438] Determination of sample size

[0439] The co-primary outcomes were change over 12 months on the 11 -item Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cogn) and on the 23-item Alzheimer's Disease Cooperative Study Activities of Daily Living Scale (ADCS-ADL23). Study size was determined based on the ADCS-ADL23which, required the larger sample size given expected decline, standard deviation (SD), and estimated treatment effect. Approximately 225 per participants treatment arm were required to achieve 90% power with two-sided alpha of 0.05 assuming a 25% withdrawal rate to detect a difference of at least 3.4 ADCS-ADL23units between HMTM 16 mg / day and control at the interim 12 month timepoint based on the exposure-dependent treatment effect of 5.0 ± 1.6 (mean ± standard error) units and decline of 7.7 units (SD 8.5) observed in the completed HMTM studies in AD,16in the control arm. For ADAS-cogn, 200 participants per treatment arm provided >90% power to detect a treatment difference of at least 2.6 units assuming a decline of 6.5 units over 52 weeks (SD 5.9) in the control arm and based conservatively on the exposure-dependent treatment effect of 5.0 ± 1.6 units observed previously.16

[0440] Assuming 160 to 170 participants entering the delayed-start treatment phase and a further 10% dropout, there was approximately 80% power to detect a noninferiority margin of -2 ADAS-Cogn units between early and late starters of the HMTM 16 mg / day dose.

[0441] Randomisation and blinding

[0442] Eligible participants were randomised at baseline in a 4:1 :4 ratio to receive HMTM 16mg / day (administered as two 4 mg tablets twice daily), 8 mg / day (administered as one 4 mg tablet and one blank tablet containing only excipients twice daily), or excipient-only tablets given twice daily among which were included 4 mg MTC tablets given every 2-4 days on a varying schedule. All subjects and study personnel were blinded to each subject’s randomization, except for the members of the DSMB who monitored safety throughout the study.

[0443] Procedures

[0444] Five post-baseline visits were scheduled during the double-blind treatment phase (Supplementary file II Table 1). Timed morning blood samples were collected pre-dose and at 1, 2, and 4 hours post dose at baseline and months 1 and 12 for pharmacokinetic and blood biomarker analyses. A single blood sample for apolipoprotein E (APOE) genotype was obtained from consenting participants prior to month 12. Eligibility and safety assessments were performed by an independent qualified medical assessor not involved in efficacy assessments. Three post-baseline visits were scheduled during the open-label treatment phase with timed morning blood samples collected pre-dose and at 1 , 2, and 4 hours post dose at month 24 for pharmacokinetic analyses.Outcomes

[0445] The co-primary interim endpoints were comparison of change over 52 weeks in ADAS-Cogn (derived from the administered 13-item ADAS-cog version) and ADCS-ADL23.

[0446] Secondary endpoints at 12 months included change in whole brain volume (WBV) and temporoparietal lobe volume quantified using the Boundary Shift Integral (BSI)21and change in Standardized Uptake Value Ratio based on temporal lobe18F-fluorodeoxyglucose positron emission tomography.

[0447] Subgroup analyses according to verified clinical diagnosis at baseline (probable AD or MCI-AD) were prespecified in the protocol and defined in the final statistical analysis plan, as were comparisons of change in plasma concentration of NfL and tau phosphorylated at threonine 181 (ptau-181) over 12 months. Analyses of change over 104 weeks included ADAS-cogn, ADAS-cog and ADCS-ADL23scales and change in WBV according to initial randomisation.

[0448] Comparisons with other patient groups

[0449] Comparisons of LUCIDITY study arms as randomised with external controls over 24 months were prespecified in the final protocol as outcomes and finalised prior to the 24-month database lock using closely matched natural history data available from the Alzheimer’s Disease Neuroimaging Program (ADNI) and comparisons with meta-analytic controls from placebo arms of clinical trials in comparable populations. For comparisons with ADNI data, propensity score matching was used to identify participants in both populations who were comparable with respect to age, sex, smoking history, education, APOE genotype, baseline MMSE score and non-treatment with cholinesterase inhibitors and / or memantine.

[0450] Results / Discussion

[0451] Between 2017, and 2023, we recruited 598 patients; the last patient visit was on 4thApril 2023. At least one dose of HMTM was taken by 537 participants either in the double-blind or open-label phase or both. The Intention-to-Treat (ITT) and Safety Populations included all 598 participants (266 in the MTC 4 mg twice weekly group, 80 in the HMTM 8 mg / day group and 252 in the HMTM 16 mg / day group) of whom 470 (79%) completed the study to 52 weeks.

[0452] Patient populations

[0453] The baseline demographic and clinical characteristics of the population are presented in Table 1. The 598 patients in the ITT population had a mean age of 72 years. Patients were predominantly Caucasian (88-1%); 60-7% were female. There was a diagnosis dementia due to AD in 56% of subjects and MCI-AD in 44%. On average, participants had been diagnosed 2-6 years prior to randomisation. Overall, 372 participants (62-2%) were treatment-naive with respect to prior use of an AChEI and / or memantine.

[0454] Primary endpoints

[0455] The co-primary efficacy results at 12 months of the double-blind phase for the co-primary clinical endpoints are summarized in Table 2. Mixed-model repeated measures (MM RM) analyses were conducted to estimate the least squares (LS) mean change from baseline (and standard deviation [SD]) up to 12 months There was no significant difference at 12months between groups receiving HMTM 16 mg / day and MTC 4 mg twice weekly (Table 2; Figure 1).

[0456] Secondary Endpoints and Pre-specified Blood Biomarker Outcomes at 12 months None of the secondary outcomes was significant at 12 months. There was a directional difference in progression from a CDR 0.5 global score to CDR > 1 in MCI-AD subjects was reduced with HMT 16 mg / day (8 / 63, 13 ± 4%) compared with controls (20 / 79, 25 ± 5%; p = 0.060, two-sided proportion test) over 12 months.

[0457] Change in plasma concentration of NfL at 12 months was prespecified as the key blood outcome. HMTM 16 mg / day reduced change in mean NfL plasma levels over 12 months by 95% to a level of change statistically indistinguishable from zero, an effect that was significantly different from control (p=0.0291). The results are shown in Figure 3A. In MCI-AD, the treatment effect on NfL was significant at both HMTM doses (p=0.0292 for 8 mg / day and p=0.0141 for 16 mg / day). In the subgroup with mild / moderate AD, there was a directionally supportive dose-response which did not reach statistical significance, suggesting that the MCI-AD subgroup is more sensitive to the treatment effects of HMTM. The results are shown in Figure 3B-C. Despite not reaching significance, there was a directionally similar overall effect on change in plasma p-tau 181 (not shown).

[0458] Baseline NfL level was highly correlated with baseline severity for the cognitive measures of severity (ADAS-cogn and MMSE) but not the functional measure (ADCS-ADL23). The two imaging parameters were also highly correlated with NfL (whole brain volume and temporal lobe18F-FDG-PET normalized with respect to pons). Likewise, change in NfL concentration over 12 months was significantly correlated with change in p-tau 181 over 12 months, and with change in ADAS-cog , ADCS-ADL23, CDR-sb and brain atrophy over 24 months. Safety profile

[0459] The overall risk / benefit profile for HMTM remains unchanged relative to earlier studies13,14. Study completion was high (79%), with no difference between the three treatment groups (76% to 81%). Overall, 328 of the 598 participants (55%) had one or more treatment-emergent adverse events (TEAEs) reported during the double-blind phase. These events were considered at least possibly related to treatment by the Investigator in only 11 % of participants exposed to HMTM. The incidences were similar across the three treatment groups (allowing for greater variability in the small HMTM 8 mg / day group). Those considered treatment related were headache (T5%), diarrhoea (T2%), and anaemia, vomiting, gastroesophageal reflux disease, fatigue, anxiety, and somnolence (each <1%).

[0460] Endpoints Over 24-months Specified in Final Statistical Analysis Plan

[0461] Analyses of outcomes over 24 months were specified in the SAP finalised prior to the 24-month database lock.

[0462] MCl-Early Alzheimer’s diseaseAlthough the expected difference between HMTM 16 mg / day and MTC 4 mg twice weekly was not seen over 12 months, a difference was demonstrated in the final analysis over 18 and 24 months in the MCI-AD subgroup. Observed case t-test comparisons showed that late-starters given HMTM 16mg / day after a delay of 12 months performed worse at 24 months on the ADAS-cog (late starters: n=101 , 4-42, 95% Cl T81 to 7-03; early starters: n=98, 0'59, 95% Cl -T68 to 2.86, p=0-030; Figure 2A, Extension Table 1), showed no difference for ADCS-ADL23(Figure 2B, Extension Table 1 ), and had greater loss of brain volume (late starters: n=101 , -17-77cm3, 95% Cl -2T12 to -14-41; early starters: n=98, -13-21cm3, 95% Cl -17-29 to -9-14, p=0-084; Figure 2C, Extension Table 1) than early starters on HMTM over24-months. Similar proportions of participants randomised to 16 mg / day (62 / 105, 59%) and MTC 4 mg twice weekly (70 / 116, 60%) completed 24 months. Early starters receiving 16 mg / day had significant cognitive improvement relative to baseline on the ADAS-cog scale at 6 (-2.24, 95% Cl -3.52 to -0.96, p=0.001), 12 (-2.11, 95% Cl -3.70 to -0.51, p=0.010), and 18 (-2.01 , 95% Cl -3.92 to -0.10, p=0.039) months, with the score at 24 months not significantly below baseline (0-586, 95% Cl -T68 to 2-86, p=0-607; Figure 3). The results were directionally similar for ADAS-cogn. Participants randomised to MTC 4 mg twice weekly had similar improvement on the ADAS-cog (-1.90, 95% Cl -3.01 to -0.80, p=0.0009) and ADCS-ADL23(2.64, 95% Cl 1.19 to 4.08, p=0.0005) scales only over the initial 6 months, but had significant decline by 24 months (ADAS-cogi3: 4.42, 95% Cl 1.81 to 7.03, p=0.0308; ADCS-ADL23: -0.68, 95% Cl -3.15 to 1.78, p=0.5829; Figure 3). Mild to moderate Alzheimer’s disease

[0463] In mild to moderate AD, late starters who received MTC 4 mg twice weekly for the first 12 months had a significantly better ADAS-cogn score at 24 months than in early starters (Extension Table 1 , Figure 2D). This effect was not seen on the ADCS-ADL23scale, nor in measurement of WBV change (Extension Table 1 ; Figure 2E).

[0464] Comparison with external patient groups

[0465] Comparisons of arms as originally randomised with external controls over 24 months were prespecified in the final protocol, with details specified in the final SAP, as can be seen in Figure 4A, there were significant differences between participants receiving HMTM 16 mg / day and closely matched ADNI participants with MCI-AD or AD at 12 and 24 months on the ADAS-cogn scale and for change in WBV. The comparisons with meta-analytic controls from placebo arms of clinical trials in comparable populations are shown in participants with MCI-AD or AD for ADAS-cogn, ADCS-ADL23and change in WBV are shown in Figure 4B. Disease progression in participants receiving HMTM 16 mg / day was significantly less than meta-analytic controls at 12 and 18 months for all outcomes.

[0466] DISCUSSION LUCIDITY was a two-phase 24-month study with an initial interim 12-month randomised double-blind phase followed by a further 12-month modified delayed-start open label phase in which all participants received HMTM 16 mg / day. Although there was no difference on the co-primary cognitive, functional or brain atropy endpoints at 12 months, change in plasma neurofilament light chain (NfL) concentration was significant overall and highly significant inthe MCI-AD subpopulation. In this group, the effect on neurodegeneration translated into statistically significant cognitive benefits at 18 and 24 months in outcomes specified prior to the 24-month database lock. Participants receiving HMTM 16 mg / day had statistically significant cognitive improvement over baseline that was sustained over 18 months and showed no significant cognitive or functional decline from baseline over 24 months.

[0467] In order to try and understand the therapeutic effects of longer term dosing of MT on subjects the inventors analysed the dose normalized trough concentration of MT (ng / ml) resulting from different dosages measured at 12 and 24 months.

[0468] The results are shown in Figure 17 which provides an explanation for the results shown in Figure 2. For example, it can be seen that dosing at 16mg with HMTM throughout the 24 month period provides a Median Cmax which drops to a level by 24 months below that needed for optimal treatment of at AD (as estimated based on NfL biomarkers). It is however enough to provide effectiveness in treating MCI.

[0469] The dissociation between the NfL biomarker measure of neurodegeneration and the primary clinical outcomes at 12 months can thus be explained by a combination of symptomatic activity of MTC in the intended control arm and atypical pharmacokinetics (PK) of HMT (see Examples 4 and 5). The plasma concentration of HMT follows a biphasic time-course at all doses, with increase over 12 months followed by reduction to half the 12-month level at 24 months.

[0470] This can be seen in Figure 18. This shows that Cmax at 24m for subjects receiving 16mg / day depends on Cmax at 12m. The higher the Cmax at 12m, the greater the reduction in Cmax at 24 months relative to Cmax for 16mg / day at 12 months.

[0471] This characteristic of HMTM having a peak concentration within this timeframe and declining after that is extremely unusual and implies a much longer period is required to reach a steady state.

[0472] Without wishing to be bound by theory it appears that the initial inhibition of clearance and recovery mechanisms may saturate at higher doses, for example a gradually improved metabolism may lead to more efficient metabolite formation or improvement in renal function or both.

[0473] Furthermore, it has previously been suggested that MT dosing demonstrates hormetic behaviour, such that very high doses may reduce therapeutic effects (K. Bruchey and F. Gonzalez-Lima, “Behavioral, Physiological and Biochemical Hormetic Responses to the Autoxidizable Dye Methylene Blue”, Am J Pharmacol Toxicol. 2008 January 1; 3(1): 72-79.

[0474] Irrespective of the precise mechanism, in the case of the MTC arm, plasma concentration of HMT increased at 12 months to a level shown to have symptomatic activity in an animal model.11,12Symptomatic activity in the intended control arm is consistent with the statistically significant cognitive and functional improvement seen at 6 months in participants with MCI-AD. This early improvement was not seen in a meta-analysis of similar amyloid-PET positive trial populations, and so is unlikely to be trial participation artefact.22Early improvement did not protect participants with MCI-AD from significantly greater cognitive decline anddirectionally greater progression of brain atrophy over 24 months, despite switching to the higher dose after 12 months. Consistent with the independence of HMT’s two modes of action, the exposure threshold for symptomatic activity is approximately 7-fold lower than required for the effect on neurodegeneration in MCI-AD.

[0475] Although both MCI-AD and mild / moderate AD groups had less disease progression than expected in comparable untreated populations, the response to the switch to HMTM 16 mg / day after 12 months differed. Subjects with mild / moderate AD did better after the switch than those continuing at the 16 mg / day dose from baseline, whereas the benefit of the switch was not seen in the MCI-AD group although the biphasic PK was the same in both groups. It is therefore likely that the thresholds for disease-modifying and symptomatic effects depend on duration of treatment and stage of disease as well as plasma levels of drug. MCI-AD participants who started HMTM 16 mg / day after a 12-month delay, and who therefore had double the plasma levels of HMT at 24 months, were unable to catch up with those who had started treatment earlier, consistent with the underlying disease-modifying effect demonstrated by the NfL result.

[0476] Example 3 - Table 1: Demographic and baseline characteristics.

[0477]

[0478]

[0479] Example 3 - Table 2: Prespecified treatment group comparisons at 12 months for study TRx-237-039.

[0480]

[0481] aOne participant did not have a baseline ADAS-cogn or ADCS-ADL23score

[0482] bThe MI-MITTv5 Population included 512 participants (230 randomized to control, 54 randomized to HMTM 8 mg / day, and 228 randomized to HMTM 16 mg / day.Example 3 - Extension Data Table 1: Twenty-four month change from baseline in the MCI-AD and mild to moderate AD subpopulations in ADAS-cog , ADCS-ADL23and whole brain volume. Two-sample t-tests were performed by visit for the treatment comparisons.

[0483]

[0484] EXAMPLE 3 REFERENCES

[0485] 11. Deiana, S., Harrington, C. R., Wischik, C. M. & Riedel, G. Methylthioninium chloride reverses cognitive deficits induced by scopolamine: comparison with rivastigmine. Psychopharmacology (Berl) 202, 53-65 (2009).

[0486] 12. Kondak, C., Riedel, G., Harrington, C. R., Wischik, C. M. & Klein, J.

[0487] Hydromethylthionine enhancement of central cholinergic signalling is blocked by rivastigmine and memantine. J Neurochem 160, 172-184 (2022).

[0488] 13. Gauthier, S. etal. Efficacy and safety of tau-aggregation inhibitor therapy in patients with mild or moderate Alzheimer’s disease: a randomised, controlled, double-blind, parallel-arm, phase 3 trial. The Lancet 388, 2873-2884 (2016).

[0489] 14. Wilcock, G. K. etal. Potential of low dose leuco-methylthioninium bis(hydromethanesulphonate) (LMTM) monotherapy for treatment of mild Alzheimer’s disease: cohort analysis as modified primary outcome in a Phase III clinical trial. Journal of Alzheimer’s Disease 61, 435-457 (2018).

[0490] 16. Schelter, B. O. etal. Concentration-dependent activity of hydromethylthionine on cognitive decline and brain atrophy in mild to moderate Alzheimer’s disease. Journal of Alzheimer’s Disease 72, 931-946 (2019).

[0491] 18. McKhann, G. M. et al. The diagnosis of dementia due to Alzheimer’s disease:

[0492] Recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimer’s and Dementia 7, 263-269 (2011 ).

[0493] 19. Albert, M. S. et al. The diagnosis of mild cognitive impairment due to Alzheimer’s disease: Recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimer’s and Dementia 7, 270-279 (2011 ).

[0494] 21. Freeborough, P. A. & Fox, N. C. The boundary shift integral: An accurate and robust measure of cerebral volume changes from registered repeat MRI. IEEE Trans Med Imaging 16, 623-629 (1997).

[0495] 22. Sommer, R. The Hawthorne dogma. Psychol Bull 70, 592 (1968).

[0496] Example 4 - Characterization of the population pharmacokinetics of hydromethylthionine mesylate (HMTM), an orally administered tau aggregation inhibitor, in healthy volunteers and Alzheimer’s Disease (AD) patients.

[0497] 1. Introduction

[0498] In this study, a population pharmacokinetic (popPK) model was constructed to describe the PK of the active HMT moiety and to identify significant covariates, based on pooled data from Phase 1 studies in healthy volunteers (TRx-237-021 , -023, -024, -035 and -036) and AD patient data from the Phase 3 study TRx -237-039 (LUCIDITY) discussed in Example 3 above. The Phase 1 studies included single doses of HMTM ranging between 4 and 100 mg and multiple dose regimens ranging between 8 and 80 mg / day. The Study TRx-237-039 was a Phase 3 trial which compared HMTM at a dose of 8 mg twice daily (BID) with a control comprising methylthioninium chloride (MTC, the oxidized form of MT) at a dose of 4 mg given twice weekly (BIW) on a varying schedule every 2-4 days

[0014] , When dosed as MTC, the oxidized form of converted to HMT in the stomach prior to absorption as HMT

[0015] . The popPK model described the PK of the active HMT moiety, whether delivered as HMTM or as MTC, evaluates the covariates and simulates their impact on HMT exposure over the courseof long-term dosing up to 2 years. These findings are of importance for interpreting the results of TRx-237-039 and informing future clinical use of HMTM.

[0499] 2. Material and Methods

[0500] 2.1 Study designs and participants

[0501] In this analysis, parent HMT data collected from subjects enrolled in six clinical studies (TRx-237-021 , -023 (PK sub study), -024, -035, -036 and -039) were used for popPK modeling. Parent HMT kinetics from all randomized subjects who received at least one dose of study treatment (HMTM or MTC) and had at least one post-dose assessment were included in the analysis.

[0502] Study TRx-237-021 was a Phase 1, open-label, parallel-group study designed to evaluate the pharmacokinetics, safety and tolerability of HMTM in subjects with varying degrees of renal function. Subjects received a single high dose of 100 mg HMTM administered orally. The sub study of the Phase 1 TRx-237-023 trial was an open label study designed to evaluate the pharmacokinetics of HMTM given as multiple doses of 8 mg twice daily (BID) for 10 days in healthy elderly participants (>61 years).

[0503] Study TRx-237-024 was a Phase 1 , open-label, parallel-group study designed to evaluate the pharmacokinetics, safety and tolerability of HMTM in subjects with varying degrees of hepatic impairment (liver cirrhosis). Subjects received a single high dose of 100 mg HMTM administered orally. Study TRx-237-035 was a Phase 1 , open-label, three-cohort study in healthy subjects designed to evaluate relative bioavailability, as well as the effect of food and gastric pH on HMTM. Depending on the cohort, subjects received HMTM as a single oral dose of 4, 8, or 30 mg. Subjects in cohort 1 also received a single oral dose of MTC for assessment of relative bioavailability.

[0504] Study TRx-237-036 was a Phase 1 , open-label, two-period, parallel-group study designed to assess the pharmacokinetics of single- and multiple-doses of HMTM in healthy older participants (>51years). Subjects received single doses of 4, 10, 40, 75, or 125 mg, and, after a washout of at least 10 days, multiple doses of 10 mg once daily (QD) or 4, 40, 75 or 125 mg BID administered orally for 14.5 days.

[0505] Study TRx-237-039 (LUCIDITY, NCT03446001; EudraCT: 2017-003558-17) is a completed Phase 3 study in participants with mild cognitive impairment (MCI)-AD and mild to moderate AD. The study evaluated HMTM as monotherapy at dose levels of 4mg BID or 8 mg BID administered orally. In order to control for potential urinary colouration, participants in the control arm received an average of 2 doses per week of 4 mg MTC on a varying schedule every 2 -4 days. Accurate MTC twice weekly dosing was not recorded; for this reason, the dosing regimen was assumed to follow a schedule of once per 3.5 days. A detailed description of the TRx-237-039 study has been described previously

[0014] ,

[0506] All Phase 1 studies included intensive PK sampling at specified times. In Phase 3 study TRx-237-039, sparse PK samples were collected at pre-dose, 1 to 2 hours and approximately 4 hours post-dose on day 1 , and at specified times throughout the treatment period (weeks 4, 52, 104), and at the end-of-treatment / treatment discontinuation visits.

[0507] Samples were collected prior to study drug administration.All studies were conducted in accordance with the ethical principles of the Declaration of Helsinki and complied with Good Clinical Practice.

[0508] 2.2 HMT and NFL measurements

[0509] Parent HMT plasma concentrations were measured using a validated liquid chromatography-tandem mass spectrometry assay (LC-MS / MS)

[0510] Long-term stability testing demonstrates that frozen plasma samples remain valid for a time period of 160 days. The method used for quantifying parent HMT has lower limits of quantification (LLOQ) of 0.04 ng / mL and is appropriate for quantitation of plasma concentrations (range 0.04 - 2 ng / mL) following high single doses or at steady state. Briefly, blood samples are collected in tubes containing sodium or lithium heparin anticoagulant and centrifuged to obtain plasma. Plasma samples are then stored at -20 C, then at -80 C. Both parent HMT and A / -desmethyl HMT are quantified with a dichloroethane (DCE) liquid-liquid extraction followed by ultra-high performance liquid chromatography and tandem mass spectroscopy (UPLC-MS / MS) detection.

[0511] Analytical standards MTC (1 mg MT7mL) and Azure B (0.1 mg desmethyl-MT7mL) are prepared as stock solutions in methanol or methanol-water (50:50), respectively. de-MT+and d4- / V-desmethyl MT+are used as internal standards.

[0512] Calibration range: 0.02-2 ng / mL

[0513] Test plasma samples are thawed at room temperature in the dark.

[0514] To 0.2 mL of plasma sample add 20 mL of sample or standard MT+.

[0515] Add 0.2 mL 10% hexanesulphonate, vortex and move to blue light.

[0516] Add 1.2 mL of 1,2-dichloroethane and rotate for 15 min at 40 rpm.

[0517] Centrifuge at 13,000 rpm for 3 min at 8 C.

[0518] Remove 0.9 mL of lower layer and dry down under nitrogen gas or vacuum at 20 C.

[0519] Return to normal light and reconstitute sample with 0.1 mL of 20 mM ammonium bicarbonate / acetonitrile (60 / 40, v / v) and vortex briefly

[0520] Transfer to vials and separate by HPLC using the following parameters:

[0521] Column: Waters Acquity UPLC CSH Fluoro Phenyl 1.7 mm, 2.1 x 50 mm (Part 186005351) Column temperature: 20 C

[0522] Mobile phase: A - 95 / 5 water / acetonitrile with 0.05% formic acid

[0523] B - 50 / 50 water / acetonitrile with 0.05% formic acid

[0524] Flow rate 0.5 mL / min

[0525] Injection system: PLNO

[0526] Injection volume: 10 mL

[0527] Run time: 5.5 min

[0528] Autosampler temperature: +6 CGradient system:

[0529]

[0530] Expected retention time for analytical and reference standards is 1.6-2.6 min

[0531] Wash parameters: weak wash - 95 / 5 water / acetonitrile with 0.05% formic acid (800 ml_) strong wash - 40 / 60 water / acetonitrile with 1 % formic acid (600 ml_) Source:

[0532] Capillary (kV) 2.2

[0533] Cone (V) 15

[0534] Source offset (V) 20

[0535] Source temperature (C) 150

[0536] Desolvation temperature (C) 500

[0537] Cone gas flow (L / hr) 150

[0538] Desolvation gas flow (L / hr) 1000

[0539] Analyser:

[0540] LM 1 resolution 3.0

[0541] HM 1 resolution 15.0

[0542] Ion energy 1 1.0

[0543] Entrance: 0.5

[0544] Collision 30

[0545] Exit: 0.5

[0546] LM 2 resolution 3.0

[0547] HM 2 resolution 15.0

[0548] Ion energy 2 1.0

[0549] Multiplier (V) 543

[0550] Collision gas flow 0.15

[0551] Example collision gas pressure read back 3.0 e-03

[0552] Detection: MRM transitions at 270.1 to 254 (±0.5) for Azure B, 284.2 to 268.07 (±0.5 for MT; 290.0 to 273.0 (±0.5) for d6-MTC and 274.1 to 255.1 (±0.5) for d4-Azure B.

[0553] A system suitability test is carried out, where the acceptance criteria include a column efficiency of >2,000 and a tailing factor <2.A calibration curve is constructed by plotting peak area ratio of the calibration standards versus the concentration of standard in control matrix and linear regression parameters of the curve determined using a 1 / x2weighting factor. Concentrations in the quality control and test samples is determined by interpolation of the peak area ratios from the calibration curve. To eliminate possible contamination in the HPLC system and to inhibit on-system reduction of MT+, a cleaning step of injection with 1,4-benzoquinone (0.05% w / v in methanol / water, 75 / 25) is included in analytical runs.

[0554] For measurement of neurofilament, light chain (NfL), plasma samples were collected (with sodium heparin anticoagulant) and NfL measured at baseline and at 12 months. If a sample for a subject at either timepoint was unavailable (sample volume, number of freeze-thaw cycles, degree of haemolysis or sample collected incorrectly), no analysis was performed for that subject. Plasma samples were initially stored at -20°C and subsequently -80°C until analysis within 3 years of sample collection. NfL was measured in duplicate for each plasma sample using a Quanterix Simoa NF-light kit on a Quanterix HD-X instrument using a fully validated and fit-for-purpose method (including long-term stability). Analysis was conducted in compliance with Good Clinical Practice (GCP) at a regulated facility (Drug Development Solutions, UK). An overview of the method is given in Table 4 below.

[0555] 2.3 Statistical methods

[0556] Plasma concentration-time data were analyzed using a nonlinear mixed effects modelling approach, using nonlinear mixed-effects modeling software (NONMEM®; version 7.4.3 or above, ICON, Hanover, MD, USA). The first-order conditional estimation method with interaction (FOCE INTER) was used as the main parameter estimation algorithm

[0016] . Log-transformed concentration data was modelled. An additive residual error on the transformed data, corresponding to an exponential variance model of the untransformed data was used.

[0557] Assessment of model adequacy and decisions about model refinement were driven by the data and guided by graphical and statistical goodness-of-fit (GOF) criteria, including the plausibility of parameter estimates, precision of parameter estimates and the likelihood ratio test [17, 18]. If models were classified as nested, one model was declared superior to the other if the minus two (-2) log-likelihood (-2LL; OFV) was decreased by 6.64 (P<0.01 x2distribution, with 1 degree of freedom).

[0558] All the covariate-parameter relationships of interest were examined within the framework of nonlinear mixed-effects modeling using a stepwise forward addition (P < 0.01) and backward elimination (P < 0.001) methodology.

[0559] The adequacy of the final model and parameter estimates was investigated with a visual predictive check (VPC) method, including prediction correction [19,18]. The final model was used to simulate 500 replicates of the analysis data. For simulation the structure of the data was retained (doses, number of patients, demographics, and covariates) but new sets of individual parameters were sampled from the population parameters. Additionally, in each replicate, new population parameters were sampled from the uncertainty distribution.Statistics of interest (5th, 50th [median], and 95th percentiles) were calculated for both data and simulation results and overlaid in a plot for comparison.

[0560] Simulations were carried out in R version 7.4.3 or higher, applying the RxODE 1.1.5 package.

[0561] Simulations of the final model were conducted to assess the impact of covariates and their impact on exposure metrics of clinical interest (AUCo-i2h, Ciast, and Cmax). This allowed assessment of whether certain covariates would lead to clinically relevant changes in exposure metrics. A fold change compared to the reference within the [0.80 - 1.25] interval was considered as not clinically relevant. The reference was a non-smoking female weighing 73kg and a creatine clearance (CrCL) of 72.4 mL / min receiving HMTM in fasted state. A dosing regimen of 4 and 8 mg BID was assumed. Simulations (n=500) were performed, taking into account parameter uncertainty and inter-individual variability. For each of the two dose groups, new population parameters were sampled from the uncertainty distribution and covariates were changed one at a time to assess the impact of each covariate in a univariate manner. Summary statistics (5thpercentile (P5), median and 95thpercentile (P95)) were calculated across all scenarios.

[0562] To evaluate the impact of the assumption of a MTC dosing regimen of once per 3.5 days on the modeling results, additional simulations were performed.

[0563] 2.4 Modeling strategy

[0564] Based on the Cohort 1 of TRx-237-035, parent HMT originating from MTC was determined to result in similar exposure (AUC) as parent HMT arising from HMTM (clinical study report TRx-237-035, v1.0, 2023) at single doses of 4 mg. Therefore, purely for the purposes of modelling only, MTC was assumed to be equivalent to HMTM, and the PK model described the PK of the active HMT moiety, whether delivered as HMTM or as MTC. The dosing regimen of MTC in TRx-237-039 was assumed, purely for the purpose of modelling, to follow a schedule of once per 3.5 days.

[0565] Due to the incremental availability of the data from the included studies, model development was performed using a stepwise approach.

[0566] The development of a population PK model started using an intermediate dataset with data from the Phase 1 studies (TRx-237-021 , TRx-237-024, and TRx-237-036) and data from the Phase 3 study (Study TRx-237-039) up to Visit 3 (week 4). These data were used to develop a base model and accounted for correction of the concentrations from Study TRx-237-036. Next, the base model was used as a starting point for a first model update, based on additional data accrued in study TRx-237-039 up to Visit 7 (12 months), and a second model update based on the complete dataset from study TRx-237-039 through to Visit 10 (24 months), as well as data from TRx-237-035 and PK sub study TRx-237-023. The final model update accounted for correction of the concentrations from Study TRx-237-039 due to the effect of sample storage time. Covariates evaluated included demographics (sex, age, body weight, ethnicity, and race), smoking history, erythrocyte count and markers of renal and liver functions namely CrCL and bilirubin, respectively. During the covariate analysis, covariates were tested with respect to specific parameters of interest based on thephysiologic / pharmacologic rationale. The effect of food on the PK of parent HMT was also evaluated based on data from study TRx-237-035. The effect was tested on parameters relating to the absorption process.

[0567] 3. Results

[0568] 3.1 Patient baseline characteristics.

[0569] Baseline characteristics and demographics were generally similar between subjects in TRx-237-021, -023 (PK sub study), -024, -035, -036 and -039, with the exception of age, CrCL and bilirubin owing to studies being conducted in specific study populations (Example 4, Table 1). Comedication information was available only from TRx-237-039. Child Pugh score was assessed only in TRx-237-023. Smoking status was recorded in all studies except for TRx-237-023.

[0570] 3.2 Parent HMT PK data

[0571] Figure6A and Figure6B display semilogarithmic plots of parent HMT dose-normalized concentrations over time in the first twenty-four hours. Doses were normalized by the total daily dose to ensure comparability between regimens. The figures suggest a linearity of exposure across the dose levels explored, linear elimination and a two compartmental disposition. The absorption phase is highly variable across studies especially in study TRx-237-021 (renally impaired participants) where the absorption appeared slower than in other studies.

[0572] Dose normalized non-BLQ (Below Limit of Quantification) trough concentration (Ctrough) of study TRx-237-039 (Figure 7), indicated a time-varying PK; the accumulation observed at Visit 7 (12 months) was reversed at Visit 10 (24 months) to approximately the trough levels of Visit 3 (1 month) in the HMTM arms.

[0573] The dose normalized non-BLQ trough concentration (Ctrough) was higher in the MTC group than the HMTM arms at Visit 3 and Visit 7. The cause of this difference was further investigated by means of simulations. These showed that the higher apparent levels in this arm were the result of exclusion of BLQ values. When BLQ values were either included or set to 0.5 x LLOQ, the increase at 12 months was comparable to that observed in the other arms.

[0574] 3.3 Model development

[0575] A linear two-com partmental disposition model, with delayed first order absorption of parent HMT using two transit compartments into the central compartment, and a time-varying (parabolic) elimination of parent HMT described the data in an adequate manner.

[0576] The time-varying PK was described by a time-varying elimination with a parabolic function:

[0577]

[0578] With CLo / F the initial apparent clearance, MaxDrop the maximal reduction of the time varying parabolic clearance, Tmaxthe time at which CUF reached the minimum.

[0579] The time-varying parabolic clearance may be interpreted as follows. The apparent clearance over time (CL / F(t)) first declines and is then followed by a symmetrical rebound, with a minimum approximately at Visit 7 (12 months). The typical CL / F decreased from 1660 L / h attime zero and reached a minimum of 782 L / h (47.1% of CLOZF) at 12 months, and then rebounded to 1550 L / h on at 24 months (Figure 8). In the final popPK model the typical values (standard errors) of CL0 / F and Vc / F were 7.41 L / hours (0.434%) and 10.5 L (0.234%) (Example 4, Table 2).

[0580] The covariate assessment identified the following covariate relationships as statistically significant at P<0.01:

[0581] • Vc / F: body weight (BW)

[0582] • CL0 / F: CrCL, smoking status and sex

[0583] • Ka: prandial status

[0584] Increased BW was associated with increased Vc / F. Increased CrCL, smoking and male sex was associated with increased CL0 / F: In a fed condition, absorption was reduced.

[0585] 3.4 Adequacy of the model

[0586] Diagnostic plots of the final model showed that the random effects were adequately normally distributed and the predicted and observed individual concentrations were generally symmetrically distributed around the line of identity. The different residuals did not show any trends either over time or over the population prediction.

[0587] Overall, the VPCs suggested that the final model was able to describe the main part of the observed data for healthy subjects and AD patients with reasonable accuracy.

[0588] VPCs showed that the final model was able to describe the long-term PK of parent HMT in patients with reasonable accuracy

[0589] 3.5 Impact of covariates on the PK

[0590] The covariate effects of BW on Vc / F, prandial status on Ka, CrCL, smoking status and sex on CL0 / F were identified to be statistically significant. At the 5th and 95th percentiles of CrCL distribution (41.8 and 130.3 mL / min, respectively) CL0 / F decreased and increased up to 1200 and 2350 L / h, respectively, compared to the typical CL0 / F of 1660 L / h. At the 5th and 95th percentiles of BW distribution (51 and 100 kg, respectively) Vc / F decreased and increased up to 27900 and 45500 L, respectively, compared to the typical Vc / F of 36200 L. In male participants, CL0 / F was increased up to 1910 L / h compared to the typical CL0 / F of 1660 L / h. In currently smoking participants, CL0 / F was increased up to 2170 L / h compared to the typical CL0 / F of 1660 L / h.

[0591] In participants in a fed condition, Kawas decreased to 3.05 h-compared to the typical Ka of 6.66 IT1. Consequently, those covariates may have an impact on the secondary PK parameters such as AUCo-i2h, Cmax, Ciastat 12 months. This was further investigated through PK simulations.

[0592] The impact of statistically significant covariates was evaluated by predicting AUCo-i2h, Cmax and Ciast at 12 months. BW, sex and prandial status did not have a clinically relevant impact on AUCo-i2h. Ciast and Cmax. The PK of parent HMT was only moderately impacted by the covariates baseline CrCL and smoking status.A subject with a baseline CrCL value of 41.8 mL / min (5thpercentile in the analysis dataset distribution) had a 38% increase in AUCo-i2h , a 43% increase in Ciast, and a 34% increase in Cmax, as compared to a typical subject with a baseline CrCL value of 72.4 mL / min.

[0593] In a subject currently smoking, AUCo-i2h decreased by 23%, Ciast decreased by 26% and Cmax decreased by 20% compared to the reference (never smoke or smoked previously) 3.6 Impact of time-varying clearance

[0594] Parent HMT Ctrough accumulation over 12 months dosing in Study TRx -237-039 was observed for each treatment arm. To investigate this further, this Ctrough was simulated including a parabolic function for clearance. This was compared to the Ctrough simulated without time dependent clearance (linear PK over time). Example 4, Table 3 summarizes the Ctrough accumulation ratio (Rac) between 12 months and first dose for time dependent clearance and linear PK over time.

[0595] For all HMTM arms there is an Rac of approximately 6.8 at 12 months relative to first dose (Example 4, Table 3) Subjects receiving MTC who had quantifiable plasma levels had a Rac of 5.6. For HMTM and MTC, the values at 12 months were respectively ~3- and ~5-fold higher than would have been expected in a model based on first principles with linear PK overtime. Differences in drug formulation, analytical assay, proportion of the below-limit-of-quantification (BLQ) concentrations, and compliance were ruled out as potential explanations for the time-varying clearance. It is important to note that the change in clearance over time is specific to parent HMT and was not observed for Total MT which measures all HMT after deconjugation.

[0596] Discussion

[0597] We developed a popPK model to describe the PK profile of parent HMT, using the data from 5 Phase 1 studies (TRx-237-021, TRx-237-023, TRx-237-024, TRx-237-035, TRx-237-036) and from the Phase 3 study TRx-237-039 including parent HMT concentration-time data up to week 104 in which participants were dosed with HMTM or MTC. The popPK model consisted of a two-compartmental disposition model, with delayed first- order oral absorption into the central compartment, and time-varying (parabolic) clearance.

[0598] Body weight, prandial status, CrCL, smoking states and sex were found to have a statistically significant impact on the PK parameters. Vc / F increased with increased weight. CLQZF increased in male and current smokers, with increasing CrCL, while Vc / F increased with increasing body weight. The absorption rate decreased with food intake. Parent HMT clearance increased with CrCL and in subjects who currently smoke with the fold change in exposure outside the range of [0.8-1.25] and therefore clinically relevant. A subject with a CrCL of 41.8 mL / min, showed a 38% increased exposure compared to a subject with a CrCL of 72.4 mL / min. In a subject currently smoking exposure decreased by 23% compared to a non-smoking subject. Covariates that influenced clearance without clinically meaningful impact on HMT exposure included sex (higher in females), bodyweight (increase in central volume) and food intake (decrease with food).

[0599] A parabolic time-varying clearance term (see Figure 8) was needed to capture both increased concentrations at Visit 7 (12 months) and less-than-anticipated concentrations atVisit 10 (24 months), for patients treated with either HMTM or MTC. This relationship suggested that the clearance is reduced by 52.9% compared to the initial clearance by 12 months and recovered by 2 months. The parabolic function remains empirical in nature and permits accurate description of the observed parent HMT concentrations across all visits. The underlying cause leading to time-dependent variation in clearance behaviour is not known. We hypothesize that it may be explained by time-dependent inhibition of conversion of parent HMT to its principal glucuronide metabolite which is subsequently restored by 104 weeks.

[0600] The unusual PK properties of HMT, whether delivered by HMTM or MTC, are clinically important for the interpretation of data from the most recent Phase 3 clinical trial (TRx-237-039). MTC was given at a dose of 4 mg twice weekly in the control arm intended as a clinically inactive urinary colorant. Based on an earlier popPK study using a linear model and data from earlier Phase 3 trials [8], the MTC dose of 4 mg twice weekly was not expected to reach therapeutic exposure

[0014] , In TRx-237-039 the MTC 4 mg twice weekly dose was found to produce temporary but nevertheless statistically significant improvement in cognitive function at 6 months. An earlier preclinical study had found that MTC was able to reverse learning deficits produced by scopolamine in a standard mouse model for symptomatic activity

[0020] . The level of exposure at 12 months predicted from the present analysis was found to correspond to the range at which symptomatic activity was observed in the mouse model. The sparse MTC dosing regimen used in TRx-237-039 is the lowest level at which blinding can be maintained, based on earlier Phase 1 studies.

[0601] A further clinical implication of the present study is the drop in exposure by about half between 12 and 24 months at the HMTM 16 mg / day dose. Since 16 mg / day was the minimum required to prevent progression of neurodegeneration determined by change in plasma levels of neurofilament light chain in TRx-237-039, it would be necessary to envisage a higher dose after 12 months to maintain prevention of progression of neurodegeneration (see Example 5).

[0602] Example 4 Table 1 Summary of Subjects Covariates (PK analysis sets)

[0603]

[0604]

[0605]

[0606] Abbreviations: N=number of participants; popPK=population pharmacokinetic(s).

[0607] Notes: For categorical covariate, the table reports sample size and associated percentage in parenthesis.

[0608] aPK sub study.

[0609] bFood / fed status can be time-varying within a participant, either due to a crossover design (TRx-237-035) or due to the altered food / fed status within the study period (TRx-237- 023, TRx-237-036). For this reason, the totality of information is collected and the sum of individuals under fasted or fed state can be > 100%.Example 4 - Table 2 Population parameter estimates for the final model (TRx -237-021,

[0610]

[0611] Abbreviations: BW=body weight; CLo / F =initial apparent clearance; CrCL=creatinine clearance; CV=coefficient of variation; MaxDrop=maximum reduction of the time-varying parabolic clearance; Tmax=time at which CL / F reach the minimum; IIV=interindividual variability; Ka=absorption constant; popPK=population pharmacokinetic(s);

[0612] Q=intercompartmental clearance; RSE(%)=percentage of the relative standard error; Vc / F =apparent central volume of distribution; Vp / F =apparent peripheral volume of distribution. Notes: log-transformed parameters were estimated and CV% are presented for I IV untransformed parameters.Example 4 - Table 3: Ctrough Accumulation Ratio for “Parabolic Function On CL” and “linear PK over time (“first principles”)

[0613]

[0614]

[0615] Example 4, Table 4: Overview of Plasma NfL Method Validation

[0616] <

[0617] <

[0618]

[0619] Abbreviations: Cmax=maximum plasma concentration; LLOQ=lower limit of quantification; MRD=Minimum Required Dilution; Nfl_=neurofilament light chain; SIMOA=single molecule array; ULOQ=upper limit of quantification

[0620] * Inferred validation parameters from original validation work performed in dipotassium ethylenediamine tetraacetic acid

[0621] Each analytical run included a 7-point calibration curve (5PL, 0.415 to 517 pg / mL) using kit-provided calibrators which were used to back-calculate sample concentrations. Three levels of established endogenous quality controls (low 2.24 pg / mL, medium 5.86 pg / mL, and high 38.0 pg / mL) were included in each sample analysis run.Example 4 References

[0622] 8. Schelter BO, Shiells H, Baddeley TC, et al. Concentration-Dependent Activity of Hydromethylthionine on Cognitive Decline and Brain Atrophy in Mild to Moderate Alzheimer's Disease. J Alzheimers Dis. 2019;72(3):931-946.

[0623] 14. 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. J Prev Alzheimers Dis 2022; 9: 780-90.

[0624] 15. Baddeley TC, McCaffrey J, Storey JM, et al. Complex disposition of methylthioninium redox forms determines efficacy in tau aggregation inhibitor therapy for Alzheimer's disease. J Pharmacol Exp Ther. 2015;352(1 ): 110-118

[0625] 16. ICON pic. NONMEM User Guides

[0626] 17. Akaike H. A new look at the statistical model identification. IEEE Trans Automatic Control.1974; 19:716-23

[0627] 18. Karlsson MO, Savic RM. Diagnosing model diagnostics. Clin Pharmacol Ther. 2007 Jul;82(1):17-20. Review.

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[0629] 20. Deiana S, Harrington CR, Wischik CM, Riedel G. Methylthioninium chloride reverses cognitive deficits induced by scopolamine: comparison with rivastigmine.

[0630] Psychopharmacology (Berl) 2009; 202: 53-655 - simulated

[0631] The developed MT “popPK” model from Example 4 was used to perform simulations with the aim of optimizing an MT dosing regimen such as to achieve Cmax > 0.74 ng / mL or / and Cavg > 0.693 ng / mL after 12 months, which are believed to be sufficienct for disease modifying effect in mild / moderate AD.

[0632] Cavg target of 0.693 ng / mL is based on the post hoc simulated median Cavg at 12m following 16 mg / day HMTM in Study 39

[0633] Table 6 Simulation of population Parent HMT Median (95% PI) Exposures at 4,

[0634] 52 and 104 Weeks for Study TRx-237-039 (run522)

[0635]

[0636] The simulation specifics were as follows:

[0637] • Simulated treatment duration: 24 months

[0638] • Simulated study population: sampling 1000 subjects from the 039 study population • Target exposure: Cmax > 0.74 ng / ml (no upper limit of Cmax), Cavg > 0.693 ng / mL

[0639] The simulation variables were as follows:

[0640] • Initial HMTM dose: 16 mg / day

[0641] • Dose increase: ramp up to ~70 mg / day (assuming 4, 8 and 30 mg tablets)

[0642] • Time of dose switch: 6 months, 9 months, 12 months

[0643] • Dose frequency: BID and QD

[0644] The simulation output is as follows:• Table of summary statistics (5th percentile, median, 95th percentile) of Cmax and Cavg at 6, 9, 12, and 24 months for each simulated scenario

[0645] • Line plots of Cmax vs time (months) with lines for median trends of each scenario • Boxplots of Cmax and Cavg at 6, 9, 12, and 24 months for each simulated scenario • Plot of (change in) Cmax at 24 months vs Cmax at 12 months

[0646] • Plot of Cmax at 12 months and 24 months vs second dose (mg / day)

[0647] Figure 9 gives an overview of the simulation strategy. The simulation was based on N=1000 virtual patients randomly drawn from Study 39 with the following variables:

[0648] N = 5 covariates sampled from study 39

[0649] • BCRCL: baseline creatinine clearance

[0650] • BW: body weight

[0651] • SEX: sex

[0652] • SMOKE: smoking status

[0653] • FED: fed status, all missing and assumed to be fasted

[0654] N=48 dosing scenarios

[0655] • N=24 QD

[0656] • N=24 BID

[0657] Simulated exposure: Cmax and Cavg

[0658] Exposure Time: 6, 7, 8, 9, 10, 11 , 12, 15, 24 month

[0659] All exposure was simulated prior of the dose switch

[0660] ***

[0661] As can seen from Figure 10, all of early dose switch, QD and higher doses increase Cmax. Under the same dose, an early dose switch results in a higher Cmax during the period between the dose switches. However, Cmax becomes similar about 1 month after the later dose switch. A corresponding analysis demonstrated the same trajectory for Cavg (not shown).

[0662] ***

[0663] The plots of Figure 11 show an increase of Daily Dose >32 mg is needed for QD and >40 mg for BID to ensure >95% patients with Cmax > 0.74 ng / mL at 24m.

[0664] ****

[0665] Figure 12 indicates that an increase of Daily Dose >32 mg is needed for QD and >40 mg for BID to ensure >95% patients with Cmax > 0.74 ng / mL after 9m switch.

[0666] ****

[0667] Figure 13 indicates an increase of Daily Dose >56 mg is needed for either QD or BID to ensure >95% patients with Cavg > 0.693 ng / mL at 24m.

[0668] ****Figure 14 indicates that increase of Daily Dose >56 mg is needed for either QD or BID to ensure >95% patients with Cavg > 0.693 ng / mL after 7m.

[0669] Figures 15 and 16 illustrate how the Median Cmax at 24m are very similar at different switching times.

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Claims

84Claims1 A method of treatment of a neurodegenerative disease in a subject,which method comprises orally administering to said subject a methylthioninium (MT) compound,at a first dosage level for a first dosage period and thenat a second dosage level for a second dosage periodwherein the second dosage level is higher than the first dosage level,and wherein the first dosage period is between 6 and 18 months,and wherein the second dosage period is at least 12 months,and wherein the first dosage level of MT is 12mg / day or more,and wherein the second dosage level of MT is 16mg / day or more.2 A method as claimed in claim 1 wherein the second dosage level is at least 25%, 30% or 33% higher than the first dosage level.3 A method as claimed in claim 1 or claim 2 wherein the first dosage level is 12 to 35 mg / day total.4 A method as claimed in claim 3 wherein the first dosage level is 23 mg / day or less.5 A method as claimed in claim 4 wherein the first dosage level is about 12, 13, 14, 15, 16, 17, 18, 19, 2021 , 22, or 23 mg / day.6 A method as claimed in any one of claims 1 to 5 wherein the second dosage level is 17 to 70 mg / day total.7 A method as claimed in claim 6 wherein the first dosage level is 32 mg / day or more.8 A method as claimed in claim 7 wherein the second dosage level is about 32, 33, 34, 35, 36, 37, 38, 39, 40 mg / day.9 A method as claimed in any one of claims 1 to 8 wherein the first dosage period is equal to or at least about 6 months, 9 months, 12 months, 15 months, or 18 months.10 A method as claimed in any one of claims 1 to 9 wherein the second dosage period is (I) equal to or least about 18 months, or longer, (ii) equal to or at least about 2, 3, 4, 5 years, or longer; (iii) between about 6 and 12 months, or (iv) between about 1 and 5 years. ***8511 A method as claimed in any one of claims 1 to 10 wherein the second dosage period immediately follows from the first dosage period.12 A method as claimed in any one of claims 1 to 11 wherein the first and second dosage periods are separated by an intermediate dosage period of up to 1 month where the dosage in the intermediate period is set at one or more intermediate levels between the first and second dosages levels.13 A method as claimed in any one of claims 1 to 12 wherein the first dosage level and / or second dosage level is selected based on one or both of the subject’s (i) baseline creatine clearance or (ii) current smoking status.14 A method as claimed in any one of claims 1 to 13 wherein the second dosage level is selected based on measurement of the subject’s parent HMT trough level Cmax after the first period.15 A method as claimed in any one of claims 1 to 14 wherein the second dosage level is selected based on measurement of the subject’s NfL level.16 A method as claimed in any one of claims 1 to 15 wherein the subject is a human who has been diagnosed as having said cognitive or CNS disorder, or wherein said method comprises making said diagnosis.17 A method of prophylactic treatment of a neurodegenerative disorder of protein aggregation in a subject, which method comprises orally administering to said patient a methylthioninium (MT) compound, wherein said administration is as defined in any one of claims 1 to 15.18 A method as claimed in claim 17 wherein the subject is a human who has been assessed as being susceptible to the cognitive or CNS disorder, optionally based on familial or genetic or other data.19 A method as claimed in any one of claims 1 to 18 wherein the disorder is a tauopathy.20 A method as claimed in any one of claims 1 to 19 wherein the disorder is selected from the list consisting of: Pick’s disease, progressive supranuclear palsy, frontotemporal dementia (frontotemporal dementia), FTD with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration syndromes; disinhibition-dementia-parkinsonism-amyotrophy complex, pallido-ponto-nigral degeneration, Guam-ALS syndrome, pallido nigro luysian degeneration, cortico-basal degeneration, dementia with argyrophilic grains, dementia pugilistica or chronic traumatic encephalopathy, Down’s syndrome, subacute sclerosing panencephalitis, mild cognitive impairment, Niemann-Pick disease, type C, Sanfilippo syndrome type B, or a myotonic dystrophy DM1 or DM2.8621 A method as claimed in any one of claims 1 to 18 wherein the disorder is Alzheimer’s disease (“AD”), which is optionally mild to moderate AD.22 A method as claimed in claim 21 wherein the MT compound is administered twice daily and the second dosage level is 40 mg or more.23 A method as claimed in claim 21 wherein the MT compound is administered once daily and the second dosage level is 32 mg or more.24 A method as claimed in any one of claims 21 to 23 wherein the first and second periods and dosages are selected to provide an amount of MT to the subject which results in a plasma MT level of at least 0.74 ng / ml throughout the entire first and second treatment periods.25 A method as claimed in claim 21 wherein the second dosage level is 56 mg / day or more.26 A method as claimed in any one of claims 1 to 18 wherein the disorder is mild cognitive impairment.27 A method as claimed in claim 26 wherein the first dosage level is between 12 and 16 mg / day and wherein the second dosage level is between 16 and 20 mg / day.28 A method as claimed in claim 27 wherein the first dosage level is 12 mg / day.29 A method as claimed in claim 27 or claim 28 wherein the second dosage level is 16 mg / day.30 A method as claimed in any one of claims 26 to 29 wherein the first and second periods and dosages are selected to provide an amount of MT to the subject which results in a plasma MT level of at least 0.40 ng / ml throughout the entire first and second treatment periods.31 A method as claimed in any one of claims 1 to 18 wherein the disorder is a polyglutamine disorder, such as Huntington’s disease, spinal bulbar muscular atrophy, dentatorubropallidoluysian atrophy or spinocerebellar ataxias; wherein the disorder is a TDP-43 proteinopathy, such as FTLD-TDP; wherein the disorder is a synucleinopathy, such as Parkinson's disease, dementia with Lewy bodies or multiple system atrophy; wherein the disorder is hereditary cerebral angiopathy; wherein the disorder is amyotrophic lateral sclerosis; or wherein the disorder is familial encephalopathy with neuronal inclusion bodies.87***32 A method as claimed in any one of claims 1 to 31 wherein the compound is a salt of either:(MT+)or a hydrate or solvate thereof.33 A method as claimed in claim 32 wherein a mixture of LMT and MT+compounds are administered.34 A method as claimed in claim 32 wherein the compound is an LMT compound. 35 A method as claimed in claim 34 wherein the compound is an LMTX compound of the following formula:wherein each of HnA and HnB (where present) are protic acids which may be the same or different,and wherein p = 1 or 2; q = 0 or 1 ; n = 1 or 2; (p + q) x n = 2.36 A method as claimed in claim 35 wherein the compound has the following formula, where HA and HB are different mono-protic acids:8837 A method as claimed in claim 35 wherein the compound has the following formula:wherein each of HnX is a protic acid.38 A method as claimed in claim 35 wherein the compound has the following formula and H2A is a di-protic acid:39 A method as claimed in claim 35 wherein the compound has the following formula and is a bis-monoprotic acid:40 A method as claimed in any one of claims 35 to 39 wherein the or each protic acid is an inorganic acid.41 A method as claimed in claim 40 wherein each protic acid is a hydrohalide acid.8942 A method as claimed in claim 40 wherein the or each protic acid is selected from HCI; HBr; HNO3;H2SO4.43 A method as claimed in any one of claims 35 to 39 wherein the or each protic acid is an organic acid.44 A method as claimed in claim 43 wherein the or each protic acid is selected from CH3COOH; methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid.45 A method as claimed in claim 44 wherein the compound is LMTM:46 A method as claimed in claim 43 wherein the compound is selected from the list consisting of:10 47 A method as claimed in claim 32 wherein the compound is an MT+salt having the formula or being a hydrate, solvate, or mixed salt thereof:91where X-is an anionic counter ion.48 A method as claimed in claim 47 wherein the compound is MTC.49 A method as claimed in claim 48 wherein the compound is MTC polymorph A pentahydrate.50 A method as claimed in any one of claims 47 to 49 wherein the compound is characterised by a purity of greater than 98%.51 A method as claimed in any one of claims 47 to 50, wherein the compound is characterised by a purity of greater than 98% and one or more of the following:(i) less than 1% Azure B as impurity;(ii) less than 0.13% MVB (Methylene Violet Bernstein) as impurity;(iii) less than 0.15% Azure A as impurity;(iv) less than 0.15% Azure C as impurity; or(v) an elementals purity better than less than 100 pg / g Aluminium (Al); less than 1 pg / g Cadmium (Cd); less than 100 pg / g Chromium (Cr); less than 300 pg / g Copper (Cu); less than 10 pg / g Tin (Sn); less than 200 pg / g Iron (Fe); less than 10 pg / g Manganese (Mn); less than 1 pg / g Mercury (Hg); less than 10 pg / g Molybdenum (Mo); less than 10 pg / g Nickel (Ni); less than 10 pg / g Lead (Pb); and less than 100 pg / g Zinc (Zn).52 A method as claimed in any one of claims 47 to 51 , wherein the compound is characterised by a purity of greater than 98% and less than 1 % Azure B as impurity.53 A method as claimed in any one of claims 47 to 52, wherein the compound is characterised by a purity of greater than 98% and an elementals purity better than less than 100 pg / g Aluminium (Al); less than 1 pg / g Cadmium (Cd); less than 100 pg / g Chromium (Cr); less than 300 pg / g Copper (Cu); less than 10 pg / g Tin (Sn); less than 200 pg / g Iron (Fe); less than 10 pg / g Manganese (Mn); less than 1 pg / g Mercury (Hg); less than 10 pg / g Molybdenum (Mo); less than 10 pg / g Nickel (Ni); less than 10 pg / g Lead (Pb); and less than 100 pg / g Zinc (Zn).54 A method as claimed in any one of claims 47 to 53, wherein the compound is characterised by:(I) at least 98% purity(i) less than 1 % Azure B as impurity; and(ii) an elementals purity better than the European Pharmacopeia limits of less than 100 pg / g Aluminium (Al); less than 1 pg / g Cadmium (Cd); less than 100 pg / g Chromium (Cr); less than 300 pg / g Copper (Cu); less than 10 pg / g Tin (Sn); less than 200 pg / g Iron (Fe); lessthan 10 pg / g Manganese (Mn); less than 1 pg / g Mercury (Hg); less than 10 pg / g Molybdenum (Mo); less than 10 pg / g Nickel (Ni); less than 10 pg / g Lead (Pb); and less than 100 pg / g Zinc (Zn).55 A method as claimed in claim 32 wherein the compound is selected from:MTC.0.5ZnCI2; MTI ; MTI.HI ; MT.NO3.56 A method as claimed in any one of claims 47 to 55 wherein the MT+salt is formulated with a reducing agent which is optionally ascorbate, and then optionally lyophilized.57 A container comprising:(i) a plurality of dosage units each of which is a composition comprising an MT compound as defined in any one of claims 32 to 56, and a pharmaceutically acceptable carrier or diluent; (ii) a label and\or instructions for their use according to a method as defined in any one of claims 1 to 56.58 A container as claimed in claim 57 wherein the label or instructions provide information regarding the disorder for which the composition is intended and the first and second dosage periods and the first and second dosage levels.59 An MT-containing compound as described in any one of claims 1 to 56, for use in a method of treatment as defined in any one of claims 1 to 56.60 Use of an MT-containing compound or composition as described in any one of claims 1 to 56, in the manufacture of a medicament for use a method of treatment as defined in any one of claims 1 to 56.

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