Therapeutic interactions of leucomethylthioninium.

Abstract

The present invention generally relates to methods for treating Alzheimer's disease or mild cognitive impairment, adapted to avoid negative interactions between combinations of therapeutic agents. In particular, such methods of treatment are disclosed in which the order of therapeutic agents is actively controlled, and homeostatic downregulation is alleviated prior to administration of an active agent, e.g., a disease-modifying agent, a therapeutic agent. In certain embodiments, treatment with a symptomatic treatment (e.g., a modulator of acetylcholine or glutamate neurotransmitter activity) can be subsequently combined with a disease-modifying or other active treatment. The present invention also applies knowledge regarding homeostatic downregulation to novel methods of clinical trial design.

Description

[Technical field]

[0001] The present invention relates generally to methods of treating Alzheimer's disease or mild cognitive impairment that are adapted to avoid negative interactions between combinations of therapeutic agents or to enhance the effects of the therapeutic agents. [Background technology]

[0002] The only treatments currently available for the treatment of Alzheimer's disease (AD) are symptomatic. The most widely used of these are acetylcholinesterase inhibitors (AChEIs), which act by chronically increasing the levels of acetylcholine (ACh) in the synaptic cleft. In experimental models, cholinergic function is primarily associated with selective attention (Botly and De Rosa, 2007;2008;Sarter et al., 2016) and is not particularly sensitive to more widespread measures of dysfunction / improvement (reviewed in (Klinkenberg and Blokland, 2010;Robinson et al., 2011)). Similar considerations apply to memantine, which also modulates brain function in a nonspecific manner (Gastambide et al., 2012;Ding et al., 2018). The therapeutic benefits of these treatments are relatively short-lived (Courtney et al., 2004), with less than 30% of patients continuing AChEIs 12 months after initiation (Mauskopf et al., 2005; Singh et al., 2005; Raina et al., 2008). A significant proportion of AD patients are not treated anyway, about 44% in the United States (Koller et al., 2016) and about 77% in the United Kingdom (Martinez et al., 2013). In France, reimbursement for these drugs has been withdrawn due to "insufficient medical benefit and risk of side effects" (Krolak-Salmon et al., 2018). Low medical use and patient adherence are due to low perceived efficacy and side effects, and the rate of decline after a temporary symptomatic improvement is not different from that occurring in the absence of treatment (Courtney et al., 2004). It is therefore generally agreed that there is a major unmet medical need to develop treatments that can slow the progression of AD.The Lancet Neurology Commission report (Winblad et al., 2016) stated that "there is still no treatment available to halt or reverse the underlying pathology of established AD. Indeed, an effective treatment for AD is perhaps the greatest unmet need facing modern medicine." The last new treatment approved for AD was memantine in 2003 (Lanctot et al., 2009). Between 2002 and 2012, 289 clinical trials in phase 2 or 3 were conducted, with an overall failure rate of 99.6% (Cummings et al., 2014). Since 2012, there have been 19 additional failed trials targeting various aspects of the pathological processing of β-amyloid (Panza et al., 2019).

[0003] Despite the limitations of available symptomatic treatments, almost all ongoing or recently completed late-stage clinical trials aimed at testing novel therapeutic approaches have been conducted in patient populations where the majority of subjects continue to receive symptomatic treatment (Panza et al., 2019). This is partly dictated by the ethical issue that patients participating in sometimes long-term clinical trials are denied access to any treatment if randomized to a placebo arm. A further consideration has been the unproven hypothesis that symptomatic treatments do not interfere with treatments targeting the underlying pathology due to their different modes of action.

[0004] Methylthioninium (MT) acts in vitro as a tau aggregation inhibitor at brain concentrations consistent with human oral dosing in transgenic mouse tau models (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 behavioral deficits (Melis et al., 2015a; Baddeley et al., 2015).

[0005] Leucomethylthioninium bis(hydromethanesulfonate) (LMTM; USAN name, hydromethylthioniine mesylate) is being developed as a treatment to target pathological aggregation of tau protein in AD (Wischik et al., 2018). The methylthioninium (MT) moiety is a cytosolic agonist that converts tau protein into its oxidized form (MT + ) and reduced (LMT) forms. LMTM is the oxidized form MT + It is a stabilized salt of LMT that has much better pharmaceutical properties than the MT form (Baddeley et al., 2015; Harrington et al., 2015). + Instead, we recently reported that LMT is the active species that blocks tau aggregation in vitro (Al-Hilaly et al., 2018). LMT blocks tau aggregation in vitro in cell-free and cell-based assays (Harrington et al., 2015;Al-Hilaly et al., 2018) and reduces tau aggregation pathology and associated behavioral impairments at clinically relevant doses in vivo in tau transgenic mouse models (Melis et al., 2015a). LMT also disaggregates tau protein in paired helical filaments (PHFs) isolated from AD brain tissue, converting tau to a form susceptible to proteases (Wischik et al., 1996;Harrington et al., 2015).

[0006] The MT portion also has a series of other potentially beneficial properties. At low concentrations (10-100 nM), it has been previously known to promote mitochondrial activity by acting as an auxiliary electron carrier in the electron transfer chain. The MT portion undergoes redox cycling catalyzed by complex I using NADH as a cofactor, whereby the MT portion accepts electrons, which are subsequently transferred to complex IV (Atamna et al., 2008;Atamna et al., 2012). The MT portion can also induce mitochondrial biogenesis and activate Nrf2-mediated oxidative stress response elements in vivo (Stack et al., 2014). Other activities include neuroprotective effects in the brain by inhibiting microglial activation and increasing autophagy (Zhao et al., 2016). The MT moiety has been shown to increase the clearance of pathological tau in vivo by enhancing autophagy in the concentration range of 10-20 nM (Congdon et al., 2012). Thus, in addition to dissolving AD tau aggregates, LMTM has a number of complementary actions that correspond to many of the pathways currently proposed to have treatment potential for AD (Oz et al., 2009;Schirmer et al., 2011).

[0007] Orally given LMTM produces brain levels sufficient for in vitro and in vivo activity (Baddeley et al., 2015), but it had minimal apparent efficacy when taken as an add-on to symptomatic treatment in two large Phase 3 clinical trials (Gauthier et al., 2016;Wilcock et al., 2018). However, in subjects receiving LMTM as monotherapy, treatment produced a significant slowing of cognitive and functional decline, a reduction in the rate of progression of brain atrophy measured by MRI, and a reduction in the decrease in glucose uptake measured by FDG-PET (Gauthier et al., 2016;Wilcock et al., 2018). When these results were analyzed in conjunction with population pharmacokinetic data available from subjects participating in the trial, it was found that LMTM produced concentration-dependent effects whether taken alone or in combination with symptomatic treatment. However, the treatment effect in monotherapy subjects was substantially greater than in those taking LMTM in combination with symptomatic treatment.

[0008] WO 2008 / 155533 relates to MT-containing compounds for treating MCI.

[0009] WO 2009 / 060191 relates to the design of clinical trials for putative therapeutic agents for neurodegenerative disorders. It is particularly anticipated that subjects receiving symptomatic treatment may be included in the trials.

[0010] WO 2018 / 019823 describes novel regimens for the treatment of neurodegenerative disorders utilizing methylthioninium (MT)-containing compounds. The publication summarizes previous disclosures of MT-containing compounds, in particular "LMTX" compounds, for treating such disorders, including AD and mild cognitive impairment (MCI). WO 2018 / 019823 identified two important factors. The first was related to the dosage of MT compounds, and the second was their interaction with symptomatic treatments.

[0011] WO 2020 / 020751 describes novel dosing regimens for LMT compounds that maximize the proportion of subjects whose in vivo MT concentrations exceed those required for therapeutic efficacy. Summary of the Invention [Problem to be solved by the invention]

[0012] Disclosure of the Invention The present studies were conducted with the objective of understanding the mechanisms involved in the reduced efficacy of LMTM as an add-on to the symptomatic treatments discussed above. In these studies, a well-characterized tau transgenic mouse model (Line 1, "L1"; (Melis et al., 2015b)) was compared to wild-type mice. [Means for solving the problem]

[0013] In tau transgenic mice expressing short tau fragments that constitute the tangle filaments of AD, we showed that LMTM given alone increased hippocampal acetylcholine (ACh) levels, glutamate release from synaptosomal preparations, synaptophysin levels in multiple brain regions, and mitochondrial complex IV activity, reduced tau pathology, restored choline acetyltransferase (ChAT) immunoreactivity in the basal forebrain, and reversed deficits in spatial learning.

[0014] Chronic pretreatment with rivastigmine was found to reduce or eliminate almost all of these effects, apart from a reduction in tau aggregation pathology and restoration of ChAT immunoreactivity in the basal forebrain. LMTM effects on hippocampal ACh and synaptophysin levels were also reduced in wild-type mice.

[0015] Thus, the clinically observed interference with LMTM efficacy by cholinesterase inhibitors can also be reproduced in tau transgenic mouse models, and to a lesser extent, in wild-type mice.

[0016] We observed that pretreatment with symptomatic drugs altered a broad range of brain responses to LMTM across different transmitter systems and cellular compartments at multiple levels of brain function. There was no single locus of negative interactions. Rather, chronic neuronal activation induced by reducing cholinesterase function produced compensatory homeostatic downregulation in multiple neural systems. This has the effect of reducing a broad range of treatment responses to LMTM that are associated with a reduction in tau aggregation pathology.

[0017] Importantly, because interference is determined by the homeostatic response to previous symptomatic treatment, it is inevitable that there will be similar interference with other drugs tested as "add-ons" to existing symptomatic treatments, regardless of the intended therapeutic target or mode of action. This summary outlines key results that will now provide a working model to explain interference by symptomatic treatments. In other words, any activating pretreatment can be expected to produce a compensatory homeostatic downregulation response, thereby attenuating the effectiveness of the second treatment if the second treatment is also activating.

[0018] The experiment we carried out was designed to mimic the clinical condition of adding LMTM in patients who have already received symptomatic treatment.However, a further implication of the model concerns treatment in which LMTM (or other therapeutic agent) is started before the above symptomatic treatment.If it is introduced later as an "add-on" treatment, it is inevitable that the treatment effect of LMTM will dominate if homeostatic downregulation is determined by the treatment that comes first, even though it can reduce the response to add-on symptomatic treatment to some extent.

[0019] Further experiments were performed to establish the effect of withdrawal of LMTM after 18 months of treatment. In these experiments, the patient group was divided according to whether they had received LMTM as an add-on to existing symptomatic treatment (AChEI, memantine "Achmem") or as monotherapy. The patient group was further divided according to whether the subjects had received higher or lower Cmax exposure to MT according to the pharmacokinetic modeling described in WO2020 / 020751.

[0020] Unexpectedly, the high Cmax add-on group actually showed unexpected improvement in ADAS-cog following withdrawal of LMTM, which is consistent with a disease-modifying effect during LMTM treatment resulting in improved response to symptomatic treatment alone.This finding has potential implications for the use of LMTM and Achmem combination therapy, for example, in patient groups that have proven unresponsive to Achmem, the disease-modifying effect of LMTX may actually enhance response to Achmem, especially when LMTX is discontinued, and especially with respect to ADAS-cog in mild AD subjects. This has utility because many patients do not respond to Ach inhibitor treatment (e.g., 30-40% or more; see, e.g., McGleenon BM, Dynan KB, Passmore AP. Acetylcholinesterase inhibitors in Alzheimer's disease. Br J Clin Pharmacol. 1999;48(4):471-480. doi:10.1046 / j.1365-2125.1999.00026.x).

[0021] Thus, the present disclosure relates to methods of treatment of Alzheimer's disease or mild cognitive impairment, in which the sequence of therapeutic agents is actively controlled, for both MT-containing and non-MT-containing compounds, e.g., to prevent homeostatic downregulation prior to administration of the active therapeutic agent. In certain embodiments, treatment with a neurotransmission-modifying compound (e.g., a modulator of acetylcholine or glutamate neurotransmitter activity) as discussed above may be subsequently combined with treatment with an MT-containing or non-MT-containing compound, or may follow treatment with an MT-containing or non-MT-containing compound.

[0022] The present disclosure also utilizes this knowledge with respect to clinical trial design. [Brief description of the drawings]

[0023] drawing [Figure 1] Pharmacokinetic-pharmacodynamic response on the ADAS-cog scale over 65 weeks in patients with mild to moderate AD taking LMTM at a dose of 8 mg / day and classified according to the status of concomitant medications with AD-labeled treatments. [Diagram 2] Effect of treatment with LMTM alone or following chronic pretreatment with rivastigmine on hippocampal levels of acetylcholine (A) or synaptophysin levels measured by immunohistochemistry as mean values ​​for the hippocampus, visual cortex, diagonal band and septal area (B) in wild-type mice. (**, p<0.01; ***, p<0.001). [Diagram 3] Effect of treatment with LMTM alone or following chronic pretreatment with rivastigmine on (A) SNARE complex proteins (SNAP25, syntaxin and VAMP2) and (B) α-synuclein levels measured by immunohistochemistry as mean values ​​for the hippocampus, visual cortex, diagonal band and septal area in tau transgenic L1 mice. (*, p<0.05; ***, p<0.001; ****, p<0.0001). [Figure 4]Effect of treatment with LMTM alone or following chronic pretreatment with rivastigmine on complex IV activity measured relative to citrate synthase activity in brain mitochondria in tau transgenic L1 mice. (*, p<0.05). [Diagram 5] Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine on levels of tau immunoreactivity (relative optical density) (A) and neurons immunoreactive for choline acetyltransferase (B) in the vertical diagonal band in tau transgenic L1 mice compared to vehicle-treated wild-type mice. (*, p<0.05; **, p<0.01; ***, p<0.001). [Figure 6] Schematic diagram of the treatment effect of LMT subjected to chronic pretreatment with acetylcholinesterase inhibitor (AChEI) rivastigmine (with a particular focus on changes in mitochondrial metabolism and presynaptic proteins) and dynamic modulation with tau aggregation inhibitor activity. Combined treatment with AChEI does not impair the effect of LMT on tau aggregation pathology. In contrast, the combination prevents the increase in synaptic proteins, ACh release and increased complex IV activity seen following treatment with LMTM alone. [Figure 7] Effects of combined administration of memantine and LMTM on problem-solving impairment in female Line 1 mice aged 5.5 months at the start of the study. Memantine was administered at 20 mg / kg and LMTM at 15 mg / kg. Mice were treated with vehicle or memantine for 5 weeks prior to 6 weeks of memantine plus LMTM treatment, and mice were tested in the water maze task over weeks 10 and 11. [Figure 8]Withdrawal (ADAS-cog) following 18 months of LMTM (8 mg / day) monotherapy. As can be seen in subjects in the high exposure (Cmax) group, they continue to decline at the same rate following withdrawal of LMTM (change=0.73; p-value=0.1651). This supports a sustained disease-modifying change in the rate of cognitive decline following the LMTX treatment period. Subjects exposed to lower exposure suffered a greater decline (change=2.29; p-value=0.0011), meaning that the benefit of LMTX in that group may be at least partially symptomatic and therefore not sustained. [Figure 9] Withdrawal (ADAS-cog) following 18 months of LMTM (8 mg / day) as an "add-on" to existing symptomatic treatment (AChEI, memantine). As can be seen in subjects in the high exposure (Cmax) group, they show improvement following withdrawal of LMTM (change = -0.98, p-value = 0.0021). This is consistent with a disease-modifying effect during LMTM treatment resulting in an improved response to symptomatic treatment alone, or a negative effect on symptomatic treatment shown by LMTM during the LMTX treatment period. Subjects exposed to lower exposure continue to decline at the same rate following withdrawal (change = 0.51, p-value = 0.0855). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Thus, in one aspect, there is provided a method of therapeutic treatment of Alzheimer's disease or mild cognitive impairment in a subject, comprising: The method includes administering to the subject a therapeutic agent, a methylthioninium (MT)-containing compound; MT-containing compounds include LMTX compounds of the formula: [ka] (In the formula, H n A and H n each B (when present) is a protic acid, which may be the same or different; p=1 or 2; q=0 or 1; n=1 or 2; (p+q)×n=2) or a hydrate or solvate thereof, The subject is one of the following groups: (i) a subject who has not previously been treated with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity; or (ii) Subjects who have previously been treated with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity, but who have discontinued such treatment for at least 3, 4, 5, 6, 7, or 8 weeks prior to treatment with the LMTX compound. Selected from; The therapeutic treatment with LMTX is maintained for a treatment window without concurrent administration of a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity; Followed by; Treatment with LMTX is accompanied by simultaneous administration of a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity; The treatment time frame prior to simultaneous administration of the therapeutic compound with the neurotransmission modifying compound is at least two months.

[0025] In another aspect, a method of therapeutic treatment of Alzheimer's disease or mild cognitive impairment in a subject is provided, comprising: The method includes administering to the subject a therapeutic compound that does not contain MT; The subject is one of the following groups: (i) a subject who has not previously been treated with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity; or (ii) Subjects who have previously been treated with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity, but who have discontinued such treatment for at least 3, 4, 5, 6, 7, or 8 weeks prior to treatment with the LMTX compound. Selected from; said therapeutic treatment with said therapeutic compound is maintained for a treatment window without concurrent administration of a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity; Optionally followed by; treatment with said therapeutic compound accompanied by simultaneous administration of a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity; The treatment time frame prior to simultaneous administration of the therapeutic compound with the neurotransmission modifying compound is at least two months.

[0026] In another aspect, a method is provided for assessing the efficacy of a non-MT containing compound that is a putative therapeutic agent for Alzheimer's disease or mild cognitive impairment in a subject, the method comprising: (a)(i) a subject who has not previously been treated with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity; and (ii) Subjects who have previously been treated with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity, but who have discontinued such treatment for at least 3, 4, 5, 6, 7, or 8 weeks prior to said assessment. selecting a subject group consisting of only (b) stratifying the subject group into at least two subgroups according to baseline indicators of possible disease progression; (c) treating each subject group member with a non-MT containing compound during a treatment window; (d) deriving psychometric and optionally physiological outcome measures for each treated patient group; (e) comparing the results in (d) with a comparison arm of said subgroup, which is optionally a placebo or a minimal efficacy comparison arm; (f) using the comparison in (e) to derive an efficacy measure for the non-MT containing compound; Includes.

[0027] In these embodiments, if previous treatment has been present, it has been discontinued 3, 4, 5, or 6 or more weeks prior to the treatment or assessment, most preferably 6 weeks prior.

[0028] In these embodiments, if there is concurrent administration, the treatment time frame prior to concurrent administration of the therapeutic compound with the neurotransmission modifying compound is preferably at least 2 months, 3 months, 4 months, 5 months or 6 months, most preferably at least 6 months.

[0029] In another aspect, a method of therapeutic treatment of Alzheimer's disease or mild cognitive impairment in a subject is provided, comprising: The method includes administering to the subject a therapeutic compound; the subject is undergoing treatment with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity; The therapeutic treatment with the therapeutic compound is maintained for a first treatment window with simultaneous administration of a neurotransmission modulating compound, during which the dosage of the neurotransmission modulating compound is reduced to zero, followed by treatment with the therapeutic compound without simultaneous administration of a neurotransmission-modifying compound during a second treatment window; The first treatment window is 2 weeks to 8 weeks, The second treatment window is at least 3 months, more preferably at least 6 months or at least 12 months.

[0030] The first treatment time frame can more preferably be 3 to 8 weeks, e.g., 3 to 6 weeks, or about 1 or 2 months. Preferably, the reduction in dosage is steady, e.g., linear, over the period, so that the subject is not exposed to abrupt withdrawal.

[0031] By "titrating off" a neurotransmission-modifying compound (e.g., an AChEI or an N-methyl-D-aspartate receptor antagonist) from a subject, it can be expected, based on the disclosures herein, that the homeostatic response due to the condition may eventually be replaced by a response to a therapeutic compound (which may be MT-containing or otherwise).

[0032] In another aspect, a method of therapeutic treatment of Alzheimer's disease or mild cognitive impairment in a subject is provided, comprising: the subject is selected as being non-responsive to treatment with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity; The method includes administering to the subject a therapeutic agent, a methylthioninium (MT)-containing compound; MT-containing compounds include LMTX compounds of the formula: [ka] (In the formula, H n A and H n each B (when present) is a protic acid, which may be the same or different; p=1 or 2; q=0 or 1; n=1 or 2; (p+q)×n=2) or a hydrate or solvate thereof, This is followed by treatment with said neurotransmission modulating compound.

[0033] Alzheimer's disease can be mild Alzheimer's disease. LTMX can be used for a treatment window with simultaneous administration of neurotransmission modulating compound, followed by treatment with neurotransmission modulating compound without LMTX, where optionally, the treatment window with simultaneous administration is at least 2 months, but can be at least 6 months, 12 months or 18 months, for example, up to 24 months.

[0034] Thus, there is provided the use of a LMTX compound (or a combination of a LMTX compound and a neurotransmission modulating compound) to increase the responsiveness of a subject to a neurotransmission modulating compound, particularly a subject that would otherwise be thought of as unresponsive to such neurotransmission modulating compounds.

[0035] Certain aspects and embodiments of the invention will now be considered in more detail.

[0036] Patient Selection As explained above, depending on the context, selection in the context of the present invention relates to subjects and their situation regarding previous exposure to neurotransmission modulating compounds that are modulators of the activity of acetylcholine or glutamate neurotransmitters. Such a compound is memantine, which is an AChEI or N-methyl-D-aspartate receptor antagonist. Examples of acetylcholinesterase inhibitors include donepezil (Aricept™), rivastigmine (Exelon™) or galantamine (Reminyl™). An example of an NMDA receptor antagonist is memantine (Exibe™, Namenda™).

[0037] In some embodiments, the subject population may be completely naive to these other treatments, having not previously received one or both of these.

[0038] In other embodiments, the subject group may have previously received one or both of these treatments, but has discontinued the medication for at least 2, 3, 4, 5, 6, 7, 8, 12, or 16 weeks, or optionally at least 1, 2, 3, 4, 5, or 6 months, etc., prior to treatment with the MT compounds according to the invention. By "discontinued" is meant either stopped at that point or steadily tapered to zero.

[0039] In other embodiments, there may be an initial period of simultaneous administration of a therapeutic agent and a neurotransmission modulating compound, during which the dosage of the neurotransmission modulating compound is "tapered" to zero as described above.

[0040] In other embodiments, subject groups may be selected as being non-responsive to treatment with neurotransmission modulating compounds that are modulators of acetylcholine or glutamate neurotransmitter activity, i.e., have not demonstrated significant clinical benefit to one or both of these symptomatic treatments; typically, in such cases, the drug is considered unsuitable for the patient group, but as described herein, previous or concomitant treatment with LMTX may create a benefit for subsequent use of these symptomatic treatments.

[0041] Aspects of the invention include active steps of selecting a group of subjects according to these criteria. Such active selection typically involves specific referral to the subject or caregiver and appropriate measures, including (1) exclusion if the subject continues treatment with the neurotransmission modulating compound without discontinuation before or during the first time window with the therapeutic agent, or (2) admission if the subject is treatment-naive or able to discontinue treatment for a sufficient time, before or during the period of simultaneous administration of the therapeutic agent when the dosage of the neurotransmission modulating compound is "tapered" to zero as described above.

[0042] Combination therapy In the present invention, there is at least one time window during which treatment with a therapeutic compound (which may or may not be MT-containing) is performed without treatment with a symptomatic treatment (neurotransmission-modifying compound).

[0043] This treatment may be a monotherapy, ie, without other active treatments, or it may be a combination therapy (with another active treatment, eg, a non-symptomatic treatment).

[0044] After the initial treatment, the therapeutic compound may be combined (or recombined) with a symptomatic treatment (neurotransmission modifying compound).

[0045] Thus, the term "treatment" includes "combination" treatments, where two or more treatments that treat related diseases are combined, for example sequentially or simultaneously.

[0046] In combination treatments, the agents (i.e., an MT-containing compound, e.g., as described herein, or a non-containing compound, plus one or more other agents) may be administered simultaneously or sequentially, with individually varying dose schedules and by different routes. For example, when administered sequentially, the agents can be administered closely spaced apart (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1 hour, 2 hours, 3 hours, 4 hours or more, or even longer periods apart, in some circumstances), with the exact dosage regime commensurate with the properties of the therapeutic agents.

[0047] Thus, in one embodiment, administration of an MT compound is initiated or continued in a subject who has not received an AChEI or memantine (for a period of time), but then treatment with such AChEI or memantine treatment is initiated or resumed after a period of treatment with the MT compound, for example, after approximately two months, or more preferably, after six months of treatment with the MT compound.

[0048] Typically, such symptomatic concurrent treatment is commenced (or resumed) when the physician determines that such would benefit the subject.

[0049] Disease-modifying and symptomatic treatment In the methods of the invention, the therapeutic MT-containing or non-containing compounds are preferably "disease-modifying" (or putatively "disease-modifying") as opposed to symptomatic in action.

[0050] This property may be inferred a priori, for example, based on a known or predicted effect on the pathogenesis of the disorder in question.

[0051] Symptomatic drugs slow or alleviate the symptoms of the disease without affecting the underlying disease process and do not change the rate of long-term decline after the initial period of treatment. A treatment is considered symptomatic if, after withdrawal, the patient returns to the situation they would have been in without treatment (Cummings, JL (2006) Challenges to demonstrating disease-modifying effects in Alzheimer's disease clinical trials. Alzheimer's and Dementia, 2:263-271).

[0052] Memantine, an AChEI and N-methyl-D-aspartate receptor antagonist, is an example of a symptomatic treatment. Tau aggregation inhibitors are examples of disease-modifying treatments.

[0053] Disease modification can also be inferred from clinical evidence, for example, if after withdrawal from treatment, the patient reverts to the situation they would have been in without the treatment, the treatment may be considered symptomatic rather than disease modifying. Alternatively (in the context of clinical trials), a treatment is considered to be disease modifying if patients randomized late to active treatment are unable to catch up with patients randomized early to active treatment.

[0054] LMTX compound Some aspects of the invention relate to "LMTX" compounds of the type described, for example, in WO 2007 / 110627 or WO 2012 / 107706.

[0055] Thus, the compound may be selected from compounds of the formula: or a hydrate or solvate thereof:

[0056] [Table 1]

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

[0058] "Protonic acid" refers to the amount of protons (H + ) donor. In the protonic acid, A - Or B - is therefore the conjugate base. Thus, protic acids have a pH of less than 7 in water (i.e., the concentration of hydronium ions is less than 10 per liter). -7 (Molar excess).

[0059] In one embodiment, the salt has the formula:

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

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

[0062] [Table 3] wherein H n Each X is a protic acid, for example a diprotic acid or a monoprotic acid.

[0063] In one embodiment, the salt has the formula:

[0064] [Table 4] where H2A is a diprotic acid.

[0065] Preferably, the salt is a bismonoprotic acid salt of the formula

[0066] [Table 5] has.

[0067] Examples of protonic acids that may be present in the LMTX compounds used herein are: Inorganic acids: hydrohalic acids (e.g., HCl, HBr), nitric acid (HNO3), sulfuric acid (H2SO4), Organic acids: Carbonic acid (H2CO3), acetic acid (CH3COOH), methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid Includes.

[0068] Preferred acids are monoprotic acids and the salts are bis(monoprotic acid) salts.

[0069] A preferred MT compound is LMTM.

[0070] [Table 6]

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

[0072] Other weight coefficients can be calculated for the example MT compounds herein, and corresponding dosage ranges can be calculated therefrom.

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

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

[0075] Other example LMTX compounds are as follows, their molecular weights (anhydrous) and weight coefficients are also given:

[0076] [Table 7]

[0077] [Table 8]

[0078] In the various aspects of the invention described herein (as they relate to MT-containing compounds), this can optionally be any of those compounds described above.

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

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

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

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

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

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

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

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

[0087] Alternatively, the compound may be a hydrate, solvate, or a mixed salt of any of these.

[0088] LMTX Dosage Based on previous and concurrent results of using LMTM in the treatment of diseases, it can be concluded that MT dosages ranging from 2 to 80 or 100 mg / day may be beneficial for the diseases described herein.

[0089] More specifically, further analysis of the concentration response for LMTM with respect to the treatment of disease supports the contention that a preferred dose is at least 2 mg / day, and that doses in the range of 20-40 mg / day, or 20-60 mg / day, are expected to maximize cognitive benefits, while nevertheless maintaining a desirable profile with respect to being well tolerated with minimal side effects.

[0090] Thus, in one embodiment, the total MT dose can be from about any of 2, 2.5, 3, 3.5, 4 mg to about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mg.

[0091] Example doses are 2-60 mg.

[0092] An example dosage is 20-40 mg.

[0093] Further example dosages are 8 or 16 or 24 mg / day.

[0094] The subject of the present invention may be an adult human, and the dosages described herein are based on that basis (typical body weight 50-70 kg). If necessary, corresponding dosages may be utilized for subjects outside this range by using a subject weight factor, thereby dividing the subject's body weight by 60 kg to provide a multiplicative factor for that individual subject.

[0095] As one of skill in the art will recognize, for a given daily dose, more frequent dosing results in greater accumulation of drug.

[0096] We derived a putative accumulation factor for MT as follows.

[0097] [Table 9]

[0098] For example, considering a total daily dose of 3.5-7 mg of MT: When given as a single daily dose, this may equate to an accumulation of MT in plasma of 4.5 to 8 When divided by bi-dilution, this may equate to an accumulation of MT in plasma of 5.1 to 10.3. When divided by tid, this may equate to an accumulation of MT in plasma of 5.8 to 11.6.

[0099] Thus, in certain embodiments of the invention, when dosed more frequently (eg, twice a day [bid] or three times a day [tid]), the total daily dosage of the MT compound may be lower.

[0100] In one embodiment, LMTM is administered at approximately 9 mg once daily; 4 mg bid; 2.3 mg tid (based on the weight of LMTM).

[0101] In one embodiment, LMTM is administered at approximately 34 mg once daily; 15 mg bid; 8.7 mg tid (based on the weight of the LMTM).

[0102] The MT compound of the present invention, or a composition containing it, is administered orally to a subject.

[0103] In some embodiments, the MT compound is administered as a composition comprising a LMTX compound as described herein and a pharma- ceutically acceptable carrier, excipient, or additive.

[0104] The term "pharmacologically acceptable," as used herein, refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable for use in contact with the tissues of a subject of interest without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, additive, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0105] Compositions comprising LMTX salts have been described in several published documents, for example, WO 2007 / 110627, WO 2009 / 044127, WO 2012 / 107706, WO 2018019823, and WO 2018041739.

[0106] In some embodiments, the compositions are compositions comprising at least one LMTX compound as described herein, together with one or more other pharma- ceutically acceptable ingredients known to those of skill in the art, including, but not limited to, pharma- ceutically acceptable carriers, excipients, additives, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweetening agents.

[0107] In some embodiments, the composition further comprises other active agents.

[0108] Suitable carriers, excipients, additives, etc. can be found in standard pharmaceutical textbooks, 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.

[0109] In some embodiments, the composition is a tablet.

[0110] In some embodiments, the composition is a capsule.

[0111] In some embodiments, the capsule is a gelatin capsule.

[0112] In some embodiments, the capsule is an HPMC (hydroxypropyl methylcellulose) capsule.

[0113] In some embodiments, the amount of MT in the unit is between 2 and 60 mg.

[0114] In some embodiments, the amount of MT in the unit is 10-40, or 10-60 mg.

[0115] In some embodiments, the amount of MT in the unit is 20-40, or 20-60 mg.

[0116] An example dosage unit may contain from 2 to 10 mg of MT.

[0117] Further example dosage units may contain 2-9 mg of MT.

[0118] Further example dosage units may contain 3-8 mg of MT.

[0119] Further preferred dosage units may contain 3.5 to 7 mg of MT.

[0120] Further preferred dosage units may contain 4-6 mg of MT.

[0121] In some embodiments, the amount is about 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg of MT.

[0122] Using the weight factors described or illustrated herein, one of ordinary skill in the art can select the appropriate amount of MT-containing compound for use in an oral formulation.

[0123] As explained above, the MT weight factor for LMTM is 1.67. Because it is convenient to use uniform or simple fractions of the active ingredient, non-limiting example LMTM dosage units may contain about 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 34, 50, 63 mg, etc.

[0124] Non-MT-containing compounds Some aspects of the present invention relate to methylthioninium (MT)-free therapeutic compounds that are used or intended for the treatment of Alzheimer's disease or mild cognitive impairment.

[0125] Such compounds may be intended to be disease-modifying or symptomatic. The primary pathologies that have been targeted for disease modification in AD are tau and amyloid. Such drugs include those intended to reduce amyloid-β (Aβ) production and promote Aβ clearance, as well as drugs that increase tau modification and inhibit aggregation.

[0126] Non-limiting examples of compounds reported to be suitable for such treatment are as follows:

[0127] Drugs targeting amyloid-β (Aβ) processing Drugs that affect Aβ include BACE inhibitors, immunotherapeutic approaches (both passive and active immunotherapy), α-secretase inhibitors or modulators, γ-secretase activators, and Aβ aggregation inhibitors including metal chelators (e.g., PBT2) ( Panza et al., 2019 ).

[0128] Drugs that target tau protein Drugs that target tau protein include tau aggregation inhibitors (e.g., daunorubicin, Congo Red, anthraquinones, benzothiazoles, cyanine dyes, phenylthiazolyl-hydrazides, N-phenylamines, rhodanines, polyphenols, porphyrins, quinoxalines, aminothienopyridazines, oleocanthal, curcumin, etc.); microtubule stabilizers (e.g., epithilone D, dictyostatin; TPI-287); kinase inhibitors that prevent phosphorylation of tau (e.g., tideglusib, sodium selenate, LiCl); acetylation inhibitors (salsalate); deglycosylation inhibitors (MK8719); and phosphodiesterase 4 inhibitors (BPN14770) (Congdon and Sigurdsson, 2015; see in particular FIG. 4). In addition, tau immunotherapy approaches are in clinical trials (e.g., AADvac1, ACI-35, RG7345).

[0129] Drugs targeting neural regeneration-based strategies include, for example, ciliary neurotrophic factor-based peptides ( Kazim and Iqbal, 2017 ).

[0130] Drugs with multiple putative AD-relevant targets, such as dimebon (or latrepirdine) ( Ustyugov et al., 2018 ).

[0131] Another proposed therapeutic approach for AD is via miRNA inhibitors that target synaptic connections, defects in assembly, and enhanced clearance of Aβ or tau, impairment in neurotrophic support, spread of neuroinflammatory signaling, and alterations in the innate immune response (Jaber et al., 2019). miRNA inhibitors are single-stranded modified RNAs that specifically inhibit endogenous miRNA function in cells. Neuronal miRNAs are involved in various stages of synapse development, including dendritogenesis (involving miR-132, miR-134, and miR-124), synaptogenesis, and synapse maturation (involving miR-134 and miR-138).

[0132] Drugs that have an effect on improving symptoms of AD Although less preferred, the present invention may also be applied in principle to non-disease modifying treatments, such as different symptomatic treatments that may otherwise be affected by the homeostatic response described herein, such as stimulants, such as amphetamines (see, e.g., Dolder, Christian R., Lauren Nicole Davis, and Jonathan McKinsey. "Use of psychostimulants in patients with dementia." Annals of Pharmacotherapy 44.10 (2010): 1624-1632.).

[0133] MCI Mild cognitive impairment is recognized by the FDA as a valid disease target. It is defined by having a slight degree of cognitive impairment that does not yet meet the clinical criteria for a diagnosis of dementia.

[0134] Representative criteria for syndromic MCI include the features listed below: A. The patient is neither normal nor demented. B. There is evidence of cognitive decline as demonstrated by objectively measured decline over time in conjunction with objective cognitive tests (e.g., memory subtests) and / or subjective reports of decline by the individual and / or informants. C. Activities of daily living are maintained and complex instrumental functions are intact or minimally impaired. (See also Winblad, B. et al. (2004) “Mild cognitive impairment - beyond controversies, towards a consensus: report of the International Working Group on Mild Cognitive Impairment.” J. Intern. Med. 256: 240-246.)

[0135] As used above, the term "dementia" refers to a mental condition in its broadest sense as defined in the American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Washington, DC, 1994 ("DSM-IV"). DSM-IV defines "dementia" as characterized by multiple cognitive deficits, including impairment in memory, and lists various dementias with presumed etiologies. DSM-IV represents the generally accepted standard for such diagnosis, classification and treatment of dementia and related psychiatric disorders.

[0136] MCI subjects in whom the present invention may preferably be used are those with an MMSE score of 24, 25, 26, 27, 28 or 29 or less, more preferably an MMSE score of 24, 25, 26 or less, and most preferably an MMSE score of 24 or 25 or less. The MMSE test is discussed in more detail herein below.

[0137] Another aspect of the invention relates to therapeutic compounds as described herein for use in such methods, e.g., methods of therapeutic treatment of Alzheimer's disease or mild cognitive impairment in a subject, or methods for assessing the effectiveness of compounds, which are putative therapeutic agents for Alzheimer's disease or mild cognitive impairment in a subject.

[0138] Another aspect of the invention pertains to the use of a therapeutic compound, as described herein, in the manufacture of a medicament for use in such a method.

[0139] Certain aspects of the present invention relate to methods for assessing the effectiveness of non-methylthioninium (MT)-containing compounds that are putative therapeutic agents for AD or MCI in a subject.

[0140] WO 2009 / 060191 relates to the design of clinical trials for putative therapeutic agents for neurodegenerative disorders, and is specifically incorporated herein by cross-reference with respect to the general conduct and procedure of analyses for such trials.

[0141] Although the methods of the invention generally relate to clinical trials for testing pharmaceuticals (or putative pharmaceuticals, e.g., investigational medicinal products (IMPs)), they may also be used to administer therapy, whereby new treatment regimens with pharmaceuticals are tested or compared for their effectiveness.

[0142] Thus, the methods herein may be used to conduct clinical trials or to provide a system for conducting said trials.

[0143] Such a system may be for assessing the efficacy of a methylthioninium (MT)-free compound that is a putative therapeutic agent for Alzheimer's disease or mild cognitive impairment in a subject, the system comprising: (a)(i) a subject who has not previously been treated with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity; and (ii) Subjects who have previously been treated with a neurotransmission-modifying compound that is a modulator of acetylcholine or glutamate neurotransmitter activity, but who have discontinued such treatment for at least 3, 4, 5, 6, 7, or 8 weeks prior to said assessment. selecting a subject group consisting of only (b) stratifying the subject group into at least two subgroups according to baseline indicators of possible disease progression; (c) selecting a treatment time window during which members of each subject group will be treated with a non-MT-containing compound; (d) selecting psychometric and optionally physiological outcome measures to be derived for each treated patient group and for a comparison arm of said subgroup, optionally a placebo or minimal efficacy comparison arm; whereby efficacy measures for a pharmaceutical product can be derived from comparisons of treated patient groups and comparison arms. Includes.

[0144] The method is particularly suitable for providing evidence of clinical efficacy suitable to meet appropriate regulatory standards for marketing, such as those required by the US Food and Drug Administration (FDA) or the European Agency for the Evaluation of Medicinal Products (EMEA).

[0145] Subject groups and possible predictive indicators of disease progression The subject population typically includes patients diagnosed with the disorder of interest using standard criteria (e.g., National Institute of Neurological and Communicative Disorders and Stroke - Alzheimer's Disease and Related Disorders Association [NINCDS-ADRDA]; DSM-IV, etc.).

[0146] DSM-IV thus describes the generally accepted standards for diagnosing, classifying and treating dementia and related psychiatric disorders.

[0147] In the methods of the invention, the subject group is itself stratified by baseline indicators of possible disease progression, which can be assessed with respect to disease severity.

[0148] Preferably, in AD, the severity of the disease is assessed using the so-called Clinical Dementia Rating (CDR) scale (Hughes, CP, Berg, L., Danziger, WL, Coben, LA, Martin, RL (1982) A new clinical scale for the staging of dementia. British Journal of Psychiatry, 140:566-572; Morris, JC (1993) The Clinical Dementia Rating (CDR): Current version and scoring rules. Neurology, 43:2412-2414).

[0149] The CDR may optionally be informed by a clinical competency test, for example a short version of CAMDEX (Roth, M., Tym, E., Mountjoy, CQ, Huppert, FA, Hendrie, H., Verma, S. & Goddard, R. (1986) CAMDEX. A standardised instrument for the diagnosis of mental disorder in the elderly with special reference to the early detection of dementia. British Journal of Psychiatry, 149:698-709). Alternatively, the CDR may be informed by a psychiatric structured interview as defined by Hughes et al. (1982) or Morris (1993).

[0150] For example, subgroups may be formed from subjects with a CDR score of 1 (mild subgroup) or 2 (moderate subgroup).

[0151] Disease severity may also be assessed using the "Braak staging" method, for example, as described in WO 02 / 075318. Subgroups may then be formed from subjects with Braak stages 1, 2, 3 and up to 4, etc.

[0152] As noted above, the subject group is further actively selected with respect to their prior exposure to certain symptomatic treatments.

[0153] Subgroups are generally tested in parallel.

[0154] Treatment Time Frame Treatment time frames for AD or MCI may be selected based on the severity of disease in subgroups. Typical time frames for clinical trials according to the present invention may be 12 weeks or more, 16 weeks or more, 24 weeks or more, 25 weeks or more, 36 weeks or more, 50 weeks or more, 100 weeks or more (or 3 months or more, 4 months or more, 6 months or more, 9 months or more, 12 months or more, 24 months or more, etc.). With such selected time frames, the present invention provides for the use of novel measures or analyses to derive more accurate measures of pharmaceutical efficacy.

[0155] Preferred trials may be of less than the above durations, for example, less than 9 months, less than 6 months, less than 5 months, less than 4 months, or less than 3 months.

[0156] For example, for shorter time scales and, for example, in patients with relatively low disease severity at baseline (where cognitive decline as measured by psychometric outcome measures may be masked by cognitive reserve), it may be preferable to use additional physiological outcome measures and / or more sensitive psychometric outcome measures.

[0157] Other trials may be longer than 6 months or longer than 12 months.

[0158] For example, for longer time scales (and in patients with relatively high disease severity at baseline, where "good survivor" artifacts are more likely to occur), it may be preferable to use linear imputation methods for each individual who discontinues treatment to correct the analysis for the effect of discontinuation.

[0159] The time frames may be the same or different for the subgroups.

[0160] Psychometric outcome measures Psychometric outcome measures for use in the methods may be any conventional one accepted by an appropriate regulatory agency.

[0161] For AD, the Alzheimer's Disease Assessment Scale-Cognitive Subscale [ADAS-cog] is preferred (Rosen WG, Mohs RC, Davis KL. A new rating scale for Alzheimer's disease. Am J Psychiatry. 1984 Nov;141(11):1356-64).

[0162] Another standardized test is the Mini-Mental State Examination [MMSE], which has been proposed as a simple and quickly administered method to grade cognitive function (Folstein MF, Folstein SE & McHugh PR. 'Mini-mental state'. A practical method for grading the cognitive state of patients for the clinician. Journal of Psychiatric Research 1975 12 189-198). The MMSE is the most widely used cognitive screening tool for detecting dementia-related cognitive impairment in elderly and psychiatric patients (Tombaugh TN & McIntyre NJ. The mini-mental state examination: a comprehensive review. Journal of the American Geriatric Society 1992 40 922-935). The MMSE assesses orientation, memory, attention and language function.

[0163] Assessment or analysis of psychometric outcome measures may include performing a linear imputation analysis on the available psychometric scores of individual subjects who discontinued treatment, which may involve subject-by-subject extrapolation of a line fitted to a graph of the scores (e.g., ADAS-cog change scores).

[0164] Physiological outcome measures As described herein, in addition to psychometric testing, we provide for the use of neurophysiological outcome measures, for example, via analysis of changes in functional brain scans, which increases the sensitivity of analysis of disease-modifying treatments when testing for relatively short periods of time, for example, 3 or 4 months.

[0165] Scan the ligand 99m SPECT (single photon emission computed tomography) with Tc-HMPAO or in the temporoparietal association neocortex in AD 18 A reduction in cerebral glucose uptake as measured by PET (positron emission tomography) using fluoro-deoxyglucose (FDG) may also be used.

[0166] For diagnosis (see, e.g., Talbot, PR, Lloyd, JJ, Snowden, JS, Neary, D., Testa, HJ (1998) A clinical role for 99mTc-HMPAO SPECT in the investigation of dementia? Journal of Neurology, Neurosurgery and Psychiatry, 64:306-313.; Masdeu, JC, Zubieta, JL, Arbizu, J. (2005) Neuroimaging as a marker of the onset and progression of Alzheimer's disease. Journal of the Neurological Sciences, 236:55-64) and in investigating response to treatment (Venneri, A., Shanks, MF, Staff, RT, Pestell, SJ, Forbes, KE, Gemmell, HG, Murray, AD (2002) Cerebral blood flow and cognitive responses to rivastigmine treatment in Alzheimer's disease. NeuroReport; 13:83-87), and the use of such scans is generally well known in the art.

[0167] Magnetic resonance-based structural imaging is an essential part of the clinical assessment of patients with suspected Alzheimer's dementia. Atrophy of medial temporal structures is considered to be a useful diagnostic marker in the mild cognitive impairment stage. Furthermore, the rate of whole brain and hippocampal atrophy is a sensitive marker of neurodegeneration (Frisoni et al., 2010, Nature Reviews Neurology 6:67-77). Conversely, ventricular enlargement detected using magnetic resonance imaging can also be used as a measure of Alzheimer's disease progression (Nestor et al., 2008, Brain 131: 2443-2454).

[0168] It will be appreciated that in addition to these techniques, any other applicable methodology that allows a direct measure of pathological burden in the brain may be used.

[0169] Comparison arm Following randomization of the subgroups, some subjects in each subgroup are selected for a comparison treatment. Preferably, this is the placebo (no treatment) or minimal efficacy comparison arm of the trial. However, alternative designs (where it is unethical to withhold an existing treatment) involve randomization to an alternative active treatment arm, either alone or in some pre-specified combination.

[0170] Final efficacy measures Typically, this will be based on a suitable statistical method for comparing the relevant treatment and placebo arms, and ultimately on psychometric results. Optionally, this may be ANCOVA, or a linear mixed-effects approach, as shown in the examples below (Petkova, E. and Teresi, J. (2002) Some statistical issues in the analysis of data from longitudinal studies of elderly chronic care populations. Psychosomatic Medicine, 64:531-547). Physiological outcome measures may also be used in the final analysis. In this regard, appropriate clinical endpoints indicating efficacy can be selected by those skilled in the art in view of the present disclosure.

[0171] Specifically, efficacy can be demonstrated when a statistically significant difference exists between subjects randomized to active treatment at some specified dose and subjects receiving a comparison treatment, dose, or placebo.

[0172] The therapeutic compounds described herein (e.g., a prescribed dose of an MT-containing compound, plus optionally other ingredients) can be provided in a labeled packet with instructions for their therapeutic or prophylactic use, e.g., their use in monotherapy or combination therapy, as discussed above.

[0173] In one embodiment, the pack is a bottle as is well known in the pharmaceutical art. A typical bottle may be made from pharmacopoeia grade HDPE (high density polyethylene) with a child-resistant HDPE push lock closure and may contain a silica gel desiccant present in a sachet or canister. The bottle itself may be packaged in a cardboard container with a label and instructions for use as described in the method, and optionally, a further copy of the label.

[0174] In one embodiment, the pack or packet is thus a blister pack (preferably one with an aluminum cavity and aluminum foil) that is substantially moisture impermeable. In this case, the pack may be packaged in a cardboard container with instructions for use and a label on the container.

[0175] The label or instructions may provide information regarding the maximum allowable daily dosage of a composition as described herein, for example, on a once daily, bid, or tid basis.

[0176] The label or instructions may provide information regarding the recommended duration of treatment.

[0177] Salts and solvates Although the LMTX-containing compounds described herein are salts, they may also be provided in the form of mixed salts (i.e., a compound of the invention in combination with another salt). Such mixed salts are intended to be encompassed by the term "and pharma-ceutically acceptable salts thereof." Unless otherwise specified, a reference to a particular compound also includes its salts.

[0178] The compound of the present invention may also be provided in the form of a solvate or hydrate. The term "solvate" is used herein in its normal sense to refer to a complex of a solute (e.g., a compound, a salt of a compound) and a solvent. When the solvent is water, the solvate may conveniently be referred to as a hydrate, for example, a monohydrate, a dihydrate, a trihydrate, a pentahydrate, etc. Unless otherwise specified, a reference to a compound also includes a solvate and any hydrate form thereof.

[0179] Of course, solvates or hydrates of the salts of the compounds are also encompassed by the present invention.

[0180] Treatment and Prevention The term "treatment", as used herein in the context of treating a condition, generally relates to the treatment and care of a human or animal (e.g., in veterinary applications) where some desired therapeutic effect is achieved, such as inhibition of the progression of the condition, including reducing the rate of progression, halting the rate of progression, regression of the condition, amelioration of the condition, and curing the condition.

[0181] The present invention also encompasses treatment as a prophylactic measure.

[0182] The term "therapeutically effective amount," as used herein, refers to that amount of a compound of the invention, or a material, composition, or dosage form comprising said compound, that is effective to produce some desired therapeutic effect commensurate with a reasonable benefit / risk ratio when administered in accordance with a desired treatment regimen. Therapeutic compounds are typically administered in a "therapeutically effective amount" or a "prophylactically effective amount."

[0183] The term "prophylactically effective amount," as used herein, refers to that amount of a compound of the invention, or a material, composition or dosage form containing said compound, that is effective, when administered in accordance with a desired treatment regimen, to produce some desired prophylactic effect commensurate with a reasonable benefit / risk ratio.

[0184] "Prophylaxis," in the context of this specification, should not be understood as addressing complete success, i.e., complete protection or complete prevention. Rather, in this context, prophylaxis refers to measures taken prior to symptomatic detection of a condition, with the goal of preserving health, by helping to delay, alleviate or avoid that particular condition.

[0185] Several 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 by reference in its entirety into this disclosure to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0186] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are 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.

[0187] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0188] Ranges are often expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

[0189] Any subheadings herein are included for convenience only and are not to be construed as limiting the disclosure in any way.

[0190] The invention will now be further described with reference to the following non-limiting figures and examples, in view of which other embodiments of the invention will occur to those skilled in the art.

[0191] The disclosures of all references cited in this specification, in so far as they may be used by those skilled in the art to carry out the present invention, are hereby expressly incorporated herein by cross-reference. EXAMPLES

[0192] Working Example Example 1 - Provision of MT-containing compounds Methods for the chemical synthesis of the MT-containing compounds described herein are known in the art. For example: Compounds 1 to 7 can be synthesized by the methods described in WO 2012 / 107706 or methods similar thereto. The synthesis of compound 8 can be carried out by methods described in WO 2007 / 110627 or methods analogous thereto.

[0193] Example 2 - Investigating the interference between LMTM and symptomatic treatment in a tau transgenic mouse model FIG. 1 illustrates an established intervention, where LMTM was then taken in combination with a symptomatic treatment.

[0194] We performed studies in a well-characterized tau transgenic mouse model (Line 1, "L1"; (Melis et al., 2015b)) with the aim of understanding the mechanisms responsible for the reduced efficacy of LMTM as an add-on to symptomatic treatment.

[0195] In summary, our findings suggest that homeostatic mechanisms downregulate multiple neural systems at different levels of brain function, compensating for chronic pharmacological activation induced by symptomatic treatments.

[0196] The effect of this downregulation is to reduce neurotransmitter release, levels of synaptic proteins, mitochondrial function and behavioral benefits when LMTM is given against a background of prior chronic exposure to an acetylcholinesterase inhibitor, compared to LMTM given alone. Thus, the interference in treatment efficacy initially seen clinically has a clear neuropharmacological basis that can be reproduced in the tau transgenic mouse model.

[0197] Importantly, the homeostatic effects we identified may have more general relevance for conducting disease-modifying trials in AD or MCI, or indeed other types of therapeutic compound trials, that need not be limited to tau aggregation inhibitors.

[0198] In the L1 mouse model used in some of the present studies, there is overexpression of a three-repeat tau fragment encompassing residues 296-390 of the 2N4R tau isoform under the control of the Thy1 promoter in the NMRI mouse strain (WO 2002 / 059150). This fragment corresponds to a segment of tau originally identified within the proteolytically stable core of PHFs (Wischik et al., 1988a;Wischik et al., 1988b) and encompasses fragment 306-378 recently identified by cryo-electron microscopy of PHFs in AD and tau filaments in Pick's disease (Fitzpatrick et al., 2017;Falcon et al., 2018).

[0199] Further characteristics of the L1 mouse model include a marked loss of neuronal immunoreactivity for choline acetyltransferase in the basal forebrain, and a corresponding reduction in acetylcholinesterase in the neocortex and hippocampus, indicating a reduction in acetylcholine.There is also a roughly 50% reduction in glutamate release for brain synaptosome preparations from L1 mice compared to those from wild-type mice.In this regard, therefore, L1 mice also mimic the neurochemical impairments in cholinergic (Mesulam, 2013; Pepeu and Grazia Giovannini, 2017) and glutamatergic (Revett et al., 2013) functions that are characteristic of AD.

[0200] Underlying these impairments in neurotransmitter function, the L1 mouse model shows impairments in the integration of synaptic proteins. Quantitative immunohistochemistry for multiple synaptic proteins in the basal forebrain (vertical diagonal band) shows that there is usually a high degree of correlation in the levels of proteins comprising the SNARE complex (e.g., SNAP-25, syntaxin, VAMP2; reviewed in Li and Kavalali, 2017) as well as the vesicle glycoproteins synaptophysin and α-synuclein in wild-type mice. These correlations are largely lost in L1 mice (Table 1). The only correlations remaining are between synaptophysin, syntaxin, and VAMP2. Thus, synaptic vesicle protein levels are no longer quantitatively linked to proteins of the SNARE complex or α-synuclein. This suggests that tau oligomer pathology in L1 mice interferes with the functional integration between vesicles and membrane docking proteins at the synapse.

[0201] [Table 10]

[0202] Example 3 - Experimental paradigm, results and discussion Experimental Paradigm The treatment schedule used to study the negative interaction between symptomatic treatment and LMTM was designed to mimic the clinical condition, where subjects were first treated chronically with a cholinesterase inhibitor or memantine, and then given LMTM. Below, we summarize some of the key results obtained with the AChEI, rivastigmine.

[0203] Wild-type and L1 mice (n=7-16 for each group) were pretreated with rivastigmine (0.1 or 0.5 mg / kg / day) or memantine (2 or 20 mg / kg / day) or vehicle by gavage for 5 weeks. For the following 6 weeks, LMTM (5 and 15 mg / kg) or vehicle was added to this daily treatment regimen, also by gavage. Animals were behaviorally tested using a problem-solving task in an open-field water maze during weeks 10 and 11 and then sacrificed for immunohistochemistry and other tissue analyses.

[0204] Converting the dose from mice to humans requires consideration of several factors. A dose of 5 mg / kg / day in mice reduces the C of parent MT in plasma by 100 mg / kg / day. max Although this corresponds to roughly 8 mg / day in humans in terms of levels, this dose is at the threshold for effects on pathology and behavior. Higher doses of 15 mg / kg / day are generally required for LMTM to be fully effective in the L1 mouse model (Melis et al., 2015a). This may be related to the much shorter half-life of MT in mice (4 h) compared to humans (37 h in aged humans). Tissues dissected for immunohistochemistry were labeled with antibodies and processed using Image J, and protein expression was determined by densitometry. Data are presented as unitless Z-score transformation.

[0205] For measurement of acetylcholine (ACh) levels in the hippocampus, animals (wild type or L1) were treated with LMTM (5 mg / kg / day, 2 weeks) after 2 weeks of prior treatment with or without rivastigmine (0.5 mg / kg / day). Rivastigmine was administered subcutaneously with an Alzet minipump, while LMTM was administered by oral gavage. ACh levels were measured in the hippocampus using an implanted microdialysis probe and HPLC analysis of the extracellular fluid.

[0206] Data are presented as group means and standard errors of the means and were analyzed using parametric statistics with alpha set at 0.05.

[0207] Experiments on animals were performed in accordance with the European Communities Council Directive (63 / 2010 / EU) with local ethical approval, a project license according to the UK Scientific Procedures Act (1986), and in accordance with the German Law for Animal Protection (Tierschutzgesetz) and the Polish Law on the Protection of Animals.

[0208] result Effect of LMTM and rivastigmine treatment in wild-type mice The effects of treatment with LMTM alone or on a background of chronic rivastigmine are summarized in Table 2.

[0209] In wild-type mice, there was a significant 2-fold increase in basal ACh levels in the hippocampus following LMTM treatment, and a 30% reduction when mice received LMTM after prior treatment with rivastigmine (FIG. 2A).

[0210] There was a three-fold increase in synaptophysin levels measured in the hippocampus, visual cortex, diagonal band, and septal area following LMTM treatment alone, and also a statistically significant reduction of the same magnitude when LMTM was given against the background of prior treatment with rivastigmine (Figure 2B).

[0211] [Table 11]

[0212] Effect of LMTM and rivastigmine treatment in tau transgenic L1 mice The activating effect of LMTM alone and the inhibitory effect of combination with rivastigmine are greater and more generalized in tau transgenic L1 mice than in wild-type mice (see Table 3). LMTM alone produces a significant increase in ACh release in the hippocampus, glutamate release from brain synaptosome preparations, synaptophysin levels, mitochondrial complex IV activity, and behavioral changes. None of these effects were seen when LMTM was preceded by chronic rivastigmine. Indeed, in the case of SNARE complex proteins (Figure 3A) and synuclein (Figure 3B), the reduction produced by the combination was at a level below that seen in the absence of LMTM treatment.

[0213] [Table 12]

[0214] LMTM given alone produced a significant increase in complex IV activity in brain mitochondria from tau transgenic L1 mice. Chronic pretreatment with rivastigmine also eliminated this effect (Figure 4).

[0215] In contrast to the effects on neurotransmitter release, synaptic protein levels and mitochondrial complex IV activity, chronic pretreatment with rivastigmine has no effect on the primary action of LMTM as a tau aggregation inhibitor. As expected, immunoreactivity against core tau units in PHFs measured by optical density was elevated in tau transgenic L1 mice, which was reduced following treatment with LMTM (Figure 5A). Conversely, the number of ChAT-positive neurons was reduced in L1 mice and restored by treatment with LMTM (Figure 5B). Both effects persist in L1 mice when LMTM is given after previous chronic treatment with rivastigmine.

[0216] Consideration of Example 3 The results presented here show that the reduction in the efficacy of LMTM can be reproduced in both wild-type and tau transgenic mouse models when given as an add-on to symptomatic treatment in humans. It is therefore based on a neuropharmacological mechanism that has the effect of changing how the brain responds to disease-modifying treatments, e.g., LMTM. The results imply that differences in clinical response to LMTM as monotherapy or add-on therapy may be explained by differences in the underlying neuropharmacology of LMTM in these two settings (Gauthier et al., 2016;Wilcock et al., 2018). Instead, explanations based on the presumption that patients prescribed symptomatic treatments are somehow different from untreated patients fail for several reasons. It has been shown that minor and variable differences in baseline severity between these two patient groups do not explain differences in treatment response (Gauthier et al., 2016;Wilcock et al., 2018). The apparent differences in the rate of decline in treated and untreated MCI patients in the ADNI program (Schneider et al., 2011) disappear when baseline severity is accounted for in the analysis (Wilcock et al., 2018). The presumption that untreated patients do not actually have AD or have a different form of AD is also inconsistent with baseline neuroimaging data from subjects participating in phase 3 trials (Wilcock et al., 2018). Finally, as summarized for the cognitive decline data in Figure 1, we have recently shown that similar concentration-response relationships exist in monotherapy and add-on therapy subjects, but the treatment effect is consistently larger for monotherapy for all clinical and neuroimaging outcomes.

[0217] The results we now report show that there are two classes of effects produced by LMTM treatment in wild-type and tau transgenic mice: those that are subject to dynamic modulation by prior exposure to a cholinesterase inhibitor, and those that are not. In tau transgenic mice, treatment effects that can be modulated include increased ACh release in the hippocampus, changes in synaptic proteins, increases in mitochondrial complex IV activity, and reversal of behavioral decline. The only treatment effects that are not subject to pharmacological modulation are the primary effects on tau aggregation pathology, as measured, for example, by restoration of choline acetyltransferase expression in the basal forebrain, and its immediate effects on neuronal function.

[0218] The two classes of LMTM treatment effects are summarized in FIG.

[0219] The effects subject to pharmacological modulation are themselves of two types: those that are augmented by the effects on tau aggregation pathology, and those that are also seen in wild-type mice. Among the outcomes we measured, the positive treatment effects of LMTM given alone in wild-type mice included increased ACh levels in the hippocampus and increased synaptophysin levels in multiple brain regions. Thus, LMTM treatment can activate neuronal function at therapeutically relevant doses in wild-type mice that lack tau aggregation pathology.

[0220] An increase in synaptophysin indicates an increase in the number or size of synaptic vesicles required for release of neurotransmitters from presynapses following activation via action potentials. Thus, increased levels of synaptophysin appear to be associated with an increase in several neurotransmitters required to support cognitive and other mental functions.

[0221] Although the MT moiety has been reported to be a weak cholinesterase inhibitor ( Pfaffendorf et al., 1997 ; Deiana et al., 2009 ), it is unlikely that this is the mechanism responsible for increasing ACh levels.

[0222] Specifically, further experiments using scopolamine, which increases ACh levels (by blocking M2 / M4 negative feedback receptors), showed that the increase produced by LMTM was less than that seen with rivastigmine alone, and the combination was again inhibitory in wild-type mice. Under the conditions of cholinesterase inhibition used in these experiments (very small amounts of the cholinesterase inhibitor, 100 nanomolar rivastigmine, added to the perfusate), ACh levels in the hippocampus increased, and when they increased strongly enough, they limited further ACh release by activating presynaptic muscarinic receptors of the M2 / M4 subtype (so-called negative feedback receptors).

[0223] In this situation, the addition of scopolamine (1 μM) to the perfusate blocks these presynaptic receptors, resulting in a 3- to 5-fold increase in ACh levels. The fact that LMTM is not additive with rivastigmine in these experiments supports the conclusion that LMTM has a different mechanism of action than rivastigmine. In other words, although LMTM has been described as a weak inhibitor of cholinesterase at high concentrations, this effect appears to be independent of cholinesterase inhibition, since there is no additive effect with small amounts of rivastigmine.

[0224] The increase in ACh and synaptophysin levels could be theoretically explained by an increase in presynaptic mitochondrial activity, since the MT portion is known to enhance mitochondrial complex IV activity (Atamna et al., 2012), and mitochondria have an important role in homeostatic regulation of presynaptic function (Devine and Kittler, 2018). In particular, the MT portion is thought to enhance oxidative phosphorylation by acting as an electron shuttle between complex I and complex IV (Atamna et al., 2012). The MT portion has a redox potential of approximately 0 mV, which is intermediate between the redox potentials of complex I (-0.4 mV) and complex IV (+0.4 mV).

[0225] However, direct measurements of complex IV activity in wild-type mice showed no increase following LMTM treatment. The activating effects of LMTM also did not correlate with improvements in spatial recognition memory in wild-type mice.

[0226] Chronic pretreatment with rivastigmine suppressed cholinergic activation in the hippocampus and reduced synaptophysin levels more generally in the brains of wild-type mice. This effect is apparently not dependent on the effect of LMTM on tau aggregation pathology, since no pathology is present in wild-type mice. Rather, they point to a general homeostatic downregulation that counteracts the effect of combining two drugs, each of which has an activating effect on neuronal function. Presumably, the primary mechanism that normally protects against excessive levels of ACh in the synaptic cleft is increased AChE activity. Because rivastigmine produces a chronic depression of this control system, pathways that are otherwise activated by LMTM are suppressed to preserve homeostasis in cholinergic and other nervous systems. Thus, when the brain is already subjected to chronic stimulation with a cholinesterase inhibitor, the effects induced by LMTM are subject to dynamic downregulation.

[0227] Although qualitatively similar, the effects of LMTM given alone are much more pronounced and broader in tau transgenic L1 mice. The most likely explanation for this is that LMTM combines an inhibitory effect on tau oligomers with an intrinsic activating effect that is not tau-dependent. The reduction in tau oligomer levels following LMTM treatment promotes more pronounced activation of synaptic function and release of neurotransmitters such as ACh and glutamate. Similarly, LMTM reverses spatial memory deficits seen in tau transgenic L1 mice (Melis et al., 2015a). The negative effects seen when LMTM is introduced against a background of chronic rivastigmine appear to simply reflect the reversal of activation seen with LMTM alone.

[0228] The deleterious effect of tau oligomers on synaptic protein function can be easily understood as a result of direct interference with synaptic vesicle docking, membrane fusion and neurotransmitter release. In tau transgenic L1 mice, synaptic vesicle protein levels are no longer quantitatively linked to proteins of the SNARE complex or α-synuclein, implying a loss of functional integration between vesicles and membrane docking proteins in the synapse. The consequences of this can be seen directly as an impairment in glutamate release from synaptosomal preparations from tau transgenic mice, and restoration of normal glutamate release following treatment with LMTM.

[0229] The mechanism involved in the mitochondrial effect of LMTM is more complex. The MT portion is thought to enhance oxidative phosphorylation by acting as an electron shuttle between complex I and complex IV (Atamna et al., 2012). The MT portion has a redox potential of approximately 0 mV, which is intermediate between the redox potentials of complex I (-0.4 mV) and complex IV (+0.4 mV). However, LMTM has no effect on complex IV activity in brain mitochondria isolated from wild-type mice. In contrast, a strong effect was seen in tau transgenic L1 mice, suggesting that tau oligomers interfere with mitochondrial metabolism. C-terminally truncated tau proteins have recently been shown to both bind to the outer mitochondrial membrane and enter the intermembrane space of mitochondria (Cieri et al., 2018). Truncated PHF-tau proteins isolated from brain tissue of AD patients form SDS-resistant complexes with voltage-dependent anion-selective channel proteins (VDACs; formerly porins) in the outer mitochondrial membrane, and also with ATP synthase subunit 9 and core protein 2 of complex III in the intermembrane space (Wischik et al., 1997). These binding interactions are likely detrimental to the functioning of the electron transport chain in mitochondria, and the effect of LMTM in reducing tau oligomer accumulation in and around mitochondria may contribute to the activation of complex IV seen in L1 mice.

[0230] It is not known how the homeostatic downregulation resulting from rivastigmine treatment may affect mitochondrial function. Mitochondria are involved in the regulation of Ca 2+ It is known that ATP is an important homeostatic regulator of synaptic function through buffering ATP levels and ATP production ( Devine and Kittler, 2018 ).

[0231] It is noteworthy that the positive effects of LMTM, and their reversal or inhibition by pretreatment with anticholinesterases, can be seen across different transmitter systems and cellular compartments at multiple levels of brain function, implying that there is no single locus responsible for the interference in the LMTM treatment response. Rather, the negative interactions appear to be part of a general homeostatic downregulation in multiple neural systems that compensates for the chronic pharmacological activation resulting from acetylcholinesterase blockade.

[0232] The results with memantine are shown in Figure 7 and show a similar depiction to the anticholinesterase pretreatment, which is expected given that the clinically seen interference in LMTM efficacy is very similar for the two drug classes.

[0233] More generally, interference that affects LMTM treatment is unlikely to be specific to LMTM: any treatment that has an activating effect on synaptic function, whether by reducing the primary pathology or by another mechanism, is likely to be subject to similar interference, since any treatment will be primarily facilitated by existing symptomatic treatments.

[0234] Thus, if clearance of amyloid aggregates leads to synaptic activation, as has been proposed by Marsh, J, Alifragis, P (2018), this can be inferred to hinder the ability of symptomatic treatments to also demonstrate this effect clinically.

[0235] A further consideration is whether the homeostatic downregulation we have shown works similarly when LMTM treatment is first and symptomatic treatment is added later.The experiments we have performed to date were originally designed to mimic clinical symptoms, but here LMTM is added to patients who are already receiving symptomatic treatment.If homeostatic downregulation is determined by the treatment that comes first, it is inevitable that the treatment effect of LMTM will be dominant, even though the response to add-on symptomatic treatment may be reduced to some extent.

[0236] In summary, our findings point to a powerful role for homeostatic control systems in the brain. Such systems are well understood and well documented in many neurophysiological contexts. It is therefore entirely plausible that treatment interventions designed to boost neuronal function induce homeostatic control that limits the degree of neuronal hyperactivation. In the case of cholinergic function, excessive activity is highly harmful, clinically resulting in convulsions, coma and death. This is entirely consistent with chronic stimulation of the brain by symptomatic treatments that alter the way the brain responds to other therapeutic interventions.

[0237] Example 4 - Withdrawal Analysis for LMTM 8 mg / day as Monotherapy or Add-on Therapy Following 18 months of treatment with 8 mg / day LMTM, subjects were required to undergo a one-month "drug holiday" followed by a cognitive assessment of change. Groups were split according to whether they had been receiving LMTM as an add-on to existing symptomatic treatment (AChEI, memantine, collectively abbreviated as "Achmem") or as monotherapy.

[0238] The groups were further analyzed for subjects who had received high or low Cmax exposure. As explained in WO2020 / 020751, pharmacokinetic modeling can be used to split the 8 mg / day treated population into groups of individuals with a "higher" estimated Cmax and groups of individuals with a "lower" estimated Cmax. WO2020 / 020751 describes the division of patients by a threshold of 0.37 ng / ml (including 35% of patients with the lowest value). The treatment difference between "high" and "low" Cmax patients receiving a dose of 8 mg / day is -3.4 ADAS-cog units.

[0239] The figures in this analysis were as follows:

[0240] [Table 13]

[0241] As shown in FIG. 8 (monotherapy), the high Cmax group continued to decline at the same rate following withdrawal of LMTM, supporting a sustained disease-modifying change in the rate of cognitive decline following the LMTX treatment period.

[0242] Subjects exposed to a lower Cmax suffered a large decline, implying that the benefit of LMTX in that group may be at least partially symptomatic and therefore not sustained.

[0243] As shown in Figure 9 (add-on), the high Cmax group actually showed an unexpected improvement following withdrawal of LMTM, consistent with a disease-modifying effect during LMTM treatment resulting in an improved response to symptomatic treatment alone, or a negative effect on symptomatic treatment shown by LMTM during the LMTX treatment period.

[0244] This finding has potential implications for the use of LMTX and Achmem combination therapy: for example, in patient populations that have proven non-responsive to Achmem, the disease-modifying effect of LMTX may actually enhance the response to Achmem, especially in relation to ADAS-cog in mild AD subjects, once LMTX is discontinued.

[0245] In contrast, subjects exposed to lower exposures continue to decline at the same rate following withdrawal.

[0246] These results support the concentration-response analysis in WO2020 / 020751. LMTM as a monotherapy has significant pharmacological activity at both high and low levels of exposure, but generally below 0.38 ng / ml, the effect may be more symptomatic than disease-modifying. As an add-on therapy, LMTM has significant pharmacological activity even at low levels of exposure, but exposure below about 0.378 ng / ml as an add-on does not provide a discernible treatment effect.

[0247] References Congdon, EE, Sigurdsson, EM (2018) Tau-targeting therapies for Alzheimer disease. Nat Rev Neurol 14:399-415 Jaber, VR, Zhao, Y, Sharfman, NM, Li, W, Lukiw, WJ (2019) Addressing Alzheimer’s disease (AD) neuropathology using anti-microRNA (AM) strategies. Molecular Neurobiology. https: / / doi.org / 10.1007 / s12035-019-1632-0. Kazim, SF, Iqbal, K. 2017. Chapter 2 - Neural Regeneration as a Disease-Modifying Therapeutic Strategy for Alzheimer’s Disease. In Neuroprotection in Alzheimer’s Disease [Ed. Gozes, I.]. pp. 3-29 Academic Press Panza, F, Lozupone, M, Logroscino, G, Imbimbo, BP (2019) A critical appraisal of amyloid-β-targeting therapies for Alzheimer disease. Nat Rev Neurol 15:73-88 Ustyugov, A, Shevtsova, E, Ashraf, GM, Tarasov, VV, Bachurin, SO, Aliev, G (2018) New therapeutic property of Dimebon as a neuroprotective agent. Current Medicinal Chemistry 25:5315-5326 Al-Hilaly, Y.K., Pollack, S.J., Rickard, J.E., Simpson, M., Raulin, A.-C., Baddeley, T., et al. (2018). Cysteine-independent inhibition of Alzheimer’s disease-like paired helical filament assembly by leuco-methylthioninium (LMT). J. Mol. Biol. 430, 4119-4131. doi: 10.1016 / j.jmb.2018.08.010. Atamna, H., Mackey, J., and Dhahbi, J.M. (2012). Mitochondrial pharmacology: electron transport chain bypass as strategies to treat mitochondrial dysfunction. Biofactors 38, 158-166. doi: 10.1002 / biof.197. Baddeley, T.C., Mccaffrey, J., Storey, J.M.D., Cheung, J.K.S., Melis, V., Horsley, D., et al. (2015). Complex disposition of methylthioninium redox forms determines efficacy in tau aggregation inhibitor therapy for Alzheimer’s disease. J. Pharmacol. Exptl. Therapeutics 352, 110-118. doi: 10.1124 / jpet.114.219352. Callaway, N.L., Riha, P.D., Bruchey, A.K., Munshi, Z., and Gonzalez-Lima, F. (2004). Methylene blue improves brain oxidative metabolism and memory retention in rats. Pharmacol. Biochem. Behav. 77, 175-181. Callaway, N.L., Riha, P.D., Wrubel, K.M., Mccollum, D., and Gonzalez-Lima, F. (2002). Methylene blue restores spatial memory retention impaired by an inhibitor of cytochrome oxidase in rats. Neurosci. Lett. 332, 83-86. Deiana, S., Harrington, C.R., Wischik, C.M., and Riedel, G. (2009). Methylthioninium chloride reverses cognitive deficits induced by scopolamine: comparison with rivastigmine. Psychopharmacology 202, 53-65. doi: 10.1007 / s00213-008-1394-2. Devine, M.J., and Kittler, J.T. (2018). Mitochondria at the neuronal presynapse in health and disease. Nat. Rev. Neurosci. 19, 63-80. doi: 10.1038 / nrn.2017.170. Fitzpatrick, A.W.P., Falcon, B., He, S., Murzin, A.G., Murshudov, G., Garringer, H.J., et al. (2017). Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature 547, 185-190. doi: 10.1038 / nature23002. Gauthier, S., Feldman, H.H., Schneider, L.S., Wilcock, G.K., Frisoni, G.B., Hardlund, J.H., et al. (2016). 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. Lancet 388, 2873-2884. doi: 10.1016 / S0140-6736(16)31275-2. Gonzalez-Lima, F., and Bruchey, A.K. (2004). Extinction memory by the metabolic enhancer improvement methylene blue. Learning & Memory 11, 633-640. Harrington, C.R., Storey, J.M.D., Clunas, S., Harrington, K.A., Horsley, D., Ishaq, A., et al. (2015). Cellular models of aggregation-dependent template-directed proteolysis to characterize tau aggregation inhibitors for treatment of Alzheimer’s disease. J. Biol. Chem. 290, 10862-10875. doi: 10.1074 / jbc.M114.616029. Husain, M., and Mehta,M.A. (2011) Cognitive enhancement by drugs in health and disease. Trends Cognitive Sci. 15, 28-36. Li, Y. and Kavalali, E.T. (2017). Synaptic vesicle-recycling machinery components as potential therapeutic targets. Pharmacol. Rev. 69, 141-160. Maier, L.J., Ferris, J.A., and Winstock, A.R. (2018). Pharmacological cognitive enhancement among non-ADHD individuals-A cross-sectional study in 15 countries.”Int. J. Drug Policy 58, 104-112. Martinez, J.L., Jr., Jensen, R.A., Vasquez, B.J., Mcguiness, T., and Mcgaugh, J.L. (1978). Methylene blue alters retention of inhibitory avoidance responses. Physiological Psychology 6, 387-390. Marsh, J, Alifragis, P (2018) Synaptic dysfunction in Alzheimer’s disease: the effects of amyloid beta on synaptic vesicle dynamics as a novel target for therapeutic intervention. Neural Regen. Res. 13:616-623. Melis, V., Magbagbeolu, M., Rickard, J.E., Horsley, D., Davidson, K., Harrington, K.A., et al. (2015a). Effects of oxidized and reduced forms of methylthioninium in two transgenic mouse tauopathy models. Behav. Pharmacol. 26, 353-368. doi: 10.1097 / fbp.0000000000000133. Melis, V., Zabke, C., Stamer, K., Magbagbeolu, M., Schwab, K., Marschall, P., et al. (2015b). Different pathways of molecular pathophysiology underlie cognitive and motor tauopathy phenotypes in transgenic models for Alzheimer’s disease and frontotemporal lobar degeneration. Cell. Mol. Life Sci. 72, 2199-2222. doi: 10.1007 / s00018-014-1804-z. Mesulam, M.M. (2013). Cholinergic circuitry of the human nucleus basalis and its fate in Alzheimer’s disease. J. Comp. Neurol. 521, 4124-4144. doi: 10.1002 / cne.23415. Pepeu, G., and Grazia Giovannini, M. (2017). The fate of the brain cholinergic neurons in neurodegenerative diseases. Brain Res. 1670, 173-184. doi: 10.1016 / j.brainres.2017.06.023. Pfaffendorf, M., Bruning, T.A., Batink, H.D., and Van Zwieten, P.A. (1997). The interaction between methylene blue and the cholinergic system. Br. J. Pharmacol. 122, 95-98. doi: 10.1038 / sj.bjp.0701355. Revett, T.J., Baker, G.B., Jhamandas, J., and Kar, S. (2013). Glutamate system, amyloid beta peptides and tau protein: functional interrelationships and relevance to Alzheimer disease pathology. J. Psychiat. Neurosci. 38, 6-23. doi: 10.1503 / jpn.110190. Riha, P.D., Bruchey, A.K., Echevarria, D.J., and Gonzalez-Lima, F. (2005). Memory facilitation by methylene blue: Dose-dependent effect on behavior and brain oxygen consumption. Eur. J. Pharmacol. 511, 151-158. Wilcock, G.K., Gauthier, S., Frisoni, G.B., Jia, J., Hardlund, J.H., Moebius, H.J., et al. (2018). 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 3 clinical trial. J. Alzheimer’s Dis. 61, 635-657. doi: 10.3233 / JAD-170560. Wischik, C.M., Edwards, P.C., Lai, R.Y.K., Roth, M., and Harrington, C.R. (1996). Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Proc. Natl. Acad. Sci. U.S.A. 93, 11213-11218. doi: 10.1073 / pnas.93.20.11213. Wischik, C.M., Novak, M., Edwards, P.C., Klug, A., Tichelaar, W., and Crowther, R.A. (1988a). Structural characterization of the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 85, 4884-4888. doi: 10.1073 / pnas.85.13.4884. Wischik, C.M., Novak, M., Thogersen, H.C., Edwards, P.C., Runswick, M.J., Jakes, R., et al. (1988b). Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 85, 4506-4510. doi: 10.1073 / pnas.85.12.4506. Wischik, C.M., Schelter, B.O., Wischik, D.J., Storey, J.M.D., and Harrington, C.R. (2018). Modeling prion-like processing of tau protein in Alzheimer’s disease for pharmaceutical development. J. Alzheimer’s Dis. 62, 1287-1303. doi: 10.3233 / JAD-170727. Wrubel, K.M., Barrett, D., Shumake, J., Johnson, S.E., and Gonzalez-Lima, F. (2007). Methylene blue facilitates the extinction of fear in an animal model of susceptibility to learned helplessness. Neurobiol. Learning Memory 87, 209-217. Zoellner, L.A., Telch, M., Foa, E.B., Farach, F.J., Mclean, C.P., Gallop, R., et al. (2017). Enhancing extinction learning in posttraumatic stress disorder wWith brief daily imaginal exposure and methylene blue: a randomized controlled trial. J. Clin. Psychiat. 78, e782-e789. doi: 10.4088 / JCP.16m10936.

Claims

1. 1. A therapeutic composition comprising a methylthioninium (MT)-containing compound for a method of therapeutic treatment of Alzheimer's disease or mild cognitive impairment in a subject, comprising: The method includes administering to the subject an MT-containing compound; The MT-containing compound is a LMTX compound of the formula: 【Chemistry 1】 (In the formula, H ｎ A and H ｎ each B, if present, is a protic acid, which may be the same or different; p=1 or 2; q=0 or 1; n=1 or 2; and (p+q)×n=2. or a hydrate or solvate thereof, The subject is one of the following groups: (i) a subject who has not previously been treated with a neurotransmission-modifying compound that is a modulator of acetylcholine activity or glutamate neurotransmitter activity; or (ii) Subjects who have previously been treated with a neurotransmission-modifying compound that is a modulator of acetylcholine activity or glutamate neurotransmitter activity, but who have discontinued such treatment for at least 3, 4, 5, 6, 7, or 8 weeks prior to treatment with said LMTX compound. Selected from: said therapeutic treatment with said LMTX compound is maintained for a treatment window without concurrent administration of a neurotransmission-modifying compound that is a modulator of acetylcholine activity or glutamate neurotransmitter activity; Followed by: treatment with the LMTX compound is accompanied by simultaneous administration of a neurotransmission-modifying compound that is a modulator of acetylcholine activity or glutamate neurotransmitter activity; the neurotransmission modulating compound which is a modulator of acetylcholine activity is an acetylcholinesterase inhibitor; the neurotransmitter modulating compound is a glutamate neurotransmitter modulator, and is a receptor antagonist of an N-methyl-D-aspartate (NMDA) receptor; A therapeutic composition, wherein the treatment time frame prior to simultaneous administration of said LMTX compound with said neurotransmission modifying compound is at least two months.

2. 1. A therapeutic composition for a method of therapeutic treatment of Alzheimer's disease (AD) or mild cognitive impairment in a subject, comprising a methylthioninium (MT)-containing compound, The subject is selected as being non-responsive to treatment with a neurotransmission-modifying compound that is a modulator of acetylcholine activity or glutamate neurotransmitter activity; The method includes administering to the subject the MT-containing compound; The MT-containing compound is a LMTX compound of the formula: 【Chemistry 2】 (In the formula, H ｎ A and H ｎ each B, if present, is a protic acid, which may be the same or different; p=1 or 2; q=0 or 1; n=1 or 2; and (p+q)×n=2. or a hydrate or solvate thereof, followed by treatment with said neurotransmission modulating compound; the neurotransmission modulating compound which is a modulator of acetylcholine activity is an acetylcholinesterase inhibitor; The therapeutic composition, wherein the neurotransmission modulating compound which is a modulator of the glutamate neurotransmitter is a receptor antagonist of the N-methyl-D-aspartate (NMDA) receptor.

3. said therapeutic treatment with LMTX is maintained for a treatment window with simultaneous administration of said neurotransmission modifying compound; Followed by: The therapeutic composition according to claim 2, wherein the treatment is with a neurotransmission modifying compound without LMTX.

4. The therapeutic composition of claim 3, wherein the treatment time frame for simultaneous administration is at least two months.

5. The therapeutic composition of any one of claims 1 to 4, wherein said therapeutic treatment with LMTX comprises a total daily dose of 2 to 100 mg MT per day to said subject.

6. The therapeutic composition of claim 5, wherein the therapeutic treatment is divided into two or more doses.

7. The therapeutic composition of claim 2, wherein the total daily dose of MT is 10 to 60 mg.

8. The LMTX compound has the formula: 【Chemistry 3】 8. The therapeutic composition of any one of claims 1 to 7, having the formula:

9. The LMTX compound has the formula: 【Chemistry 4】 (In the formula, H ｎ Each of X is a protonic acid.

10. The LMTX compound has the formula: 【Chemistry 5】 and H ２ 10. The therapeutic composition of claim 9, wherein A is a diprotic acid.

11. The LMTX compound has the formula: 【Chemistry 6】 10. The therapeutic composition of claim 9, which is a bis-monoprotonic acid salt.

12. A therapeutic composition according to any preceding claim, wherein the or each protic acid is an inorganic acid.

13. A therapeutic composition according to claim 12, wherein the or each protic acid is selected from a hydrohalic acid or HCl; HBr; HNO3; H2SO4.

14. A therapeutic composition according to any preceding claim, wherein the or each protic acid is an organic acid.

15. A therapeutic composition as claimed in claim 14, wherein the or each protonic acid is selected from: H 2 CO 3 ; CH 3 COOH; methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid.

16. The LMTX compound is LMTM: 【Chemistry 7】 The therapeutic composition according to any one of claims 1 to 7,

17. The LMTX compound is 【Chemistry 8】 【Chemistry 9】 The therapeutic composition of any one of claims 1 to 7, selected from the group consisting of:

18. The therapeutic composition according to any one of claims 1 to 17, wherein the neurotransmission modifying compound is an acetylcholinesterase inhibitor and is selected from donepezil; rivastigmine; and galantamine.

19. The therapeutic composition according to any one of claims 1 to 17, wherein the neurotransmission modifying compound is a receptor antagonist of the N-methyl-D-aspartate (NMDA) receptor and is memantine.

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