METHYLTHIONINIUM AS ENHANCERS OF THE COGNITIVE FUNCTION

Abstract

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

Claims

1. Non-therapeutic use of a methylthioninium (MT) containing compound to stimulate cognitive function in a healthy subject, wherein said use comprises administering between 2 and 100 mg of MT to the subject per day, optionally split into 2 or more doses, wherein the MT compound is an LMTX compound of the following formula: ##STR00015## wherein each of H.sub.nA and H.sub.nB (where present) are protic acids which may be the same or different, and wherein p=1 or 2; q=0 or 1; n=1 or 2; (p+q)×n=2 or a hydrate or solvate thereof.

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

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

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

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

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

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

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

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

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

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

12. The non-therapeutic use as claimed in any one of claims 1 to 11 wherein the compound has the following formula, where HA and HB are different mono-protic acids: ##STR00016## wherein HA and HB are different mono-protic acids:

13. The non-therapeutic use as claimed in any one of claims 1 to 11 wherein the compound has the following formula: ##STR00017## wherein each of H.sub.nX is a protic acid.

14. The non-therapeutic use as claimed in any one of claims 1 to 11 wherein the compound has the following formula and H.sub.2A is a di-protic acid: ##STR00018##

15. The non-therapeutic use as claimed in claim 13 wherein the compound has the following formula and is a bis-monoprotic acid: ##STR00019##

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

17. The non-therapeutic use as claimed in claim 16 wherein each protic acid is a hydrohalide acid.

18. The non-therapeutic use as claimed in claim 16 wherein the or each protic acid is selected from HCl; HBr; HNO.sub.3; H.sub.2SO.sub.4.

19. The non-therapeutic use as claimed in any one of claims 1 to 15 wherein the or each protic acid is an organic acid.

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

21. The non-therapeutic use as claimed in claim 20 wherein the compound is LMTM: ##STR00020##

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

23. The non-therapeutic use as claimed in claim 22 wherein the dose of LMTM is around 34 mg/once per day; 15 mg b.i.d.; 8.7 mg t.i.d.

24. The non-therapeutic use as claimed in claim 20 wherein the compound is selected from the list consisting of: ##STR00021##

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

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

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

28. The non-therapeutic use as claimed in claim 25 or claim 26 which is a tablet or capsule.

29. A container comprising: (i) a plurality of dosage units as defined in any of claims 25 to 28; (ii) a label and\or instructions for their non-therapeutic use as defined in any one of claims 1 to 24.

30. A container as claimed in claim 29, wherein the dosage units are present in a blister pack which is substantially moisture-impervious.

31. A container as claimed in claim 29 or claim 30 wherein the label or instructions provide information regarding the stimulation of cognitive function for which the composition is intended.

32. A container as claimed in any one of claims 29 to 31 wherein the label or instructions provide information regarding the maximum permitted daily dosage of the dosage units.

33. A non-therapeutic method of treating a healthy human subject to stimulate their cognitive function, the treatment comprising orally administering to the subject a nootropically effective amount of methylthioninium (MT) containing LTMX compound according to the use defined in any one of claims 1 to 28.

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

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

Description

FIGURES

[0140] FIG. 1. Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine in wild-type mice on hippocampal levels of acetylcholine (A) or synaptophysin levels measured immunohistochemically as the mean in hippocampus, visual cortex, diagonal band and septum (B). (**, p<0.01; ***, p<0.001).

EXAMPLES

Example 1—Provision of MT-Containing Compounds

[0141] Methods for the chemical synthesis of the MT-containing compounds described herein are known in the art. For example:

[0142] Synthesis of compounds 1 to 7 can be performed according to the methods described in WO2012/107706, or methods analogous to those.

[0143] Synthesis of compound 8 can be performed according to the methods described in WO2007/110627, or a method analogous to those.

Example 2—Features of the Tau Transgenic Mouse Model Used for Interference Studies

[0144] In the L1 mouse model which was used in some of the present studies, there is over-expression of a three-repeat tau fragment encompassing residues 296-390 of the 2N4R tau isoform under the control of the Thy 1 promotor in an NMRI mouse strain (WO2002/059150). This fragment corresponds to the segment of tau first identified within the proteolytically stable core of the PHF (Wischik et al., 1988a; Wischik et al., 1988b) and recently confirmed by cryo-electronmicroscopy of PHFs in AD and tau filaments in Pick's disease (Fitzpatrick et al., 2017; Falcon et al., 2018).

[0145] Further features of the L1 mouse model include a prominent loss of neuronal immunoreactivity for choline acetyltransferase in the basal forebrain region, and a corresponding reduction in acetylcholinesterase in neocortex and hippocampus, indicative of reduction in acetylcholine. There is also an approximate 50% reduction in glutamate release for brain synaptosomal preparations from L1 mice compared with those from wild-type mice. In these respects, therefore, L1 mice also model the neurochemical impairments in cholinergic (Mesulam, 2013; Pepeu and Grazia Giovannini, 2017) and glutamatergic (Revett et al., 2013) function that are characteristic of AD.

[0146] Underlying these impairments in neurotransmitter function, the L1 mouse model shows a disturbance in integration of synaptic proteins. Quantitative immunohistochemistry for multiple synaptic proteins in the basal forebrain (vertical diagonal band) shows that there is normally a high degree of correlation in levels of proteins comprising the SNARE complex (e.g. SNAP-25, syntaxin, VAMP2; reviewed in Li and Kavalali, 2017), and the vesicular glycoprotein synaptophysin and α-synuclein in wild-type mice. These correlations are largely lost in L1 mice (Table 1). The only correlations that remain are between synaptophysin, syntaxin and VAMP2. Therefore, synaptic vesicular protein levels are no longer linked quantitatively to the proteins of the SNARE complex or α-synuclein. This suggests that the tau oligomer pathology of the L1 mice interferes with the functional integration between vesicular and membrane-docking proteins in the synapse.

Example 3—Experimental Paradigms, Results and Discussion

Experimental Paradigms

[0147] The treatment schedule used to study the negative interaction between symptomatic treatments and LMTM was designed to model the clinical situation in which subjects are first treated chronically with a cholinesterase inhibitor or memantine before receiving LMTM. In what follows, we summarise some of the key results obtained for the AChEI, rivastigmine. Wild-type and L1 mice (n=7-16 for each group) were pre-treated with rivastigmine (0.1 or 0.5 mg/kg/day) or memantine (2 or 20 mg/kg/day) or vehicle for 5 weeks by gavage. For the following 6 weeks, LMTM (5 and 15 mg/kg) or vehicle were added to this daily treatment regime, also by gavage. Animals were tested behaviourly during weeks 10 and 11 using a problem solving task in the open field water maze and then sacrificed for immunohistochemical and other tissue analyses.

[0148] Translating doses from mice to humans requires consideration of a number of factors. Although 5 mg/kg/day in mice corresponds approximately to 8 mg/day in humans in terms of C.sub.max levels of parent MT in plasma, this dose is at the threshold for effects on pathology and behaviour. The higher dose of 15 mg/kg/day is generally required for LMTM to be fully effective in the L1 mouse model (Melis et al., 2015a). This may relate to the much shorter half-life of MT in mice (4 hours) compared to humans (37 hours in elderly humans). Tissue sectioned for immunohistochemistry was labelled with antibody and processed using Image J to determine protein expression densitometrically. Data are presented as Z-score transformations without units.

[0149] For measurement of acetylcholine (ACh) levels in hippocampus, animals (wild-type or L1) were treated with LMTM (5 mg/kg/day for 2 weeks) after prior treatment for 2 weeks with or without rivastigmine (0.5 mg/kg/day). Rivastigmine was administered subcutaneously with an Alzet minipump whereas LMTM was administered by oral gavage. Levels of ACh were measured in hippocampus using an implanted microdialysis probe and HPLC analysis of the extracellular fluid.

[0150] Data are presented as group averages and standard errors of mean and were analysed using parametric statistics, with alpha set to 0.05.

[0151] Experiments on animals were carried out in accordance with the European Communities Council Directive (63/2010/EU) with local ethical approval, a project license under 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.

Results

[0152] Effects of Treatment with LMTM and Rivastigmine in Wild-Type Mice

[0153] The effects of treatment with LMTM alone or on a chronic rivastigmine background are summarised in Table 2.

[0154] In wild-type mice, there was a significant, 2-fold increase in basal ACh levels in hippocampus following LMTM treatment, and a 30% reduction when mice received LMTM after prior treatment with rivastigmine (FIG. 1A).

[0155] There was also a 3-fold increase in mean synaptophysin levels measured in hippocampus, visual cortex, diagonal band and septum following LMTM treatment alone and a statistically significant reduction of the same magnitude when LMTM was given against a background of prior treatment with rivastigmine (FIG. 1B).

TABLE-US-00003 TABLE 2 Summary of treatment effects of LMTM given alone (5 or 15 mg/kg/day) or following chronic pretreatment with rivastigmine (0.1 or 0.5 mg/kg/day) in wild-type mice, given as approximate rounded percentages to indicate scale and direction of change. Numbers in black signify treatment effects which reached statistical significance, those in grey were directional, ‘—’ indicates no effect. Effects in wild-type mice LMTM alone Rivastigmine + LMTM ACh release ↑ × 200% ↓ × 30%  SNARE complex — — Synaptophysin ↑ × 300% ↓ × 300% α-Synuclein — — Mitochondrial complex IV — — Behaviour — —
Effects of Treatment with LMTM and Rivastigmine in Tau Transgenic L1 Mice

[0156] The activating effects of LMTM alone and the inhibitory effects of the combination with rivastigmine are larger and more generalised in the tau transgenic L1 mice than in the wild-type mice (results not shown).

Discussion of Example 3

[0157] The results presented here demonstrate that the reduction in efficacy of LMTM when given as an add-on to a symptomatic treatment in humans can be reproduced both in wild-type mice and in a tau transgenic mouse model.

[0158] The results we now report demonstrate that there are two classes of effect produced by LMTM treatment in wild-type and tau transgenic mice: those that are subject to dynamic modulation by prior exposure to cholinesterase inhibitor and those which are not. In tau transgenic mice, the treatment effects that can be modulated include increase in ACh release in the hippocampus, changes in synaptic proteins, increase in mitochondrial complex IV activity and reversal of behavioural impairment. The only treatment effects that are not subject to pharmacological modulation are the primary effect on tau aggregation pathology and its immediate effect on neuronal function, as measured for example by restoration of choline acetyltransferase expression in the basal forebrain.

[0159] Effects that are subject to pharmacological modulation are themselves of two types: those which are augmented by the effect on tau aggregation pathology and those which are also seen in wild-type mice. Of the outcomes we have measured, positive treatment effects of LMTM given alone in wild-type mice included an increase in ACh levels in hippocampus, and an increase in synaptophysin levels in multiple brain regions. Therefore, LMTM treatment is able to activate neuronal function at therapeutically relevant doses in wild-type mice lacking tau aggregation pathology.

[0160] In experimental models, cholinergic function is associated primarily with selective attention (Botly and De Rosa, 2007; 2008; Sarter et al., 2016), and the improvements in cognitive function resulting from cholinesterase inhibitors in AD are thought to be the result of elevated levels of acetylcholine in the synaptic cleft. However, these drugs are believed not to increase acetylcholine levels in wild-type mice because of efficient homeostatic adaptations which mitigate the inhibition of acetylcholinesterase inhibitors (e.g. by reducing levels of synaptic vesicles in the presynapse).

[0161] By contrast, LMTM does produce a significant increase in acetylcholine levels in the hippocampus, which is known to be important for cognitive function.

[0162] Likewise, an increase in synaptophysin signals an increase in number or size of the synaptic vesicles that are required for release of neurotransmitters from the presynapse following activation via an action potential. Therefore, an increase in synaptophysin levels appears to be associated with an increase in a number of neurotransmitters needed to support cognitive and other mental functions.

[0163] Although it has been reported that the MT moiety is a weak cholinesterase inhibitor (Pfaffendorf et al., 1997; Deiana et al., 2009), this is unlikely to be the mechanism responsible for the increase in ACh levels.

[0164] Specifically, further experiments using scopolamine to increase 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 that the combination was again inhibitory in wild type mice. Under the condition of cholinesterase inhibition used in these experiments (a very small amount of a cholinesterase inhibitor, 100 nanomolar rivastigmine, added to the perfusion fluid), ACh levels in the hippocampus rise, and when they rise strongly enough, they limit additional ACh release by activating pre-synaptic muscarinic receptors of the M2/M4 subtype (so-called negative feedback receptors).

[0165] In this situation, adding scopolamine (μM) to the perfusion fluid blocks these presynaptic receptors and, as a consequence, ACh levels rise by 3-5 fold. The fact that LMTM is not additive with rivastigmine in these experiments supports the conclusion that LMTM has a different mechanism of action from rivastigmine. In other words, although LMTM has been described as being a weak inhibitor of cholinesterases in high concentrations, the present effects seem to be unrelated to cholinesterase inhibition, because there is no additive effect with small quantities of rivastigmine.

[0166] The increase in ACh and synaptophysin levels might theoretically be explained by an increase in presynaptic mitochondrial activity, since the MT moiety 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 moiety is thought to enhance oxidative phosphorylation by acting as an electron shuttle between complex I and complex IV (Atamna et al., 2012). The MT moiety has a redox potential of approximately 0 mV, midway between the redox potential of complex I (−0.4 mV) and complex IV (+0.4 mV).

[0167] However, direct measurement of complex IV activity in wild type mice did not show any increase following LMTM treatment. The activating effects of LMTM were also not associated with improvement in spatial recognition memory in wild-type mice.

[0168] Chronic pretreatment with rivastagmine suppressed the cholinergic activation in the hippocampus and reduced synaptophysin levels more generally in the brains of wild-type mice. This effect is clearly not dependent on the effects of LMTM on tau aggregation pathology, since there is no pathology in wild-type mice. Rather, they point to a generalised homeostatic downregulation that counteracts the effect of combining two drugs which each have activating effects on neuronal function. Presumably, the primary mechanism that would normally protect against excessive levels of ACh in the synaptic cleft would be an increase in AChE activity. Since rivastigmine produces chronic impairment of this control system, pathways that would otherwise be activated by LMTM are suppressed in order to preserve homeostasis in cholinergic and other neuronal systems. Thus, LMTM-induced effects are subject to dynamic downregulation if the brain is already subject to chronic stimulation by a cholinesterase inhibitor.

[0169] A further consideration is whether the homeostatic downregulation that we have demonstrated would operate in the same way if LMTM treatment were primary and symptomatic treatment were added at a later date. The experiments we have conducted to date were originally designed to mimic the clinical situation in which LMTM is added in patients already receiving symptomatic treatments. If homeostatic downregulation is determined by the treatment that comes first, it is logical that the treatment effects of LMTM would dominate, albeit that the response to add-on symptomatic treatment could be reduced to some extent.

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