THERAPEUTIC INTERACTIONS OF LEUCOMETHYLTHIONINIUM

Abstract

The present invention relates generally to methods of treatment of Alzheimer's Disease or Mild Cognitive Impairment which are adapted to avoid negative interactions between combinations of therapeutics. In particular there are disclosed such methods of treatment in which the order of therapeutics is actively controlled to mitigate homeostatic downregulation prior to administration of active, for example disease modifying, therapeutic agents. In certain embodiments therapy with symptomatic treatments (such as modifiers of the activity of acetylcholine or glutamate neurotransmitters) may subsequently be combined with the disease modifying or other active treatment. The invention also applies the findings in relation to homeostatic downregulation to novel methods of clinical trial design.

Claims

1. A method of therapeutic treatment of Alzheimer's disease or Mild Cognitive Impairment in a subject, which method comprises administering to said subject a therapeutic methylthioninium (MT)-containing compound, wherein the MT-containing compound is an LMTX compound of the following formula: ##STR00019## 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; and (p+q)×n=2, or a hydrate or solvate thereof, wherein said subject is selected from the following groups: (i) a subject who has not historically received treatment with a neurotransmission modifying compound which is a modifier of the activity of acetylcholine or glutamate neurotransmitters; or (ii) a subject who has historically received treatment with a neurotransmission modifying compound which is a modifier of the activity of acetylcholine or glutamate neurotransmitters, but ceased that treatment at least 3, 4, 5, 6, 7, or 8 weeks prior to treatment with the LMTX compound; wherein said therapeutic treatment with the LMTX compound is maintained for treatment timeframe without co-administration with a neurotransmission modifying compound which is a modifier of the activity of acetylcholine or glutamate neurotransmitters, followed by; treatment with the LMTX compound with co-administration of a neurotransmission modifying compound which is a modifier of the activity of acetylcholine or glutamate neurotransmitters. wherein the treatment timeframe prior to co-administration of the LMTX compound with the neurotransmission modifying compound is at least 2 months.

2. The method as claimed in claim 1 wherein said therapeutic treatment with the LMTX compound comprises a total daily dose of between 2 and 100 mg of MT to the subject per day, optionally split into 2 or more doses.

3. The method as claimed in claim 2 wherein the total daily dose of MT is from 10-60 mg.

4. (canceled)

5. The method as claimed in claim 2 wherein the total daily dose is between 20 and 40 mg.

6. The method as claimed in claim 1 wherein the LMTX compound has the following formula, where HA and HB are different mono-protic acids: ##STR00020##

7. The method as claimed in claim 1 wherein the LMTX compound has the following formula: ##STR00021## wherein each of H.sub.nX is a protic acid.

8. The method as claimed in claim 1 wherein the LMTX compound has the following formula and H.sub.2A is a di-protic acid: ##STR00022##

9. The method as claimed in claim 7 wherein the LMTX compound has the following formula and is a bis-monoprotic acid salt: ##STR00023##

10. The method as claimed in claim 1 wherein the or each protic acid is an inorganic acid.

11. The method as claimed in claim 10 wherein each protic acid is a hydrohalide acid.

12. (canceled)

13. The method as claimed in claim 1 wherein the or each protic acid is an organic acid.

14. The method as claimed in claim 13 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, and p-toluenesulfonic acid.

15. The method as claimed in claim 1 wherein the LMTX compound is LMTM: ##STR00024##

16-17. (canceled)

18. The method as claimed in claim 1 wherein the LMTX compound is selected from the group consisting of: ##STR00025## ##STR00026##

19-29. (canceled)

30. The method as claimed in claim 1 wherein: (i) the treatment timeframe prior to co-administration of the LMTX compound with the neurotransmission modifying compound is at least 6 months, and/or (ii) the treatment prior to co-administration of the LMTX compound with the neurotransmission modifying compound is a monotherapy, and/or (iii) the subject who has historically received treatment with a neurotransmission modifying compound which is a modifier of the activity of acetylcholine or glutamate neurotransmitters has ceased that treatment at least 6 weeks prior to administration of the LMTX compound, or assessment.

31-36. (canceled)

37. A method of therapeutic treatment of Alzheimer's disease or Mild Cognitive Impairment in a subject, which subject has been selected as being non-responsive to treatment with a neurotransmission modifying compound which is a modifier of the activity of acetylcholine or glutamate neurotransmitters, which method comprises administering to said subject a therapeutic methylthioninium (MT)-containing compound, wherein the MT-containing compound is an LMTX compound of the following formula: ##STR00027## 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; and (p+q)×n=2, or a hydrate or solvate thereof, followed by treatment with said neurotransmission modifying compound.

38. The method as claimed in claim 37, wherein the Alzheimer's disease is mild Alzheimer's disease.

39. The method as claimed in claim 37 wherein said therapeutic treatment with the LMTX compound is maintained for treatment timeframe with co-administration with the neurotransmission modifying compound, followed by; treatment with the neurotransmission modifying compound without the LMTX compound, wherein optionally the treatment timeframe with co-administration is at least 2 months.

40-41. (canceled)

42. The method as claimed in claim 1, wherein the neurotransmission modifying compound is an acetylcholinesterase inhibitor.

43. The method as claimed in claim 42, wherein the neurotransmission modifying compound is selected from donepezil; rivastigmine; and galantamine.

44. The method as claimed claim 1, wherein the neurotransmission modifying compound is an N-methyl-D-aspartate receptor (NMDA) receptor antagonist.

45. The method as claimed in claim 44, wherein the neurotransmission modifying compound is memantine.

46-47. (canceled)

48. The method as claimed in claim 37 wherein the LMTX compound has the following formula, where HA and HB are different mono-protic acids: ##STR00028##

49. The method as claimed in claim 37 wherein the LMTX compound has the following formula: ##STR00029## wherein each of H.sub.nX is a protic acid.

50. The method as claimed in claim 37 wherein the LMTX compound has the following formula and H.sub.2A is a di-protic acid: ##STR00030##

51. The method as claimed in claim 49 wherein the LMTX compound has the following formula and is a bis-monoprotic acid salt: ##STR00031##

52. The method as claimed in claim 37 wherein the or each protic acid is an inorganic acid, which is optionally a hydrohalide acid.

53. The method as claimed in claim 52, wherein the or each inorganic acid is a hydrohalide acid.

54. The method as claimed in claim 37 wherein the or each protic acid is an organic acid.

55. The method as claimed in claim 54, wherein the or each organic 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.

56. The method as claimed in claim 37 wherein the LMTX compound is LMTM: ##STR00032##

57. The method as claimed in claim 37 wherein the LMTX compound is selected from the group consisting of: ##STR00033##

Description

FIGURES

[0261] FIG. 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 categorized by co-medication status with AD-labelled treatments.

[0262] FIG. 2. 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 value for hippocampus, visual cortex, diagonal band and septum (B). (**, p<0.01; ***, p<0.001).

[0263] FIG. 3. Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine in tau transgenic L1 mice on levels of (A) SNARE complex proteins (SNAP25, syntaxin and VAMP2) and (B) α-synuclein measured immunohistochemically as the mean value for hippocampus, visual cortex, diagonal band and septum. (*, p<0.05; ***, p<0.001; ****, p<0.0001).

[0264] FIG. 4. Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine in tau transgenic L1 mice on complex IV activity measured relative to citrate synthetase activity in brain mitochondria. (*, p<0.05).

[0265] FIG. 5. Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine in tau transgenic L1 mice compared with vehicle-treated wild-type mice on levels of tau immunoreactivity (relative optical density) (A) and neurons immunoreactive for choline acetyltransferase (B) in vertical diagonal band. (*, p<0.05; **, p<0.01; ***, p<0.001).

[0266] FIG. 6. Summary schema of treatment effects of LMT which are subject to dynamic modulation by chronic pretreatment with the acetylcholinesterase inhibitor (AChEI) rivastigmine (with particular focus on changes in mitochondrial metabolism and presynaptic proteins) and tau aggregation inhibitor activity. Combined treatment with AChEI does not impair LMT effects on tau aggregation pathology. By contrast, the combination prevents the increases in synaptic proteins, ACh release and increased complex IV activity that are seen following treatment with LMTM alone.

[0267] FIG. 7. Effect of combined administration of Memantine and LMTM on problem solving deficits in female Line 1 mice, aged 5.5 months at the beginning 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 Memantine plus LMTM treatment for 6 weeks, with the mice being tested in the water maze task over weeks 10 and 11.

[0268] FIG. 8. Withdrawal following 18 month LMTM 8 mg/day monotherapy (ADAS-cog). As can be seen in the subjects in the high exposure (Cmax) group continue to decline at same rate following LMTM withdrawal (change=0.73; p-value=0.1651). This supports a persistent disease-modifying change in rate of cognitive decline following the LMTX treatment period.

[0269] The subjects exposed to a lower exposure suffered a large decline (change=2.29; p-value=0.0011) implying that the LMTX benefit in that group may be at least partly symptomatic, and hence not persistent.

[0270] FIG. 9. Withdrawal following 18 month LMTM 8 mg/day as an “add on” to existing symptomatic therapy (AChEI, Memantine) (ADAS-cog). As can be seen in the subjects in the high exposure (Cmax) group show improvement following LMTM withdrawal (change=−0.98 p-value=0.0021). This is consistent with either a disease-modifying effect during LMTM treatment which results in an improved response to symptomatic treatment alone, or a negative effect on the symptomatic treatment exhibited by LMTM during the LMTX treatment period.

[0271] The subjects exposed to a lower exposure continue to decline at same rate following withdrawal (change=0.51, p-value=0.0855).

EXAMPLES

Example 1—Provision of MT-Containing Compounds

[0272] Methods for the chemical synthesis of the MT-containing compounds described herein are known in the art. For example:

[0273] Synthesis of compounds 1 to 7 can be performed according to the methods described in WO2012/107706, or methods analogous to those.

[0274] Synthesis of compound 8 can be performed according to the methods described in WO2007/110627, or a method analogous to those.

Example 2—Investigation of Interference Between LMTM and Symptomatic Treatments in a Tau Transgenic Mouse Model

[0275] FIG. 1 illustrates the interference which was established then LMTM was taken in combination with symptomatic treatments.

[0276] We have undertaken work in a well-characterised 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 treatments.

[0277] In summary, our findings suggest that homeostatic mechanisms downregulate multiple neuronal systems at different levels of brain function to compensate for the chronic pharmacological activation induced by symptomatic treatments.

[0278] Compared with LMTM given alone, the effect of this downregulation is to reduce neurotransmitter release, levels of synaptic proteins, mitochondrial function and behavioural benefits if LMTM is given against a background of chronic prior exposure to acetylcholinesterase inhibitor. Therefore, the interference in treatment efficacy, first seen clinically, has a clear neuropharmacological basis that can be reproduced in a tau transgenic mouse model.

[0279] Importantly, the homeostatic effects we have identified are likely to have more general relevance for the conduct of disease-modifying trials, or indeed other kinds of therapeutic compound-trials, in AD or MCI, that need not be restricted to tau aggregation inhibitors.

[0280] 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 encompasses the fragment 306-378 recently confirmed by cryo-electronmicroscopy of PHFs in AD and tau filaments in Pick's disease (Fitzpatrick et al., 2017; Falcon et al., 2018).

[0281] 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.

[0282] 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.

TABLE-US-00004 TABLE 1 Correlations between levels of a range of presynaptic proteins in basal forebrain (vertical diagonal band) measured immunochemically in (A) wild-type mice or (B) tau transgenic L1 mice. α-Synuclein SNAP25 Syntaxin VAMP2 Synaptophysin A Wild-type mice α-Synuclein SNAP25 * Syntaxin — ** VAMP2 — * * Synaptophysin — ** * — Synapsin — — — — — B L1 mice α-Synuclein SNAP25 — Syntaxin — — VAMP2 — — — Synaptophysin — — — * Synapsin — — * — — Significance of correlations, by linear regression analysis, are denoted as * p < 0.05; ** p < 0.01; — no significance at p = 0.05.

Example 3—Experimental Paradigms, Results and Discussion

[0283] Experimental Paradigms

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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.

[0288] Data are presented as group averages and standard errors of mean and were analysed using parametric statistics, with alpha set to 0.05.

[0289] 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.

[0290] Results

[0291] Effects of Treatment with LMTM and Rivastigmine in Wild-Type Mice

[0292] The effects of treatment with LMTM alone or on a chronic rivastigmine background are summarised in Table 2.

[0293] 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. 2A).

[0294] There was also a 3-fold increase in 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. 2B).

TABLE-US-00005 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. Rivastigmine + Effects in wild-type mice LMTM alone LMTM ACh release ↑ x 200% ↓ x 30%  SNARE complex — — Synaptophysin ↑ x 300% ↓ x 300% α-Synuclein — — Mitochondrial complex — — IV Behaviour — — Numbers in black signify treatment effects which reached statistical significance, ‘—’ indicates no effect.

[0295] Effects of Treatment with LMTM and Rivastigmine in Tau Transgenic L1 Mice

[0296] 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 (see Table 3). LMTM alone produces significant increases in ACh release in the hippocampus, in glutamate release from brain synaptosomal preparations, in synaptophysin levels, in mitochondrial complex IV activity and in behavioural changes. None of these effects were seen when LMTM was preceded by chronic rivastigmine. Indeed, in the case of SNARE complex proteins (FIG. 3A) and synuclein (FIG. 3B), the reduction produced by the combination was to levels below those seen in the absence of LMTM treatment.

TABLE-US-00006 TABLE 3 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 L1 mice, given as approximate rounded percentages to indicate scale and direction of change. Rivastigmine + Effects in L1 mice LMTM alone LMTM ACh release ↑ x 200% ↓ x 30% Glutamate release ↑ x 200% n/a SNARE complex — ↓ x 300% Synaptophysin ↑ x 400% ↓ x 300% α-Synuclein — ↓ x 200% Mitochondrial complex ↑ x 50%  ↓ x 30%  IV Behaviour ↑ x 30%  ↓ x 20%  Numbers in black signify treatment effects that reached statistical significance, those in grey were directional and n/a signifies that results are not yet available.

[0297] LMTM given alone produced significant enhancement of complex IV activity in brain mitochondria from tau transgenic L1 mice. Chronic pretreatment with rivastigmine also eliminated this effect (FIG. 4).

[0298] 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 the core tau unit of the PHF measured by optical density is elevated in tau transgenic L1 mice, and this was reduced following treatment with LMTM (FIG. 5A). Conversely, counts of ChAT-positive neurons are reduced in L1 mice and restored by treatment with LMTM (FIG. 5B). Both effects persist in L1 mice if LMTM is given after prior chronic treatment with rivastigmine.

[0299] Discussion of Example 3

[0300] 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. Therefore, it is based on neuropharmacological mechanisms that have the effect of altering how the brain responds to a disease-modifying treatment such as LMTM. The results imply that the differences in clinical response to LMTM as monotherapy or add-on therapy are likely to be explained by differences in the underlying neuropharmacology of LMTM in these two contexts (Gauthier et al., 2016; Wilcock et al., 2018). Alternative explanations based on the presumption that patients who are prescribed symptomatic treatments are somehow different from untreated patients fail for a number of reasons. The minor and variable differences in baseline severity between these two patient groups have been shown not to account for differences in treatment response (Gauthier et al., 2016; Wilcock et al., 2018). Apparent differences in rate of decline in treated and untreated MCI patients in the ADNI program (Schneider et al., 2011) disappear when severity at baseline is accounted for in the analysis (Wilcock et al., 2018). The presumption that untreated patients do not really have AD, or have a different form of AD, is also inconsistent with baseline neuroimaging data from subjects participating in the Phase 3 trials (Wilcock et al., 2018). Finally, as summarised for cognitive decline data in FIG. 1, we have shown recently that there are similar concentration-response relationships in monotherapy and add-on therapy subjects, but that the treatment effects are consistently larger for monotherapy on all clinical and neuroimaging outcomes.

[0301] 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.

[0302] The two classes of LMTM treatment effect are summarised in FIG. 6.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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).

[0307] In this situation, adding scopolamine (1 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.

[0308] 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).

[0309] 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.

[0310] Chronic pretreatment with rivastagmine suppressed the cholinergic activation in the hippocampus and reduced synaptophysin levels more generally in the brain in 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.

[0311] Although qualitatively similar, the effects of LMTM given alone are much more prominent and more broad-ranging in tau transgenic L1 mice. The most likely explanation for this is that LMTM combines an inhibitory effect on tau oligomers together with inherent activating effects which are not tau-dependent. The reduction in tau oligomer levels following LMTM treatment facilitates a more pronounced activation of synaptic function and release of neurotransmitters such as ACh and glutamate. Likewise, LMTM reverses the spatial memory deficit seen in tau transgenic L1 mice (Melis et al., 2015a). The negative effects seen when LMTM is introduced on a chronic rivastigmine background appears simply to reflect the reversal of the activation seen with LMTM alone.

[0312] A deleterious effect of tau oligomers on functioning of synaptic proteins is readily understandable as being the result of direct interference with docking of synaptic vesicles, membrane fusion and release of neurotransmitter. In tau transgenic L1 mice, synaptic vesicular protein levels are no longer linked quantitatively to either the proteins of the SNARE complex or α-synuclein, implying a loss of functional integration between vesicular and membrane-docking proteins at the synapse. The consequence of this can be seen directly as an impairment in glutamate release from synaptosomal preparations from tau transgenic mice, and a restoration of normal glutamate release following treatment with LMTM.

[0313] The mechanisms responsible for the mitochondrial effects of LMTM are more complex. 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). However, LMTM has no effect on complex IV activity in brain mitochondria isolated from wild-type mice. By contrast, a strong effect was seen in tau transgenic L1 mice. This suggests that tau oligomers interfere with mitochondrial metabolism. It has been shown recently that C-terminally truncated tau protein is bound both to the mitochondrial outer membrane and also enters the intermembrane space of mitochondria (Cieri et al., 2018). Truncated PHF-tau protein isolated from brain tissues of AD patients forms SDS-resistant complexes with the voltage-dependent anion-selective channel protein (VDAC; formerly porin) in the mitochondrial outer membrane, and also with ATP synthase subunit 9 and core protein 2 of complex Ill in the intermembrane space (Wischik et al., 1997). These binding interactions are likely to be deleterious 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.

[0314] It is not known how homeostatic downregulation resulting from rivastigmine treatment might affect mitochondrial function. Mitochondria are known to be important homeostatic regulators of synaptic function via buffering of Ca.sup.2+ levels and ATP generation (Devine and Kittler, 2018).

[0315] It is striking that the positive effects of LMTM and their reversal or suppression by pretreatment with anticholinesterase can be seen across different transmitter systems and cellular compartments at multiple levels of brain function. This implies that there is no single locus responsible for the interference in the LMTM treatment response. Rather, the negative interaction appears to be part of a generalised homeostatic downregulation in multiple neuronal systems that compensate for the chronic pharmacological activation resulting from blockade of acetylcholinesterase.

[0316] The results with memantine are shown in FIG. 7, and show a similar picture to pretreatment with anticholinesterase. This is as expected, given that the interference in LMTM efficacy seen clinically is very similar for the two drug classes.

[0317] More generally, it would be unlikely that the interference affecting LMTM treatment is specific to LMTM. Any treatment that has an activating effect on synaptic function, whether by reducing primary pathology or by another mechanism, is likely to be subject to similar interference, since it is driven primarily by the pre-existing symptomatic treatment.

[0318] Thus if clearance of amyloid aggregates results in synaptic activation, as has been proposed Marsh, J, Alifragis, P (2018), then it can be inferred that symptomatic treatments would also interfere with the ability to demonstrate this effect clinically.

[0319] 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.

[0320] In summary, our findings point to the powerful role of 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 controls that limit the extent of neuronal over-activation. In the case of cholinergic function, excessive activity is highly deleterious and results clinically in convulsions, coma and death. This is entirely consistent with chronic stimulation of the brain by symptomatic treatments altering the way in which it responds to other therapeutic interventions.

Example 4—Withdrawal Analysis for LMTM 8 mg/Day as Monotherapy or Add-On Therapy

[0321] Following 18 months of treatment with 8 mg/day LMTM, subjects were required to have a 1 month “washout” followed by cognitive assessment of change. The group was split according to whether they has received LMTM as an add-on to existing symptomatic therapy (AChEI, Memantine, collectively abbreviated to “Achmem”) or as a monotherapy.

[0322] The group was further analysed in terms of subjects who had received a high Cmax exposure or a low one. As explained in WO2020/020751, using pharmacokinetic modelling, the 8 mg/day treated population can be split into a group of individuals with “higher” estimated Cmax and a group of individuals with “lower” estimated Cmax. WO2020/020751 explains that splitting of patients according to the threshold of 0.37 ng/ml (that encompasses the 35% of patients with the lowest values) the treatment difference in “high” and “low” Cmax patients receiving the 8 mg/day dose is −3.4 ADAS-cog units.

[0323] The numbers in the present analysis were as follows:

TABLE-US-00007 Cmax (exposure) Add-on to Achmem Monotherapy Low 120 24 High 242 47

[0324] As shown in FIG. 8 (monotherapy) the high Cmax group continue to decline at same rate following LMTM withdrawal supporting a persistent disease-modifying change in rate of cognitive decline following the LMTX treatment period.

[0325] The subjects exposed to a lower Cmax suffered a large decline implying that the LMTX benefit in that group may be at least partly symptomatic, and hence not persistent.

[0326] As shown in FIG. 9 (add-on) the high Cmax group actually showed an unexpected improvement following LMTM withdrawal. This is consistent with either a disease-modifying effect during LMTM treatment which results in an improved response to symptomatic treatment alone, or with a negative effect on the symptomatic treatment exhibited by LMTM during the LMTX treatment period.

[0327] This finding has potential implications for the use of LMTM and Achmem combination therapies—for example in patient groups who have proved non-responsive to Achmem, the disease modifying effects of LMTX may actually enhance the response to Achmem, particularly once the LMTX is discontinued, and particularly in relation to the ADAS-cog in mild AD subjects.

[0328] By contrast the subjects exposed to a lower exposure continued to decline at same rate following withdrawal.

[0329] These results support the concentration-response analysis in WO2020/020751. LMTM as monotherapy has significant pharmacological activity at both high and low levels of exposure, but below around 0.38 ng/ml the effects may be more symptomatic than disease-modifying. As an add-on therapy, LMTM has significant pharmacological activity even at low levels of exposure, but an exposure to below ˜0.378 ng/ml as add-on does not give a discernible treatment effect.

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