Tau protein aggregation (Ki = 0.12 μM)

In addition to lowering tau and p-tau expression levels, colorless methylene blue (100 nM, 48 h) methanesulfonate also undid the encouraging effects of Aβ25-35 on beta-A and adenosine A1R expression levels [2].

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A stabilized, reduced form of MTC, TRx 0237 (LMTX™), is being developed by TauRx Therapeutics (Singapore, Republic of Singapore). An in vitro study showed the ability of TRx 0237 in disrupting PHFs isolated from AD brain tissues at the concentration at 0.16 μM. This value is identical to what found for MT (0.16 μM)[3].

The in vivo effects of MTC and TRx 0237 (5–75 mg/kg orally for 3–8 weeks) were compared in two novel mouse models overexpressing different human tau-protein constructs (L1 and L66) Both MTC and TRx 0237 dose-dependently rescued the learning impairment and restored behavioral flexibility in a spatial problem-solving water-maze task in L1 (minimum effective dose: 35 mg MT/kg for MTC, 9 mg MT/kg for TRx 0237) and corrected motor learning in L66 (effective doses: 4 mg MT/kg). Both compounds reduced the number of tau-reactive neurons, particularly in the hippocampus and entorhinal cortex in L1 and in a more widespread manner in L66[3].

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In recently completed Phase 3 trials testing the tau aggregation inhibitor leuco-methylthioninium bis (hydromethane-sulfonate) (LMTM ), we found significant differences in treatment response according to whether patients were taking LMTM either as monotherapy or as an add-on to symptomatic treatments.[4]


Methods[4]
We have examined the effect of either LMTM alone or chronic rivastigmine prior to LMTM treatment of tau transgenic mice expressing the short tau fragment that constitutes the tangle filaments of AD. We have measured acetylcholine levels, synaptosomal glutamate release, synaptic proteins, mitochondrial complex IV activity, tau pathology and Choline Acetyltransferase (ChAT) immunoreactivity.

Results[4]
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, partially restored ChAT immunoreactivity in the basal forebrain and reversed deficits in spatial learning. Chronic pretreatment with rivastigmine was found to reduce or eliminate almost all these effects, apart from a reduction in tau aggregation pathology. LMTM effects on hippocampal ACh and synaptophysin levels were also reduced in wild-type mice.

Conclusion[4]
The interference with the pharmacological activity of LMTM by a cholinesterase inhibitor can be reproduced in a tau transgenic mouse model and, to a lesser extent, in wild-type mice. Long-term pretreatment with a symptomatic drug alters a broad range of brain responses to LMTM across different transmitter systems and cellular compartments at multiple levels of brain function. There is, therefore, no single locus for the negative interaction. Rather, the chronic neuronal activation induced by reducing cholinesterase function produces compensatory homeostatic downregulation in multiple neuronal systems. This reduces a broad range of treatment responses to LMTM associated with a reduction in tau aggregation pathology. Since the interference is dictated by homeostatic responses to prior symptomatic treatment, it is likely that there would be similar interference with other drugs tested as add-on to the existing symptomatic treatment, regardless of the intended therapeutic target or mode of action. The present findings outline key results that now provide a working model to explain interference by symptomatic treatment.

Western Blot Analysis[2]
Cell Types: human SH-SY5Y cell line.
Tested Concentrations: 100 nM.
Incubation Duration: 48 hrs (hours).
Experimental Results: Co-treatment of Aβ25-35 and TRx 0237 Dramatically reversed the promoting effect of Aβ25-35 on the expression of tau, p-tau, orexin A and adenosine A1R.

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SH-SY5Y cells were treated with or without the tau inhibitor TRx 0237. SH-SY5Y cells were grouped into negative control (SH-SY5Y) and Aβ 25-35 (SH-SY5Y+Aβ 25-35) or TRx 0237 (SH-SY5Y+Aβ 25-35+TRx 0237). SH-SY5Y cells (1 ×105 cells/well) were seeded in 6-well plates and transfected with vehicle, Aβ 25-35 or TRx 0237 for 48 h. Before use, Aβ 25-35 was diluted in sterile saline to a concentration of 0.5 mM and was maintained at 37°C for 7 days to pre-age the peptide (24). The aged Aβ solution was diluted to 40 µM for use[2].

Processing of specimen batches[1]
Extraction specimens were processed in batches consisting of six calibration standards (10.0 to 20,000 ng/mL), QC samples, extract storage stability samples, and control (blank) samples. Following processing, each standard was split into two aliquots, one of which was run at the beginning and the other at the end of each analytical run. A total of 48 samples/standards were processed in each run. Analysis preparation for each animal sample consisted of two separate extractions, one extraction for the quantitation of excreted methylene blue, and the second extraction for the quantitation of leucomethylene blue/TRx 0237. In order to prepare samples for extraction of methylene blue, 200 lJL of 1M NaCl solution and 100 IJL of Basic Blue 3 internal standard solution were added to 0.5 mL of urine in polypropylene tubes. Tube contents were vortex mixed gently. A 4-mL aliquot of 1,2-dichloroethane was added to each tube, and the contents were shaken for 15 min and then centrifuged for 5 min with a table-top centrifuge. The 1,2-dichloroethane layer was transferred to a clean 4-mL polypropylene vial using a polyethylene pipette and evaporated to dryness with a Savant Speedvac sample concentrator set to medium heat. A 200-~L aliquot of 0.1% trifluoroacetic acid solution and 100 ~L of acetonitrile were added to each vial, and the contents were sonicated for 6 min and transferred to a polypropylene autosampler vial insert. A second extraction was then performed to remove the leucomethylene blue/TRx 0237 from the samples following conversion of the leucomethylene blue present to methylene blue. To convert the leucomethylene blue to methylene blue, 1001JL of 1N HC1 was added to the tubes. The tubes were immersed in boiling water for 20 min and allowed to cool to room temperature. The methylene blue which formed from oxidation of leucomethylene blue was then extracted and prepared for analysis using the same technique used for the original sample extraction.

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Acetylcholine Measurement in Hippocampus[4]
Animals were treated with LMTM (leuco-methylthioninium bis (hydromethane-sulfonate) (5 mg/kg/day for 2 weeks, gavage) after prior treatment for 2 weeks with or without rivastigmine (0.5 mg/kg/day subcutaneous Alzet minipump). Levels of ACh were measured in the hippocampus via indwelling microdialysis probes and HPLC analysis of the extracellular fluid. After the experiment, brains were harvested and histologically assessed for correct cannula placement.

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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 Fig. (1). After five weeks of daily gavaging with vehicle or rivastigmine, combination treatment proceeded in some groups while others received only LMTM monotherapy. [4]

Wild-type and L1 female mice (n = 7-16 for each group) were pre-treated with rivastigmine (0.1 or 0.5 mg/kg/day) or vehicle for 5 weeks by gavage. For the following 6 weeks, LMTM (5 and 15 mg/kg) was added to this daily treatment regime, also administered by gavage Fig. (1). Animals were then sacrificed for immunohistochemical and other tissue analyses, as described in a study [36]. Although 5 mg/kg/day in mice corresponds approximately to 8 mg/day in humans in terms of Cmax 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. This may relate to the much shorter half-life of MT in mice (4 hours) compared to humans (37 hours in elderly humans).

TRx0237 is claimed to have superior pharmacokinetics and tolerability compared to MTC, but there is currently no convincing evidence to support this claim. In healthy volunteers, oral absorption of TRx0237 in the presence of food is superior to that of MTC, but this result does not translate into higher central nervous system drug concentrations, as in miniature pigs, the intracranial drug concentrations of both were almost identical after administration of 33 mg/kg (approximately 5 μM) of MT or TRx0237. On the other hand, there are currently no data on the concentration of TRx0237 in human cerebrospinal fluid. Reliable data on the safety and tolerability of TRx0237 in humans are also lacking, making direct comparison with MTC impossible. In vitro comparative data show that the therapeutic index (LD50/EC50 ratio) of LMT-dihydrobromide is 92, the therapeutic index of LMT-dihydromethanesulfonate is 179, and the therapeutic index of MTC is 110. We believe that these in vitro differences are not significant enough to translate into pharmacological or clinical differences. In terms of efficacy, pharmacological studies in transgenic mouse tau proteinosis models did not show a significant difference between the two compounds. In fact, 45 mg/kg doses of MTC or TRx0237 produced the same behavioral effects. [3]

safety and tolerability study of TRx0237 (250 mg/day for 4 weeks) in 9 patients with mild to moderate Alzheimer's disease was initiated in September 2012 but terminated in April 2013, reportedly for administrative reasons (ClinicalTrials.gov registration number: NCT01626391) (Table 1). Currently, three Phase III placebo-controlled studies of TRx0237 are underway (Table 1). The first study is evaluating the effect of a 200 mg/day dose on 700 patients diagnosed with all-cause dementia or likely to have Alzheimer's disease, using the ADAS-Cog 11 Cognitive Function Scale and the Clinical Alzheimer's Disease Collaborative Study - Clinical Global Impression Change Scale (ADCS-CGIC) as the primary efficacy endpoints (ClinicalTrials.gov registration number: NCT01689233). The second study is evaluating the efficacy of daily doses of 150 mg and 250 mg in 833 patients with mild to moderate Alzheimer's disease, with ADAS-Cog 11 and ADCS-CGIC as the primary endpoints (ClinicalTrials.gov registration number: NCT01689246). The third phase III clinical trial is evaluating the efficacy of a daily dose of 200 mg in 220 patients with behavioral variant frontotemporal dementia (bvFTD) (ClinicalTrials.gov registration number: NCT01626378). This trial uses a modified ADCS-CGIC scale as the clinical efficacy endpoint and a revised Ardenbrook Cognitive Assessment scale as the cognitive function endpoint. Finally, an open-label extension study in subjects who have completed phase II or III clinical trials of TRx0237 is evaluating the long-term safety of this compound (ClinicalTrials.gov registration number: NCT02245568) (Table 1). To maintain blinding, the Phase III clinical trials used “active placebo” tablets containing 4 mg TRx0237 as a urine and stool staining agent. Overall, these Phase III clinical trials are recruiting 1,753 patients at 250 centers in 22 countries, with one trial (ClinicalTrials.gov registration number: NCT01689246) expected to release results in the first half of 2016, and the other two trials (ClinicalTrials.gov registration numbers: NCT01689233 and NCT01626378) expected to release results in the second half of 2016. [3]

[1]. Determination of methylene blue and leucomethylene blue in male and female Fischer 344 rat urine andB6C3F1 mouse urine. J Anal Toxicol. 2005 Jan-Feb;29(1):28-33.

[2]. Amyloid β and tau are involved in sleep disorder in Alzheimer's disease by orexin A and adenosine A(1) receptor. Int J Mol Med. 2019 Jan;43(1):435-442.

[3]. Tau aggregation inhibitors: the future of Alzheimer's pharmacotherapy? Expert Opin Pharmacother. 2016;17(4):457-61.

[4]. Mechanisms of Anticholinesterase Interference with Tau Aggregation Inhibitor Activity in a Tau-Transgenic Mouse Model. Curr Alzheimer Res. 2020 Mar;17(3):285–296.

Hydromethylthionine mesylate is a small molecule drug with the most completed Phase III clinical trials (covering all indications) and two investigational indications. This study developed a liquid chromatography method for the determination of methylene blue and colorless methylene blue in the urine of male and female Fischer 344 rats and male and female B6C3F1 mice to support toxicokinetic studies. The method was validated and used to analyze urine samples for preliminary dose-range exploration studies. The method was validated in urine at concentrations ranging from 10.0 to 20,000 ng/mL. Samples at concentrations up to 75,000 ng/mL showed good recoveries when diluted to the calibration curve range. To validate the reproducibility and robustness of the method, six sets of urine calibration standards from male F344 rats were prepared. The stability of the sample extracts was determined under different storage conditions. Potential metabolic processes were observed during the analysis of animal samples. Despite the presence of numerous impurities in methylene blue trihydrate, the levels of methylene blue B detected in the urine samples of rodents that had taken methylene blue trihydrate were significantly higher than those in the urine samples of rodents that had taken methylene blue directly. This observation suggests that methylene blue may be converted to methylene blue B through N-demethylation metabolism. [1] Sleep disorders have been confirmed as a core component of Alzheimer's disease (AD), and the accumulation of β-amyloid protein (Aβ) in brain tissue is an important pathological feature of AD. However, how Aβ affects AD-related sleep disorders is unclear. This study used animal and cell models to conduct experiments to detect the association between sleep disorders and Aβ. The results showed that administration of Aβ25-35 significantly reduced non-rapid eye movement sleep in mice while increasing their wakefulness. In addition, reverse transcription-quantitative polymerase chain reaction and Western blot analysis showed that, compared with control mice, the expression levels of tau protein, phosphorylated tau protein (p-tau), orexin A, and adenosine A1 receptor (A1R) expressed by orexin neurons were significantly upregulated in the brain tissue of AD mice. In addition, in vitro studies have shown that, compared with control cells, the expression levels of tau protein, p-tau protein, orexin A and adenosine A1R in human neuroblastoma SH-SY5Y cells treated with Aβ25-35 were significantly increased. Furthermore, the tau protein inhibitor TRx 0237 significantly reversed the promoting effect of Aβ25-35 on the expression levels of tau protein, p-tau protein, orexin A and adenosine A1R, while knockdown of adenosine A1R or orexin A also inhibited the expression levels of tau protein and p-tau protein in Aβ25-35-mediated AD. These results suggest that Aβ and tau proteins may be considered as novel biomarkers of sleep disorders in the pathology of Alzheimer's disease (AD), and they play a role by regulating the expression levels of orexin A and adenosine A1R. [2]

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In the past decade, many clinical trials of anti-Aβ drugs have failed, which challenges the hypothesis that Aβ accumulation is the initiating event of the AD pathological cascade and highlights the need to develop new treatments and targets. Among TAI drugs, MT belongs to the diaminophenothiazine class of compounds, which exhibit TAI activity in vitro. MTC (MT administered in its oxidized form MT+) was studied in an exploratory phase II double-blind dose-range clinical trial enrolling 321 patients with mild to moderate AD. At 24 weeks, both clinical and molecular imaging endpoints showed that the lowest effective dose of MT was 138 mg/day. The study found that this dose prevented regional cerebral blood flow loss, particularly in the medial temporal lobe structures and temporoparietal lobe region. Because the delivery of the highest dose of MT is subject to dose-dependent dissolution and absorption limitations, four phase I studies and two preclinical in vitro and in vivo studies are required to fully address the bioavailability limitations of the MT form tested in the phase II trial, thus laying the foundation for a phase III trial of TRx0237 for AD treatment. TRx0237 is claimed to have superior pharmacokinetics and tolerability compared to MTC, but convincing evidence to support this claim has not yet been provided. In healthy volunteers, TRx0237 showed better oral absorption after ingestion compared to MTC, but this did not translate into higher central nervous system drug concentrations, as the intracranial drug concentrations were almost identical in miniature pigs after administration of 33 mg/kg (approximately 5 μM) of either MT or TRx0237. On the other hand, there are currently no data on the concentration of TRx0237 in human cerebrospinal fluid. Reliable data on the safety and tolerability of TRx0237 in humans are also lacking, making direct comparison with MTC impossible. In vitro comparative data showed that the therapeutic index (LD50/EC50 ratio) of LMT-dihydrobromide was 92, that of LMT-dihydromethanesulfonate was 179, and that of MTC was 110. We consider these in vitro differences to be insignificant and insufficient to translate into pharmacological or clinical differences. Regarding efficacy, pharmacological studies in a transgenic mouse tau proteinosis model did not show a significant difference between the two compounds. In fact, a dose of 45 mg/kg of MTC or TRx0237 produced the same behavioral effects[3].

C17H21N3O3S2

379.493

477.106

C, 45.27; H, 5.70; N, 8.80; O, 20.10; S, 20.14

1236208-20-0

613-11-6;1236208-20-0 (mesylate); 951131-15-0 (HBr); 61-73-4 (chloride);

60150609

Light green to green solid powder

3

10

2

30

396

0

SPCMQFLNOVTUBM-UHFFFAOYSA-N

InChI=1S/C16H19N3S.2CH4O3S/c1-18(2)11-5-7-13-15(9-11)20-16-10-12(19(3)4)6-8-14(16)17-132*1-5(2,3)4/h5-10,17H,1-4H32*1H3,(H,2,3,4)

3,7-bis(dimethylamino)phenothiazin-5-ium methanesulfonate

TRX-0237 LMTX; L-MTx LMT-X; TRX 0237 TRX0237; Leuco-MTx; Methylene blue mesylate; 1236208-20-0; Leucomethylene blue Mesylate; Leucomethylene Blue dimesylate; Hydromethylthionine mesylate; Leucomethylene blue (Mesylate); Leucomethylene Blue bismesylate; TRX-0237 dimesylate; LMTM; Leuco-methylonium bis(hydromethanesulfonate);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~110 mg/mL (~230.31 mM)
H2O : ~83.33 mg/mL (~174.47 mM)

Solubility in Formulation 1: ≥ 7.5 mg/mL (15.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 75.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (5.76 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 50 mg/mL (104.69 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)