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 a better pharmacokinetic and tolerability profile than MTC, but not convincing evidences have been provided to support this. The better oral absorption of TRx0237 compared to MTC in the presence of food showed in healthy volunteers did not translate in higher CNS levels since drug brain levels in minipigs are almost identical after 33 mg/kg (about 5 μM) of MT or TRx0237. On the other hand, no data on TRx0237 CSF concentrations in humans are available. No robust data on safety and tolerability of TRx0237 in humans are available to make direct comparison with MTC. Comparative in vitro data showed a therapeutic index (ratio of LD50/EC50) of 92 for LMT-dihydrobromide and 179 for LMT-dihydromesylate compared to value of 110 for MTC. We believe that these in vitro differences were not so dramatic to necessarily translating in pharmacological or clinical differences. In terms of efficacy, pharmacological studies in transgenic mouse tauopathy models did not show dramatic differences between the two compounds. Indeed, a dose of 45 mg/kg of MTC or TRx0237 produced identical behavioral effects.[3]

A safety and tolerability study of TRx0237 (250 mg/day for 4 weeks) in nine patients with mild-to-moderate AD began in September 2012 but it was terminated in April 2013, reportedly for administrative reasons (ClinicalTrials.gov Identifier: NCT01626391) (Table 1). Three Phase III placebo-controlled studies with TRx0237 are ongoing (Table 1). The first study is evaluating the 200-mg/day dose in 700 patients with a diagnosis of either all-cause dementia and probable AD and adopted the cognitive ADAS-Cog 11 scale and the clinical Alzheimer’s Disease Cooperative Study – Clinical Global Impression of Change (ADCS-CGIC) scale as primary efficacy variables (ClinicalTrials.gov Identifier: NCT01689233). The second study is evaluating the doses of 150 and 250 mg/day in 833 patients with mild-to-moderate AD and is using the ADAS-Cog 11 and ADCS-CGIC as primary endpoints (ClinicalTrials.gov Identifier: NCT01689246). The third Phase III trial is evaluating the 200 mg/day dose in 220 patients affected by the behavioral variant of frontotemporal dementia (bvFTD) (ClinicalTrials.gov Identifier: NCT01626378). This trial adopted a modified version of the ADCS-CGIC scale as measure of clinical efficacy and the revised Addenbrooke’s Cognitive Examination as cognitive measure. Finally, an open-label extension study in subjects who have completed participation in a Phase II or Phase III trials with TRx0237 is evaluating the long-term safety of the compound (ClinicalTrials.gov Identifier: NCT02245568) (Table 1). With the hope of maintaining blinding, the Phase III studies are using ‘active placebo’ tablets that include 4 mg of TRx0237 as a urinary and fecal colorant. Overall, these Phase III trials are recruiting 1753 patients at 250 centers in 22 countries and results are expected in the first half of 2016 for one of these trials (ClinicalTrials.gov Identifier: NCT01689246) and in the second half of 2016 for the other two studies (ClinicalTrials.gov Identifiers: NCT01689233 and NCT01626378).[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 a maximum clinical trial phase of III (across all indications) and has 2 investigational indications.

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A liquid chromatographic method for the determination of methylene blue and leucomethylene blue in male and female Fischer 344 rat urine and male and female B6C3F1 mouse urine was developed for use in supporting toxicokinetic studies, validated, and used to analyze urine samples for a preliminary dose level range-finding study. The method was validated for a concentration range of 10.0 to 20,000 ng/mL in urine. Samples up to 75,000 ng/mL demonstrated good recoveries when diluted into the range of the calibration curve. Six sets of calibration standards were prepared in F344 male rat urine for analysis to demonstrate reproducibility and ruggedness. The stability of sample extracts was determined under various storage conditions. During the course of analyses of the animal samples, a possible metabolic process was observed. Although Azure B is a significant impurity in methylene blue trihydrate, the amount of Azure B seen in urine samples collected from rodents dosed with methylene blue trihydrate is significantly greater than the amount seen in rodent urine spiked directly with methylene blue. This observation suggests possible N-demethylation of the methylene blue as a metabolic transformation to Azure B.[1]

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Sleep disorder is confirmed as a core component of Alzheimer's disease (AD), while the accumulation of amyloid β (Aβ) in brain tissue is an important pathological feature of AD. However, how Aβ affects AD‑associated sleep disorder is not yet well understood. In the present study, experiments on animal and cell models were performed to detect the association between sleep disorder and Aβ. It was observed that Aβ25‑35 administration significantly decreased non‑rapid eye movement sleep, while it increased wakefulness in mice. In addition, reverse transcription‑quantitative polymerase chain reaction and western blot analysis revealed that the expression levels of tau, p‑tau, orexin A and orexin neurons express adenosine A1 receptor (A1R) were markedly upregulated in the brain tissue of AD mice compared with that in samples obtained from control mice. Furthermore, the in vitro study revealed that the expression levels of tau, p‑tau, orexin A and adenosine A1R were also significantly increased in human neuroblastoma SH‑SY5Y cells treated with Aβ25‑35 as compared with the control cells. In addition, the tau inhibitor TRx 0237 significantly reversed the promoting effects of Aβ25‑35 on tau, p‑tau, orexin A and adenosine A1R expression levels, and adenosine A1R or orexin A knockdown also inhibited tau and p‑tau expression levels mediated by Aβ25‑35 in AD. These results indicate that Aβ and tau may be considered as novel biomarkers of sleep disorder in AD pathology, and that they function by regulating the expression levels of orexin A and adenosine A1R. [2]

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In the last 10 years, several clinical trials with anti-Aβ agents failed, challenging the hypothesis that Aβ accumulation is the initiating event in the pathological AD cascade, and underscoring the need for novel therapeutic approaches and targets. Among TAIs, MT belongs to a class of diaminophenothiazines that have TAI activity in vitro. MTC, in which MT is dosed as the oxidized form MT+, was investigated in an exploratory Phase II dose-ranging double-blind clinical trial in 321 patients with mild-to-moderate AD. The minimum effective dose was identified as 138 mg MT/day at both clinical and molecular imaging endpoints at 24 weeks. Treatment at this dose was found to prevent the decline in regional cerebral blood flow, particularly in medial temporal lobe structures and temporoparietal regions. Given that the delivery of the highest dose of MT was impaired due to dose-dependent dissolution and absorption limitations, four Phase I studies and two preclinical in vitro and in vivo studies were required to get to the bottom of the bioavailability limitations of the form of MT tested in the Phase II trial, setting out the basis for proceeding into Phase III trials with TRx0237 for AD treatment. TRx0237 is claimed to have a better pharmacokinetic and tolerability profile than MTC, but not convincing evidences have been provided to support this. The better oral absorption of TRx0237 compared to MTC in the presence of food showed in healthy volunteers did not translate in higher CNS levels since drug brain levels in minipigs are almost identical after 33 mg/kg (about 5 μM) of MT or TRx0237. On the other hand, no data on TRx0237 CSF concentrations in humans are available. No robust data on safety and tolerability of TRx0237 in humans are available to make direct comparison with MTC. Comparative in vitro data showed a therapeutic index (ratio of LD50/EC50) of 92 for LMT-dihydrobromide and 179 for LMT-dihydromesylate compared to value of 110 for MTC. We believe that these in vitro differences were not so dramatic to necessarily translating in pharmacological or clinical differences. In terms of efficacy, pharmacological studies in transgenic mouse tauopathy models did not show dramatic differences between the two compounds. Indeed, a dose of 45 mg/kg of MTC or TRx0237 produced identical behavioral effects [3]

C16H21BR2N3S

447.233

444.982

C, 42.97; H, 4.73; Br, 35.73; N, 9.40; S, 7.17

951131-15-0

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

23651551

Solid powder

3

4

2

22

304

0

Br.Br.S1C2C=C(C=CC=2NC2C=CC(=CC1=2)N(C)C)N(C)C

JAUPSVVTGFBHTN-UHFFFAOYSA-N

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

N3,N3,N7,N7-Tetramethyl-10H-phenothiazine-3,7-diamine dihydrobromide

TRX-0237; Leucomethylene Blue dihydrobromide; TRX-0237 dihydrobromide; TRX0237; TRX 0237; 951131-15-0; Leucomethylene Blue dihydrobromide; TRx0237; E79ZM68IOZ; Leukomethylene Blue dihydrobromide; Hydromethylthionine (dihydrobromide); Reduced methylene Blue dihydrobromide; N3,N3,N7,N7-Tetramethyl-10H-phenothiazine-3,7-diamine dihydrobromide; TRX-0237 HBr; Leukomethylene Blue dihydrobromide; Reduced methylene Blue dihydrobromide; Hydromethylthionine HBr

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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