Arylamine Analogs of Methylene Blue: Substituent Effect on Aggregation Behavior and DNA Binding.

The synthesis of new phenothiazine derivatives, analogs of Methylene Blue, is of particular interest in the design of new drugs, as well as in the development of a new generation of agents for photodynamic therapy. In this study, two new derivatives of phenothiazine, i.e., 3,7-bis(4-aminophenylamino)phenothiazin-5-ium chloride dihydrochloride (PTZ1) and 3,7-bis(4-sulfophenylamino)phenothiazin-5-ium chloride (PTZ2), are synthesized for the first time and characterized by NMR, IR spectroscopy, HRMS and elemental analysis. The interaction of the obtained compounds PTZ1 and PTZ2 with salmon sperm DNA is investigated. It is shown by UV-Vis spectroscopy and DFT calculations that substituents in arylamine fragments play a crucial role in dimer formation and interaction with DNA. In the case of PTZ1, two amine groups promote H-aggregate formation and DNA interactions through groove binding and intercalation. In the case of PTZ2, sulfanilic acid fragments prevent any dimer formation and DNA binding due to electrostatic repulsion. DNA interaction mechanisms are studied and confirmed by UV-vis and fluorescence spectroscopy in comparison with Methylene Blue. The obtained results open significant opportunities for the development of new drugs and photodynamic agents.

Photoacoustic Imaging Quantifies Drug Release from Nanocarriers via Redox Chemistry of Dye-Labeled Cargo.

We report a new approach to monitor drug release from nanocarriers via a paclitaxel-methylene blue conjugate (PTX-MB) with redox activity. This construct is in a photoacoustically silent reduced state inside poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PTX-MB@PLGA NPs). During release, PTX-MB is spontaneously oxidized to produce a concentration-dependent photoacoustic signal. An in vitro drug-release study showed an initial burst release (25 %) between 0-24 h and a sustained release between 24-120 h with a cumulative release of 40.6 % and a 670-fold increase in photoacoustic signal. An in vivo murine drug release showed a photoacoustic signal enhancement of up to 649 % after 10 hours. PTX-MB@PLGA NPs showed an IC50 of 78 µg mL-1 and 44.7±4.8 % decrease of tumor burden in an orthotopic model of colon cancer via luciferase-positive CT26 cells.

Synthesis and antimicrobial photodynamic effect of methylene blue conjugated carbon nanotubes on E. coli and S. aureus.

Catheter-related bloodstream infections (CRBSIs) are one of the leading causes of high morbidity and mortality in hospitalized patients. The proper management, prevention and treatment of CRBSIs rely on the understanding of these highly resistant bacterial infections. The emergence of such a challenge to public health has resulted in the development of an alternative antimicrobial strategy called antimicrobial photodynamic therapy (aPDT). In the presence of a photosensitizer (PS), light of the appropriate wavelength, and molecular oxygen, aPDT generates reactive oxygen species (ROS) which lead to microbial cell death and cell damage. We investigated the enhanced antibacterial and antibiofilm activities of methylene blue conjugated carbon nanotubes (MBCNTs) on biofilms of E. coli and S. aureus using a laser light source at 670 nm with radiant exposure of 58.49 J cm-2. Photodynamic inactivation in test cultures showed 4.86 and 5.55 log10 reductions in E. coli and S. aureus, respectively. Biofilm inhibition assays, cell viability assays and EPS reduction assays showed higher inhibition in S. aureus than in E. coli, suggesting that pronounced ROS generation occurred due to photodynamic therapy in S. aureus. Results from a study into the mechanism of action proved that the cell membrane is the main target for photodynamic inactivation. Comparatively higher photodynamic inactivation was observed in Gram positive bacteria due to the increased production of free radicals inside these cells. From this study, we conclude that MBCNT can be used as a promising nanocomposite for the eradication of dangerous pathogens on medical devices.

Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease.

Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 µM. The Ki value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 µM. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 µM) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 µM) required to reverse behavioral deficits and pathology in Tau transgenic mice.

Azure B and a synthetic structural analogue of methylene blue, ethylthioninium chloride, present with antidepressant-like properties.

AIMS: The phenothiazinium compound, methylene blue (MB), possesses diverse pharmacological actions and is attracting attention for the treatment of bipolar disorder and Alzheimer's disease. MB acts on both monoamine oxidase (MAO) and the nitric oxide (NO)-cGMP pathway, and possesses antidepressant activity in rodents. The goal of this study was to synthesise a structural analogue of MB, ethylthioninium chloride (ETC), and to evaluate the effects of the structural changes on the MAO inhibitory and antidepressant properties of MB. This study also investigated the antidepressant properties of azure B, the major metabolite of MB, versus MB and imipramine as active comparators. MAIN METHODS: ETC and azure B were firstly evaluated as inhibitors of human MAO, and secondly for antidepressant-like activity in the acute forced swim test (FST) in rats, and compared to saline, imipramine and MB. KEY FINDINGS: The results document that ETC is a reversible inhibitor of MAO-A and MAO-B with IC50 values of 0.510 µM and 0.592 µM, respectively, and that it is a weaker MAO-A inhibitor than MB and azure B. ETC and azure B were more effective than imipramine and MB in reversing immobility in the FST without inducing locomotor effects, with evidence supporting a serotonergic action. Of interest is the finding that ETC is more toxic for cultured cells than MB. CONCLUSION: Azure B may therefore be a contributor to the antidepressant effect of MB. Small structural changes made to MB retain its antidepressant effect, even though the resulting phenothiazinium compound possesses reduced MAO-A inhibitory potency.

Fully-automated radiosynthesis and in vitro uptake investigation of [N-methyl-¹¹C]methylene blue.

Malignant melanoma is a type of skin cancer which can spread rapidly if not detected early and left untreated. Positron Emission Tomography (PET) is a powerful imaging technique for detecting cancer but with only a limited number of radiotracers available the development of novel PET probes for detection and prevention of cancer is imperative. In the present study we present the fully-automated radiosynthesis of [N-methyl-(11)C]methylene blue and an in vitro uptake study in metastasic melanoma cell lines. Using the GE TRACERlab FXc Pro module [N-methyl-(11)C]methylene blue was isolated via solid-phase extraction in an average time of 36 min after end of bombardment and formulated with a radiochemical purity greater than 95%. The in vitro uptake study of [N-methyl-(11)C]methylene blue in SK-MEL28 melanin-expressing melanoma cell line demonstrated in site-specific binding of 51% promoting it as a promising melanoma PET imaging agent.

Structure-based optimization of FDA-approved drug methylene blue as a c-myc G-quadruplex DNA stabilizer.

G-quadruplexes are non-canonical DNA secondary structures putatively present in the promoter regions of oncogenes in the human genome. The targeting of promoter G-quadruplex structures to repress oncogene transcription represents a potential anticancer strategy. Here, we have used high-throughput virtual screening to identify FDA-approved drug methylene blue (MB) as a promising scaffold for binding the c-myc oncogene G-quadruplex DNA. Based on molecular docking analysis of MB to the c-myc G-quadruplex, we designed and screened 50 MB derivatives containing side chains that could interact with the G-quadruplex grooves. As a proof-of-concept, the highest-scoring compounds were synthesized and the interactions with the c-myc G-quadruplex were investigated using the FID assay. The results showed that the methylene blue derivatives 6a-c were able to bind to the c-myc G-quadruplex with greater binding affinity compared to the known G-quadruplex binding ligand, crystal violet. The activity of the most potent compound identified from the FID assay, 6b, as an inhibitor for polymerase-drive DNA extension was examined using a PCR-stop assay and compared against that of the parent compound methylene blue. The results of the PCR-stop assay showed that the addition of the side chain improved the activity of the derivatives as an inhibitor compared to the parent compound. The MB derivative 6b was shown to be highly selective towards c-myc G-quadruplex over double-stranded DNA and other biologically relevant G-quadruplexes using UV-visible spectroscopy and mass spectrometry, respectively. The MB derivative 6b could induce or stabilize c-myc G-quadruplex formation in both cell-free and cellular biological models, and displayed higher cytoxicity against human hepatocarcinoma cells compared to the parent compound, MB.

Phenothiazinium photosensitisers VII: novel substituted asymmetric N-benzylphenothiaziniums as photoantimicrobial agents.

The synthesis of asymmetrical analogues of methylene blue, in which one of the dimethylamino groups is replaced by a diethylamino or di-n-propylamino group, and the other by benzylamino or 4-substituted benzylamino, is reported, the substituents being alkyl, alkoxyl or halogen. As expected, because of their longer alkyl chains these diethylamino- and di-n-propylamino derivatives proved to be considerably more lipophilic than the parent compound methylene blue, while maintaining suitable maximum absorption wavelengths and singlet oxygen efficiencies for photoantimicrobial use. Also as expected, in screening tests against Gram-positive and Gram-negative bacteria, the substituted benzylamino derivatives were highly active on illumination, presumably via singlet oxygen damage, and exhibited considerably increased activity against both classes relative to that of the standard, methylene blue. In addition, the more lipophilic derivatives exhibited greater activity against Escherichia coli. This may be due to increased interaction with the lipid-rich outer membrane of this Gram-negative bacterium. DNA binding of the derivatives was also increased, relative to methylene blue, showing large bathochromic shifts (>10nm) on binding typical of strong intercalators.

Síntese e estudo das propriedades fotoinduzidas de derivados fenotiazínicos em sistemas biomiméticos / Synteses and study of photoinduced properties of phenothiazine derivatives on biomimetic systems

Neste trabalho são apresentados estudos do efeito de interfaces nas propriedades fotofísicas e fotoquímicas do azul de metileno (AM) e de derivados fenotiazínicos com o intuito de avaliar o potencial destes compostos como fotossensibilizadores (FS) em terapia fotodinâmica. As propriedades físico-químicas do AM foram estudadas em soluções de SDS e observou-se que a presença do AM em solução altera o equilíbrio entre as micelas de SDS, -diminuindo o valor da concentração micelar crítica de 7mmolL-1 para 70µmoIL-1. A presença das micelas em solução também interfere nas propriedades do AM. Em baixas concentrações de SDS há formação de dímeros de AM, constatados pelo aumento da absorbância em 580nm e diminuição da emissão de fluorescência. A caracterização das espécies transientes mostrou a existência de moléculas de azul de metileno no estado triplete (3AM) e de oxigênio singlete em soluções com altas concentrações de SDS e a formação de espécies radicalares do AM em baixas concentrações do tensoativo. Esta observação sugere que o mecanismo fotoquímico do AM é dependente da sua concentração local próxima de interfaces carregadas. As interações do AM e de alguns de seus derivados fenotiazínicos (tionina, azure A e azure B) com vesículas e com células...

Radioiodinated methylene blue--a promising agent for melanoma scintigraphy: labelling, stability and in vitro uptake by melanoma cells.

Melanoma is a tumor of continuously increasing incidence for which new methods of imaging and targeted therapy are widely sought. Radioiodinated methylene blue is a promising tracer, showing selective uptake in human pigmented melanoma cells. We performed 131I-labeling of the tracer using 1% methylene blue injection United States Pharmacopeia (USP) and 131I sodium iodide. For quality control, a Merck high performance liquid chromatography (HPLC) system was used. We developed a new HPLC procedure using 0.1% trifluoroacetic acid, 90% acetonitrile and 10% water as solvent for isocratic elution of the tracer and applied a TLC method using ITLC-SG strips and the same solvent. The stability of the preparation was studied for 15 min, 3 h and 6 h. In order to evaluate the potential relevance of 131I-labeled methylene blue for melanoma detection, the in vitro uptake of 131I-methylene blue was investigated in SK-MEL 28 and 518A2 melanoma cells. Time and a temperature influence on uptake of 1311 methylene blue by these two melanoma cells were investigated. The radiochemical purity obtained by the HPLC method was 99.97 +/- 0.08% (n=8), while that by the TLC method was 99.88 +/- 0.16% (n=8). This indicates the excellent agreement between these two methods. The stability was persistent over 6 h and amounted to 99.75% +/- 0.21% (n=8). The uptake of 131I methylene blue was time and temperature dependent by both melanoma cells lines. The net cellular uptake on incubation at 37 degrees C of 131I methylene blue by SK-MEL 28 cells was high at 56.3-61.8% and that by 518A2-cells was 36.3-56.0%. Uptake by these cells was also investigated at 22 degrees C. The uptake by both cell types was also high at this temperature, but lower than that at 37 degrees C, amounting to 45.0-51.7% and 25.6-36.3%, respectively. Due to its easy handling and quite high uptake by melanoma cells, we expect that this tracer could be successfully used in routine application for melanoma imaging or eventual radiotherapy.

NMR studies on self-complementary oligonucleotides conjugated with methylene blue.

A carboxyl-functionalized methylene blue (MB) derivative was synthesized and covalently coupled to three CG-rich self-complementary 2'-deoxyoligonucleotides at their 5'-end. Thermodynamic and structural details about the interactions between the dye and oligonucleotide duplexes were investigated employing ultraviolet (UV) melting and (1)H nuclear magnetic resonance (NMR) experiments. In contrast to previous findings on MB binding, no specific intercalation or binding in the minor or major groove of the double helix was found in a 100 mM NaCl buffer. Rather, proton chemical shift changes in the conjugates provide ample evidence for weak dye-DNA interactions largely through external MB stacking on the terminal base pairs.

O comportamento do Mycobacterium lepraemurium nos testes de redução do azul de metileno e do tetrazólio / The behavior of \"Mycobacterium lepraemurium in tests to reduce the methylene blue and tetrazolium

Using M. lepraemurium suspensions obtained from leproma of rats inoculated by subcutaneous route or from nodules of the peritoneal cavity, the AA. have tested the capacity of these bacilli in reduce the methylene blue and formation of formazan from the tetrazolium. The time or intensity of reduction of methylene blue or formation of formazan are proportional to the concentration of bacilli in the suspensions and is increased with the storage in the refrigerator at 1øC. In comparison with tests perfomed upon other acid fast bacilli the ones with M. lepraemurium are developed very slowly.