Mark Wainwright

Methylene Blue--a therapeutic dye for all seasons?

Abstract Since it was first synthesised in 1876, Methylene Blue (MB) has found uses in many different areas of clinical medicine, ranging from dementia to cancer chemotherapy. In addition, MB formed the basis of antimicrobial chemotherapy - particularly in the area of antimalarials – and eventually led to the discovery of the neuroleptic drug families. More recently, the photosensitising potential of MB and its congeners has been recognised, and these are being applied in various antimicrobial fields, especially that of blood disinfection. The range of activities of MB is due to the combination of its simple chemical structure and facility for oxidation-reduction reactions in situ.

Increased cytotoxicity and phototoxicity in the methylene blue series via chromophore methylation.

The cytotoxic and photodynamic activities of the commercially-available biological stains methylene blue (MB), 1,9-dimethyl MB (Taylors Blue) and a newly synthesised compound, 1-methyl MB, were measured against the murine mammary tumour cell line, EMT-6. Both 1-methyl MB and 1,9-dimethyl MB exhibited increased dark toxicity with concomitant higher phototoxicity compared to MB at a light dose of 7.2 J cm-2. While increasing the light dose as a function of the fluence rate increased the photocytotoxicity of MB, this had little effect on the methylated derivatives. In vitro chemical testing proved that successive methylation rendered the phenothiazinium chromophore both more resistant to reduction to its inactive leuco form, and also led to increased levels of singlet-oxygen production, thus providing a possible explanation for the increased toxicities of the methylated derivatives. Comparisons are made with the benzo[a]phenothiazinium photosensitizer, EtNBS.

Methylene blue derivatives--suitable photoantimicrobials for blood product disinfection?

Photodynamic antimicrobial agents based on the well-established phenothiazinium biological stain methylene blue offer a simple method for the inactivation or destruction of pathogens contained in donated blood and blood products. The technique is currently concentrated on viruses and the disinfective procedure can be carried out in blood bags using basic low-power light sources. Pathogens of the bacterial, yeast and protozoal classes are also susceptible to phenothiaziniums. The photoantimicrobial mode of action is usually via oxidative damage to cellular components, either due to redox reactions between the agent and a biomolecular target or by the action of reactive oxygen species generated in situ by photodynamic action. The targeting of various microbial species is discussed in relation to the physicochemical make-up of the photosensitizers, and future directions are suggested.

Phenothiazinium photosensitisers: choices in synthesis and application

The use of phenothiazinium dyes in the photodynamic therapy of cancer and its related antimicrobial protocols, e.g. blood product decontamination, has been mainly limited to a few commercially available dyes, such as Methylene Blue, the Azure stains and Toluidine Blue O. Novel Methylene Blue derivatives are scarce in the literature, and yet there are various synthetic routes available to furnish sufficient candidate compounds for properly organised programmes of photosensitiser design and development to be carried out. In this review, consideration is given to the types of phenothiazinium derivative required for the various photodynamic applications currently under examination, and also to the synthetic strategies involved in their preparation.

Photodynamic Therapy: The Development of New Photosensitisers

The first 20 years of anticancer photodynamic therapy (PDT) were based on the utility of the oligomeric mixture haematoporphyrin derivative (HpD) in various forms. More recently new derivatives have become available, both porphyrin-derived and employing new chromophores, for example from the phthalocyanine and phenothiazinium families. In addition, a major research effort has been rewarded with the clinical acceptance of the porphyrin precursor 5-aminolaevulinic acid (ALA). New photosensitisers intended for clinical use must exhibit advantageous drug performance profiles compared to the first-generation porphyrin derivatives. This can be seen, in vitro, in improved photophysical properties such as the extension of the useful light absorption spectrum into the near infrared - offering greater tissue penetration - as well as in the synthesis of pure compounds rather than mixtures. In this review, recent developments in photosensitiser families are discussed with respect to in vitro performance indicators and to potential application in oncology.

Review: The phenothiazinium chromophore and the evolution of antimalarial drugs

The phenothiazinium salt methylene blue [3,7‐bis(dimethylamino)phenothiazinium chloride] is the oldest known synthetic antimalarial drug, its clinical efficacy having been reported in 1891. The role of methylene blue in the evolution of the modern antimalarial armoury is often unappreciated, yet it can be linked directly to standard drugs such as chloroquine and its congeners. Also, in the face of increasing plasmodial resistance to modern antimalarials, phenothiazinium derivatives have again featured as lead compounds in drug research. The precise mode of action of methylene blue and its commercial analogues against Plasmodium spp. remains a cause for conjecture, having been variously described as nucleic acid intercalation, food vacuole basification, parasite redox cycle interference and haem polymerization inhibition. That the activity of the series may be due to more than one route – i.e. a multifactorial activity – underlines the utility of these compounds in antimalarial research either as single drugs or as adjuvants (partners in a drug combination), particularly in the face of resistant parasitic strains.

The development of phenothiazinium photosensitisers.

Methylene blue has been widely used since the late 19th century in biomedical research, and was the lead compound in several important clinical areas, including therapeutics for malaria and schizophrenia. The photodynamic therapy (PDT) of cancer and, more recently, of microbial infection (photodynamic antimicrobial chemotherapy (PACT)) has also employed methylene blue and its congeners, among other chemical types, due to the low human toxicities and efficient photosensitising properties of the group. However, little work has been carried out in terms of derivative and structure-activity development, most reports covering standard, commercially available compounds. This review deals with the evolution of phenothiazinium photosensitisers for both PACT and PDT use.

Some observations on the synthesis of polysubstituted zinc phthalocyanine sensitisers for photodynamic therapy

The syntheses and properties of a number of polysubstituted zinc phthalocyanines are described which are suitable as sensitisers for photodynamic therapy. Zinc phthalocyanine tetrasulphonic acid, itself a useful photosensitiser, has been used to prepare tetrasulphonamides by converting it to the tetrasulphonyl chloride and reacting this with various amino compounds. This has afforded lipophilic and water-soluble anionic and neutral tetra-sulphonamido derivatives. Chlormethylation of zinc phthalocyanine under carefully defined conditions followed by reaction with pyridine affords a water-soluble cationic dye with an average of two methylenepyridinium groups per molecule. Hydrolysis of zinc phthalocyanine tetracarboxyamide could not be carried out to completion to afford the tetracarboxylic acid, and the resultant product had an average composition corresponding to a dicarboxylic acid-dicarboxyamide structure. Visible absorption spectroscopic properties and singlet oxygen sensitising efficiencies of the dyes (measured relative to methylene blue) are described. All the dyes are photocytotoxic in vitro and show anti-tumour photoactivity in vivo.

Photodynamic antimicrobial chemotherapy (PACT) for the treatment of malaria, leishmaniasis and trypanosomiasis

A photodynamic effect occurs when photosensitiser molecules absorb light and dissipate the absorbed energy by transferring it to biological acceptors (usually oxygen), generating an excess of reactive species that are able to force cells into death pathways. Several tropical diseases present physiopathological aspects that are accessible to the application of a photosensitiser and local illumination. In addition, disease may be transmitted through infected blood donations, and many of the aetiological agents associated with tropical diseases have been shown to be susceptible to the photodynamic approach. However, there has been no systematic investigation of the application of photoantimicrobial agents in the various presentations, whether to human disease or to the disinfection of blood products or even as photo-insecticides. We aim in this review to report the advances in the photoantimicrobial approach that are beneficial to the field of anti-parasite therapy and also have the potential to facilitate the development of low-cost/high-efficiency protocols for underserved populations.

Photoinactivation of viruses

Although the photodynamic effect was demonstrated against viral targets more than seventy years ago, the use of photosensitisers as antivirals in vivo has been slow in gaining acceptance. From a clinical viewpoint, this may be due to the pronounced side effects produced in several cases of the phototreatment of herpes genitalis in the early 1970s, the unfortunate patients presenting with post-treatment Bowens disease. Currently, the clinical use of photosensitisers in this field is limited to the treatment of laryngeal papillomata. However, considerable progress has been made in the photodynamic disinfection of blood products. Photoantivirals have traditionally been targeted at viral nucleic acid, in many cases via an intercalative mechanism. However, given the potential for deleterious sequelae associated with this route, the design of new photosensitisers should encourage alternative targets, such as viral enzymes or the cell envelope (where this exists). Targeting is obviously determined by the chemistry of the photosensitiser employed and there are many different structural types available. The chemistry, photochemistry and cellular effects of the various agents are discussed, along with future prospects for this exciting area of medicine.