Mechanistic Insight into the Photodynamic Effect Mediated by Neutral Red and a New Azine Compound in Staphylococcus aureus Cells.

The photodynamic activity of Neutral Red and the new monobrominated Neutral Red was studied in suspensions of Staphylococcus aureus. The effect of mannitol and sodium azide in the presence of 25 µm photosensitizer on lethal photosensitization were investigated. The results of the mechanistic evaluation of Neutral Red showed that both mannitol and sodium azide produced a completed protective effect after irradiation without significant differences between them. The evaluation of monobrominated Neutral Red also showed a protective effect of microorganisms with the addition of mannitol. Although sodium azide produced a protective effect of the photoinactivation, it was incomplete and less than that exhibited by mannitol. The results indicate that the starting reagent, Neutral Red, is a producer of radical species, acting through a type I mechanism, whereas the halogenated derivative of Neutral Red produced reactive oxygen species and a contribution of singlet molecular oxygen cannot be discarded in the photoinactivation of Staphylococcus aureus cells. These results, analyzed together with the previously evaluated properties of the dyes, allow us to explain the differences observed in the photoinactivation of Staphylococcus aureus mediated by both azine photosensitizers.

Evaluation of physicochemical properties and bacterial photoinactivation of phenothiazine photosensitizers.

We report herein the physicochemical properties and antimicrobial activity of a new monobrominated derivative of Azure B and its parent compound. These dyes are used as photosensitizers for photodynamic therapy and photodynamic antimicrobial chemotherapy. Relevant pharmaceutical properties (pKa, chemical and photochemical stability, and in vitro antimicrobial activity) were determined. A UV-visible spectrophotometry method was developed and validated according to the International Conference on Harmonization (ICH) guidelines for use in stability indicating studies and determination of the acid dissociation constant of Azure B and its monobrominated derivative. The results showed that both dyes were chemically stable. In addition, bromination of the phenothiazine dye decreased its photochemical stability and pKa value without affecting the ionization rate at physiological pH. The analytical parameters for validation of the method were linearity (r2 > 0.9981), limit of detection (LOD) (0.2-0.9 µM), limit of quantification (LOQ) (0.6-2.7 µM), and intra-day precision (0.76-1.40%) expressed as relative standard deviation (RSD). Recoveries ranging from 99.5 to 100.9% were obtained for the two dyes. Thus, this method provides a simple, sensitive, accurate, and precise assay for the determination of all compounds. The effect of photosensitizer concentration and visible irradiation time on lethal photosensitization against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was investigated. Both photosensitizers were active against the evaluated bacteria. However, the new monobrominated derivative was more effective than its predecessor and managed to eradicate these microorganisms by using different doses of the dye and light. In other words, a lower concentration of AzBBr and irradiation time were required to cause bacterial death equal to or greater than its precursor. The photodynamic efficacy of the two photosensitizers presented the following order: S. aureus > E. coli > P. aeruginosa. These studies indicated that the tested dyes satisfy the conditions of potential photosensitizers in terms of physicochemical and antimicrobial properties.

Thiazine dyes: Evaluation of monomeric and aggregate forms.

The aggregation phenomenon of Azure B, monobrominated Azure B, Thionine and Methylene Blue was studied by UV-Visible spectrophotometry in different media as a function of dye concentration and temperature variations. The tests carried out in organic solvents allowed the identification of monomeric species of these compounds, which have not been reported in literature and have been wrongly assigned for years. The results obtained in water allowed demonstrating that different kinds of aggregates are present in this medium. In addition, the aggregation tendency of these dyes in organic solvent and aqueous media was established. Several parameters such as lipophilicity, effect of bulky substituents and interactions with media were considered to interpret the aggregation behavior of thiazine dyes.

Photodynamic properties and photoinactivation of Candida albicans mediated by brominated derivatives of triarylmethane and phenothiazinium dyes.

The photodynamic activity of brominated derivatives of New Fuchsin and Azure B was studied in solution and in cell suspensions of Candida albicans. The spectroscopic and photodynamic properties of these photosensitizers were compared with those of Crystal Violet and Azure B, which represent active photosensitizer related to each family of compounds. Triarylmethane derivatives absorb intensely with a band centered at ∼ 570 nm, while the phenothiazinium dyes at ∼ 650 nm. Photooxidation of 9,10-dimethylanthracene was observed using phenothiazinium compounds indicating the formation of singlet molecular oxygen, while it was not detected using triarylmethane agents. However, triarylmethane dyes were able to photooxidize l-tryptophan. In yeast cell suspensions, the photosensitized inactivation of C. albicans increases with photosensitizer concentration, causing a ∼ 5 log decrease of cell survival, when the cultures are treated with 20 µM of Crystal Violet and irradiated for 60 min. Under these conditions, the photodynamic activity of 50 µM Azure B induced a ∼ 3 log decrease of cell survival. Studies of photodynamic action mechanism indicated that photoinactivation of C. albicans cells induced by triarylmethane compounds involves mainly type I photoprocess. Although, phenothiazinium derivatives produce singlet molecular oxygen, a contribution of other reactive oxygen species cannot be discarded in the photoinactivation of C. albicans.

Physicochemical properties and photodynamic activity of novel derivatives of triarylmethane and thiazine.

Triarylmethane and thiazine dyes have attracted attention as anticancer and antimicrobial agents, due to their structural features and selective localizations. Although these dyes have been initially explored in the context of photodynamic therapy, some of these such as New Fuchsin and Azure B have still not been extensively investigated. For this reason, we evaluated the chemical stability, aggregation effect, and lipophilicity, as well as the photodynamic activity against LM-2 murine mammary carcinoma cells of five new brominated dyes of triarylmethane and thiazine. These cationic compounds were obtained at high purities and unequivocally characterized by conventional techniques. The introduction of bromine atoms into the chromophoric system of New Fuchsin and Azure B dyes gave rise to a moderate bathochromic shift and increased the lipophilicity, thereby improving their photophysical and photochemical properties for biomedical applications. Moreover, the in vitro photodynamic activity demonstrated that, as the degree of bromination increased, the phototoxicity remained unchanged or decreased. The lower efficiency to inactivate cultured tumor cells may be attributed to the formation of the colorless carbinol pseudobase and aggregation effects for triarylmethane and thiazine dyes, respectively. A promising strategy to reverse the biological activity decrease observed might be the design of third-generation photosensitizers.

Separation, purification, and characterization of analogues components of a commercial sample of new Fuchsin.

New Fuchsin (NF), also known as Magenta III, has potential applications in photodynamic therapy. The commercial product labeled NF contains two other dye components in different proportions, Magenta II and Magenta I (Rosaniline). The proportions of NF, Magenta II, and Magenta I determined by reversed-phase high-performance liquid chromatography (RP-HPLC) in the commercial sample used were 71.6 +/- 0.4%, 25.2 +/- 0.2%, and 2.8 +/- 0.1% (n = 7), respectively. The isolation, purification, and characterization of commercial NF dye components were carried out applying different techniques, such as preparative column liquid chromatography (PCLC), thin layer chromatography (TLC), RP-HPLC, absorption spectrophotometry, nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry (ESI-MS), and tandem electrospray ionization mass spectrometry (ESI-MS-MS). After separation and isolation, the degree of purity obtained for NF compound was higher than 95% and 92% for Magenta II and Magenta I compounds, respectively. Therefore, it is essential to ensure a high degree of purity of these dyes as raw material to obtain new drugs intended for therapeutic treatments.

Simultaneous determination of bifonazole and tinctures of calendula flower in pharmaceutical creams by reversed-phase liquid chromatography.

The aim of this research was to develop and validate a sensitive, rapid, easy, and precise reversed-phase liquid chromatography (LC) method for stability studies of bifonazole (I) formulated with tinctures of calendula flower (II). The method was especially developed for the analysis and quantitative determination of I and II in pure and combined forms in cream pharmaceutical formulations without using gradient elution and at room temperature. The influence on the stability of compound I of temperature, artificial radiation, and drug II used for the new pharmaceutical design was evaluated. The LC separation was carried out using a Supelcosil LC-18 column (25 cm x 4.6 mm id, 5 microm particle size); the mobile phase was composed of methanol-0.1 M ammonium acetate buffer (85 + 15, v/v) pumped isocratically at a flow rate of 1 mL/min; and ultraviolet detection was at 254 nm. The analysis time was less than 10 min. Calibration graphs were found to be linear in the 0.125-0.375 mg/mL (rI = 0.9991) and 0.639-1.916 mg/mL (rII = 0.9995) ranges for I and II, respectively. The linearity, precision, recovery, and limits of detection and quantification were satisfactory for I and II. The results obtained suggested that the developed LC method is selective and specific for the analysis of I and II in pharmaceutical products, and that it can be applied to stability studies.

Simultaneous spectrophotometric determination of phenilpropanolamine HCL, caffeine and diazepam in tablets.

A rapid, reliable and specific UV spectrophotometric method was developed to determine Phenilpropanolamine Hydrochloride (I), Caffeine (II) and Diazepam (III) formulated in tablets. This method was validated and compared with a liquid chromatography (LC) procedure used for the simultaneous quantitative analysis of the drugs. The established linearity ranges by both methods for compounds I, II and III were 0.36-0.88, 0.012-0.028 and 0.036-0.084 mg/ml, respectively. The correlation coefficients by HPLC were r(I)(2)=0.997, r(II)(2)=0.999, r(III)(2)=0.999 and by the UV spectrophotometric method were r(I)(2)=0.998, r(II)(2)=0.996, r(III)(2)=0.999. LC and UV methods showed excellent precision and accuracy. As regards precision, LC showed CV values range of 0.2-0.9 and UV 0.15-0.72. On the other hand, accuracy was obtained with CV values range of 0.1-1.8 and 0.32-1.11 for LC and UV, respectively. The recoveries of I, II and III were >98.04% for both methods over the linear range. The UV and HPLC methods have been successfully used to determine the I, II and III content in tablets of different origin.

Qualitative and quantitative reversed-phase liquid chromatography of a new bisisoxazolylnaphthoquinone.

The main objective of this study was to develop and test the applicability of a sensitive, accurate, and precise liquid chromatographic (LC) method for evaluating the stability characteristics of a new bisisoxazolylnaphthoquinone, 2-(3,5-dimethyl-4-isoxazolylamino)-N-(3,5-dimethyl-4-isoxazolyl)-1,4-naphthoquinone-4-imine compound 1. The method was shown to be selective and stability-indicating. Isocratic elution with a mobile phase of methanol-water (75 + 25, v/v) on a reversed-phase column with UV detection at ambient temperature completely resolved compound 1 from its degradation products. The LC system was calibrated by plotting peak responses versus known concentrations of a reference standard by using an internal standardization procedure. Complete elution occurred after 12 min with a peak symmetry factor of 0.95 for the drug peak. The kinetic degradation of compound 1 was studied over a pH range of 0.88-14.00 to determine the kinetic parameters involved in its decomposition path in aqueous solution.