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The main aim of the present study was to synthetize polyacrylamide nanoparticles and to use them as photosensitizer carriers. The new monobrominated derivatives (monobrominated neutral red and monobrominated azure B) were the photosensitizers used for antimicrobial photodynamic therapy. They were loaded into the nanocarriers and their antibacterial and oxidative activities were evaluated. The polyacrylamide nanoparticles were evaluated and prepared by inverse microemulsion polymerization. The nanoparticles obtained were characterized by size, polydispersity index, and zeta potential analysis. The Dynamic Light Scattering indicated that the diameter of the particle (z-average) was optimal, with an acceptable polydispersity index. The antibacterial activity of the polyacrylamide nanoparticles loaded with photosensitizers was evaluated against Staphylococcus aureus. Both photosensitizers loaded into the nanoparticles showed great potential as antibacterial agents since they suppressed the bacterial growth. The maximum percentage of growth reduction was 35.5% (>2 Log CFU/mL), with the monobrominated azure B loaded into the nanocarrier with 2 hydroxyethyl methacrylate against methicillin resistant S. aureus. The improved physicochemical and photophysical properties of these photosensitizers were accompanied by a significant increase in the photoantimicrobial action, in conventional-sensitive and-methicillin resistant S. aureus. The results obtained clearly suggest that polyacrylamide nanoparticles loaded with photosensitizers have great potential for further application in antimicrobial photodynamic therapy. Fil: Gualdesi, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Aiassa, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Vara, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Alvarez Igarzabal, Cecilia Ines. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina

The aim of the present paper was to optimize the properties of phenothiazine photosensitizers (PSs), Azure B and its monobrominated derivative. They were entrapped in multilamellar liposomes of egg phosphatidylcholine (EPC) and dipalmitoylphosphatidylcholine (dpPC). The higher partition coefficient of both PSs in the EPC/water system compared with that of both PSs in the dpPC/water system allowed the selection of EPC to be used for the development of third generation photosensitizers. The optimal phospholipid/phenothiazine ratio and the location of these dyes in the lipid bilayer systems were established. These dyes were found to be located within the polar head group region of the lipid bilayer, which would enhance the effectiveness of the irradiation procedure. In addition, the encapsulation of both photosensitizers in liposomes led to a decrease in the aggregation of these compounds, and consequently to an increase in their singlet oxygen quantum yield (ϕΔ). The encapsulation of the PSs in lipid vesicles significantly increased their photochemical reactivity, doubling the ϕΔ value of AzBBr and increasing that of AzB by 60%. The results obtained demonstrate that the vehiculization of these PSs in liposomes allowed the development of third generation photosensitizers with better photochemical properties to be used in potential therapeutic applications. Fil: Vara, Jimena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Sanchez, Julieta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; Argentina Fil: Perillo, Maria Angelica. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina

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.

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. Fil: Vara, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Gualdesi, María Soledad. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina Fil: Aiassa, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina

The aim of this work was to investigate the aggregation effect of Azure B and monobrominated Azure B on their singlet oxygen generation. The monomeric and dimeric forms of these dyes were evaluated to select the species with better properties as photosensitizers. The results demonstrate that the monomeric forms are better producers of singlet oxygen than their aggregated species. However, the dimeric species of monobrominated Azure B is also a good producer of singlet oxygen. The bromination of this phenothiazine dye significantly increases the singlet oxygen quantum yields of the monomeric and dimeric species, enhancing its properties like photosensitizer and its application in Photodynamic Therapy. In addition, significant differences were observed in the singlet oxygen production of the monomeric forms of both photosensitizers determined by phosphorescence decay of singlet oxygen and photooxidation of 9,10-dimethylanthracene (DMA). The physicochemical interaction between the excited species of the phenothiazine monomers and the molecules of DMA could be responsible for the lower values of singlet oxygen production because the disaggregated forms did not present any activity as physical or chemical attenuators. The dimeric form of phenothiazine dyes did not show this interaction. In conclusion, there is a need for developing vehiculization systems that stabilize the monomers of phenothiazine dyes because they have better properties than their aggregates in terms of singlet oxygen production. Fil: Vara, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Gualdesi, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Bertolotti, Sonia Graciela. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina

There are promising data on the use of poly(acrylamide) nanoparticles (PAA-NPs) in the development of third generation photosensitizers for application in antimicrobial photodynamic therapy. These nanoparticle delivery systems are suitable for biological applications and have potential for drug delivery due to their biocompatibility, chemical flexibility, easy preparation and low cost. The objective of this work was to obtain new PAA-NPs for the vehiculization of the monobrominated derivatives of Azure B (AzBBr) and Neutral Red (NRBr). These two dyes were found to have promising properties as photosensitizers. However, the use of nanotechnology was required to overcome difficulties in solubility, aggregation and stability in order to optimize their photodynamic efficiency. The combination of different monomers and crosslinkers allowed us to develop two new PAA-NPs. These systems were loaded with AzBBr and NRBr and characterized according to their morphology, size, polydispersity index and zeta potential. The new third generation photosensitizers showed better photochemical reactivity, chemical stability and photodynamic efficacy against Gram-positive and Gram-negative bacteria than the free photosensitizers. The use of this vehiculization strategy improved the properties of the second generation photosensitizers (AzBBr and NRBr) studied. This improvement in their properties makes them good candidates for potential phototherapeutic applications against numerous pathologies.

To face the challenges of optimizing the properties of the photosensitizers currently used in the photodynamic inactivation of various microorganisms, we propose the bromination of thionine and the use of polymeric nanoparticles for the entrapment of halogenated derivative. Within this framework, the dibrominated derivative of thionine was synthesized and characterized. This new compound showed a high chemical and photochemical stability. In addition, bromination decreased the ionization constant and increased the aggregation of this photosensitizer in an aqueous medium. For this reason, the new compound showed a lower quantum yield of singlet oxygen production. However, the dibrominated thionine showed greater cell damage after photodynamic treatment. In order to prevent or decrease the aggregation of the photosensitizer in an aqueous medium, it was vehiculized in different polymeric polyacrylamide nanoparticles. In general, the vehiculization of the photosensitizers (PSs) in nanosized systems increased the production of singlet oxygen and enhanced their phototoxic activity against the microorganisms evaluated (methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus). The nanoparticle formed with the monomer 2 hydroxyethyl methacrylate (HEMA) showed the best photodynamic efficacy. The results obtained clearly suggest that polyacrylamide nanoparticles loaded with photosensitizers of the thiazine family have great potential for further application in antimicrobial photodynamic therapy.

Cresyl violet and cresyl red, components of commercial cresyl violet acetate, were separated and purified using preparative column liquid chromatography. The stationary phase was silica gel and gradient elution was carried out using chloroform:methanol. The purified dyes were obtained in high yield; 51% of the original lot was recovered as cresyl violet and 40% as cresyl red. Separated materials were characterized by nuclear magnetic resonance and mass spectroscopy; UV-visible and Fourier-transform infrared spectra also were obtained for samples of pure cresyl violet and cresyl red. The colored constituents of the commercial dye lot were identified using thin layer chromatography and reverse phase high performance liquid chromatography. Both methodologies were suitable for routine testing; reverse phase high performance liquid chromatography is an appropriate tool for quality control and high resolution identification of these compounds.

A thiazine derivative of AzB, the monoiodinated Azure B (AzBI), was designed, characterized, chemical and photochemically evaluated, and screened for antibacterial activity. The monoiodinated derivative showed better chemical and photochemical properties compared to the leading compound Azure B (AzB). Also, AzBI exhibited good antibacterial activity against methicillin-sensitive Staphylococcus aureus (MSSA) ATCC 29213 and methicillin-resistant S. aureus (MRSA) ATCC 43300. The assayed compound represents a promising structure for the development of a new synthetic photosensitizer with improved properties and biological activity. Based on the results obtained for AzBI, this compound was loaded in the polymeric nanoparticles synthesized by our working group in order to improve some of its physicochemical properties. In this way, it was possible to successfully load the monoiodinated derivative and yield a promising drug transport system. The latter presented better characteristics and antimicrobial activity than AzBI against the different strains tested. All these results make AzBI an excellent candidate for application in photodynamic therapy.

This research evaluated the synthesis and suitability of synthetic and commercial photosensitizers for use as potential photosensitizers in photodynamic therapy and photodynamic antimicrobial chemotherapy. Spectroscopic properties, experimental aggregation behavior in ethanol, N,N-dimethylformamide, water and water:polyethylene glycol 400, computational details, the ability to generate singlet oxygen, logPHPLC and theoretical logP were determined. All the compounds evaluated formed aggregates in different media. Novel brominated tested compounds exhibited higher lipophilicity and generation of singlet oxygen in comparison with the corresponding starting reagent. The studies showed that the tested oxazine and oxazone dyes satisfy the conditions of a potential drug in terms of physicochemical and photochemical properties. Fil: Urrutia, María Noel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

At present, polyacrylamide nanoparticles are attractive to drug delivery. However, some physicochemical characteristics of these nanoparticles still need to be further improved in practice. Polyacrylamide nanoparticles with an average size of 80 nm and a zeta potential of 30 mV were synthesized and used as photosensitizer carriers. The new monobrominated derivatives and parent compounds were the photosensitizers for the photodynamic therapy loaded in the nanocarrier. The physicochemical characterization of the prepared nanoparticles, drug loading, the ability to generate singlet oxygen and chemical stability were investigated. The novel tested nanoparticles exhibited a loading percentage of between 80 and 99%, higher generation of singlet oxygen and good stability in comparison with the corresponding starting reagent. According to these results, the novel polyacrylamide nanoparticles are excellent candidates for drug vehiculization. Fil: Gualdesi, María Soledad. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Alvarez Igarzabal, Cecilia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; Argentina Fil: Vara, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina

The formation of the inclusion complexes of Basic Violet 2 (New Fuchsin) with hydroxypropyl-β-cyclodextrin (HP-β-CD) was studied by UV-Vis absorption and fluorescence spectroscopy. The binding constants of the inclusion complexes, KB, were obtained considering the maximum shift of the absorption spectra, the absorption areas deconvoluted as the sum of two gaussian constituents, the relative fluorescence quantum yield and the fluorescence maximum shift. The values achieved were 2.7 × 103 M -1, 0.95 × 103 M-1 and 2.8 × 103 M-1, 2.2 × 103 M-1, respectively. Ab-initio calculations using B3LYP/6-31G level of approach and the natural bond orbital (NBO) analysis were performed to gain a deeper understanding of the inclusion phenomena. The result showed a high complexation in aqueous media. © 2011 Elsevier Ltd. All rights reserved. Fil: Aiassa Martínez, Ivana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Montes de Oca, María Noel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina Fil: Iriarte, Ana Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Ortiz, Cristina Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina Fil: Arguello, Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina

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. Fil: Vara, Jimena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

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 × 4.6 mm id, 5 μm 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.

Aggregation phenomena of Neutral Red and a new derivative monobrominated Neutral Red were examined in different buffer solutions as a function of dye concentration. The dyes were chemically and photochemically stable and the lipophilicity of the new bromo-compound was characterized by experimental and computer prediction data. In addition, the acid dissociation constant and the singlet molecular oxygen production were determined spectrophotometrically yielding values of 1.16 and 3.14 for Neutral Red and the monobrominated derivative, respectively. The effect of photosensitizer concentration, visible irradiation time and cellular density on lethal photosensitization against Staphylococcus aureus were investigated. The improved physicochemical and photophysical properties of monobrominated Neutral Red were accompanied by a significant increase in the photoantimicrobial action, in conventional-sensitive and -resistant isolates. Based on these results, the dyes exhibited favorable properties; therefore, the new derivative compound could be a promising sensitizer with applications in the photoinactivation of S. aureus. Fil: Urrutia, María Noel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alovero, Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

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.

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.

The goal of this study was to determine the kinetic parameters involved in the decomposition of 2-(5-methyl-4-isoxazolylamino)-N-(5-methyl-4-isoxazolyl)-1,4-naphthoquinone-4-imine (1) in aqueous solution and to identify the main degradation products. An isocratic HPLC assay was used to study the degradation rate of 1. The products of hydrolysis were identified by comparison of their retention times with those of authentic samples. The amount of 1 and the two degradation products resulting from storage of 1 in various buffer solutions was followed in function of time by a reversed-phase HPLC stability-indicating method. The observed degradation rates followed pseudo-first-order kinetics at constant pH, temperature and ionic strength. The log k–pH-profile was constructed at 35°C from the first-order rate constants obtained from studies at pH values ranging from 0.88 to 10.80 (μ=0.5 M). Hydrolysis in the acidic and alkaline media resulted in the formation of two degradation products in each case. The pH-rate profile of 1 in buffer solution was adequately described using a four-term rate equation. The obtained pH-rate profile indicated specific acid–base catalysis with a region of maximum stability between pH 6.40 and 7.40 which can be adequate for formulations of 1.

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. Fil: Alvarez, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina Fil: Durantini, Edgardo Néstor. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina Fil: Milanesio, María Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina Fil: Durantini, Edgardo Néstor. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina

A simple low pressure liquid chromatographic method is reported that can separate the basic fuchsine homologues, rosaniline, magenta II and new fuchsine from an impure commercial dye. The chromatographic purity of the separated dyes is > 90%. All homologues were obtained in multi-milligram amounts per chromatographic run; precise yields depend on the composition of the starting material and potentially may be greater. This is a useful preparative procedure for generating chromatographically pure samples of basic fuchsine homologues, especially those that cannot be obtained in pure form by direct synthesis.

In this study, we have reported for the first time a complete experimental investigation on the compound Neutral Red and the new monobrominated derivative in different media as a function of the concentration. These compounds belong to the quinone-imine class of dyes and have good potential to be applied as photosensitizers in Photodynamic Therapy. Although the aggregation of Neutral Red has been reported by several authors, this has not been thoroughly evaluated due to spectral changes occurring depending on the solvent and concentration of dye. We investigated and compared the effects of aggregation properties in water, ethanol, water–ethanol mixtures, water-polyethylene glycol 400 (75:25 v/v) and N,N-dimethylformamide. An analysis of the changes in the absorption spectra with respect to the solvent as a function of dye concentration together with theoretical calculations confirmed the monomeric species and the formation of different types of aggregates in this class of compounds. Fil: Urrutia, María Noel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

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. Fil: Montes de Oca, María Noel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vara, Jimena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Milla Sanabria, Laura Natalia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rivarola, Viviana. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

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. Fil: Montes de Oca, María Noel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Aiassa Martínez, Ivana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Urrutia, María Noel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Arguello, Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Ortiz, Cristina Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

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.

The kinetics of enolization and degradation of N ‐(5‐methyl‐4‐isoxazolyl)‐4‐amino‐1,2‐naphthoquinone (1) was investigated in aqueous solutions over a pH range of 7.30 to 12.25, at 35 °C and at constant ionic strength (μ = 0.5) using reversed‐phase HPLC. Pseudo‐first‐order kinetics was observed throughout the pH range studied. The rate of enolization ( k e ), the keto–enol equilibrium constant ( k t ), and specific base catalysis rate constant ( k OH ) were determined. Good agreement between the theoretical pH–rate profile and the experimental data supports the proposed transformation process. The average recovery for 1 and its tautomerization product 2‐hydroxy‐ N ‐(5‐methyl‐4‐isoxazolyl)‐1,4‐naphthoquinone 4‐imine ( 2 ) from mixtures of different composition was evaluated.

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.

A reverse phase high performance liquid chromatography assay was carried out for the simultaneous determination of three active principles present in tablets of different origin and wide commercial use in the Province of Cordoba (Argentina). Prescriptions, commercially available as appetite suppressants, very often include the active principles Phenylpropanolamine Hydrochloride ( I ), Caffeine ( II ) and Diazepam ( III ). Simultaneous determination of these three drugs: anorexic, central nervous stimulant and tranquilizer, respectively, in pharmaceutical dosage forms has not been reported. In this study these active principles are quantified. The only sample preparation necessary for the analysis was their dilution with acetonitrile. The resulting solution was filtered and analyzed on a column packed with Supelcosil LC-18 (5 μm) with acetonitrile:water (30:70 v/v) as initial mobile phase (0.4 ml min −1 ) and the detection was performed at 254 nm. Then a linear gradient up to 100% acetonitrile in 18 min (3.0 ml min −1 ) was applied. The procedure was simple and suitable for quality control. The calibration function was established in the ranges of 0.072–0.168 mg ml −1 for I , 0.036–0.084 mg ml −1 for II and 0.06–0.196 mg ml −1 for III . The detection limits of these compounds were 12.8, 4.1 and 11.0 μg ml −1 , respectively with linear response. No chromatographic interference from the tablet excipients was found. The method described in this paper was validated following the analytical performance parameters required by the USP XXIV, and was successfully applied to the commercial tablets.

A reverse phase high performance liquid chromatographic assay was carried out for the simultaneous determination of two out of three active principles present in a pharmaceutical preparation. This method was developed to assess the quality of the product.