FTIR spectroscopy of chronic venous leg ulcer exudates: an approach to spectral healing marker identification.

Chronic venous leg ulcer (CVLU) arises as a chronic venous insufficiency complication and is a major cause of morbidity throughout the world. Our hypothesis is that the CVLU exudate composition is a biochemical representation of the wound clinical state. Then, Fourier Transform Infrared (FTIR) spectroscopy could be a useful and less-invasive technique to study the clinical state of the ulcer. For this, the aim of this work was to perform a spectral characterization of the exudate from CVLU using FTIR spectroscopy to identify potential healing markers. 45 exudate samples from CVLU, 95% of the strains isolated from CVLU in planktonic and biofilm phenotypes and other related biological samples such as human plasma, serum, urine, blood cells, urea, creatinine, glucose and albumin were studied by FTIR spectroscopy. According to the vibration frequency of biomolecules' (lipids, proteins, nucleic acids and carbohydrates) characteristic bonds in the infrared region, different spectral windows were selected and spectral areas of each window were measured. Besides, Savitzky-Golay second derivatives were obtained for all spectra and peaks from each standardized window were detected. FTIR spectroscopy allowed identification of sample types (exudate, plasma, serum, urine) as each one presents a unique relative composition and ratios range. Also, this technique could be useful to identify bacteria in the phenotypic-ulcer state and allows differentiation of whether bacteria are in the biofilm or planktonic form which is unlikely by conventional methods. In this work we found some spectral markers (areas, peaks) that allow identification of several parameters in the exudate such as (a) total cellularity, (b) inflammatory cell load, (c) bacterial load, (d) fibrin amount, and (e) inflammatory proteins. Because the measured areas or founded peaks are concentration-dependent this method could also serve to measure them. Therefore, FTIR spectroscopy could be useful to evaluate patient evolution as all these exudate parameters represent critical negative markers for wound healing.

Novel topical formulation for ischemic chronic wounds. Technological design, quality control and safety evaluation.

Ulceration of the foot in diabetes is common and disabling, and frequently leads to amputation of the leg. The pathogenesis of foot ulceration is complex, clinical presentation variable and management requires early expert assessment. Despite treatment, ulcers readily become chronic wounds. Chronic wounds are those that remain in a chronic inflammatory state failing a normal healing process patterns. This is partially caused by inefficient eradication of opportunistic pathogens like Pseudomonas aeruginosa. We propose its control or eradication will promote wound healing. Lactobacillus plantarum cultures supernatants (LAPS) shows antipathogenic and pro-healing properties. The main objective was to design two pharmaceutical dosage forms by using LAPS as active pharmaceutical ingredient and to perform its quality control, in vitro activity conservation tests and human trials (safety evaluation). Both selected formulations reach the technological quality expected for 120 days, shows adequate occlusive characteristics and proper adhesion to human skin. From the in vitro release assays were found that LAPS shows adequate release from matrix and maintain its antimicrobial and anti-biofilm activity. First human trials were developed and neither edema nor erythema on healthy skin voluntaries was found. We conclude that C80 and C100 are adequate for their use in future clinical trials to demonstrate a comprehensive therapeutic effectiveness in ischemic chronic wounds.

Equipment and method for in vitro release measurements on topical dosage forms.

CONTEXT: In countries where research budgets are meager as Argentina, the tendency to innovation and improvements in the designs prototypes "made in Argentina" marks a growing trend adopted by researchers. This article presents a diffusion cell of original design, for release studies of Active Pharmaceutical Ingredient (API) from classical topical dosage forms, also includes the methodology for its optimization and validation. The objective was to evaluate and validate a system designed and to compare it to the Franz cells system. METHODS: Parameters, reproducibility and robustness were performed included factors as, stirring conditions, membrane stabilization treatment and temperature variation. Release and retention on membrane assay were performed using two different API and formulations. RESULTS: The method is reproducible and robust for the parameters tested. Release assays show that no significative difference with the Franz Cells system. Our system allows the simultaneous measurement of different parameters, representing an innovation on these methodologies. The LMC was used for assays of in vitro retention on membrane and the values obtained were reproducible and coincident whit values obtained for other authors. CONCLUSIONS: The system designed and the methodology employed, are acceptable for in vitro release studies. The device and method has the characteristics required.

Formulation and quality control of semi-solid containing harmless bacteria by-products: chronic wounds pro-healing activity.

Chronic wounds are those that remain in a chronic inflammatory state and fail to follow normal healing process. Infection is one of the most important causes of chronicity. A frequent pathogen isolated from chronic infections is Pseudomonas aeruginosa; refractory to therapy and host immune attack in its biofilm phenotype. Lactobacillus plantarum cultures supernatants (LAPS) interfere with its pathogenic capacity. In addition, LAPS showed bacteriostatic and bactericide properties and is neither cytotoxic nor an inductor of necrosis-apoptosis. LAPSs chemical composition was determined; allowing us to propose a correlation between its constituents and their biological activity. This article shows a pharmaceutical dosage form designed by using LAPS as an API with pro-healing activity and its quality control. Pharmacotechnical and anti-microbial assays were adapted to demonstrate that the vehicle used does not modify LAPS activities. Selected formulation (F100) showed fair mechanical and technological properties. From the in vitro release assays was found an adequate release from the carrier matrix and maintains its anti-pathogenic activity for 6 months. We propose F100 for chronic wounds treatment. The use of harmless bacteria by-products, such as LAPS, to antagonize infectious pathogens that have ability to form biofilm is an efficient and economic approach to treat infected chronic wounds.

Actividad antifúngica sinérgica de combinaciones de estatinas y azólicos revelada mediante bioanálisis con Saccharomyces cerevisiae y Candida utilis y cuantificación de ergosterol / Synergistic antifungal activity of statin–azole associations as witnessed by Saccharomyces cerevisiae- and Candida utilis-bioassays and ergosterol quantification

Fundamento. La frecuencia de micosis oportunistas y la resistencia a los antimicóticos convencionales han fomentado la búsqueda de nuevas alternativas terapéuticas, como las combinaciones de antimicóticos. Objetivos. El presente estudio trató de detectar el sinergismo antifúngico entre las estatinas y los azólicos mediante un bioanálisis de difusión en pocillos de agar, utilizando Saccharomyces cerevisiae (S. cerevisiae) ATCC 32051 y Candida utilis (C. utilis) PR1-2 como cepas de control. Métodos. Los efectos antifúngicos sinérgicos se examinaron mediante la adición simultánea de una concentración sub-inhibitoria (CSI) de estatina (atorvastatina, lovastatina, pravastatina, rosuvastatina o simvastatina) más una concentración mínima inhibitoria (CMI) de un azólico (clotrimazol, fluconazol, itraconazol, ketoconazol o miconazol) a placas de agar YNB con las levaduras sembradas por inclusión. Un resultado positivo correspondió a un diámetro del halo de inhibición del crecimiento de la levadura mayor que el producido por la CMI del azólico exclusivo. Para confirmar el sinergismo estatina-azólico, se cuantificó el ergosterol de la membrana celular de las levaduras con cromatografía líquida de alto rendimiento (HPLC-RP). Para valorar la inhibición de la síntesis de ergosterol inducida por estatinas, se emplearon bioanálisis de rescate de ergosterol. Resultados. La inhibición del crecimiento aumentó significativamente cuando se combinaron clotrimazol, fluconazol, itraconazol, ketoconazol y miconazol con atorvastatina, lovastatina, rosuvastatina y simvastatina. Los mayores incrementos de la inhibición del crecimiento se observaron en S. cerevisiae (77,5%) y C. utilis (43,2%) con una CSI de simvastatina y una CMI de miconazol de 4+2,4mg/ml o 20+4,8mg/ml, respectivamente. Para pravastatina apenas se identificaron efectos significativos (incremento de la inhibición del 0-7,6%). Los mayores cocientes de interacción correspondieron a la combinación de simvastatina y miconazol y fueron indicativos de sinergismo. Este también se confirmó por la mayor disminución de los niveles celulares de ergosterol (S. cerevisiae, 40% y C. utilis, 22%). La inhibición de la síntesis de ergosterol inducida por estatinas se corroboró mediante bioanálisis de rescate de ergosterol, donde la inhibición por pravastatina se abolió con facilidad, mientras que la de rosuvastatina fue la más refractaria. Conclusiones. Las combinaciones seleccionadas de estatinas y azólicos podrían ser alternativas viables para el manejo terapéutico de las micosis, en dosis más bajas o con una mayor eficiencia(AU)

Background. Frequent opportunist fungal infections and the resistance to available antifungal drugs promoted the development of new alternatives for treatment, like antifungal drug combinations. Aims. This work aimed to detect the antifungal synergism between statins and azoles by means of an agar-well diffusion bioassay with Saccharomyces cerevisiae ATCC 32051 and Candida utilis Pr1–2 as test strains. Methods. Synergistic antifungal effects were tested by simultaneously adding a sub inhibitory concentration (SIC) of statin (atorvastatin, lovastatin, pravastatin, rosuvastatin or simvastatin) plus a minimal inhibitory concentration (MIC) of azole (clotrimazole, fluconazole, itraconazole, ketoconazole or miconazole) to yeast-embedded YNB agar plates, and a positive result corresponded to a yeast growth inhibition halo higher than that produced by the MIC of the azole alone. Yeast cell ergosterol quantification by RP-HPLC was used to confirm statin–azole synergism, and ergosterol rescue bioassays were performed for evaluating statin-induced ergosterol synthesis blockage. Results. Growth inhibition was significantly increased when clotrimazole, fluconazole, itraconazole, ketoconazole and miconazole were combined with atorvastatin, lovastatin, rosuvastatin and simvastatin. Highest growth inhibition increments were observed on S. cerevisiae (77.5%) and C. utilis (43.2%) with a SIC of simvastatin plus a MIC of miconazole, i.e. 4+2.4mg/ml or 20+4.8mg/ml, respectively. Pravastatin showed almost no significant effects (0–7.6% inhibition increase). Highest interaction ratios between antifungal agents corresponded to simvastatin–miconazole combinations and were indicative of synergism. Synergism was also confirmed by the increased reduction in cellular ergosterol levels (S. cerevisiae, 40% and C. utilis, 22%). Statin-induced ergosterol synthesis blockage was corroborated by means of ergosterol rescue bioassays, pravastatin being the most easily abolished inhibition whilst rosuvastatin being the most ergosterol-refractory. Conclusions. Selected statin–azole combinations might be viable alternatives for the therapeutic management of mycosis at lower administration doses or with a higher efficiency(AU)

Synergistic antifungal activity of statin-azole associations as witnessed by Saccharomyces cerevisiae- and Candida utilis-bioassays and ergosterol quantification.

BACKGROUND: Frequent opportunist fungal infections and the resistance to available antifungal drugs promoted the development of new alternatives for treatment, like antifungal drug combinations. AIMS: This work aimed to detect the antifungal synergism between statins and azoles by means of an agar-well diffusion bioassay with Saccharomyces cerevisiae ATCC 32051 and Candida utilis Pr(1-2) as test strains. METHODS: Synergistic antifungal effects were tested by simultaneously adding a sub inhibitory concentration (SIC) of statin (atorvastatin, lovastatin, pravastatin, rosuvastatin or simvastatin) plus a minimal inhibitory concentration (MIC) of azole (clotrimazole, fluconazole, itraconazole, ketoconazole or miconazole) to yeast-embedded YNB agar plates, and a positive result corresponded to a yeast growth inhibition halo higher than that produced by the MIC of the azole alone. Yeast cell ergosterol quantification by RP-HPLC was used to confirm statin-azole synergism, and ergosterol rescue bioassays were performed for evaluating statin-induced ergosterol synthesis blockage. RESULTS: Growth inhibition was significantly increased when clotrimazole, fluconazole, itraconazole, ketoconazole and miconazole were combined with atorvastatin, lovastatin, rosuvastatin and simvastatin. Highest growth inhibition increments were observed on S. cerevisiae (77.5%) and C. utilis (43.2%) with a SIC of simvastatin plus a MIC of miconazole, i.e. 4 + 2.4 µg/ml or 20 + 4.8 µg/ml, respectively. Pravastatin showed almost no significant effects (0-7.6% inhibition increase). Highest interaction ratios between antifungal agents corresponded to simvastatin-miconazole combinations and were indicative of synergism. Synergism was also confirmed by the increased reduction in cellular ergosterol levels (S. cerevisiae, 40% and C. utilis, 22%). Statin-induced ergosterol synthesis blockage was corroborated by means of ergosterol rescue bioassays, pravastatin being the most easily abolished inhibition whilst rosuvastatin being the most ergosterol-refractory. CONCLUSIONS: Selected statin-azole combinations might be viable alternatives for the therapeutic management of mycosis at lower administration doses or with a higher efficiency.