Técnicas analíticas para la caracterización químico-farmacéutica de lacas para el tratamiento de la onicomicosis

  1. Pérez Rodríguez, Zenia
Supervised by:
  1. María Pilar Viñas López-Pelegrín Director
  2. Natalia Campillo Seva Director
  3. Zenaida Rodríguez Negrín Director

Defence university: Universidad de Murcia

Fecha de defensa: 19 November 2024

Committee:
  1. Manuel Hernández Córdoba Chair
  2. Natalia Arroyo Manzanares Secretary
  3. Mirtha Mayra González Bedia Committee member

Type: Thesis

Abstract

Fungal infections of the hands and feet are very common in humans. Dermatophytes, yeasts and moulds are the three main fungi that affect the skin and can cause onychomycosis. Currently, there are many commercially available oral antifungal agents that have been used for the treatment of fungal infections of the fingernails and toenails, but these show several disadvantages such as toxicity and high active pharmaceutical ingredient (API) contents. The API 2-bromo-5-(2-bromo-2-nitrovinyl)furan (also known as Furvina®) is an efficient antimicrobial agent with broad spectrum activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi. The drug in the form of a dermatological ointment based on this 0.15% IFA for the treatment of fungal diseases of skin and nails is active against onychomycosis but requires prolonged treatments due to its poor penetration into the nail, as it is an ointment. The general objective of this Thesis is to develop different analytical methodologies for the chemical-pharmaceutical characterisation of nail lacquers for the treatment of onychomycosis containing Furvina® as an IFA. The development and validation of a UV/VIS spectrophotometric analytical technique for the determination of Furvina® in different lacquers has been proposed. The validation of the method using international guidelines showed excellent results of specificity, linearity, precision (coefficients of variation (CV) lower than 5%), trueness (97-103%) and sensitivity (limit of detection (LOD) and limit of quantification (LQ) equal to 0.038 and 0.156 µg/mL, respectively). Thus, the proposed methodology is an interesting tool easily accessible to most laboratories for the determination of Furvina®. A method based on the combination of capillary liquid chromatography with diode array detection (LCcapillary-DAD) is developed and validated for the determination of Furvina® in lacquers. Method validation provided very favourable values for linearity (0.1-10 µg/mL), precision (CV< 5%), trueness (99-101%) and sensitivity (0.01 and 0.035 µg/mL for LD and LQ, respectively). On the other hand, the application of the miniaturised dispersive liquid-liquid microextraction technique (DLLME) in the treatment of urine samples prior to their analysis by LCcapillary-DAD has allowed not only to increase the sensitivity of the methodology, but also to simultaneously improve selectivity, as DLLME acts by pre-concentrating the analyte and cleaning the sample matrix. Thus, the DLLME with LCcapillary-DAD developed method allows reaching high levels of analyte preconcentration in very short times with excellent values for the different analytical parameters. The characterisation of nail lacquers is proposed using different analytical methodologies including X-ray diffraction (XRD), scanning electron microscopy (SEM), infrared spectroscopy (IR) and differential scanning calorimetry (DSC), which are shown to be very useful tools. Thus, diffractograms obtained by XRD suggest that the lacquers do not comply with Bragg's Law, as they are not penetrated by X-rays. SEM analyses have shown morphological and typographical changes of the IFA crystals in the formulations. The detection of the characteristic IFA bands by IR analysis of the formulations corroborates the presence of Furvina® in the lacquers. Likewise, the results obtained by DSC have allowed establishing the optimum storage temperature conditions of the formulations (87.57-95.46 ºC). In addition, the evaluation of the penetrability of the lacquers using Franz cells concludes the need to recommend contact times with the nail longer than 11 days for them to be effective. The use of the atomic absorption spectrophotometry technique with high resolution continuous source electrothermal atomisation for the determination of the levels of the metals lead, arsenic and copper in different nail lacquers and in Furvina® IFA is also addressed. The methodologies developed for the metals have allowed very good results to be achieved for the validation parameters, confirming that they comply with the maximum limits established by international standards, so that the products can be considered safe in terms of the metals studied. Finally, the dermal single dose toxicity in rabbits is studied and the dermal irritant and/or corrosive effects on rabbits are determined for the nail lacquers. The results obtained show that the lacquers do not produce demonstrable dermal toxicity when administered at a limit dose of 2000 mg/kg; furthermore, they have no dermal irritant potential and are classified as non-irritating to the skin.