Nanoparticles as carrier for improve therapeutic efficacy of pioglitazone in ocular inflammatory disorders: development and validation of a high throughput LC-MS/MS method for quantitation in ocular tissues

Abstract: Pioglitazone is an oral anti-hyperglycemic agent and it is used for the treatment of diabetes mellitus type 2. The anti-inflammatory activity has also been demonstrated in the literature. Pioglitazone belongs to Class II of Biopharmaceutical Classification System, i.e., slightly soluble and highly permeable. Polymeric nanoparticle formulations play an important role in the improvement of the efficacy of ocular therapies. These systems are non-toxic and biodegradable, show appropriate physicochemical characteristics as well as prolonged release profile suitable for ocular delivery. An accurate, sensitive, selective, reproducible and high throughput HPLC-MS/MS method was validated to quantitate pioglitazone in ocular tissues (cornea, sclera, lens, aqueous humour and vitreous humour). The chromatographic separation was achieved in 10 min on a Kinetex C18 column. Linear response of pioglitazone was observed over the range of 5-100 ng/ml. The limit of quantitation was 10 ng/ml (in extract). The recovery of pioglitazone or pioglitazone encapsulated in nanoparticles of polylactic-co-glycolic acid-polyethylene glycol (PLGA-PEG) was in the range 85-115 % in all tissues and levels tested. The intra-day and inter-day precision were <5 % and <10 % respectively. The obtained extracts demonstrated to be stable under various experimental conditions in all the studied matrices. This method can be applied to in vivo and ex vivo biodistribution studies related to the ocular administration of pioglitazone nanoparticles

Ex-Vivo and In-Vivo Assessment of Cyclamen europaeum Extract After Nasal Administration

Rhinosinusitis is a prevalent disorder with a severe impact on the health-related quality of life. Saponins of Cyclamen europaeum exert a clinically proven curative effect on rhinosinusitis symptoms when instilled into the nasal cavity, however, more extensive preclinical assessment is required to better characterize the efficacy of this botanical extract. This work evaluates the potential use of a natural freeze-dried extract of C. europaeum given as topical nasal administration. Permeation experiment on porcine nasal mucosa was performed with Franz diffusion cells. Experiments in rabbits were performed to test for any toxicological, hematological, biochemical or histological evidence of systemic action. No theoretical levels of saponins were found in the receptor chamber of Franz diffusion cells. Hematological data did not show significant differences between control and experimental animals (p > 0.05). Histological studies also showed that enhanced secretory activity in response to intranasal administration was not accompanied by any visible signs of injury. An examination of the brain, lungs, liver, kidneys, spleen, and gastrointestinal organs did not reveal any abnormality. The absence of mucosal permeation of saponins and negligible probability of C. europaeum saponins absorption in the course of a therapeutic application was demonstrated

Assessment of Efficacy and Safety Using PPAR-γ Agonist-Loaded Nanocarriers for Inflammatory Eye Diseases

Drug-loaded nanocarriers (NCs) are new systems that can greatly improve the delivery and targeting of drugs to specific tissues and organs. In our work, a PPAR-γ agonist loaded into polymeric NCs was prepared, stabilized by spray-drying, and tested in vitro, ex vivo, and in vivo (animal models) to provide a safe formulation for optical anti-inflammatory treatments. The NCs were shown to be well tolerated, and no signs of irritancy or alterations of the eye properties were detected by the in vitro HET-CAM test and in vivo Draize test. Furthermore, no signs of cytotoxicity were found in the NC formulations on retinoblastoma cells (Y-79) analyzed using the alamarBlue assay, and the transmittance experiments evidenced good corneal transparency with the formulations tested. The ocular anti-inflammatory study confirmed the significant prevention efficacy using the NCs, and these systems did not affect the corneal tissue structure. Moreover, the animal corneal structure treated with the NCs was analyzed using X-ray diffraction using synchrotron light. Small-angle X-ray scattering (SAXS) analysis did not show a significant difference in corneal collagen interfibrillar spacing after the treatment with freshly prepared NCs or NCs after the drying process compared to the corresponding negative control when inflammation was induced. Considering these results, the PPAR-γ agonist NCs could be a safe and effective alternative for the treatment of inflammatory ocular processes

Liquid chromatography - Mass spectrometry

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32166In this article, selected examples of applications of liquid chromatography coupled to mass spectrometry are given. The examples include the analysis of i) impurities in manufactured, pharmaceutical or synthesis products, ii) polyphenols in natural products, and iii) phytohormones in plant extracts. Finally, examples of applications of molecular characterization via flow injection analysis by electron spray ionization mass spectrometry (ESI-MS) are also given

Basics of mass spectrometry

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32166This article summarizes the basic principles of mass spectrometry instrumentation with special emphasis in sample introduction methods, ionization techniques and mass analyzers used in the different mass spectrometry techniques

Taula rodona

Taula rodona duta a terme dins el marc de l'edició del Face2Face 2013 a l'EPSEVG.Taula rodona duta a terme dins el marc de l'edició del Face2Face 2013 a l'EPSEVG

Assessment of Efficacy and Safety Using PPAR-&gamma; Agonist-Loaded Nanocarriers for Inflammatory Eye Diseases

Drug-loaded nanocarriers (NCs) are new systems that can greatly improve the delivery and targeting of drugs to specific tissues and organs. In our work, a PPAR-&gamma; agonist loaded into polymeric NCs was prepared, stabilized by spray-drying, and tested in vitro, ex vivo, and in vivo (animal models) to provide a safe formulation for optical anti-inflammatory treatments. The NCs were shown to be well tolerated, and no signs of irritancy or alterations of the eye properties were detected by the in vitro HET-CAM test and in vivo Draize test. Furthermore, no signs of cytotoxicity were found in the NC formulations on retinoblastoma cells (Y-79) analyzed using the alamarBlue assay, and the transmittance experiments evidenced good corneal transparency with the formulations tested. The ocular anti-inflammatory study confirmed the significant prevention efficacy using the NCs, and these systems did not affect the corneal tissue structure. Moreover, the animal corneal structure treated with the NCs was analyzed using X-ray diffraction using synchrotron light. Small-angle X-ray scattering (SAXS) analysis did not show a significant difference in corneal collagen interfibrillar spacing after the treatment with freshly prepared NCs or NCs after the drying process compared to the corresponding negative control when inflammation was induced. Considering these results, the PPAR-&gamma; agonist NCs could be a safe and effective alternative for the treatment of inflammatory ocular processes

Optimization of a liquid chromatography-tandem mass spectrometry method for the quantification of traces of taxanes in a Corylus avellana cell suspension medium

Since the recent discovery of taxol and other taxanes in Corylus avellana, this plant species has attracted interest as a potential new source of these compounds. However, its low taxane content in comparison with Taxus spp. has restricted research to analytical identification or global quantitation. A feasible and sensitive method based on liquid chromatography-tandem mass spectrometry using a triple quadrupole analyzer was developed for the analysis of taxol and four other taxanes in a Corylus avellana cell suspension medium. Taxanes were extracted from the cell culture medium with dichloromethane and analyzed using electrospray ionization and quantified by multiple-reaction monitoring mode. Methanol and matrix-matching calibration curves using docetaxel as the internal standard were analyzed. Linearity was confirmed over the whole calibration range (0.3–2.1 μg mL−1). The inter- and intra-day precision of taxanes ranged from 80% to 120% and the recovery rates were higher than 80%. Limits of detection were between 0.24–38 ng mL−1 and the limits of quantification were between 0.8–125 ng mL−1. The low detection and quantitation values obtained allowed us to detect small quantities of the released taxanes (120 ng mL−1 of B, 151 ng mL−1 of CF and 105 ng mL−1 of T), which correspond to about 0.5 ng mL−1 of each taxane, in the 20 mL Corylus avellana cell suspension culture medium extracted, even at the beginning of the culture. These results were confirmed by high resolution mass spectrometry.This research has been supported by a grant from the Spanish MEC (BIO2011-29856-CO2-01) and a grant from the Catalan Government (2014SGR215). A. Gallego was supported by a fellowship from the University Pompeu Fabra

Optimization of a liquid chromatography-tandem mass spectrometry method for the quantification of traces of taxanes in a Corylus avellana cell suspension medium

Since the recent discovery of taxol and other taxanes in Corylus avellana, this plant species has attracted interest as a potential new source of these compounds. However, its low taxane content in comparison with Taxus spp. has restricted research to analytical identification or global quantitation. A feasible and sensitive method based on liquid chromatography-tandem mass spectrometry using a triple quadrupole analyzer was developed for the analysis of taxol and four other taxanes in a Corylus avellana cell suspension medium. Taxanes were extracted from the cell culture medium with dichloromethane and analyzed using electrospray ionization and quantified by multiple-reaction monitoring mode. Methanol and matrix-matching calibration curves using docetaxel as the internal standard were analyzed. Linearity was confirmed over the whole calibration range (0.3-2.1 μg mL−1). The inter- and intra-day precision of taxanes ranged from 80% to 120% and the recovery rates were higher than 80%. Limits of detection were between 0.24-38 ng mL−1 and the limits of quantification were between 0.8-125 ng mL−1. The low detection and quantitation values obtained allowed us to detect small quantities of the released taxanes (120 ng mL−1 of B, 151 ng mL−1 of CF and 105 ng mL−1 of T), which correspond to about 0.5 ng mL−1 of each taxane, in the 20 mL Corylus avellana cell suspension culture medium extracted, even at the beginning of the culture. These results were confirmed by high resolution mass spectrometry

Alternative methods for sampling and preservation of photosynthetic pigments and tocopherols in plant material from remote locations

Current methods for the study of pigments involve freezing in liquid nitrogen and storage at -80°C or lyophilization until HPLC analysis. These requirements greatly restrict ecophysiological research in remote areas where such resources are hardly available. We aimed to overcome such limitations by developing several techniques not requiring freezing or lyophilization. Two species with contrasting foliar characteristics (Olea europaea and Taraxacum officinale) were chosen. Seven preservation methods were designed, optimized and tested in a field trial. These protocols were compared with a control immediately frozen after collection. Pigments and tocopherols were analysed by HPLC. Main artefacts were chlorophyll epimerization or phaeophytinization, carotenoid isomerization, altered de-epoxidation index and tocopherol degradation. Among all methods, sample desiccation in silica gel provides robust samples (pigment composition was unaffected by storage time or temperature) and almost unaltered pigment profiles, except for a shift in epoxidation state. Although liquid nitrogen freezing and subsequent lyophilization or freezer storage were preferred, when these facilities are either not available or not suitable for long-distance transport, desiccation with silica gel, passive extraction in acetone and/or storage of fresh samples in water vapour saturated atmospheres enable a complete pigment characterization. Silica gel is advisable for long-term sample conservation