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Laboratory rabbits are fed foods rich with cationic metals, and while fasting cannot empty gastric contents because of their coprophagic habits. This implies that, in rabbits, the oral bioavailability of chelating drugs could be modulated by the slow gastric emptying rates and the interaction (chelation, adsorption) with gastric metals. In the present study, we tried to develop a rabbit model with low amounts of cationic metals in the stomach for preclinical oral bioavailability studies of chelating drugs. The elimination of gastric metals was achieved by preventing food intake and coprophagy and administering a low concentration of EDTA 2Na solution one day before experiments. Control rabbits were fasted but coprophagy was not prevented. The efficacy of rabbits treated with EDTA 2Na was evaluated by comparing the gastric contents, gastric metal contents and gastric pH between EDTA-treated and control rabbits. The treatment with more than 10 mL of 1 mg/mL EDTA 2Na solution decreased the amounts of gastric contents, cationic metals and gastric pH, without causing mucosal damage. The absolute oral bioavailabilities (mean values) of levofloxacin (LFX), ciprofloxacin (CFX) and tetracycline hydrochloride (TC), chelating antibiotics, were significantly higher in EDTA-treated rabbits than those in control rabbits as follows: 119.0 vs. 87.2%, 9.37 vs. 13.7%, and 4.90 vs. 2.59%, respectively. The oral bioavailabilities of these drugs were significantly decreased when Al(OH)₃ was administered concomitantly in both control and EDTA-treated rabbits. In contrast, the absolute oral bioavailabilities of ethoxycarbonyl 1-ethyl hemiacetal ester (EHE) prodrugs of LFX and CFX (LFX-EHE, CFX-EHE), which are non-chelating prodrugs at least in in vitro condition, were comparable between control and EDTA-treated rabbits irrespective of the presence of Al(OH)₃, although some variation was observed among rabbits. The oral bioavailabilities of LFX and CFX from their EHE prodrugs were comparable with LFX and CFX alone, respectively, even in the presence of Al(OH)₃. In conclusion, LFX, CFX and TC exhibited higher oral bioavailabilities in EDTA-treated rabbits than in control rabbits, indicating that the oral bioavailabilities of these chelating drugs are reduced in untreated rabbits. In conclusion, EDTA-treated rabbits were found to exhibit low gastric contents including metals and low gastric pH, without causing mucosal damage. Ester prodrug of CFX was effective in preventing chelate formation with Al(OH)₃ in vitro and in vivo, as well as in the case of ester prodrugs of LFX. EDTA-treated rabbits are expected to provide great advantages in preclinical oral bioavailability studies of various drugs and dosage formulations. However, a marked interspecies difference was still observed in the oral bioavailability of CFX and TC between EDTA-treated rabbits and humans, possibly due to the contribution of adsorptive interaction in rabbits. Further study is necessary to seek out the usefulness of the EDTA-treated rabbit with less gastric contents and metals as an experimental animal. Full article

Cell injection therapy is emerging as an alternative to treat corneal endothelial dysfunction (CED) and to avoid corneal scarring due to bullous keratopathy. However, establishing a standardized culture procedure that provides appropriate cell yield while retaining functional features remains a challenge. Here, we describe a detailed framework obtained from in vitro culture of human corneal endothelial cells (HCECs) and comparative in vivo experimental models for CED treatment with a new cell tracking approach. Two digestion methods were compared regarding HCEC morphology and adhesion. The effect of Y-27632 (ROCKi) supplementation on final cell yield was also assessed. Cell adhesion efficacy with two cell delivery systems (superparamagnetic embedding and cell suspension) was evaluated in an ex vivo human cornea model and in an in vivo rabbit CED model. The injection of supplemented culture medium or balanced salt solution (BSS) was used for the positive and negative controls, respectively. HCEC isolation with collagenase resulted in better morphology and adhesion of cultured HCEC when compared to EDTA. Y-27632 supplementation resulted in a 2.6-fold increase in final cell yield compared to the control. Ex vivo and in vivo adhesion with both cell delivery systems was confirmed by cell tracker fluorescence detection. Corneal edema and opacity improved in both animal groups treated with cultured HCEC. The corneas in the control groups remained opaque. Both HCEC delivery systems seemed comparable as treatments for CED and for the prevention of corneal scarring. Full article

The purpose of this study was to evaluate the effectiveness and safety of a novel buffered riboflavin solution approved for corneal cross-linking (CXL) in progressive keratoconus and secondary corneal ectasia. Following the in vivo preclinical study performed on New Zealand rabbits comparing the novel 0.25% riboflavin solution (Safecross^®) containing 1% hydroxypropyl methylcellulose (HPMC) with a 0.1% riboflavin solution containing 0.10% EDTA, accelerated epithelium-off CXL was performed on 10 patients (10 eyes treated, with the contralateral eye used as control) through UV-A at a power setting of 9 mW/cm² with a total dose of 5.4 J/cm². Re-epithelialization was evaluated in the postoperative 7 days by fluorescein dye test at biomicroscopy; endothelial cell count and morphology (ECD) were analyzed by specular microscopy at the 1st and 6th month of follow-up and demarcation line depth (DLD) measured by anterior segment optical coherence tomography (AS-OCT) one month after the treatment. We observed complete re-epithelization in all eyes between 72 and 96 h after surgery (88 h on average). ECD and morphology remained unchanged in all eyes. DLD was detected at a mean depth of 362 ± 50 µm, 20% over solutions with equivalent dosage. SafeCross^® riboflavin solution chemically-boosted corneal cross-linking seems to optimize CXL oxidative reaction by higher superoxide anion release, improving DLD by a factor of 20%, without adverse events for corneal endothelium. Full article