Comparison of In Vitro Corneal Permeation and In Vivo Ocular Bioavailability in Rabbits of Three Marketed Latanoprost Formulations.

BACKGROUND AND OBJECTIVE: All latanoprost formulations currently available for the treatment of glaucoma or ocular hypertension contain the same concentration of latanoprost (0.005%) but differ in excipients, which may affect corneal drug permeability or stability. This study aimed at comparing corneal penetration of three marketed latanoprost solutions with different excipient formulations in in vitro and in vivo drug permeability studies. METHODS: Three latanoprost formulations were tested under good laboratory practice conditions: a formulation containing benzalkonium chloride (BAK) but no surfactant (Preserved latanoprost); the same formulation except preservative-free (PF) without BAK or surfactant (SF) (PF SF latanoprost); and a different formulation without BAK but containing a non-ionic surfactant (MGHS 40 at 5%) combined with thickening agents (Carbomer 974P, Macrogol 4000) (PF latanoprost). Corneal permeation of latanoprost acid (LAT) was first determined in vitro using a reconstructed human corneal epithelium tissue. Then, in vivo pharmacokinetic studies were performed on pigmented rabbits, for which LAT concentration was measured in the aqueous humour (AH) and iris-ciliary body (ICB). RESULTS: In vitro, the cumulative transport of LAT was linear between 1 h and 4 h for preserved latanoprost and PF SF latanoprost, and LAT concentrations matched exactly at each timepoint. By contrast, the permeation of PF latanoprost was linear between 2 h and 12 h and was significantly lower than that of preserved latanoprost and PF SF latanoprost at 4 and 8 h (p < 0.001). In rabbits, the concentrations of LAT in AH and ICB were not statistically different between preserved latanoprost and PF SF latanoprost at each timepoint, except at 1 h in ICB (p = 0.005). By comparison, the LAT concentration of PF latanoprost was statistically (p < 0.05) lower than that of preserved latanoprost and PF SF latanoprost in AH and ICB from 0.5 to 3 h. CONCLUSION: BAK did not influence the corneal penetration of latanoprost in in vitro and in vivo studies. The formulation containing a non-ionic surfactant resulted in lower and slower ocular penetration compared with preserved or PF SF formulations. This raises questions about the relevance of BAK and some surfactants in enhancing corneal penetration of ocular formulations.

Impact of Latanoprost Antiglaucoma Eyedrops and Their Excipients on Toxicity and Healing Characteristics in the Ex Vivo Eye Irritation Test System.

INTRODUCTION: Corneal epithelial toxicity and delayed healing process have already been attributed to preservatives or some excipients. We study the effects of galenic components in antiglaucoma drugs such as benzalkonium chloride (BAC) or surfactants like macrogolglycerol hydroxystearate 40 (MGHS 40) on corneal toxicity in an ex vivo system mimicking chronic use. METHODS: Latanoprost-containing eyedrops are available with and without preservatives on the market. Unpreserved, they are available in different formulations with various excipients like MGHS at different concentrations (0%, 2.5%, and 5%). We studied these in the ex vivo bioreactor (EVEIT) on initially injured rabbit corneas. The drugs were applied six times daily for observation periods of 3 or 5 days. BAC, 5% MGHS 40 solution, and 0.18% hyaluronic acid served as controls. Macroscopic photographic, biochemical methods and corneal integrity quantification were used for evaluation. Toxicity was assessed by measuring wound healing and corneal fluorescein sodium permeability and was confirmed by histology studies. RESULTS: The BAC-preserved formulation resulted in high corneal toxicity, which was expected. Interestingly, the preservative-free (PF) formulation containing 5% MGHS 40, carbomer, macrogol 4000, and sorbitol showed the highest corneal toxicity, followed by the control formulation with equal MGHS 40 concentration, which presented significantly less damage. No toxicity was shown by eyedrops containing 2.5% MGHS 40 or salts only. CONCLUSION: Our study demonstrates a significant corneal toxicity of certain formulations of PF antiglaucoma ophthalmic drugs containing 5% MGHS 40 with other excipients compared to other formulations with lower MGHS 40 concentrations (2.5% or 0%), or even compared to the solution containing 5% MGHS alone. This suggests a concentration-dependent toxicity of MGHS 40, especially in interaction with other excipients, which may increase its epithelial toxicity, and that has to be considered in clinical glaucoma therapy. Further single-component formulation trials are needed to support this interpretation.

Corneal neuroepithelial compartmentalized microfluidic chip model for evaluation of toxicity-induced dry eye.

Part of the lacrimal functional unit, the cornea protects the ocular surface from numerous environmental aggressions and xenobiotics. Toxicological evaluation of compounds remains a challenge due to complex interactions between corneal nerve endings and epithelial cells. To this day, models do not integrate the physiological specificity of corneal nerve endings and are insufficient for the detection of low toxic effects essential to anticipate Toxicity-Induced Dry Eye (TIDE). Using high-content imaging tool, we here characterize toxicity-induced cellular alterations using primary cultures of mouse trigeminal sensory neurons and corneal epithelial cells in a compartmentalized microfluidic chip. We validate this model through the analysis of benzalkonium chloride (BAC) toxicity, a well-known preservative in eyedrops, after a single (6h) or repeated (twice a day for 15 min over 5 days) topical 5.10-4% BAC applications on the corneal epithelial cells and nerve terminals. In combination with high-content image analysis, this advanced microfluidic protocol reveal specific and tiny changes in the epithelial cells and axonal network as well as in trigeminal cells, not directly exposed to BAC, with ATF3/6 stress markers and phospho-p44/42 cell activation marker. Altogether, this corneal neuroepithelial chip enables the evaluation of toxic effects of ocular xenobiotics, distinguishing the impact on corneal sensory innervation and epithelial cells. The combination of compartmentalized co-culture/high-content imaging/multiparameter analysis opens the way for the systematic analysis of toxicants but also neuroprotective compounds.

Benzalkonium chloride-induced direct and indirect toxicity on corneal epithelial and trigeminal neuronal cells: proinflammatory and apoptotic responses in vitro.

Benzalkonium chloride (BAK), a quaternary ammonium compound widely used as disinfecting agent as well as preservative in eye drops is known to induce toxic effects on the ocular surface with inflammation and corneal nerve damage leading to dry eye disease (DED) in the medium-to-long term. The aim of this study was to evaluate in vitro the toxicity of a conditioned medium produced by corneal epithelial cells previously exposed to BAK (BAK-CM) on trigeminal neuronal cells. A human corneal epithelial (HCE) cell line was exposed to 5.10-3% BAK (i.e. 0.005% BAK) for 15 min and let recover for 5 h to prepare a BAK-CM. This BAK concentration is the lowest one found in eye drops. After this recovery period, BAK effect on HCE cells displayed cytotoxicity, morphological alteration, apoptosis, oxidative stress, ATP release, CCL2 and IL6 gene induction, as well as an increase in CCL2, IL-6 and MIF release. Next, a mouse trigeminal ganglion primary culture was exposed to the BAK-CM for 2 h, 4 h or 24 h. Whereas BAK-CM did not alter neuronal cell morphology, or induced neuronal cytotoxicity or oxidative stress, BAK-CM induced gene expression of Fos (neuronal activation marker), Atf3 (neuronal injury marker), Ccl2 and Il6 (inflammatory markers). Two and 4 h BAK-CM exposure promoted a neuronal damage (ATF-3, phospho-p38 increases; phospho-Stat3 decreases) while 24 h-BAK-CM exposure initiated a prosurvival pathway activation (phospho-p44/42, phospho-Akt increases; ATF-3, GADD153, active Caspase-3 decreases). In conclusion, this in vitro model, simulating paracrine mechanisms, represents an interesting tool to highlight the indirect toxic effects of BAK or any other xenobiotic on corneal trigeminal neurons and may help to better understand the cellular mechanisms that occur during DED pathophysiology.

Sensory and physicochemical evolution of tropical cooked peeled shrimp inoculated by Brochothrix thermosphacta and Lactococcus piscium CNCM I-4031 during storage at 8°C.

This study investigated the sensory quality and physicochemical evolution (pH, glucose, l-lactic acid, biogenic amine, free amino-acids and volatile compounds) during storage at 8°C of cooked peeled shrimp inoculated with the specific spoilage bacteria Brochothrix thermosphacta alone or mixed with the protective strain Lactococcus piscium CNCM I-4031. Growth of both bacteria was monitored at regular intervals during storage by microbial counts and the thermal temperature gradient gel electrophoresis (TTGE) technique. Bacterial counts showed that L. piscium and B. thermosphacta inoculated at 7 log CFU/g and 3 log CFU/g were well adapted to shrimp, reaching a maximum level of 9 log CFU/g after 4days and 10days respectively. In mixed culture, the growth of B. thermosphacta was reduced by 3.2±0.1 log CFU/g. The TTGE technique allowed monitoring the colonisation of the strains on the shrimp matrix and confirming the dominance of L. piscium in mixed culture throughout the experiment. Sensory analysis confirmed that B. thermosphacta spoiled the product after 11days, when its cell number attained 8 log CFU/g with the emission of strong butter/caramel off-odours. This sensory profile could be linked to the production of 2,3 butanedione, cyclopentanol, 3-methylbutanol, 3-methylbutanal, 2-methylbutanal, 4-methyl-3-chloro-3-pentanol and ethanol, which were produced in more significant quantities in the B. thermosphacta batch than in the batches in which the protective strain was present. On the contrary, TVBN and TMA were not suitable as quality indicators for B. thermosphacta spoilage activity. In the products where the protective L. piscium strain was present, no adverse effect on sensory quality was noted by the sensory panels. Moreover, biogenic amine assessment did not show any histamine or tyramine production by this strain, underlining its safety profile. Both strains produced lactic acid (1850mg/kg in L. piscium and B. thermosphacta batch on days 3 and 10 respectively; 3830mg/kg on day 7 in mixed culture) and the pH decrease from 6.6±0.0 to 5.9±0.1 was similar in all batches. Lactic acid production or competition for free amino-acid was not involved in the inhibition mechanism; however rapid glucose consumption by L. piscium could partially explain the growth limitation of the spoilage micro-organism. This study demonstrated the spoilage characteristic of B. thermosphacta and the usefulness of L. piscium as a bioprotective culture for tropical cooked peeled shrimp without any adverse effect on the sensory quality of the product.