RESUMEN
This study investigates the interaction of two approved and one newly developed latanoprost formulation with in vitro and in silico models of the tear film and tear film lipid layer (TFLL). Latanoprost, a prostaglandin analogue used for intraocular elevated pressure treatment, is topically delivered by nanocarriers within aqueous solutions or emulsions. The study focuses on the impact of these carriers on drug interactions with the tear film and their effect on the TFLL. Three different types of latanoprost carriers, micellar, nanoemulsion, and polymer-based, were compared, and each revealed distinct interaction patterns with the TFLL. Surface pressure kinetics demonstrated a rapid increase for the benzalkonium chloride formulation and a slow rise for the preservative-free variants. Visualization of the acellular in vitro TFLL model revealed different patterns of incorporation for each formulation, indicating unique interaction mechanisms. Molecular dynamics simulations further revealed different mechanisms of drug release in the TFLL between micellar and nanoemulsion formulations. In-depth examination highlighted the role of triglyceride molecules in replenishing the nonpolar layer of the TFLL, which suggests potential improvements in ocular surface compatibility by adjusting the quality and concentration of the oily phase. These findings suggest the potential for optimizing latanoprost formulations by tuning the oily phase-to-surfactant ratio and selecting suitable surfactants.
Asunto(s)
Ojo , Glaucoma , Humanos , Latanoprost/uso terapéutico , Presión Intraocular , Glaucoma/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Antihipertensivos/uso terapéuticoRESUMEN
Glaucoma is a leading cause of blindness worldwide, with elevated intraocular pressure being a major risk factor for its development and progression. First-line treatment for glaucoma relies on the administration of prostaglandin analogs, with latanoprost being the most widely used. However, before latanoprost reaches the cornea, it must pass through the tear film and tear film lipid layer (TFLL) on the ocular surface. Given the significant lipophilicity of latanoprost, we hypothesize that TFLL could, to a certain extent, act as a reservoir for latanoprost, releasing it on longer time scales, apart from the fraction being directly delivered to the cornea in a post-instillation mechanism. We investigated this possibility by studying latanoprost behavior in acellular in vitro TFLL models. Furthermore, we employed in silico molecular dynamics simulations to rationalize the experimental results and obtain molecular-level insight into the latanoprost-TFLL interactions. Our experiments demonstrated that latanoprost indeed accumulates in the TFLL models, and our simulations explain the basis of the accumulation mechanism. These results support the hypothesis that TFLL can serve as a reservoir for latanoprost, facilitating its prolonged release. This finding could have significant implications for optimizing glaucoma treatment, especially in the development of new drug delivery systems targeting the TFLL.
Asunto(s)
Glaucoma , Humanos , Latanoprost/uso terapéutico , Glaucoma/tratamiento farmacológico , Lágrimas , Córnea , Simulación por Computador , Antihipertensivos/uso terapéutico , Presión IntraocularRESUMEN
Prostaglandin analogue topical medications are one of the most effective therapeutic approaches for the chronic management of glaucoma and ocular hypertension, through the reduction of elevated intra ocular pressure (IOP). While many of the first generations of anti-glaucoma eye drops were preserved with benzalkonium chloride, their repeated use may induce chronic ocular surface toxicity that leads to ocular surface disease (OSD) signs and symptoms. As a result, soft-preservatives and preservative-free formulations have been developed with the goal to avoid the long-term iatrogenic toxicity of the preservative agents. In addition, it has been suggested that OSD and its associated inflammation may negatively impact the efficacy of the IOP-lowering medications, including treatment adherence and compliance. Hence, it may be particularly interesting that glaucoma medications can concomitantly protect and "heal" the ocular surface and its environment while lowering elevated IOP, for the greater benefit of glaucoma patients. The objective of the present review is to briefly present the preclinical data of the cationic oil-in-water emulsion of latanoprost (latanoprost-CE) to shed some light on its mechanisms of action. It overall supports the following hypothesis: the restoration of a healthy ocular surface environment and treatment of the OSD signs and symptoms will allow for an improved elevated IOP reduction and glaucoma management. This would be achieved with a once daily dosing regimen to preserve glaucoma patients' vision, ocular surface, and quality-of-life and wellness.
Asunto(s)
Glaucoma de Ángulo Abierto , Glaucoma , Hipertensión Ocular , Prostaglandinas F Sintéticas , Humanos , Latanoprost/uso terapéutico , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Emulsiones/uso terapéutico , Presión Intraocular , Prostaglandinas F Sintéticas/efectos adversos , Antihipertensivos/efectos adversos , Glaucoma/tratamiento farmacológico , Hipertensión Ocular/tratamiento farmacológico , Soluciones Oftálmicas/uso terapéutico , Conservadores Farmacéuticos/efectos adversosRESUMEN
Benzalkonium chloride (BAK) compounds are commonly used in topical ophthalmic products as preservatives and stabilizers. BAK mixtures containing several compounds with different alkyl chain lengths are typically used. However, in chronic eye conditions, such as dry eye disease and glaucoma, the accumulation of adverse effects of BAKs was observed. Hence, preservative-free eye drops formulations are preferred. On the other hand, selected long-chain BAKs, particularly cetalkonium chloride, exhibit therapeutic functions, promoting epithelium wound healing and tear film stability. Nevertheless, the mechanism of BAKs influence on the tear film is not fully understood. By employing in vitro experimental and in silico simulation techniques, we elucidate the action of BAKs and demonstrate that long-chain BAKs accumulate in the lipid layer of the tear film model, stabilizing it in a concentration-dependent fashion. In contrast, short-chain BAKs interacting with the lipid layer compromise the tear film model stability. These findings are relevant for topical ophthalmic drug formulation and delivery in the context of selecting proper BAK species and understanding the dose dependency for tear film stability.
Asunto(s)
Síndromes de Ojo Seco , Conservadores Farmacéuticos , Humanos , Conservadores Farmacéuticos/farmacología , Compuestos de Benzalconio/efectos adversos , Lágrimas , Síndromes de Ojo Seco/tratamiento farmacológico , Soluciones Oftálmicas , Lípidos/farmacologíaRESUMEN
BACKGROUND: Benzalkonium chloride (BAK)-containing antiglaucoma therapies alter the ocular surface over the long term. We used an in vitro scraping model to compare the effects of preserved and unpreserved topical commercial prostaglandins (PGs) in a wound-healing model. METHODS: Standardized mechanical scraping was performed in confluent immortalized human corneal/conjunctival epithelial cell layers. Cytotoxicity, cell migration and proliferation, as well as the percentage of closure, were analyzed 2 h and 1/2/3/6 days after a 30-min exposure to 1/10 dilutions in phosphate buffered saline (PBS) used also as control, BAK solutions at concentrations ranging from 0.0001% to 0.1%, latanoprost-0.02%BAK, travoprost-0.015%BAK, bimatoprost-0.005%BAK, BAK-free Tafluprost, latanoprost in cationic emulsion, and travoprost (Polyquad® and SofZia®). RESULTS: PG eyedrop preparations with BAK preservative delayed corneal healing, which is primarily related to the presence of BAK, in a dose-dependent manner, especially at day 1, as evidenced through actin disorganization and decreased Ki-67-positive cell numbers. The PGs (BAK-free tafluprost, latanoprost in cationic emulsion,travoprost (Polyquad® and SofZia®)) maintained a normal healing process with results similar to those of control. Conjunctiva-derived cell layers healed more slowly than corneal cell layers and were more sensitive in in vitro cytotoxicity tests. CONCLUSIONS: This novel in vitro scraping model mimics the damaged ocular surface epithelia observed in glaucoma patients affected by ocular surface disease, such as toxic-induced dry eye (TIDE) and offers a tool to assess the potential cytotoxic effects of PG formulations with or without BAK.
Asunto(s)
Prostaglandinas F Sintéticas , Antihipertensivos , Cloprostenol , Emulsiones , Humanos , Latanoprost/farmacología , Travoprost/farmacologíaRESUMEN
Dry eye disease (DED) is the most common ocular surface disease, characterized by insufficient production and/or instability of the tear film. Tear substitutes are usually the first line of treatment for patients with DED. Despite the large variety of tear substitutes available on the market, few studies have been performed to compare their performance. There is a need to better understand the specific mechanical and pharmacological roles of each ingredient composing the different formulations. In this review, we describe the main categories of ingredients composing tear substitutes (e.g., viscosity-enhancing agents, electrolytes, osmo-protectants, antioxidants, lipids, surfactants and preservatives) as well as their effects on the ocular surface, and we provide insight into how certain components of tear substitutes may promote corneal wound healing, and/or counteract inflammation. Based on these considerations, we propose an approach to select the most appropriate tear substitute formulations according to the predominant etiological causes of DED.
Asunto(s)
Síndromes de Ojo Seco/tratamiento farmacológico , Gotas Lubricantes para Ojos/uso terapéutico , Composición de Medicamentos , Humanos , Gotas Lubricantes para Ojos/química , Gotas Lubricantes para Ojos/farmacología , ViscosidadRESUMEN
BACKGROUND: Cyclosporine A (CsA) has been used as a topical treatment for various ocular surface diseases including dry eye disease (DED). Several CsA formulations are available as solutions or emulsions. PURPOSE: This review describes the development and the preclinical testing of a cationic oil-in-water emulsion of CsA (CE-CsA) in terms of pharmacodynamics, pharmacokinetics, and ocular tolerance. Due to the cationic charge, CE electrostatically interacts with the negatively-charged ocular surface, improving its residence time. Compared to other CsA formulations, CE-CsA and CE itself were found to reduce the signs and symptoms of DED, by restoring tear film stability and properties, and inhibiting the expression and secretion of pro-inflammatory factors. No delay in wound healing nor ocular toxicity were observed using CE formulations. CONCLUSION: these findings indicate that the type of vehicle can significantly affect the performance of eye drops and play an ancillary role in DED treatment. CE appears as a promising strategy to deliver drugs to the ocular surface while maintaining its homeostasis.
Asunto(s)
Ciclosporina , Oftalmopatías , Humanos , Ciclosporina/uso terapéutico , Oftalmopatías/tratamiento farmacológicoRESUMEN
The goal of this study was to explore the specific signaling pathways related to inflammation in two experimental mouse dry eye (EDE) models. Female C57BL/6 mice housed for 10 days in a controlled desiccative environment were either treated with scopolamine (EDE-1; n = 18) or subjected to extraorbital lacrimal gland excision bilaterally (EDE-2; n = 10). Non-induced mice (n = 20) served as healthy controls. A corneal fluorescein staining (CFS) scoring was used at baseline through to day (D) 10 to evaluate epitheliopathy. At D10, corneas and conjunctivas were collected for multiplexed transcriptomic analysis with the NanoString® mouse inflammatory CodeSet. Both EDE-1 and EDE-2 mice presented a change in corneal integrity, with a significant increase in CFS scores at D10. More gene transcripts were identified in EDE-2 compared with EDE-1 (116 vs. 96, respectively), and only a few were common to both models, 13 for the cornea and 6 for the conjunctiva. The gene functional annotation analysis revealed that the same inflammatory pathways were involved in both models. Comparative profiling of gene expression in the two EDE models leads to the identification of various targets and signaling pathways, which can be extrapolated to and confirmed in human disease.
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Modelos Animales de Enfermedad , Síndromes de Ojo Seco/patología , Regulación de la Expresión Génica , Mediadores de Inflamación/metabolismo , Aparato Lagrimal/cirugía , Transcriptoma , Adyuvantes Anestésicos/toxicidad , Animales , Córnea/metabolismo , Córnea/patología , Síndromes de Ojo Seco/etiología , Síndromes de Ojo Seco/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Escopolamina/toxicidadRESUMEN
Recent clinical findings suggest that mucomimetic polymers (MMP) can alter not only the texture of the aqueous tear but also the spreading and structure of the tear film (TF) lipid layer, thereby allowing for their synchronized performance in vivo. Thus, we aimed to evaluate in vitro (i) the capability of pharmaceutically applicable MMP to ensure the formation of post-evaporative ferning patterns (a characteristic feature of the "healthy" tear colloid) and (ii) the MMP interactions with human meibum films accessed in the course of blink-like deformations via Langmuir surface balance and Brewster angle microscopy (BAM). Four MMP were used- hyaluronic acid (HA), cross-linked hyaluronic acid (CHA), carboxymethyl cellulose (CMC) and gellan gum (GG)- at the concentrations of 0.0001%, 0.001%, 0.01%, 0.05% and 0.1%. Significant differences were observed in the MMP fern formation capability: CHA (≥0.001%) > HA (≥0.01%) = CMC (≥0.01%) > GG (≥0.05%). All MMP affected the spreading of meibum, with BAM micrographs revealing thickening of the films. CHA was particularly efficient, showing concentration-dependent enhancement of tear ferning and of meibomian layer structure, surfactant properties and viscoelasticity. Thus, endogenous and exogenous MMP may play key roles for the concerted action of the TF layers at the ocular surface, revealing novel routes for TF-oriented therapeutic applications.
Asunto(s)
Materiales Biomiméticos/química , Glándulas Tarsales/química , Lágrimas/química , Adulto , Carboximetilcelulosa de Sodio/química , Femenino , Humanos , Ácido Hialurónico/química , Glándulas Tarsales/metabolismo , Lágrimas/metabolismo , ViscosidadRESUMEN
Tiny nanoparticles of dexamethasone palmitate (DXP) were designed as transparent suspensions for intravitreal administration to treat age-related macular degeneration (AMD). The influence of three surfactants (PEG-40-stearate and Pluronic block copolymers F68 and F127) on nanoparticles size and stability was investigated and led to an optimal formulation based on Pluronic F127 stabilizing DXP nanoparticles. Size measurements and TEM revealed tiny nanoparticles (around 35 nm) with a low opacity, compatible with further intravitreal injection. X-Ray powder diffraction (XRPD) and transmission electronic microscopy (TEM) performed on freeze-dried samples showed that DXP nanoparticles were rather monodisperse and amorphous. The efficacy of DXP nanoparticles was assessed in vivo on pigmented rabbits with unilateral intravitreal injections. After breakdown of the blood-retinal barrier (BRB) induced by injection of rhVEGF165 with carrier protein, DXP nanoparticles induced a restoration of the BRB 1 month after their intravitreal injection. However, their efficacy was limited in time most probably by clearance of DXP nanoparticles after 2 months due to their small size.
Asunto(s)
Dexametasona , Nanopartículas , Animales , Glucocorticoides , Inyecciones Intravítreas , Palmitatos , ConejosRESUMEN
PURPOSE/AIM: Dry eye (DE) disease is a multifactorial disease in which uncontrolled inflammation can lead to corneal epithelium lesions and symptoms of discomfort. The aim of the present study was to evaluate the efficacy of two cyclosporine emulsions in a mouse model of DE with corneal epithelium lesions. MATERIALS AND METHODS: Six- to 9-week-old female C57BL/6 N mice were housed in a controlled-environment room to induce DE. Following DE development, mice were instilled with: QD 0.1%CsA cationic emulsion (CaEm), BID 0.05%CsA anionic emulsion (AEm), or left untreated. Aqueous tear production and corneal epithelium lesions were assessed throughout the experiment. At the end of the treatment period, left eyes were sampled, fixed, and stained for histology, while the cornea, conjunctiva, and lacrimal gland of right eyes were sampled for transcriptomic analysis. RESULTS: Corneal lesion scores were reduced by 10.4%, 18.4%, and 10.9% at day 6, 10, and 14, respectively, with CaEm (QD), and by 2.6%, 3.0%, and 5.5% at day 6, 10, and 14, respectively, with AEm (BID). Histology demonstrated that 7 out of 10 DE mice presented moderate to severe ocular lesions, while only 2 and 5 out of 10 mice presented slight to moderate ocular lesions when treated with the CaEm (QD) and AEm (BID), respectively. The transcriptomic profile analysis suggests that a different set of inflammatory genes are modulated in the cornea, conjunctiva, and lacrimal gland upon DE development. In addition, the two emulsions distinctively modulate the gene expression profile. CONCLUSIONS: This study demonstrates that both emulsions were effective at reducing corneal lesions, with the CaEm (QD) being slightly better than the AEm (BID). Interestingly, this study suggests that ocular tissues may not respond similarly to a dry environment and that a different set of genes is modulated by the two formulations in the ocular tissues.
Asunto(s)
Ciclosporina/administración & dosificación , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/tratamiento farmacológico , Emulsiones , Inmunosupresores/administración & dosificación , Administración Oftálmica , Animales , Conjuntiva/efectos de los fármacos , Conjuntiva/metabolismo , Conjuntiva/patología , Córnea/efectos de los fármacos , Córnea/metabolismo , Córnea/patología , Síndromes de Ojo Seco/metabolismo , Proteínas del Ojo/metabolismo , Femenino , Fluorofotometría , Aparato Lagrimal/efectos de los fármacos , Aparato Lagrimal/metabolismo , Aparato Lagrimal/patología , Ratones , Ratones Endogámicos C57BL , Soluciones Oftálmicas , Conservadores Farmacéuticos , Lágrimas/fisiología , Transcriptoma , Resultado del TratamientoRESUMEN
BACKGROUND: Vernal keratoconjunctivitis (VKC) is a chronic, potentially blinding ocular allergic disease affecting children with uncertain pathogenic mechanisms. OBJECTIVE: To identify differences in gene expression between VKC and normal subjects (CT) and to evaluate the expression of pattern recognition receptors (PRRs). METHODS: Conjunctival cells were collected by impression cytology device from 25 VKC patients and 10 CT. Isolated RNA was assayed with the NanoString human immunology codeset to evaluate the expression levels of immunology-related genes. RESULTS: Of the 579 genes, 398 were detected and 58 were significantly differently expressed in VKC compared to CT. The number of significantly differentially expressed genes (DEG) in the 3 different phenotypes vs CT were 149 in tarsal, 17 in limbal and 68 in the mixed form of VKC. The list of the most overexpressed genes included several chemokines (CCL24, CCL18, CCL22, CXCL1), proinflammatory cytokines (IL-1ß, IL-6, IL-8, TGFß-1) and genes related to Th2- and Th17-signaling families. Toll like receptors (TLR)4 and TLR8, Dectin-1/CLEC7A, mincle/CLEC4E, MCR1, NOD2 and NLRP3 and several of their pathway-related genes were significantly overexpressed in VKC. The number of DEG increased with the disease severity either in IgE+ or IgE- patients. Immunohistochemistry analysis of VKC conjunctival tissues confirmed an increased expression of these molecules at protein level. CONCLUSIONS: The increased expression of several chemotactic factors and co-stimulatory signals required for T-cell activation, confirms that VKC is mostly cell-mediated with local eosinophilia. The multiple expression of PRRs suggests a role of host-pathogens interaction in VKC development.
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Conjuntivitis Alérgica , Niño , Conjuntiva , Conjuntivitis Alérgica/genética , Citocinas/genética , Perfilación de la Expresión Génica , HumanosRESUMEN
The tear film at the ocular surface is covered by a thin layer of lipids. This oily phase stabilizes the film by decreasing its surface tension and improving its viscoelastic properties. Clinically, destabilization and rupture of the tear film are related to dry eye disease and are accompanied by changes in the quality and quantity of tear film lipids. In dry eye, eye drops containing oil-in-water emulsions are used for the supplementation of lipids and surface-active components to the tear film. We explore in detail the biophysical aspects of interactions of specific surface-active compounds, cetalkonium chloride and poloxamer 188, which are present in oil-in-water emulsions, with tear lipids. The aim is to better understand the macroscopically observed eye drops-tear film interactions by rationalizing them at the molecular level. To this end, we employ a multi-scale approach combining experiments on human meibomian lipid extracts, measurements using synthetic lipid films, and in silico molecular dynamics simulations. By combining these methods, we demonstrate that the studied compounds specifically interact with the tear lipid film enhancing its structure, surfactant properties, and elasticity. The observed effects are cooperative and can be further modulated by material packing at the tear-air interface.
Asunto(s)
Lípidos/química , Microscopía Fluorescente/métodos , Simulación de Dinámica Molecular , Películas Cinematográficas , Alcoholes Grasos/química , Humanos , Glándulas Tarsales/metabolismo , Modelos Teóricos , Poloxámero/química , Compuestos de Amonio Cuaternario/químicaRESUMEN
Dry eye disease (DED) is a complex multifactorial disease that affects an increasing number of patients worldwide. Close to 30% of the population has experienced dry eye (DE) symptoms and presented with some signs of the disease during their lifetime. The significant heterogeneity in the medical background of patients with DEs and in their sensitivity to symptoms renders a clear understanding of DED complicated. It has become evident over the past few years that DED results from an impairment of the ocular surface homeostasis. Hence, a holistic treatment approach that concomitantly addresses the different mechanisms that result in the destabilization of the tear film (TF) and the ocular surface would be appropriate. The goal of the present review is to compile the different types of scientific evidence (from in silico modeling to clinical trials) that help explain the mechanism of action of cationic emulsion (CE)-based eye drop technology for the treatment of both the signs and the symptoms of DED. These CE-based artificial tear (AT) eye drops designed to mimic, from a functional point of view, a healthy TF contribute to the restoration of a healthy ocular surface environment and TF that leads to a better management of DE patients. The CE-based AT eye drops help restore the ocular surface homeostasis in patients who have unstable TF or no tears.
Asunto(s)
Síndromes de Ojo Seco/tratamiento farmacológico , Emulsiones/química , Gotas Lubricantes para Ojos/uso terapéutico , Fenómenos Fisiológicos Oculares/efectos de los fármacos , Adulto , Emulsiones/farmacología , Voluntarios Sanos/estadística & datos numéricos , Homeostasis , Humanos , Gotas Lubricantes para Ojos/administración & dosificación , Gotas Lubricantes para Ojos/química , Propiedades de Superficie/efectos de los fármacos , Lágrimas/fisiologíaRESUMEN
Purpose/Aim: Inflammation is recognized as playing an etiological role in dry eye disease. This study aimed to assess the efficacy of various topical cyclosporine A (CsA) formulations on cornea inflammatory markers in a mouse model of dry eye. MATERIAL AND METHODS: Six- to 7-week-old mice treated with scopolamine were housed in a controlled environment room to induce dry eye. Following dry eye confirmation by corneal fluorescein staining (CFS), the mice were treated three times a day with: 0.05%CsA (Restasis, Allergan), 0.1%CsA (Ikervis, Santen), 1%CsA oil solution, and 0.5% loteprednol etabonate (LE, Lotemax, Baush+Lomb), or left untreated. Aqueous tear production and CFS scores were assessed during the treatment period, and corneas were collected to measure the expression profile of a selection of inflammatory genes. RESULTS: After 7 days of treatment, the CFS scores were reduced by 21%, 31%, and 44% with 0.05%CsA, 0.1%CsA, and 1%CsA eye drops, respectively. By contrast, 0.5% LE did not decrease corneal fluorescein staining at day 10. A statistically significant dose-dependent CFS reduction was observed only between the 0.05% and 1%CsA formulations. The gene expression profiles indicated that 12, 18, 17 genes were downregulated by 0.05%CsA, 0.1%CsA, 1%CsA, respectively. Among them, the genes significantly downregulated were: IL1A, IL1R1, and TLR4 with 0.05%CsA; H2-Eb1, IL1A, IL1B, IL1RN, IL6, TGFB2, TGFB3, TLR2, TLR3, and TLR4 with 0.1%CsA; IL1B, IL6, TGFB3, and TLR4 with 1%CsA. TGFB1 and TGFBR1 were the only genes upregulated in all groups, but only TGFB1 upregulation reached significance. IL6RA was significantly upregulated by 0.05%CsA. CONCLUSIONS: This study indicates that the three CsA formulations effectively modulated TLR4, TGFß1, IL1, and IL6 pathways to reduce corneal epithelium lesions in a mouse model of severe dry eye. The study also suggests that the different anti-inflammatory eye drops modulated inflammatory genes in a slightly different manner.
Asunto(s)
Ciclosporina/uso terapéutico , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/tratamiento farmacológico , Regulación de la Expresión Génica/fisiología , Marcadores Genéticos/genética , Inmunosupresores/uso terapéutico , Inflamación/genética , Administración Oftálmica , Animales , Córnea/metabolismo , Citocinas/genética , Síndromes de Ojo Seco/metabolismo , Femenino , Fluoresceína/metabolismo , Colorantes Fluorescentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Soluciones Oftálmicas , ARN Mensajero/genética , Receptores de Citocinas/genética , Lágrimas/metabolismoRESUMEN
Purpose: In several multicenter clinical trials, HLA-DR was found to be a potential biomarker of dry eye disease (DED)'s severity and prognosis. Given the fact that HLA-DR receptor is a heterodimer consisting in an alpha and a beta chain, we intended to investigate the correlation of inflammatory targets with the corresponding transcripts, HLA-DRA and HLA-DRB1, to characterize specific targets closely related to HLA-DR expressed in conjunctival cells from patients suffering from DED of various etiologies. Methods: A prospective study was conducted in 88 patients with different forms of DED. Ocular symptom scores, ocular-staining grades, tear breakup time (TBUT) and Schirmer test were evaluated. Superficial conjunctival cells were collected by impression cytology and total RNAs were extracted for analyses using the new NanoString® nCounter technology based on an inflammatory human code set containing 249 inflammatory genes. Results: Two hundred transcripts were reliably detected in conjunctival specimens at various levels ranging from 1 to 222,546 RNA copies. Overall, from the 88 samples, 21 target genes showed a highly significant correlation (R > 0.8) with HLA-DRA and HLA-DRB1, HLA-DRA and B1 presenting the highest correlation (R = 0.9). These selected targets belonged to eight family groups, namely interferon and interferon-stimulated genes, tumor necrosis factor superfamily and related factors, Toll-like receptors and related factors, complement system factors, chemokines/cytokines, the RIPK enzyme family, and transduction signals such as the STAT and MAPK families. Conclusions: We have identified a profile of 21 transcripts correlated with HLA-DR expression, suggesting closely regulated signaling pathways and possible direct or indirect interactions between them. The NanoString® nCounter technology in conjunctival imprints could constitute a reliable tool in the future for wider screening of inflammatory biomarkers in DED, usable in very small samples. Broader combinations of biomarkers associated with HLA-DR could be analyzed to develop new diagnostic approaches, identify tighter pathophysiological gene signatures and personalize DED therapies more efficiently.
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Síndromes de Ojo Seco/genética , Perfilación de la Expresión Génica/métodos , Cadenas alfa de HLA-DR/genética , Cadenas HLA-DRB1/genética , Inflamación/genética , Biomarcadores/metabolismo , Conjuntiva/metabolismo , Conjuntiva/patología , Síndromes de Ojo Seco/diagnóstico , Femenino , Humanos , Masculino , Estudios Prospectivos , Transducción de Señal/genéticaRESUMEN
Purpose: Preservative-free cationic emulsion-based artificial tear (AT) is an innovative eye drop based on the Novasorb® technology with cetalkonium chloride (CKC) as the cationic agent. The cationic emulsion Cationorm is designed for the management of mild-to-moderate dry eye disease (DED) patients that present cornea epithelium alterations. The aim of the present study was to evaluate the safety and tolerability of overdosed ATs by altered corneal epithelium in vivo and assess the usefulness of the ex vivo eye irritation test (EVEIT) as a predictive alternate toxicity test method. Methods: The experimental procedure, treatment duration, and instillation frequency closely mimic in vivo the ex vivo protocol described by Pinheiro et al. and discussed in the Discussion and Conclusion section of this article. Two to 3-month-old female New Zealand white rabbits, n = 6 per group, were treated with ATs (21 instillations/day over 3 days) following corneal abrasion. Corneal fluorescein staining, in vivo confocal microscopy (IVCM), and slit lamp examinations were performed to assess corneal epithelium recovery and the ocular tolerability of the overdosed ATs. Results: All abraded eyes experienced almost complete epithelium recovery within 3 days following treatments with Cationorm, Optive, Vismed, and Saline. Benzalkonium chloride (BAK, 0.02%) treatment resulted in 82.4% reepithelialization. IVCM data illustrated corneal epithelium normal recovery. Acute local tolerability of the overdosed ATs was confirmed using Draize and McDonald-Shadduck's test scales. Conclusions: The different ATs were demonstrated to be well tolerated by abraded corneas in vivo, and the extreme overdosing regimen did not hamper the wound healing process of the rabbit eye in comparison to saline. These data did not confirm the ones obtained with the nonvalidated ex vivo eye irritation test.
Asunto(s)
Lesiones de la Cornea , Síndromes de Ojo Seco/tratamiento farmacológico , Epitelio Corneal , Gotas Lubricantes para Ojos/farmacología , Alcohol Polivinílico/farmacología , Povidona/farmacología , Animales , Lesiones de la Cornea/diagnóstico , Lesiones de la Cornea/tratamiento farmacológico , Lesiones de la Cornea/etiología , Técnicas de Diagnóstico Oftalmológico , Combinación de Medicamentos , Síndromes de Ojo Seco/complicaciones , Síndromes de Ojo Seco/diagnóstico , Emulsiones , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/patología , Alcoholes Grasos/farmacología , Femenino , Soluciones Oftálmicas/farmacología , Compuestos de Amonio Cuaternario/farmacología , Conejos , Tensoactivos/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Purpose: Preservative-free cationic emulsion-based artificial tears (ATs) or drug vehicles are innovative eye drop formulations with tear film stabilization and drug delivery properties, and valuable in vivo anti-inflammatory and wound healing properties. These ATs have recently reached the market as ATs for the management of dry eye disease (DED) symptoms (i.e., Cationorm) or as a drug vehicle for cyclosporine (Ikervis). The aim of the present study was to explore the mechanism of action underlying the intrinsic anti-inflammatory and wound-healing efficacies harbored by the cationic emulsions of cetalkonium chloride (CE-CKC). Methods: The anti-inflammatory activity of two CE-CKC (0.002% and 0.005% CKC) emulsions was evaluated by assessing the expression of proinflammatory genes and the secretion of various markers in the following human cell types stressed by different agents: peripheral blood mononuclear cells (PBMCs; stimulation with anti-CD3/anti-CD28 or lipopolysaccharide (LPS)), CD4+ T lymphocytes (TCD4; stimulation with anti-CD3/anti-CD28), and a human corneal epithelial cell line (HCE-2; stimulation with LPS). The cells were incubated for 30 min with a 10% dilution of CE-CKC emulsions and then cultured without the emulsions for 24 h or 72 h in the presence of the various challenging agents. The supernatant was collected, and the secreted markers quantitated with flow cytometry or an enzyme-linked immunosorbent assay (ELISA). Gene expression of inflammatory markers was evaluated only in the PBMCs and HCE-2 cells stimulated with LPS. The in vitro protein kinase C (PKC) binding assay for IC50 determination was performed using standard procedures. Results: The CE-CKC emulsions decreased inflammatory gene expression in LPS-stimulated PBMCs (IFN-γ, IL-17A, CXCL-9, and TNFα) and LPS-stimulated HCE-2 cells (THBS1 and CCL2). Both CE-CKC emulsions inhibited the secretion of IL-17 (from anti-CD3/anti-CD28-stimulated TCD4), TNFα, IFN-γ, and IL-2 (from anti-CD3-/anti-CD28-stimulated PBMCs), and IL-6 and IL-8 (from LPS-stimulated HCE-2). The in vitro PKC binding assay revealed that CKC, the cationic agent, is a specific PKCα inhibitor. In addition, tyloxapol, another excipient, showed some anti-inflammatory activity on IL-6 and IL-8 in the LPS-stimulated HCE-2 cells. Conclusions: This study indicates that the CE-CKC emulsions are able to directly modulate the secretion and expression of proinflammatory cytokines and chemokines. The results also suggest that CKC and tyloxapol are pharmacologically active excipients with potentially beneficial effects in vivo. These data shed new light on the efficacy observed on the DED signs of these CE-CKC emulsions in clinical trials.