RESUMO
Maroteaux - Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is a lysosomal storage disease resulting from insufficient enzymatic activity for degradation of the specific glycosaminoglycans (GAG) chondroitin sulphate (CS) and dermatan sulphate (DS). Among the most pronounced MPS VI clinical manifestations caused by cellular accumulation of excess CS and DS are eye disorders, in particular those that affect the cornea. Ocular manifestations are not treated by the current standard of care, enzyme replacement therapy (ERT), leaving patients with a significant unmet need. Using in vitro and in vivo models, we previously demonstrated the potential of the ß-D-xyloside, odiparcil, as an oral GAG clearance therapy for MPS VI. Here, we characterized the eye phenotypes in MPS VI arylsulfatase B deficient mice (Arsb-) and studied the effects of odiparcil treatment in early and established disease models. Severe levels of opacification and GAG accumulation were detected in the eyes of MPS VI Arsb- mice. Histological examination of MPS VI Arsb- eyes showed an aggregate of corneal phenotypes, including reduction in the corneal epithelium thickness and number of epithelial cell layers, and morphological malformations in the stroma. In addition, colloidal iron staining showed specifically GAG accumulation in the cornea. Orally administered odiparcil markedly reduced GAG accumulation in the eyes of MPS VI Arsb- mice in both disease models and restored the corneal morphology (epithelial layers and stromal structure). In the early disease model of MPS VI, odiparcil partially reduced corneal opacity area, but did not affect opacity area in the established model. Analysis of GAG types accumulating in the MPS VI Arsb- eyes demonstrated major contribution of DS and CS, with some increase in heparan sulphate (HS) as well and all were reduced with odiparcil treatment. Taken together, we further reveal the potential of odiparcil to be an effective therapy for eye phenotypes associated with MPS VI disease.
Assuntos
Oftalmopatias/tratamento farmacológico , Glicosídeos , Mucopolissacaridose VI , N-Acetilgalactosamina-4-Sulfatase , Animais , Modelos Animais de Doenças , Olho/patologia , Oftalmopatias/genética , Glicosídeos/uso terapêutico , Humanos , Camundongos , Mucopolissacaridose VI/tratamento farmacológico , Mucopolissacaridose VI/genética , N-Acetilgalactosamina-4-Sulfatase/genética , FenótipoRESUMO
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.
Assuntos
Modelos Animais de Doenças , Síndromes do Olho Seco/patologia , Regulação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Aparelho Lacrimal/cirurgia , Transcriptoma , Adjuvantes Anestésicos/toxicidade , Animais , Córnea/metabolismo , Córnea/patologia , Síndromes do Olho Seco/etiologia , Síndromes do Olho Seco/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Escopolamina/toxicidadeRESUMO
Dry eye disease (DED) is a complex, multifactorial pathology characterized by corneal epithelium lesions and inflammation. The aim of the present study was to evaluate the efficacy of a cationic emulsion of cyclosporine A (CsA) in a mouse model that mimics severe dry eye. Eight to 12-week-old female C57BL/6N mice with tail patches of scopolamine were housed in controlled environment chambers to induce dry eye. At day three, following dry eye confirmation by corneal fluorescein staining (CFS, score 0-15) and phenol red thread (PRT) lacrimation test, the mice (n = 10/gp) were either treated 3 times a day in both eyes with drug-free cationic emulsion, a 0.1% CsA cationic emulsion, or 1% methylprednisolone (positive control), or non-treated. Aqueous tear production and CFS scores were evaluated at baseline and throughout the treatment period. The lacrimation test confirmed the scopolamine-induced decrease in aqueous production by the lacrimal gland. A reduction of 59% in induced-CFS was observed following topical treatment with 0.1% CsA. The beneficial effect of the cationic emulsion vehicle itself on keratitis was also clearly evidenced by its better performance over 1% methylprednisolone, -36%, vs. -28% on the CFS scores, respectively. This study indicates that the cationic emulsion of CsA (0.1%) was a very effective formulation for the management of corneal epithelium lesions in a severe DED mouse model. In addition, it performed better than a potent glucocorticosteroid (1% methylprednisolone). This cationic emulsion of CsA (0.1%), combining CsA and a tear film oriented therapy (TFOT), i.e. with vehicle properties that mechanically stabilize the tear film, represents a promising new treatment strategy for the management of the signs of dry eye.
Assuntos
Córnea/efeitos dos fármacos , Ciclosporina/administração & dosagem , Síndromes do Olho Seco/tratamento farmacológico , Administração Tópica , Animais , Córnea/metabolismo , Córnea/patologia , Modelos Animais de Doenças , Síndromes do Olho Seco/diagnóstico , Síndromes do Olho Seco/metabolismo , Emulsões , Imunossupressores/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Lágrimas/metabolismo , Resultado do TratamentoRESUMO
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.
Assuntos
Ciclosporina , Oftalmopatias , Humanos , Ciclosporina/uso terapêutico , Oftalmopatias/tratamento farmacológicoRESUMO
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.
Assuntos
Ciclosporina/administração & dosagem , Modelos Animais de Doenças , Síndromes do Olho Seco/tratamento farmacológico , Emulsões , Imunossupressores/administração & dosagem , Administração Oftálmica , Animais , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Córnea/efeitos dos fármacos , Córnea/metabolismo , Córnea/patologia , Síndromes do Olho Seco/metabolismo , Proteínas do Olho/metabolismo , Feminino , Fluorofotometria , Aparelho Lacrimal/efeitos dos fármacos , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Soluções Oftálmicas , Conservantes Farmacêuticos , Lágrimas/fisiologia , Transcriptoma , Resultado do TratamentoRESUMO
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.
Assuntos
Ciclosporina/uso terapêutico , Modelos Animais de Doenças , Síndromes do Olho Seco/tratamento farmacológico , Regulação da Expressão Gênica/fisiologia , Marcadores Genéticos/genética , Imunossupressores/uso terapêutico , Inflamação/genética , Administração Oftálmica , Animais , Córnea/metabolismo , Citocinas/genética , Síndromes do Olho Seco/metabolismo , Feminino , Fluoresceína/metabolismo , Corantes Fluorescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Soluções Oftálmicas , RNA Mensageiro/genética , Receptores de Citocinas/genética , Lágrimas/metabolismoRESUMO
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.
Assuntos
Lesões da Córnea , Síndromes do Olho Seco/tratamento farmacológico , Epitélio Corneano , Lubrificantes Oftálmicos/farmacologia , Álcool de Polivinil/farmacologia , Povidona/farmacologia , Animais , Lesões da Córnea/diagnóstico , Lesões da Córnea/tratamento farmacológico , Lesões da Córnea/etiologia , Técnicas de Diagnóstico Oftalmológico , Combinação de Medicamentos , Síndromes do Olho Seco/complicações , Síndromes do Olho Seco/diagnóstico , Emulsões , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/patologia , Álcoois Graxos/farmacologia , Feminino , Soluções Oftálmicas/farmacologia , Compostos de Amônio Quaternário/farmacologia , Coelhos , Tensoativos/farmacologia , Cicatrização/efeitos dos fármacosRESUMO
PURPOSE: Artificial tears (ATs) are used routinely to alleviate the symptoms of mild to moderate dry eye. Preservative-free cationic emulsions (eg, Cationorm(®)) are an innovative approach for the management of signs and symptoms of dry eye. The aim of the present exploratory experiment was to evaluate the efficacy of this cetalkonium chloride (CKC)-containing cationic emulsion on debrided cornea and to characterize its effects on scraping-induced inflammation. METHODS: Four ATs were assessed in a rat model of corneal scraping. The upper part of the corneal epithelium was scraped before a 5-day treatment, followed by clinical evaluations and fluorescein staining to evaluate cornea recovery. The anti-inflammatory efficacy of the ATs was assessed in vivo and in vitro. RESULTS: In vivo confocal microscopy (IVCM) revealed a trend toward better corneal clinical signs (lower IVCM scores) for the animals treated with the unpreserved ATs. Benzalkonium chloride treatment decreased goblet cell count by 37.5%. While the soft-preserved Systane Balance(®) and Optive(®) and the preservative-free Vismed(®) had no effect on the goblet cell count, Cationorm increased this count by almost 40%. Interestingly, inflammatory cell infiltration in the stroma was at its lowest following treatment with the preservative-free Cationorm. Cationorm is also the only AT decreasing IL6- and IL8-stimulated secretion by 59% and 74%, respectively. CONCLUSION: By restoring an adequately hydrated ocular surface environment, the different ATs promote corneal epithelium healing. These data position Cationorm as a promising AT for the management of signs and symptoms of dry eye in patients with mild to moderate dry eye disease presenting chronic subclinical levels of ocular inflammation.
Assuntos
Túnica Conjuntiva/efeitos dos fármacos , Modelos Animais de Doenças , Epitélio Corneano/efeitos dos fármacos , Inflamação/tratamento farmacológico , Lubrificantes Oftálmicos/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Células Cultivadas , Emulsões , Masculino , Microscopia Confocal , Ratos , Ratos WistarRESUMO
The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9'-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9'-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial.
Assuntos
Carotenoides/administração & dosagem , Degeneração Macular/tratamento farmacológico , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Retinoides/efeitos adversos , Transportadores de Cassetes de Ligação de ATP/genética , Oxirredutases do Álcool/genética , Animais , Bixaceae/química , Carotenoides/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Técnicas In Vitro , Injeções Intravítreas , Degeneração Macular/induzido quimicamente , Degeneração Macular/genética , Degeneração Macular/metabolismo , Camundongos , Camundongos Knockout , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Ratos , Epitélio Pigmentado da Retina/citologia , SuínosRESUMO
Melanin-concentrating hormone (MCH) is a cyclic neuropeptide, with a major role in stimulation of feeding behavior in mammals. MCH signals in the brain occur via two seven-transmembrane G protein-coupled receptors, namely MCH1 (SLC-1, MCH(1), MCH-R1, or MCH-1R) and MCH2 (SLT, MCH(2), MCH-R2, or MCH-2R). In this study, we demonstrate that the pro-MCH(131-165) peptide neuropeptide-glutamic acid-isoleucine (NEI)-MCH is more potent than MCH in stimulating feeding in the rat. Using rat MCH1-expressed human embryonic kidney 293 cells, we show that NEI-MCH exhibits 5-fold less affinity in a binding assay and 2-fold less potency in a cAMP assay than MCH. A similar 7- to 8-fold shift in potency was observed in a Ca(2+)(i) assay using rat MCH1 or human MCH2-transfected Chinese hamster ovary cell models. This demonstrates that NEI-MCH is not a better agonist than MCH at either of the MCH receptors. Then, we compared the proteolysis resistance of MCH and NEI-MCH to rat brain membrane homogenates and purified proteases. Kinetics of peptide degradation using brain extracts indicated a t(1/2) of 34.8 min for MCH and 78.5 min for NEI-MCH with a specific pattern of cleavage of MCH but not NEI-MCH by exo- and endo-proteases. Furthermore, MCH was found highly susceptible to degradation by aminopeptidase M and endopeptidase 24.11, whereas NEI-MCH was fully resistant to proteolysis by these enzymes. Therefore, our results strongly suggest that reduced susceptibility to proteases of NEI-MCH compared with MCH account for its enhanced activity in feeding behavior. NEI-MCH represents therefore the first MCH natural functional "superagonist" so far described.