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Purpose: This analysis investigated potential associations between gene variants and clinical end points in the VIEW 1 and 2 randomized clinical trials of intravitreal aflibercept and ranibizumab in neovascular age-related macular degeneration (AMD). Methods: A genome-wide association analysis was conducted in a subgroup of patients from VIEW 1 and 2 consenting to the optional pharmacogenetic analysis. Results: Data were pooled from 780 samples from patients representative of the overall VIEW 1 and 2 populations. After Bonferroni correction for multiplicity and statistical adjustment for baseline risk factors, no significant associations were found between previously identified prognostic AMD gene variants and treatment response according to key prespecified VIEW 1 and 2 end points. Genome-wide, there were no significant genetic associations in patients experiencing gains of ≥15 Early Treatment of Diabetic Retinopathy Study letters after 1 or 2 years of treatment. A cluster of variants in ANO2 (encoding anoctamin 2, a calcium-activated chloride channel expressed on photoreceptor cells) on chromosome 12 reached the level of significance for loss of ≥5 letters after 1 year of treatment (P < 5 × 10-8), with the ANO2 rs2110166 SNP demonstrating highly significant association (P = 1.99 × 10-8). Carriers of the ANO2 rs2110166 TT genotype showed a robust increase in visual acuity versus baseline compared with a small decrease in those with the TC genotype. Conclusions: None of the potential prognostic candidate genes were associated with the clinical end points for treated patients. Preliminary analyses suggest an association of ANO2 with retinal function, with a potential impact on vision of approximately one line over at least the first year. Further investigation of the function of ANO2 in retinal pathophysiology is merited.
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Inibidores da Angiogênese , Anoctaminas , Estudo de Associação Genômica Ampla , Injeções Intravítreas , Polimorfismo de Nucleotídeo Único , Ranibizumab , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão , Fator A de Crescimento do Endotélio Vascular , Acuidade Visual , Humanos , Masculino , Feminino , Inibidores da Angiogênese/uso terapêutico , Idoso , Acuidade Visual/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ranibizumab/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Anoctaminas/genética , Degeneração Macular Exsudativa/genética , Degeneração Macular Exsudativa/tratamento farmacológico , Idoso de 80 Anos ou mais , Genótipo , Resultado do Tratamento , Farmacogenética , Testes FarmacogenômicosRESUMO
INTRODUCTION: AURIGA is the largest real-world study to date to evaluate intravitreal aflibercept (IVT-AFL) treatment of diabetic macular edema or macular edema secondary to retinal vein occlusion (RVO) in routine clinical practice. Here, we report the 24-month outcomes in the RVO cohort from France, Germany, Italy, and Taiwan. METHODS: AURIGA (NCT03161912) was a prospective observational study. Eligible patients with RVO were enrolled for whom the decision to treat with IVT-AFL had already been made by the attending physician. Patients were treated with IVT-AFL for up to 24 months at physician discretion according to local practice. The primary endpoint was mean change in visual acuity (VA; Early Treatment Diabetic Retinopathy Study [ETDRS] letters) from baseline to month (M) 12. All statistical analyses were descriptive. RESULTS: In 554 treatment-naïve and 65 previously treated patients with RVO, the respective mean (95% confidence interval) change in VA from baseline was + 12.5 (10.8, 14.3) and + 7.9 (3.3, 12.6) letters by M12 and + 11.4 (9.4, 13.3) and + 4.4 (- 0.6, 9.5) letters by M24 (baseline mean ± standard deviation: 51.0 ± 21.9 and 51.9 ± 20.4 letters); 44.0% of treatment-naïve and 27.9% of previously treated patients reported ≥ 15-letter gains by M24. By M24, the mean change in central retinal thickness from baseline was - 247 (- 267, - 227) µm in treatment-naïve patients and - 147 (- 192, - 102) µm in previously treated patients. From baseline to M6, M12, and M24, treatment-naïve patients received a total of 4.0 ± 1.3, 5.5 ± 2.5, and 6.9 ± 4.2 injections, respectively, and previously treated patients received 3.8 ± 1.5, 5.0 ± 2.2, and 6.3 ± 3.7 injections, respectively. The safety profile of IVT-AFL was consistent with that of previous studies. CONCLUSIONS: In AURIGA, patients with RVO experienced clinically relevant functional and anatomic improvements following IVT-AFL treatment in routine clinical practice. These improvements were largely maintained in treatment-naïve patients over the 24-month study despite the decreasing treatment frequency, suggesting long-term durability of IVT-AFL treatment outcomes. Infographic available for this article. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03161912 (May 19, 2017). INFOGRAPHIC.
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Purpose: Anti-vascular endothelial growth factor (anti-VEGF) therapies, which attenuate the capacity of VEGF to bind to VEGF receptors, are standard-of-care options for various retinal disorders that are characterized by pathologic retinal angiogenesis and vascular permeability. Multiple receptors and ligands have also been reported as being involved in these pathways, including angiopoietin-1 (ANG1) and angiopoietin-2 (ANG2). Methods: Electrochemiluminescence immunoassays were used to detect human VEGF (hVEGF), as well as rabbit ANG2 and basic fibroblast growth factor protein levels in vitreous samples derived from a study evaluating the efficacy of the anti-VEGF agents ranibizumab, aflibercept, and brolucizumab in an hVEGF165-induced rabbit retinal vascular hyperpermeability model. Results: hVEGF was completely suppressed in rabbit vitreous after anti-VEGF treatment for 28 days. ANG2 protein in vitreous and ANGPT2 mRNA in retina tissue were similarly suppressed, although the anti-VEGF agents do not directly bind to ANG2. Aflibercept demonstrated the greatest inhibitory effect in ANG2 levels in vitreous, which correlated with strong, durable suppression of intraocular hVEGF levels. Conclusions: This study explored the effects of anti-VEGF therapies beyond direct binding of VEGF by evaluating protein levels and the expression of target genes involved in angiogenesis and associated molecular mechanisms in the rabbit retina and choroid. Translational Relevance: In vivo data suggest that anti-VEGF agents currently used for the treatment of retinal diseases could provide beneficial effects beyond direct binding of VEGF, including suppression of ANG2 protein and ANGPT2 mRNA.
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Angiopoietina-2 , Fator A de Crescimento do Endotélio Vascular , Animais , Coelhos , Humanos , Fator A de Crescimento do Endotélio Vascular/genética , Angiopoietina-2/genética , Angiopoietina-2/metabolismo , Fatores de Crescimento do Endotélio Vascular , Receptores de Fatores de Crescimento do Endotélio Vascular , Neovascularização Patológica , RNA Mensageiro/metabolismoRESUMO
Purpose: To evaluate the molecular, pharmacokinetic, and pharmacological properties of three anti-vascular endothelial growth factor (VEGF) agents-aflibercept, brolucizumab, and ranibizumab-and to provide a prediction of the optimal design of an intravitreal VEGF challenge in rabbits to assess the preclinical in vivo activity of the different anti-VEGF agents. Methods: Biochemical analyses and cellular and animal models of retinopathy were used to characterize anti-VEGF efficacy. Anti-VEGF biochemical binding affinity was determined through a kinetic exclusion assay. The in vitro potency was investigated by a calcium mobilization assay. Pharmacokinetic parameters were estimated for each drug to predict intraocular exposure relationships among the agents. The in silico modeling efforts informed the design of an in vivo rabbit model of VEGF-induced retinal hyperpermeability to determine the extent of VEGF neutralization in vivo. Consequently, data generated from the in vivo study enabled pharmacokinetic analysis and the generation of a logistical model describing the impact of the anti-VEGF agents on the VEGF-induced vascular leakage in rabbits. Results: The three anti-VEGF agents ranked from most efficacious to least efficacious as aflibercept, brolucizumab, and ranibizumab, with results consistent and significant within each individual characterization experiment. Conclusions: This composite study demonstrated how the molecular properties of aflibercept, brolucizumab, and ranibizumab translate into differences of in vivo efficacy, with results in line with the reported literature. Translational Relevance: In silico, in vitro, and in vivo integrated studies provide information that enables the enhanced characterization of translational properties of anti-VEGF agents currently used for the treatment of retinal diseases.
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Cálcio , Ranibizumab , Animais , Coelhos , Ranibizumab/farmacologia , Ranibizumab/uso terapêutico , Fatores de Crescimento Endotelial , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Injeções Intravítreas , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Purpose: To investigate whether vascular endothelial growth factor (VEGF)-suppression durations contribute to our understanding of clinical trial outcomes by simulating vitreous molar concentrations (Cvm) of intravitreal aflibercept (IVT-AFL) and brolucizumab (IVT-BRO) using pharmacokinetic (PK) modeling. Methods: A PK model simulated Cvm after single-dose IVT-AFL, IVT-BRO, and ranibizumab (IVT-RAN), and extrapolated intraocular VEGF-suppression thresholds and durations. Vitreous PK after multidose regimens used in studies of IVT-AFL versus IVT-BRO were simulated and compared with best-corrected visual acuity (BCVA) data. Results: Cvm peaked higher (Cmax) and decreased more quickly to the VEGF-suppression threshold and minimum (Cmin) levels with IVT-BRO than with IVT-AFL, consistent with their molar doses calculated using molecular weights and vitreous half-lives (26 kDa and 115 kDa; 4.4-5.1 and 9.1-11 days, respectively). The mean VEGF suppression durations were 71 days for IVT-AFL 2 mg and 51 (48-59) days for IVT-BRO 6 mg. Based on dosing in OSPREY (matched dosing to week [w]32 for both agents; thereafter, IVT-AFL every eight weeks [q8w] and IVT-BRO q12w for the last two doses [w32âw44 and w44âw56]), IVT-BRO showed wider Cmax-Cmin fluctuations than IVT-AFL. The IVT-BRO Cmin fell below the VEGF-suppression threshold at timepoints near w56, when decreases in BCVA were also observed. The IVT-AFL vitreous Cmin remained above the suppression threshold through w56, where BCVA gains were maintained. Conclusions: The PK-modeled mean VEGF-suppression duration for IVT-BRO was substantially shorter than that published for IVT-AFL and may not be sufficient to effectively suppress VEGF throughout q12w dosing. Translational Relevance: The PK modeling suggests that more patients may be maintained on ≥q12w dosing with IVT-AFL than with IVT-BRO.
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Inibidores da Angiogênese , Fator A de Crescimento do Endotélio Vascular , Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais Humanizados , Humanos , Injeções Intravítreas , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão , Acuidade VisualRESUMO
Five vascular endothelial growth factor receptor (VEGFR) ligands (VEGF-A, -B, -C, -D, and placental growth factor [PlGF]) constitute the VEGF family. VEGF-A binds VEGF receptors 1 and 2 (VEGFR1/2), whereas VEGF-B and PlGF only bind VEGFR1. Although much research has been conducted on VEGFR2 to elucidate its key role in retinal diseases, recent efforts have shown the importance and involvement of VEGFR1 and its family of ligands in angiogenesis, vascular permeability, and microinflammatory cascades within the retina. Expression of VEGFR1 depends on the microenvironment, is differentially regulated under hypoxic and inflammatory conditions, and it has been detected in retinal and choroidal endothelial cells, pericytes, retinal and choroidal mononuclear phagocytes (including microglia), Müller cells, photoreceptor cells, and the retinal pigment epithelium. Whilst the VEGF-A decoy function of VEGFR1 is well established, consequences of its direct signaling are less clear. VEGFR1 activation can affect vascular permeability and induce macrophage and microglia production of proinflammatory and proangiogenic mediators. However the ability of the VEGFR1 ligands (VEGF-A, PlGF, and VEGF-B) to compete against each other for receptor binding and to heterodimerize complicates our understanding of the relative contribution of VEGFR1 signaling alone toward the pathologic processes seen in diabetic retinopathy, retinal vascular occlusions, retinopathy of prematurity, and age-related macular degeneration. Clinically, anti-VEGF drugs have proven transformational in these pathologies and their impact on modulation of VEGFR1 signaling is still an opportunity-rich field for further research.
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Inflamação/patologia , Neovascularização Patológica , Retina/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Células Endoteliais , Humanos , Fator de Crescimento Placentário , Transdução de Sinais , Fator A de Crescimento do Endotélio VascularRESUMO
PURPOSE: To evaluate the impact of baseline retinal capillary nonperfusion (RNP) and macular retinal capillary nonperfusion (MNP) status on outcomes at week 24 (W24). DESIGN: Post hoc analyses of 2 phase 3, randomized, double-masked, multicenter, sham-controlled studies. PARTICIPANTS: Three hundred sixty-six patients with macular edema secondary to central retinal vein occlusion randomized in COPERNICUS and GALILEO. METHODS: We randomized patients 3:2 to receive intravitreal aflibercept 2 mg every 4 weeks or sham injections until W24. RNP and MNP were assessed by a masked independent reading center. MAIN OUTCOME MEASURES: Proportion of patients with 10 disc areas (DA) or more of RNP and any degree of MNP at W24, relative risks of 10 DA or more of RNP or any degree of MNP at W24 developing, change from baseline in best-corrected visual acuity (BCVA) and central retinal thickness (CRT) by baseline RNP and MNP status, and relationship between baseline RNP and MNP status. RESULTS: At baseline, 24.6% of patients showed 10 DA or more of RNP and 72.6% showed MNP, regardless of baseline RNP status. At W24, the pooled proportions of patients in the intravitreal aflibercept and sham groups with 10 DA or more of RNP were 11.6% and 29.0%, respectively (P = 0.0001); the respective proportions with any degree of MNP were 61.2% and 79.5% (P = 0.0008). Relative risks and 95% confidence intervals for intravitreal aflibercept versus sham were 0.4 (0.25-0.62) for 10 DA or more of RNP and 0.8 (0.68-0.90) for MNP, indicating a lower risk for these outcomes with intravitreal aflibercept than with sham. Mean BCVA change was greater in intravitreal aflibercept- versus sham-treated eyes, with less than 10 DA and 10 DA or more of RNP at baseline (+17.5 vs. +0.8 letters and +18.3 vs. -4.1 letters, respectively) and with and without baseline MNP (+15.7 vs. +0.3 letters and +17.1 vs. +0.4 letters, respectively). Agreement between baseline RNP and MNP status was low (κ = 0.12). The proportions of patients with 1 or more ocular serious adverse event in the intravitreal aflibercept- and sham-treated groups, respectively, were 3.2% and 11.3%. CONCLUSIONS: At W24, visual and anatomic improvements, including perfusion status, were greater in eyes treated with intravitreal aflibercept than in eyes treated with sham, regardless of baseline RNP or MNP status.
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Inibidores da Angiogênese/uso terapêutico , Edema Macular/fisiopatologia , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Oclusão da Veia Retiniana/fisiopatologia , Veia Retiniana/fisiopatologia , Idoso , Barreira Hematorretiniana/fisiologia , Capilares/fisiopatologia , Permeabilidade Capilar/fisiologia , Método Duplo-Cego , Feminino , Humanos , Injeções Intravítreas , Edema Macular/tratamento farmacológico , Edema Macular/etiologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Oclusão da Veia Retiniana/complicações , Oclusão da Veia Retiniana/tratamento farmacológico , Tomografia de Coerência Óptica , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Acuidade Visual/fisiologiaRESUMO
OBJECTIVES: To report on the efficacy and safety of intravitreal aflibercept in patients with macular edema secondary to central retinal vein occlusion (CRVO) in an integrated analysis of COPERNICUS and GALILEO. PATIENTS AND METHODS: Patients were randomized to receive intravitreal aflibercept 2 mg every 4 weeks or sham injections until week 24. From week 24 to week 52, all intravitreal aflibercept-treated patients in both studies and sham-treated patients in COPERNICUS were eligible to receive intravitreal aflibercept based on prespecified criteria. In GALILEO, sham-treated patients continued to receive sham treatment through week 52. RESULTS: At week 24, mean gain in best-corrected visual acuity and mean reduction in central retinal thickness were greater for intravitreal aflibercept-treated patients compared with sham, consistent with individual trial results. At week 52, after 6 months of intravitreal aflibercept as-needed treatment in COPERNICUS, patients originally randomized to sham group experienced visual and anatomic improvements but did not improve to the extent of those initially treated with intravitreal aflibercept, while the sham group in GALILEO did not improve over week 24 mean best-corrected visual acuity scores. Ocular serious adverse events occurred in <10% of patients. CONCLUSION: This analysis of integrated data from COPERNICUS and GALILEO confirmed that intravitreal aflibercept is an effective treatment for macular edema following CRVO.
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PURPOSE: To investigate the plasma levels of amyloid beta (Aß) and select inflammatory mediators in patients with various stages of AMD compared to that of age-matched controls, and discern a relationship to disease severity. METHODS: Plasma samples were obtained from AMD subjects at various stages of disease-early (drusen only), geographic atrophy (GA), neovascular AMD (CNV)-and from controls of similar age without AMD. Samples were analyzed using a commercially available ELISA kit (sixteen cytokines) or LC/MS/MS (Aß isotypes). Descriptive statistics were compiled on all analytes. Analysis of covariance (ANCOVA) was conducted to compare each analyte across AMD groups while adjusting for sex and age of the patients, and in comparison to the control group. Receiver operating characteristics plots were generated for the strongest predictor variables. RESULTS: Levels of alternative spliced CC3 proteins were significantly different between controls and CNV groups (p < 0.05), with median levels almost twice higher in CNV than in controls. There was an increasing trend for plasma levels of Αß isotypes across AMD progressive stages (p values ranged from 0.052 to 0.0012) (ANCOVA). When adjusted for multiple comparisons analysis, plasma Aß 1-42 levels, and its ratio with Aß 1-40 were the most significantly associated with late AMD stages. Consistently with the ANCOVA results for Αß isotypes, the ROC curve showed a moderate prediction (AUC = - ~ 0.78) of AMD vs control using the Aß 1-42 isotype. CONCLUSION: Plasma Aß 1-42 may have utility as a systemic biomarker for AMD.
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Peptídeos beta-Amiloides/sangue , Biomarcadores/sangue , Citocinas/sangue , Atrofia Geográfica/sangue , Degeneração Macular Exsudativa/sangue , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Cromatografia Líquida , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Projetos Piloto , Drusas Retinianas/sangue , Espectrometria de Massas em TandemRESUMO
The aim of this study was to assess the feasibility of using a commercially available high-resolution adaptive optics (AO) camera to image the cone mosaic in Japanese macaques (Macaca fuscata) with dominantly inherited drusen. The macaques examined develop drusen closely resembling those seen in humans with age-related macular degeneration (AMD). For each animal, we acquired and processed images from the AO camera, montaged the results into a composite image, applied custom cone-counting software to detect individual cone photoreceptors, and created a cone density map of the macular region. We conclude that flood-illuminated AO provides a promising method of visualizing the cone mosaic in nonhuman primates. Future studies will quantify the longitudinal change in the cone mosaic and its relationship to the severity of drusen in these animals.
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Modelos Animais de Doenças , Fundo de Olho , Macaca , Degeneração Macular/patologia , Drusas do Disco Óptico/patologia , Células Fotorreceptoras Retinianas Cones/citologia , Animais , Comprimento Axial do Olho/patologia , Contagem de Células/instrumentação , Contagem de Células/métodos , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Neoplasia de Células Basais , Oftalmoscopia/métodosRESUMO
PURPOSE: The intraocular pharmacodynamics of PF-04523655, a small-interfering RNA (siRNA) directed against RTP801, was characterized using rat models of retinopathy. METHODS: Rat models of streptozotocin-induced diabetes and wet AMD were used to determine the onset, extent, and duration of siRNA inhibition of retinal RTP801 expression by PF-04523655, and this inhibition was characterized by pharmacokinetic/pharmacodynamic (PK/PD) modeling. A rat model of wet AMD was also used to examine PF-04523655 dose-dependent effects on the incidence of clinical grade 3 or 4 choroidal neovascularization lesions. Whole homogenate versus laser-capture microdissected (LCM) retinal samples were analyzed by quantitative PCR for RTP801 expression. RESULTS: RTP801 expression in RPE/choroid (RPE/C) increased in diabetic rats by up to 70% above nondiabetic rat levels. Inhibition of retinal RTP801 expression by PF-04523655 began 1 day after intravitreous injection and was observed through day 7 in the neurosensory retina and through day 14 or longer in RPE/C. PF-04523655 inhibition of RTP801 expression was maintained well after clearance of PF-04523655 from the eye and was best characterized by an effect compartment PK/PD model. Moreover, PF-04523655 administration decreased the incidence of clinical grade 3 or 4 lesions by approximately 60% (P = 0.053), and dose-dependently inhibited retinal RTP801 expression (P < 0.01). RTP801 expression was enriched in the outer nuclear layer in LCM samples. CONCLUSIONS: In rodent retinopathy models, administration of the siRNA, PF-04523655, reduced RTP801 expression in the retina, consistent with the RNA-induced silencing complex (RISC) mechanism of action. The pharmacodynamic profile from the animal models could be useful to elucidate dose and exposure dependency of RTP801 expression inhibition by siRNA.
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Retinopatia Diabética/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/genética , Regulação para Cima/efeitos dos fármacos , Animais , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/farmacocinética , Ratos , Ratos Long-Evans , Proteínas Repressoras/biossíntese , Proteínas Repressoras/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Fatores de TranscriçãoRESUMO
PURPOSE: We characterized tear protein markers in dry eye disease (DED). METHODS: In this prospective study, based on the ocular surface disease index (OSDI) and corneal staining (CS), 95 DED patients (OSDI ≥13) with increasing CS were enrolled into 3 severity groups: DE1 (CS <4), DE2 (CS 4-7), and DE3 (CS >7), while 25 asymptomatic subjects with no CS were enrolled into the control group (OSDI <13 and CS = 0). Tear fluid was collected at day 0 and day 7 visits, and concentrations of 43 protein markers were measured by multiplexed immunoassay. RESULTS: We analyzed 22 control and 80 DED subjects. Among 33 markers detectable, good inter-visit repeatability was observed with 25 markers, with intraclass correlation coefficients (ICC) ranging from 0.85-0.60; ICCs were <0.60 in the other 8. Correlation with clinical measures was found with two markers, with absolute partial correlation coefficients >0.40: Interleukin-1 receptor antagonist (IL-1Ra) and IL-8. IL-1Ra and IL-8 correlated with conjunctival staining (0.43, P < 0.001 and 0.35, P < 0.01, respectively), and with Schirmer test (-0.58 and -0.42, P < 0.001). IL-1Ra and IL-8 in DE3 were 4.4- and 2.1-fold higher than in DE1 (P = 0.0001 and 0.0007), and 1.9- and 1.6-fold higher than in DE2 (P = 0.022 and 0.017). IL-1Ra in DE2 was 2.3-fold higher than in DE1 (P = 0.038). CONCLUSIONS: Tear levels of many immune mediators were highly repeatable between visits in DED. Among them, IL-1Ra and IL-8 were associated with clinical signs and disease severity defined by corneal staining.
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Síndromes do Olho Seco/diagnóstico , Proteínas do Olho/análise , Lágrimas/química , Adulto , Biomarcadores/análise , Síndromes do Olho Seco/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reprodutibilidade dos Testes , Índice de Gravidade de DoençaRESUMO
OBJECTIVE: To evaluate the immunomodulatory effect of topical ophthalmic tofacitinib (CP-690,550) after an 8-week treatment period in patients with dry eye disease (DED). DESIGN: Biomarker substudy of a phase 1/2 prospective, randomized, vehicle- and comparator-controlled clinical trial (NCT00784719). PARTICIPANTS: A total of 82 patients with moderate to severe DED enrolled. METHODS: Patients received 1 of 5 doses of tofacitinib (0.0003%, 0.001%, 0.003%, or 0.005% twice daily [BID] or 0.005% once daily [QD]), active comparator (cyclosporine ophthalmic emulsion, 0.05% [Restasis, Allergan Inc., Irvine, CA]), or vehicle control BID for 8 weeks. Conjunctival impression cytology and tear fluid samples were collected at baseline and after an 8-week treatment period. Conjunctival cells were analyzed by flow cytometry for human leukocyte antigen DR-1 (HLA-DR). Tear fluids were analyzed by microsphere-based immunoassays for tear levels of cytokines and inflammation markers. MAIN OUTCOME MEASURES: Reduction in inflammation assessed by change from baseline in conjunctival cell surface level of HLA-DR and tear level of cytokines and inflammation markers. RESULTS: At week 8, a decrease in conjunctival cell surface expression of HLA-DR was observed in patients treated with tofacitinib 0.005% QD and 0.003% BID: 71% and 67% of baseline, respectively, compared with 133% of baseline in patients treated with vehicle (P=0.023 and P=0.006, compared with vehicle, respectively). Matrix metalloproteinase (MMP)-3 in tears was reduced from baseline at week 8 (40% of baseline, P=0.035) in the tofacitinib 0.005% QD group, whereas the vehicle group showed 77% of baseline (P>0.20). Interleukin (IL)-1ß in tears was 36% of baseline (P=0.053) in the tofacitinib 0.005% QD group and 95% of baseline (P > 0.20) in the vehicle group. Several other cytokines and inflammation markers in tears, including MMP-9, IL-15, IL-17A, and IL-12p70, were markedly reduced in the tofacitinib 0.005% QD group but not the vehicle group. There was an association between the changes in HLA-DR and the tear inflammation markers (P<0.05): HLA-DR with IL-12p70 (r=0.49) and IL-1ß (r=0.46), IL-12p70 with IL-1ß (r=0.90), and IL-17A with MMP-9 (r=0.82). CONCLUSIONS: Topical ophthalmic tofacitinib may act as an immunomodulator in patients with DED. Treatment for 8 weeks showed a promising reduction of conjunctival cell surface HLA-DR expression and tear levels of proinflammatory cytokines and inflammation markers.
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Síndromes do Olho Seco/tratamento farmacológico , Janus Quinase 3/antagonistas & inibidores , Pirimidinas/administração & dosagem , Pirróis/administração & dosagem , Administração Tópica , Túnica Conjuntiva/metabolismo , Ciclosporina/administração & dosagem , Citocinas/metabolismo , Síndromes do Olho Seco/metabolismo , Feminino , Citometria de Fluxo , Antígeno HLA-DR1/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Soluções Oftálmicas , Piperidinas , Lágrimas/metabolismoRESUMO
PURPOSE: PF-655, a synthetic 19-mer siRNA, targeting the RTP801 gene is currently in clinical trials for the treatment of wet age-related macular degeneration and diabetic macular edema. Preclinical studies have shown a dose-related suppression of RTP801 expression in rat disease models. Investigative studies were conducted with PF-655 to validate the Dutch-Belted rabbit as a biologically relevant species for gene silencing to support nonclinical ocular toxicity and continual dosing studies. METHODS: Cross-species comparison and DNA sequencing was done to determine the level of homology between PF-655 and rabbit RTP801. Human (HEK 293) and rabbit (SIRC cornea) cell lines were stimulated with CoCl(2) to mimic hypoxic stress (an inducer of RTP801 expression) and treated with PF-655. Taqman-polymerase chain reaction and immunoblot analysis were performed to gauge RTP801 expression in cell culture and rabbit retinas. RESULTS: Sequence analysis showed a 1-base mismatch in the PF-655 targeting site from genomic DNA of Dutch-Belted rabbit and the SIRC cell line, a cornea cell derived from the New Zealand White rabbit. HEK and SIRC CoCl(2)-stressed cells induced RTP801 expression 10-20-fold above control conditions. Treatment with 20 or 100 nM PF-655 showed a decrease in gene expression, 40%-50% relative to appropriate controls. RTP801 mRNA was detectable in primary rabbit retina tissues, with cycle threshold values showing a large linear range for the assay. CONCLUSION: These results support our investigation into cross-species validation of gene suppression by a therapeutic siRNA designed to a human gene. The SIRC cell line was utilized as a surrogate to test the degree of RTP801 gene silencing induced by PF-655 in vitro. With a 1-base mismatch, the level of silencing in a rabbit ocular cell line was comparable to that of a human cell line. Sequence analysis and expression data confirmed the relevance of the RTP801 target gene in rabbits and the utility of this species as a relevant animal model. Additionally, our work outlines a tractable method that validates relevant larger non-rodent species for ophthalmic drug testing.
Assuntos
Oftalmopatias/genética , Oftalmopatias/terapia , RNA Interferente Pequeno/genética , Retina/metabolismo , Fatores de Transcrição/genética , Animais , Linhagem Celular Transformada , Córnea/metabolismo , Oftalmopatias/metabolismo , Expressão Gênica , Inativação Gênica , Terapia Genética/métodos , Células HEK293 , Humanos , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , Coelhos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , TransfecçãoRESUMO
It is concluded from immunohistochemical that all four types of prostaglandin-E(2) (PGE(2)) (EP1, EP2, EP3 and EP4) receptors are associated with specific cell-types in primary rat retinal cultures. Analysis specifically of EP2 receptor immunoreactivity shows it to coexist with some neurones expressing Thy-1 and calbindin immunoreactivities as well as with vimentin-positive Müller cells. Moreover, exposure of cultures to the EP2 specific agonist butaprost (100 nM) for a period of 24h results in a generation of cAMP thus providing support for the functionality of EP2 receptors. Cell survival was significantly affected in cultures where the serum concentration was reduced from 10 to 1% for 24h. This was reflected by a reduction in the number of GABA-positive neurons and an elevation of released lactate dehydrogenase (LDH) into the culture medium. Moreover, a number of cells displayed a clear generation of reactive oxygen species (ROS) and a staining for the breakdown of DNA by the TUNEL procedure as an indicator for apoptosis. These negative effects were attenuated when butaprost (100 nM) was present during the serum reduction and 30 min before the insult. The present studies provide evidence to show that all PGE(2) receptor types exist in the retina of rat pups, remain functional when the retinal cells are cultured and that specific activation of EP2 receptors with butaprost can attenuate a detrimental insult caused by insufficient serum that may occur in situ by reduced trophic support.
Assuntos
Alprostadil/análogos & derivados , Dinoprostona/agonistas , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de Prostaglandina E/agonistas , Retina/efeitos dos fármacos , Alprostadil/sangue , Alprostadil/farmacologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Calbindinas , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura Livres de Soro/farmacologia , AMP Cíclico/metabolismo , Dano ao DNA/efeitos dos fármacos , Dinoprostona/metabolismo , Marcação In Situ das Extremidades Cortadas , L-Lactato Desidrogenase/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/sangue , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Prostaglandina E/metabolismo , Retina/citologia , Retina/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Antígenos Thy-1/metabolismo , Vimentina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
PURPOSE: To localize different prostaglandin E(2) receptors in rat retinas of varying age, deduce how they are affected by acute stress insult, and determine whether the negative effect of ischemia/reperfusion is attenuated by the EP2 agonist butaprost. METHODS: Ischemia was induced by the elevation of intraocular pressure. Butaprost was injected intravitreally immediately after ischemia. Standard methods were used for recording of electroretinograms (ERGs) and processing of immunohistochemistry. Extracts of whole retinas were analyzed for specific proteins by Western blotting or by RT-PCR for defined mRNAs. RESULTS: The localization of different EP receptor types is similar in retinas of all aged rats. However, differences exist in the monomer/dimer ratios in retinas of different age. Acute stress insult (48 hours after ischemia) affects the ratio of monomer/dimer of all EP receptor types and increases EP2 and EP3 immunoreactivities in Müller cells of the adult retina. Ischemia and 5 to 7 days of reperfusion to the retina caused the normal ERG and the localization of nNOS and ChAT immunoreactivities to be affected. Certain proteins and mRNAs were lowered in content, whereas other proteins and mRNAs were upregulated. In addition, specific optic nerve proteins were drastically reduced. Most of these changes induced by ischemia/reperfusion were significantly blunted by butaprost. CONCLUSIONS: All subtypes of EP receptors exist primarily in the inner retina at different ages, but their monomer/dimer ratios vary. Stress affects the monomer/dimer ratio and EP2 and EP3 immunoreactivities in Müller cells. Butaprost injected intravitreally significantly blunts the detrimental influence of ischemia/reperfusion to the retina.
Assuntos
Envelhecimento/fisiologia , Alprostadil/análogos & derivados , Estresse Oxidativo , Receptores de Prostaglandina E/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Doenças Retinianas/prevenção & controle , Alprostadil/uso terapêutico , Animais , Western Blotting , Primers do DNA/química , Eletrorretinografia , Imuno-Histoquímica , Injeções , Proteínas do Tecido Nervoso/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Prostaglandinas E Sintéticas/uso terapêutico , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E Subtipo EP2 , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Retina/metabolismo , Retina/fisiopatologia , Doenças Retinianas/metabolismo , Doenças Retinianas/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Corpo VítreoRESUMO
Posterior segment drug delivery challenges inherent in the treatment of many sight-threatening diseases have become increasingly apparent. Therapeutic interventions for ocular diseases such as neovascular retinopathies, inflammatory and/or infectious diseases may involve drug delivery to vitreoretinal targets. An important part of successful therapeutic strategies for such diseases involves verification that efficacious concentrations of the pharmacological agent are achieved within relevant intraocular regions. Microdialysis has been effectively employed for characterizing intraocular disposition in both anterior and posterior segments, providing important documentation of successful drug delivery to desired targets. Recent papers that showcase the maturation in the model development of microdialysis approaches for estimating posterior segment pharmacokinetics and further validation of the methodology are described in this review. Special problems examined include anterior and posterior ocular clearance mechanisms, intraocular metabolism and active transport of drugs.