RESUMO
Three different types of sunitinib-loaded (SUN-loaded) nanocarriers were compared, aiming at the topical treatment of corneal neovascularization (CNV): polymeric nanospheres (NS), liposomes (LIP), and solid lipid nanoparticles (SLN). Three out of eleven formulations prepared for an optimization study - the best SUN-loaded nanocarrier of each assessed type (NS, LIP, and SLN) - were selected, based on their size, polydispersity index (PdI), drug load (DL), and encapsulation efficiency (EE). These three optimal formulations were further characterized by nanoparticle tracking analysis (NTA), electron paramagnetic resonance (EPR) spectroscopy, and zeta potential. In vitro SUN release profiles were obtained for the optimal formulations, along with ex vivo corneal permeability/retention studies, and ocular tolerance assays, namely: the bovine corneal opacity and permeability (BCOP) assay, the HET-CAM test (hen's egg test - chorioallantoic membrane), and hemolytic potential (HP) assay. None of the optimal formulations exhibited toxicity or potential for ocular irritation. SLN showed higher surface fluidity, drug release more suitable for topical ocular applications, besides greater SUN corneal retention. Our results suggest that SLN are the best CNV-targeting SUN-loaded nanocarriers for clinical translation when compared to their NS and LIP analogues.
Assuntos
Neovascularização da Córnea , Nanopartículas , Nanosferas , Animais , Bovinos , Feminino , Neovascularização da Córnea/tratamento farmacológico , Sunitinibe , Galinhas , Nanopartículas/química , Polímeros , Lipídeos/química , Portadores de Fármacos/químicaRESUMO
ABSTRACT Purpose: The aim of this study was to compare the effects of topical cyclosporine 0.1% and bevacizumab on experimentally induced corneal neovascularization in a rat model. Methods: A total of 30 adult Sprague-Dawley rats were used in this experimental study. The central cornea of the rats was cauterized chemically. The rats were randomly enrolled into three groups as follows: Group 1 received bevacizumab 1%, Group 2 received cyclosporine 0.1%, and Group 3 received isotonic saline twice a day for 28 days. Slit-lamp examination of all rats was performed at the 3rd and 28th day. The rats were then sacrificed, and the corneas were excised. The number of blood vessels, state of inflammation, and collagen formation were evaluated histopathologically in the corneal sections. Results: Corneal opacity and edema grades were significantly lower in Group 2 than in Group 3 (p=0.04 and 0.00, respectively). In the histopathological examination, Group 2 demonstrated significantly lesser number of blood vessels than Group 3 (p=0.001). Regarding collagen formation, Group 2 exhibited more regular collagen formation than Groups 1 and 3 (p=0.03). Inflammation grades were significantly lower in Groups 1 and 2 than in Group 3 (p=0.014 and 0.001, respectively). Conclusion: Topical bevacizumab is effective in inhibiting newly formed corneal neovascularization. The topical cyclosporine 0.1% treatment appears to be more effective than the topical bevacizumab treatment.
RESUMO Objetivo: Comparar os efeitos da ciclosporina tópica 0,1% e do bevacizumabe na neovascularização da córnea produzida experimentalmente em um modelo com ratos. Métodos: Trinta ratos Sprague-Dawley adultos foram usados neste estudo experimental. A córnea central dos ratos foi cauterizada quimicamente. Os ratos foram distribuídos aleatoriamente em três grupos. O grupo 1 recebeu bevacizumabe a 1%, o grupo 2 recebeu ciclosporina tópica a 0,1% e o grupo 3 recebeu solução salina isotônica duas vezes ao dia durante 28 dias. O exame de lâmpada de fenda de todos os ratos foi realizado no terceiro e no vigésimo oitavo dias. Os ratos foram então sacrificados e as córneas excisadas. Nos cortes da córnea, o número de vasos sanguíneos, o estado de inflamação e a formação de colágeno foram avaliados em uma análise anatomopatológica. Resultados: No Grupo 2, os graus de opacidade e de edema da córnea foram significativamente menores que no Grupo 3 (p=0,04 e 0,00, respectivamente). No exame histopatológico, o Grupo 2 apresentou um número significativamente menor de vasos sanguíneos do que o Grupo 3 (p=0,001). Em relação à avaliação da formação de colágeno, esta mostrou-se mais regular no Grupo 2 que no Grupo 1 e no Grupo 3 (p=0,03). Os graus de inflamação foram significativamente menores no Grupo 1 e no Grupo 2 em comparação com o Grupo 3 (p=0,014 e 0,001, respectivamente). Conclusão: O bevacizumabe tópico é eficaz na inibição da neovascularização da córnea recém-formada. O tratamento tópico com ciclosporina a 0,1% parece ser mais eficaz em comparação ao tratamento tópico com bevacizumabe.
RESUMO
Purpose: To evaluate the effect of topical instillation of pegaptanib sodium upon inflammatory angiogenesis induced in the rabbit cornea by alkaline cauterization. Methods: Inflammatory angiogenesis was induced by alkaline (sodium hydroxide) cauterization in the corneas of 29 male New Zealand rabbits. The animals were divided into 4 groups: a control group treated with 0.5% carboxymethylcellulose sodium eye drops, a group treated with 1.0% prednisolone acetate eye drops, a group treated with 0.5% pegaptanib sodium diluted in 15 mL 0.5% carboxymethylcellulose sodium, and a group treated with 1.0% pegaptanib sodium diluted in 15 mL 0.5% carboxymethylcellulose sodium. After cauterization, eye drops were administered every 12 hours for 21 days. The animals were evaluated every 3 days after cauterization, and the newly formed vessels were quantified from photographs. The treatment effectiveness was analyzed with 3 parameters of antiangiogenic response: neovascularization area (NA), total vascular length (TVL), and number of blood vessels (BVN). Results: Average NA, TVL, and BVN values were significantly higher in both pegaptanib groups than in the prednisolone group. A nonstatistically significant reduction in parameters on days 18 and 21 was the minimum achieved in both pegaptanib groups. The efficacy of the treatments in relation to the control was significantly greater in the prednisolone group than in the 0.5% pegaptanib group or the 1.0% pegaptanib group (P < 0.001). Conclusion: Topical instillation of 0.5% and 1.0% pegaptanib sodium diluted in 15 mL 0.5% carboxymethylcellulose sodium had no inhibitory effect on corneal neovascularization in this rabbit model.
Assuntos
Inibidores da Angiogênese/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Inflamação/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Administração Tópica , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/uso terapêutico , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Aptâmeros de Nucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos/uso terapêutico , Cáusticos/administração & dosagem , Cáusticos/efeitos adversos , Córnea/patologia , Lesões da Córnea/induzido quimicamente , Neovascularização da Córnea/induzido quimicamente , Modelos Animais de Doenças , Inflamação/diagnóstico , Instilação de Medicamentos , Masculino , Prednisolona/administração & dosagem , Prednisolona/farmacologia , Coelhos , Hidróxido de Sódio/administração & dosagem , Hidróxido de Sódio/efeitos adversos , Resultado do TratamentoRESUMO
The present study aimed to demonstrate the possibility to treat corneal neovascularization using the combination of anti-VEGF injection and argon laser photocoagulation.
Assuntos
Neovascularização da Córnea , Inibidores da Angiogênese , Argônio , Neovascularização da Córnea/diagnóstico , Neovascularização da Córnea/tratamento farmacológico , Neovascularização da Córnea/etiologia , Humanos , Fotocoagulação a Laser , Lasers , Neovascularização Patológica/terapiaRESUMO
The aim of this study was to examine the expression of the cytokines and chemokines receptor-3 (CCR3) molecule in endothelial cells and vascular structures in a murine model of corneal neovascularization and in samples of neovascularized human corneas. An immunofluorescence assay using the murine model showed a greater proportion and intensity of CCR3 in the epithelium and corneal subepithelial regions in corneas with neovascularization. In the absence of vascularization, no CCR3 was found. Of the 32 studied tissues, eight were vascularized and 24 were avascular. In the human corneas, vascularized corneas showed positive labeling for CD31 in all the analzedtissues, as well as positive labeling for CCR3. Therefore, all vascularized tissues showed positive coexpression of CCR3 and CD31, whereas none of the avascular corneas showed immunolabeling for either of these receptors. These results suggest that CCR3 could be a possible marker for corneal neovascularization with potential to be a therapeutic target.
Assuntos
Córnea/metabolismo , Neovascularização da Córnea/genética , Regulação da Expressão Gênica , RNA/genética , Receptores CCR3/genética , Animais , Córnea/patologia , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR3/biossínteseRESUMO
Corneal neovascularization (CNV) is a common sight-threatening pathology that can be induced by a variety of inflammatory and angiogenic stimuli. Current CNV treatments include anti-inflammatory drugs and antibody-based inhibitors of vascular endothelial growth factor (VEGF). However, these are not always effective and novel therapeutic approaches are needed. Previous work has indicated a role for nucleolin (NCL) in VEGF-mediated neoangiogenesis in a suture-induced CNV model. The major goal for this current study is to test the effect of AS1411, a NCL-binding DNA aptamer that has reached human clinical trials, on neovascularization in a murine model of VEGF-mediated CNV. Our results show that topical administration of AS1411 can significantly inhibit corneal neovascularization in this model. Mechanistic studies indicate that AS1411 reduces the VEGF-stimulated proliferation, migration, and tube formation of primary cells obtained from human limbus stroma (HLSC). AS1411 treatment also significantly reduced VEGF-stimulated induction of miR-21 and miR-221 in HLSC, suggesting a role for these pro-angiogenic miRNAs in mediating the effects of AS1411 in this system. In sum, this new research further supports a role for NCL in the molecular etiology of CNV and identifies AS1411 as a potential anti-angiogenic CNV treatment that works by a novel mechanism of action.
Assuntos
Córnea/patologia , Neovascularização da Córnea/tratamento farmacológico , Oligodesoxirribonucleotídeos/farmacologia , Animais , Aptâmeros de Nucleotídeos , Movimento Celular , Proliferação de Células , Células Cultivadas , Córnea/efeitos dos fármacos , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
PURPOSE: The objective is to analyze the antiangiogenic mechanism of suramab, a pharmaceutical compound of bevacizumab and suramin, in a rabbit model of corneal angiogenesis. MATERIAL AND METHODS: Corneal neovascularization was induced in four groups of six New Zealand White rabbits by applying a filter paper disk soaked in 1 M Na (OH) on the central cornea. Group one was treated after injury with intravenous suramab at a dose equivalent to 3 mg/kg of bevacizumab and 10 mg/kg of suramin. Group two was treated with intravenous bevacizumab (5 mg/kg). Group three was treated with 10 mg/kg of suramin while the control group received no treatment. Digital photographs were taken at days 9, 15, 21, and 35. Neovessel formation was quantified giving a 0-4 score to each quadrant according to the centripetal growth of the longest vessel (neovessel index, NVI). Animals were sacrificed at day 35. Corneas were processed for histology, immunohistochemistry, and Western-blot using primary antibodies against P2X2, basic fibroblast growth factor (bFGF), LYVE-1, PECAM-1, and vascular endothelial growth factor-A (VEGF-A). RESULTS: Suramab significantly reduced neovessel growth (mean NVI: 4.2) compared to bevacizumab (8.4), suramin (7.22), and control animals (12.2) at 35 days post-injury (p < 0.01). A lower protein expression of P2X2, bFGF, LYVE-1, PECAM-1, and VEGF-A was found in the cornea of suramab animals than in the other groups of animals. CONCLUSIONS: Joint downregulation of bFGF, P2X2, bFGF, and LYVE-1 constitutes a mechanism that induces greater and longer inhibition of corneal angiogenesis. Results might be relevant to ophthalmic care. Ocular administration of suramab is currently being investigated.
Assuntos
Bevacizumab/farmacologia , Córnea/patologia , Neovascularização da Córnea/tratamento farmacológico , Regulação para Baixo/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/biossíntese , Receptores Purinérgicos P2X2/biossíntese , Suramina/farmacologia , Animais , Western Blotting , Córnea/metabolismo , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Combinação de Medicamentos , Imuno-Histoquímica , CoelhosRESUMO
ABSTRACT Purpose: To evaluate and compare the effects of topical application and subconjunctival injection of bevacizumab on corneal neovascularization (CNV) in rabbits' eyes after chemical burning of the cornea. Methods: The animals were randomly distributed into four groups of five animals. In one group, the drug was instilled, while in another, it was administered by subconjunctival injection. The two procedures using bevacizumab were compared with instillation and subconjunctival injection of saline solution (S). Neovascularization was evaluated according to the size of the invasion area of new blood vessels and through computerized analysis of this area. The data were analyzed using the Kruskal-Wallis test followed by Dunn's test for two-by-two comparison of the groups, to assess the external examination of CNV. Analysis of variance was used to assess the area of CNV. P<0.05 was considered statistically significant. Results: Assessing both the external examination and the invasion area of neovessels on the 5th and 10th days, there was a clear difference between the groups. The group to which saline solution had been applied showed higher scores for CNV, as well as increases in the invasion area of neovessels. Two-by-two comparison of groups revealed no significant differences. However, an analysis of the factors involved (injection vs. instillation and bevacizumab vs. saline solution) showed that injection did not differ from instillation, but that bevacizumab differed from saline solution. Conclusion: Bevacizumab showed an inhibitory effect on CNV in rabbits' eyes after chemical burning of the cornea. There was no difference between the topical or subconjunctival administration of bevacizumab in the inhibition of CNV.
RESUMO Objetivos: Avaliar e comparar o efeito do uso tópico e da injeção subconjuntival do bevacizumabe na neovascularização corneana de olhos de coelhos após queimadura química. Métodos: Os animais foram distribuídos de forma aleatória em quatro grupos de cinco animais. Em um grupo de coelhos a droga foi instilada, enquanto em outro foi aplicada injeção subconjuntival, sendo os dois procedimentos comparados com a instilação e injeção subconjuntival de soro fisiológico 0,9% (SF) e entre si. A neovascularização foi avaliada conforme o tamanho da área de invasão dos neovasos e com análise computadorizada da mesma. Na análise de dados aplicou-se o teste de Kruskal-Wallis seguido do teste de Dunn com p<0,05 para comparação dos grupos dois a dois na análise do exame externo da neovascularização corneana. Na análise da área de neovascularização corneana, aplicou-se o teste F de análise de variância. A significância estatística foi definida como valor de p<0.05. Resultados: A avaliação do exame externo e da área de invasão de neovasos, no 5º e 10º dia, mostrou nítida diferença entre os grupos. Com o uso de soro fisiológico houve maior graduação na escala de neovascularização corneana e também na área de invasão dos nevasos, o que demonstrou o efeito inibitório do bevacizumabe. Nas comparações dos grupos dois a dois não foram detectadas diferenças significativas, porém, ao analisar os fatores envolvidos (procedimentos: injeção ou instilação, e as drogas: bevacizumabe ou soro fisiológico), verificou-se que a injeção não diferiu da instilação, mas o bevacizumabe diferiu do soro fisiológico. Conclusão: O bevacizumabe apresentou efeito inibitório na neovascularização corneana de olhos de coelhos após queimadura química, tanto por via tópica como por via subconjuntival e não houve diferença entre a via tópica e a via subconjuntival de administração do bevacizumabe na inibição da neovascularização corneana.
Assuntos
Animais , Masculino , Ratos , Neovascularização da Córnea/tratamento farmacológico , Córnea/efeitos dos fármacos , Inibidores da Angiogênese/administração & dosagem , Bevacizumab/administração & dosagem , Soluções Oftálmicas , Queimaduras Químicas , Distribuição Aleatória , Queimaduras Oculares , Administração Tópica , Neovascularização da Córnea/patologia , Córnea/inervação , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Modelos Animais de Doenças , Injeções Intraoculares , Lesões da Córnea/induzido quimicamenteRESUMO
Abstract Purpose: To establish and compare protocols of alkaline cauterization for inducing corneal angiogenesis in murine models. Methods: Twenty-four adult Wistar rats were distributed into four groups (G1, G2, G3, and G4). The right eye cornea from each rat was cauterized using filter paper (3 mm), soaked in a solution of silver and potassium nitrates (3:1). Cauterization times were 10 (G1 and G4), or 20 seconds (G2 and G3). Cauterized corneas were washed with Ringer's lactate solution. The filter paper was either removed before washing (G1 and G2), or kept on the corneas (G3 and G4). Corneas were photographed at multiple time points (2, 4, 6, 8, 11, 13, and 15 days after the procedure), and neovascularization parameters were assayed. Results: Neovascularization was observed in 66% of G1 corneas, and 100% of G2, G3, and G4 corneas. On day 15, G1 corneas showed smaller vascularized areas (12.63 ± 12.59%) compared to those in the G3 (41.95 ± 17.32%) and G4 (33 ± 11.74%) (P < 0.05) groups. Conclusions: The silver and potassium nitrate solution effectively induced corneal angiogenesis. The G2, G3, and G4 protocols showed excellent reproducibility, and induced vascularization in 100% of corneas.
Assuntos
Animais , Masculino , Cauterização/métodos , Neovascularização da Córnea/etiologia , Compostos de Potássio , Modelos Animais de Doenças , Neovascularização Patológica/etiologia , Nitratos , Valores de Referência , Fatores de Tempo , Reprodutibilidade dos Testes , Ratos Wistar , Córnea/cirurgia , Córnea/irrigação sanguíneaRESUMO
Purpose: To establish and compare protocols of alkaline cauterization for inducing corneal angiogenesis in murine models. Methods: Twenty-four adult Wistar rats were distributed into four groups (G1, G2, G3, and G4). The right eye cornea from each rat was cauterized using filter paper (3 mm), soaked in a solution of silver and potassium nitrates (3:1). Cauterization times were 10 (G1 and G4), or 20 seconds (G2 and G3). Cauterized corneas were washed with Ringers lactate solution. The filter paper was either removed before washing (G1 and G2), or kept on the corneas (G3 and G4). Corneas were photographed at multiple time points (2, 4, 6, 8, 11, 13, and 15 days after the procedure), and neovascularization parameters were assayed. Results: Neovascularization was observed in 66% of G1 corneas, and 100% of G2, G3, and G4 corneas. On day 15, G1 corneas showed smaller vascularized areas (12.63 ± 12.59%) compared to those in the G3 (41.95 ± 17.32%) and G4 (33 ± 11.74%) (P 0.05) groups. Conclusions: The silver and potassium nitrate solution effectively induced corneal angiogenesis. The G2, G3, and G4 protocols showed excellent reproducibility, and induced vascularization in 100% of corneas.(AU)