Age-related macular degeneration (ARMD) is the leading cause for visual impairment and blindness in the elder population of developed countries. The primary underlying cause for significant visual loss is the choroidal neovascularization (CNV). CNV is also generated in high myopia ; angioid streaks and some inflammatory diseases and rarely after laser photocoagulation. Current treatment strategies for ARMD include laser photocoagulation , photodynamic therapy , but neither treatment addresses underlying stimuli for blood vessel growth. Therefore, recurrent disease is a problem of clinical significant relevance. Surgical excision of submacular neovascular membrane lead to the additional damage of the retinal pigment epithelium (RPE). Consequently, use of therapeutic agents that directly inhibit the angiogenic stimuli may be able to provide a more effective and permanent treatment. Vasostatin (VS) , the N-terminal domain (amino acid 1-180) of a calcium-binding protein , is a potent angiogenesis inhibitor , isolated from culture supernatants of an Epstein-Barr virus-immortalized cell line. In previous studies, we demonstrated that gene delivery of angiogenesis inhibitor vasostatin attenuated the corneal neovascularization in animals. The recombinant vasostatin also prevented or apparently reduced growth of human Burkitt lymphoma and human colon carcinoma in animal model. The primary objective of this study was to vasostatin attenuated the choroidal neovascularization in animals. Retinal and visual function will be evaluated. The above experiments would enable us to test the hypothesis that the topical application of VS delivery might be a promising strategy for the treatment of ARMD and other retinal disorders. Furthermore, the results from animal studies might be extrapolated for future clinical application.

Age-related macular degeneration (AMD) is one of the leading causes of blindness in adults over the age of 60. Currently, at least 11 million patients in the United States have some form of macular degeneration and this number is projected to grow as the population ages. The more severe form of the disease – neovascular (wet) AMD, is characterized by intraocular neovascularization, inflammation, and retinal damage; however, the disease progression can be deterred through intraocular injections of anti-angiogenic agents. The complications and burden that arise from repetitive injections as well as the difficulty posed by targeting the posterior segment of the eye make this an interesting territory for the development of novel drug delivery systems. New methods for drug delivery are being investigated exploring the use of nanoparticles and other polymeric materials. The goal of this project is to study the potential use of poly(lactide-co-glycolic acid)-polyethylene glycol (PLGA-PEG) nanoparticles in thermoreversible gels as localized sustained intraocular drug delivery. We prepared stable and reproducible corticosteroid-encapsulated nanoparticles in thermoreversible gels to inhibit vascular endothelial growth factor (VEGF) overexpression characteristic of neovascular AMD. We characterized the drug delivery system by obtaining size, shape, and drug encapsulation data. We also demonstrated that the polymer could be injected into the vitreous as a solution and transition to a gel phase based on the temperature difference between regular indoor environment and the vitreous body. The drug delivery system was tested on human retinal pigment epithelial cells (ARPE-19), for cytotoxicity, uptake and VEGF expression. We also examined the drug delivery system's ability to mitigate the disease progression in a mouse model of choroidal neovascularization (CNV). The effect on blood vessel area was shown and the changes in the mRNA expression of angiogenesis mediators were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). These results indicate that the proposed drug delivery systems has the promise to be developed for retinal diseases, involving CNV, including neovascular AMD. Further studies are warranted in developing this promising intraocular drug delivery system for wet AMD and similar ophthalmic diseases. / Ph. D.

Age-related macular degeneration (AMD) is the leading cause for visual impairment and blindness in the elder population of developed countries. The primary underlying cause for significant visual loss is the choroidal neovascularization (CNV). Current treatment strategies for AMD include laser photocoagulation, photodynamic therapy (PDT) and excision of neovascular membranes, but have met with limited success. In our previous studies, we demonstrated that gene delivery of angiogenesis inhibitor vasostatin (VS) attenuated the corneal neovascularization in animals. The primary objective of this study was to investigate gene delivery of vasostatin (VS) attenuated the choroidal neovascularization in animals. Retinal and visual function will be evaluated. However, systematic expression of angiogenesis inhibitor may bring adverse effects to physiological processes. The feasibility, efficiency and safety of gene delivery with systemic and local routes were evaluated. Intramuscular polymer-based gene delivery had no side effect such as virus vector and revealed the safety. Recombinant adenovirus (Ad) was used gene delivery system because of its high titer, wide host range, and transduction efficiency. Adeno-associated virus (AAV) represents highly efficient that can facilirate long-term transduction. We propose to improve the efficacy and safety of VS gene delivery, and to search for the effective delivery route and other adjuvant therapy in conjunction with VS for treatment of CNV. Recently, PDT with veteporfin is an established treatment for subfoveal CNV secondary to AMD. We tried to compare the effect and safety of standard and reduced-dose light application PDT in an animal mocel of CNV. The 180-residue VS and its 48-residue (VS48) inhibited the migration and tube formation in cultured endothelial cells. Topical VS application suppresses the progression of laser-induced CNV via angiogenesis ihhibition, as well as in VS48. VS-48 inhibited the growthof vessels in arota rings. Electroretinograms (ERG) analysis revealed that topical VS-48 application for 21 days had no effect on rat retinal functions. Topical VS-48 treatment significantly reversed the CNV-induced alterations in ERG. Transfection of pCMV3-VS into muscle cells resulted in increased production and release of exogenous VS, which specifically inhibited the proliferation of endothelial cells. Rats treated with intrmuscular injection with PVP-VS also showed a significant reduction in the CNV lesions for at least 42 days. Subconjunctival injection with Ad vector revealed no retinal toxicity in ERG. Ad-luciferase via subconjunctival injections showed ocular expression for as long as 112 days by using bioluminescence image analysis in rodent. AAV-luciferase via subconjunctival injections showed ocular expression for as long as 365 days by using bioluminescence image analysis in mice, and AAV serotype 5-luciferase even showed expression lasting for 2 years. Suppression of laser photocoagulation¡Vinduced CNV by Ad-VS was documented in rat model. Combination therapies are important as treatment options. We demonstrated that PDT could effectively attenuate CNV in a rat model, and reduced doses, worked just as well as the standard dose. In the preliminary study of PDT combined topical VS application, treatment led to CNV attenuation more than alone with PDT. The above experiments would enable us to demonstrate that the vasostatin delivery might be a promising strategy for the treatment of AMD and other retinal or ocular disorders. Furthermore, the results from animal studies might be extrapolated for future clinical application.

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Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / CD-ROM i ficka. Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Orientador: José Paulo Cabral de Vasconcellos / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T03:32:34Z (GMT). No. of bitstreams: 1 Medina_FlavioMacCord_D.pdf: 2571030 bytes, checksum: 442e6e86f7bd3a0d6fae088740a1e85e (MD5) Previous issue date: 2013 / Resumo: Introdução: O fator de complemento H (CFH) é um componente do sistema imunológico que possui ação imunomoduladora sobre a resposta inflamatória. A gravidade da degeneração macular relacionada à idade (DMRI) é determinada em parte por um estado inflamatório sustentado por atividade aberrante da via alternativa do complemento. As evidências na literatura da relação entre o polimorfismo Y402H do gene CFH e a resposta ao tratamento da DMRI exsudativa permanecem controversas. Objetivo: Avaliar a associação entre as variantes do polimorfismo Y402H do gene CFH e os efeitos funcional e morfológico a curto prazo, assim como a evolução a longo prazo, dos antiangiogênicos em pacientes com DMRI exsudativa. Métodos: Vinte e cinco pacientes recém diagnosticados com DMRI exsudativa foram avaliados em um estudo de curto prazo com acuidade visual medida pela tabela do ETDRS e espessura retiniana central por tomografia de coerência óptica (OCT) de alta resolução, submetidos a injeção intravítrea de bevacizumabe e prospectivamente reexaminados em 7 e 28 dias. Quarenta e seis pacientes previamente submetidos ao tratamento com antiangiogênicos tiveram seus prontuários e exames retrospectivamente avaliados em um estudo de longo prazo quanto às evoluções funcional e morfológica ao longo de um ano. Esses parâmetros foram comparados com o genótipo do CFH, cuja análise molecular do polimorfismo Y402H foi realizada por meio da reação em cadeia da polimerase (PCR) e sequenciamento direto. Resultados: No estudo de curto prazo, houve melhora da acuidade visual no dia 28 em relação ao valor inicial (D0 vs. D28) em todos os genótipos. Entretanto, no grupo homozigoto para o alelo de risco (CC), ocorreu diferença apenas no dia 28 em relação ao dia 7 (D7 vs. D28), enquanto nos grupos CT e TT, a acuidade visual melhorou mais precocemente, no dia 7 em relação ao valor inicial (D0 vs. D7). A espessura retiniana central apresentou redução nos grupos CT (D0 vs. D7 e D0 vs. D28) e TT (D0 vs. D28), enquanto não houve mudança significativa no grupo CC. No estudo de longo prazo, foi evidenciada melhora da acuidade visual ao longo de um ano de acompanhamento apenas no grupo de pacientes sem o alelo C, sem diferença significativa no grupo de pacientes com o alelo de risco. A espessura retiniana central apresentou redução nos genótipos CT e TT, enquanto que no grupo CC não houve significância. Número de injeções, persistência de atividade neovascular e percepção subjetiva de melhora não diferiram entre os genótipos. Conclusão: O perfil de genótipo do CFH parece influenciar o efeito funcional e morfológico da injeção intravítrea de bevacizumabe com uma ação mais precoce em pacientes sem o genótipo de risco. A presença do alelo de risco parece estar relacionada à ausência de melhora visual ao longo de um ano de tratamento com inibidores do VEGF. Esses resultados sugerem que o perfil do genótipo do CFH possa exercer efeito farmacogenético nesse grupo de pacientes brasileiros, influenciando negativamente a resposta ao tratamento da DMRI exsudativa com antiangiogênicos / Abstract: Introduction: The complement factor H (CFH) is a component of the immune system that has immunomodulatory action on the inflammatory response. The severity of age-related macular degeneration (AMD) is determined in part by an inflammatory state sustained by aberrant activity of the alternative complement pathway. Evidences in the literature of the relationship between the Y402H polymorphism of CFH gene and response to treatment of wet AMD remain controversial. Purpose: To evaluate the association between variants of the Y402H polymorphism of CFH gene polymorphism and the short-term functional and morphological effects, as well as long-term evolution, of antiangiogenic drugs in patients with exudative AMD. Methods: Twenty-five patients with newly diagnosed exudative AMD were evaluated in a short-term study with visual acuity on ETDRS chart and central retinal thickness measured with high resolution optical coherence tomography (OCT), underwent intravitreal injection of bevacizumab and were prospectively reviewed in 7 and 28 days. Forty-six patients previously submitted to treatment with VEGF inhibitors had their medical charts retrospectively evaluated in a long-term study about the functional and morphological evolutions over one year. These parameters were compared with the CFH genotype, whose molecular analysis of Y402H polymorphism was performed by polymerase chain reaction (PCR) and direct sequencing. Results: In the short-term study, there was improvement in visual acuity at day 28 compared to baseline (D0 vs. D28) in all genotypes. However, in the group homozygous for the risk allele (CC), differences occurred only on day 28 compared to day 7 (D7 vs. D28), while the CT and TT groups, visual acuity improved earlier in the day 7 compared the initial value (D0 vs. D7). The central retinal thickness decreased in groups CT (D0 vs. D7, D0 vs. D28) and TT (D0 vs. D28), while there was no significant change in group CC. In the long-term study, it was noticed improvement in visual acuity over one year of follow-up in the group of patients without the C allele and no significant difference in the group of patients with the risk allele. The central retinal thickness decreased in the CT and TT genotypes, whereas in the CC group the difference was not significant. Number of injections, persistent neovascular activity and subjective perception of improvement did not differ between genotypes. Conclusion: The profile of the CFH genotype seems to influence the functional and morphological effect of intravitreal injection of bevacizumab with an earlier action in patients without the risk genotype. The presence of the risk allele seems to be related to the lack of visual improvement over one year of treatment with inhibitors of VEGF. These results suggest that the profile of the CFH genotype may present pharmacogenetic effect in this group of Brazilian patients, negatively influencing the response to treatment of exudative AMD with antiangiogenic drugs / Doutorado / Oftalmologia / Doutor em Ciências Médicas

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Made available in DSpace on 2014-07-29T15:29:14Z (GMT). No. of bitstreams: 1 Dissertacao Livia C de S N Bianchi - Oftalmologia.pdf: 3291987 bytes, checksum: bf690044678ae1c1ecf40768b3ef062a (MD5) Previous issue date: 2012-03-27 / Purpose: To evaluate the efficacy and safety of strontium-90 epimacular brachytherapy combined with intravitreal bevacizumab for treating subfoveal choroidal neovascularization secondary to exudative age-related macular degeneration. Its effects on visual acuity and macular thickness at 36 months of follow up were also studied. Methods: Sixteen patients with predominantly classic, minimally classic and occult choroidal neovascularization were treated with a single 24 Gy dose of radiation during pars plana vitrectomy, associated to two injections of bevacizumab at baseline and one injection at the one-month visit. The patients underwent a complete ophthalmologic examination, which included fluorescein angiography and optical coherence tomography, at the initial visit, every three months during the first year, and every six months during the second and third years of follow up. Results: Of the 16 cases, 2 patients (12.5%) lost more than 15 letters at the end of 36 months, and 3 (18.75%) lost fewer than 15 letters. Among the patients with improved visual acuity, 3 (18.75%) gained less than 15 letters and 8 (50%) gained more than 15 letters at the end of treatment. The mean best corrected visual acuity showed a gain of 16.0 letters at 12 months. After 36 months (n = 16), the mean best corrected visual acuity showed a gain of 10.4 letters. Of the 16 eyes included in the study, six required further therapy. The evaluation of macular thickness showed decreased thickness in 10, among the 16 patients (62.5%). Three eyes (18.75%) had similar values at the beginning and the end of 36 months, and 3 eyes (18.75%) showed increased macular thickness. Conclusion: Epimacular brachytherapy combined with bevacizumab may be considered a possible therapeutic option for choroidal neovascularization in age-related macular degeneration. The procedure was thought to be safe and was well tolerated by the patients. The treatment resulted in anatomical and functional improvements, compatible with currently available therapies. / Objetivo: Avaliar a eficácia e segurança da braquiterapia epimacular com estrôncio 90 e bevacizumabe intravítreo para tratamento da neovascularização coróidea subfoveal secundária à degeneração macular relacionada à idade exsudativa, e observar seus efeitos na acuidade visual e espessura macular, em 36 meses de seguimento. Metodologia: Foram estudados 16 pacientes portadores de neovascularização coróidea, (predominantemente clássicas, minimamente clássicas e ocultas) tratados com dose única de radiação de 24Gray, durante vitrectomia via pars plana, associada a duas injeções de bevacizumabe uma no início do tratamento e a outra um mês após. Foram realizados exame oftalmológico completo, angiofluoresceinografia e tomografia de coerência óptica na visita inicial, a cada três meses durante o primeiro ano e a cada seis meses durante o segundo e o terceiro anos de seguimento. Resultados: Dos 16 casos, 2 (12,5%) perderam mais de 15 letras ao final de 36 meses, e 3 pacientes (18,75%) perderam menos de 15 letras. Dentre os pacientes que melhoraram a acuidade visual, 3 (18,75%) ganharam menos de 15 letras e 8 (50%) ganharam mais de 15 letras ao final do tratamento. A média da melhor acuidade visual corrigida demonstrou ganho de 16,0 letras ao final de 12 meses. Após 36 meses (n = 16), a média da melhor acuidade visual corrigida apresentou um ganho de 10,4 letras pela tabela ETDRS. Dos 16 olhos observados, 6 necessitaram de tratamento adicional. Na avaliação da espessura macular, 10 (62,5%) dos 16 pacientes apresentaram diminuição da espessura, 3 olhos (18,75%) mantiveram valores semelhantes no início e ao final de 36 meses, e 3 olhos (18,75%) apresentaram aumento da espessura macular. Conclusão: A braquiterapia epimacular combinada ao uso de bevacizumabe se mostra como uma possível opção terapêutica para a neovascularização de coróide na degeneração macular relacionada à idade. O procedimento foi considerado seguro e bem tolerado, com melhora antômica-funcional compatível aos tratamentos atualmente disponíveis.