Higiene Palpebral en el Tratamiento de la Blefaritis

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1 Volumen V No.2 Junio 2006 Terapia Genética nas Doenças Corneanas Lauro A. Oliveira, MD, Mark I. Rosenblatt, MD, Ph.D. Higiene Palpebral en el Tratamiento de la Blefaritis David Díaz Valle MD, Jose Manuel Benitez-Del-Castillo Sánchez, MD. Current Role of Anti-VEGF Therapy in the Management of Choroidal Neovascularization J. Fernando Arévalo, MD FACS, Reinaldo A. García, MD, Juan G. Sánchez, MD. Estandarización de una técnica de cultivo de epitelio, endotelio y fibroblastos corneales y evaluación de su crecimiento sobre mallas de colágeno tipo I. Carlos H. Triana S. MD; Carlos A. Chiriboga N. MD; Marta R. Fontanilla D. PhD

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3 MENSAJE DEL PRESIDENTE l aprendizaje de la oftalmología como una especialidad ha cambiado con el tiempo. Hace menos de un siglo se aprendía al lado de un maestro, E sin un programa definido y en tiempos variables. De ahí evolucionamos a las residencias médicas, en ellas, a través de un programa definido, el aspirante a oftalmólogo va cumpliendo en forma gradual y progresiva objetivos en conocimientos y destrezas. Este sistema de residencias es el que ha predominado en prácticamente todo el mundo occidental. Los programas de residencia varían en su calidad; los hay excelentes, muchos buenos y unos más que dejan que desear. Su diversidad sucede por condiciones socioeconómicas, patología variable y tecnología distintas. Estas diferencias existen y continuarán existiendo. Los oftalmólogos que en ellas se forman pueden, en consecuencia, tener distintas capacidades. Sin embargo, es importante buscar que estas diferencias sean las menores y que un oftalmólogo que egresa de cualquier centro tenga un mínimo de conocimientos necesarios para ejercer una oftalmología moderna y así cumplir con las Enrique Graue Presidente de la Asociación Panamericana de Oftalmología. demandas de salud del país en el que va a ejercer. Por lo tanto, el disminuir estas diferencias educativas entre los distintos centros formadores es preocupación constante de los distintos organismos nacionales e internacionales. En el modelo de residencias médicas un personaje de gran importancia es el encargado de los residentes (program director). Este puede ser el profesor titular o bien, algún oftalmólogo joven que tiene una inclinación especial para la enseñanza. De él, con frecuencia, depende toda la organización de la estructura curricular de la residencia de oftalmología. La Asociación Panamericana de Oftalmología ha considerado que uno de sus programas prioritarios es el mejorar la calidad de la enseñanza en los programas de residencia en Latinoamérica. Para tal efecto el 23 y 24 de marzo del presente año se llevó a cabo en la ciudad de Lima, Perú, el segundo curso latinoamericano encaminado a enseñar a enseñar, específicamente dirigido a los encargados y responsables de las residencias de Oftalmología del Perú, Bolivia y Ecuador. El curso fue un gran éxito tanto en la asistencia (51 directores) como en la participación activa y discusión de los temas más importantes relacionados a la enseñanza de la oftalmología, y en curso se trataron: las formas de superar los desarrollos curriculares y metas del aprendizaje; las técnicas para mejorar la efectividad de la enseñanza; los distintos métodos para evaluar las competencias adquiridas; la retroalimentación de las virtudes y defectos de los residentes; los métodos de enseñanza clínica y quirúrgica y los programas de certificación. Para desarrollar un curso de esta naturaleza deben orquestarse una serie de organizaciones. Se contó con la decidida colaboración del Internacional Council de Oftalmología (ICO); de la Academia Americana de Oftalmología (AAO); de la Asociación de Profesores Universitarios de Oftalmología en los Estados Unidos de Norteamérica (AUPO) y de la Organización de Profesores de Oftalmología de la APAO (PACUPO ). Gracias al esfuerzo de todos ellos bajo excelente la coordinación del Dr. José Antonio Roca y del Dr Karl Golnik el curso logró sus objetivos. Participaron como profesores del curso en forma muy destacada los Dres: Bradley R. Straatsma, Anthony Arnold, Karl Golnik, Andrew Lee, Peter A. Quiros, Juan Cuadros, Victor Dulanto y Guillermo Barriga. Una especial mención debe hacerse a la industria. En esta ocasión Laboratorios Alcon, con discreción y elegancia, cargó con todos los gastos de traslado y hospedaje de los asistentes mientras que el Internacional Council hizo lo propio con los profesores. Una aportación adicional fue efectuada por la Fundación Panamericana de Oftalmología. De esta forma, entre todos, vamos construyendo un mejor futuro para la oftalmología de este continente. Junio 2006 PRESIDENT S MESSAGE he training in the field of ophthalmology as a specialty has changad with T time. In less than a century, one learned in an apprentice model at the side of a teacher, without a defined program and with varied schedules. From there we developed medical residencies and in those programs, along with a defined program, the aspiring ophthalmologist continues gradually with progre-ssive objectives in knowledge and skill. This system of residencies has become standard in practically all of the Western Hemisphere. The different residency programs vary in their level of quality; there are excellent programs, many good ones and some leave something to be desired. Its diversity is a result of socio-economic conditions, varied backgrounds and different technologies. These differences exist and will continue to exist. The ophthalmologists that train in different residencies, consequently, can have different capacities. Nevertheless, it is important to make sure that these differences are minimum and that the ophthalmologist that returns from whatever training center has had at the least minimum standards necessary to be able to do modern ophthalmology and thereby fulfill the health care demands in the country in which he/she is going to practice. It is a constant concern, therefore, of the different national and internacional training and educational organizations that they make sure that the educational differences are as minimal as posible. In the medical residencies model, the person of great importance is the program director. This person can be a degreed professor or perhaps a young ophthalmologist that has a special inclination towards teaching. The program director is often responsible for the organization of the curricular structure for the ophthalmological residency program. The Pan-American Association of Ophthalmology has determined that one of its program priorities is to standardize the teaching quality of the residency programs in Latin America. As such, on March 23-24, 2006, in Lima, Peru, the 2nd Latin American "Teaching the Teachers" Course took place directed to those responsible for ophthalmological residencies in Peru, Bolivia and Ecuador. The course was a great success. 53 program directors actively participated and they discussed the most important topics related to teaching in ophthalmology: Development of the curriculum and training goals; different methods of evaluating acquired skills; the nourishment of the virtues and the culling of the defects of the residents; methods of clinical and surgical training and certification programs. To organize a course of this type one needs the collaboration of several organizations. We had the committed collaboration of the Internacional Council of Ophthalmology (ICO), the American Academy of Ophthalmology (AAO) and the Association of University Professors in Ophthalmology (AUPO), and the Pan-American Council of University Professors in Ophthalmology (PACUPO). The course achieved its objectives, thanks to the efforts of everyone involved and the excellent coordination of José Antonio Roca MD and Dr. Karl Golnik MD. Also participating as instructors in the course were the esteemed Bradley R. Straatsma MD, Anthony Arnold MD, Karl Golnik MD, Andrew Lee MD, Peter A. Quiros MD, Juan Cuadros MD, Victor Dulanto MD and Guillermo Barriga MD. A very special mention needs to be made regarding industry support. On this occasion, Alcon Laboratorios, with discretion and elegance, took care of transportation and housing of the attendees and meanwhile the International Council of Ophthalmology offered an educational grant in support of the instructors. Additional support was given by an educational grant by the Pan-American Ophthalmology Foundation. Because of this course, and with the collaboration of many individuals and organizations, we are continuing to construct a better future for Ophthalmology on this continent. : 1 :

4 REVIEW Junio 2006 Terapia Genética nas Doenças Corneanas Lauro A. Oliveira, MD I, Mark I. Rosenblatt, MD, Ph.D. II I- Doutorando em Oftalmologia - Universidade Federal de São Paulo - UNIFESP/EPM. Research Fellow na Universidade da Califórnia - Davis. II- Assistant Professor of the Department of Ophthalmology & Vision Science - University of California - Davis. ABSTRACT Technological advances in the field of molecular biology and especially recombinant DNA techniques have enabled scientists to develop gene therapy techniques for the treatment of human diseases. The potential therapeutic applications of gene transfer technology are enormous. The cornea is an excellent candidate for gene therapy because of its accessibility and immune-privileged nature. Studies examining the feasibility and optimal methods for vector-mediated gene transfer to the cornea have been performed using histochemical and fluorescent marker genes. These studies have utilized various viral vectors, such as adenovirus, adeno-associated virus, retrovirus, lentivirus, and herpes simplex virus, as well as non-viral methods to transfer heterologous genes into corneal cells in vitro, in vivo and ex vivo. In this review, we will discuss viral and non-viral approaches of gene delivery into the cornea as well as the limitations of these approaches. Additionally, we will discuss some potential applications for gene-based interventions which have been analyzed in specific corneal disorders such as allograft rejection, corneal wound healing, and corneal dystrophies using experimental models. INTRODUÇÃO Terapia genética em seres humanos é um dos mais atuais e excitantes campos de pesquisa abordados pelas áreas médicas e de biologia molecular. Os avanços tecnológicos nesta área podem ser comprovados pelo grande número de ensaios clínicos nas fases I e II para o tratamento de câncer, AIDS, degeneração macular, doenças cardiovasculares e outras doenças monogênicas. Além de sua utilização no tratamento de inúmeras doenças, a transferência de genes está sendo utilizada no desenvolvimento de vacinas tanto preventivas como terapêuticas para hepatite A, B e C, tuberculose e outras doenças. É uma abordagem que carrega consigo o potencial de curar doenças ou condições médicas anormais através da reposição de genes defeituosos por outro normal. É uma promessa para tratar tanto doenças adquiridas como hereditárias em todo o organismo. Distingue-se dos métodos convencionais de tratamento por atuar na fisiopatologia das doenças a nível molecular, assim proporcionando benefícios terapêuticos a longo prazo e distinguindo-se dos efeitos transitórios proporcionados pela terapia convencional com drogas. Apesar do seu enorme potencial terapêutico, existem algumas limitações fundamentais com esta forma de medicina molecular. Tais limitações precisam ser sanadas antes que esta tecnologia torne-se terapeuticamente aplicável e viável aos pacientes com desordens genéticas. Os dois principais desafios são: como transferir material genético de maneira eficiente para as células alvo; e, talvez ainda mais difícil, como fazer este material genético expressar-se ou tornar-se disponível para o hospedeiro. Vários estudos têm sido desenvolvidos para analisar sistemas de deliberação de genes que facilitem a transferência de material genético para as células alvo, porém ainda não se encontrou a solução mais segura e eficaz para tal problema. Algumas características da córnea conferem-lhe significante potencial para aplicação de tratamento genético nas doenças corneanas. Isto inclui sua relativa simplicidade na estrutura histológica o que permite fácil acesso para manipulação e exame; outro fator importante é que ela também permite monitorização visual da deliberação e expressão dos genes através da utilização de marcadores genéticos fluorescentes (EGFP). Além disto, usufrui de relativo privilégio imunológico na câmara anterior1 e pode ser mantida em cultura por algumas semanas. Fato este que a qualifica como potencial candidata a provedora de transferência "ex vivo" de material genético. Sistemas de deliberação de genes U ma modalidade eficiente de liberação de genes para tecidos vivos é essencial para terapia e pesquisas genéticas. As técnicas de transferência de genes se diferenciam de a- cordo com certas características dos vetores. Primeiramente, os vetores podem ou não possuir habilidades para integrar o material genético no DNA das células alvo. Isto define se a transferência será estável, caso consigam integrar o material genético, ou se será transitória, caso os mesmos não sejam capazes de integrá-lo na célula alvo. O segundo fator é o tropismo dos vetores por células e ou tecidos específicos. Alguns podem infectar apenas células com capacidade de divisão, enquanto outros podem infectar tanto células divisoras como células não divisoras ao mesmo tempo. Isto, teoricamente, reflete na expressividade de certos vetores. Assim sendo, a escolha do vetor baseia-se na necessidade terapêutica, na cronicidade da doença a ser tratada e nos riscos potenciais associados a uma super expressão do gene desejado. Por exemplo: em condições crônicas como síndrome de olho seco, desordens metabólicas e inflamações crônicas, seria interessante um vetor que promovesse uma expressão estável (com integração de material genético). Por outro lado, processos agudos como nas erosões recorrentes ou alterações pós-operatórias em cirurgia refrativa, seria interessante um vetor que : 2 :

5 REVIEW Junio 2006 promovesse uma expressão estável (com integração de material genético). Por outro lado, processos agudos como nas erosões recorrentes ou alterações pós-operatórias em cirurgia refrativa, seria interessante um vetor que promovesse expressividade inicialmente elevada e que diminuísse com o tempo (expressividade transitória). Basicamente os sistemas de deliberação de genes podem ser divididos em duas categorias. Existem os métodos que utilizam vírus como vetores e métodos mediados por vetores não virais 2-4. Vetores virais Os sistemas de vetores virais tem sido desenvolvidos utilizando-se técnicas de recombinação de DNA que permitem a produção de elevados títulos de vírus sem condições de replicação. Os genes virais são expressos por elementos genéticos separados do gene a ser transferido, garantindo assim um aceitável nível de segurança. Os primeiros vetores virais utilizados em terapia genética foram os retrovírus, os quais infectam apenas células com capacidade de divisão. O gene desejado é inserido no genoma da célula alvo e é expresso estavelmente pela célula infectada e por todas as suas células filhas. Os vetores retrovirais não têm habilidade para infectar células sem capacidade de divisão. A sua segurança é questionável pelo fato de muitos dos vetores estarem intimamente relacionados a patógenos e também pelo fato de promoverem uma integração de material genético de forma randomizada, o que aumenta o risco transformação celular maligna 5,6. A mais nova geração de vetores lentivirus, os quais são intimamente relacionados aos retrovírus, podem infectar tanto células divisoras quanto células não divisoras. Eles também promovem uma transferência genética estável pelo fato de se integrarem ao genoma hospedeiro. E da mesma maneira que os retrovirus impõem riscos de transformação maligna. Atualmente constitutem a tecnologia viral líder 7,8. Os virus adenovirus-associado também infectam células divisoras e não divisoras, porém com o benefício adicional de se integrarem ao genoma hospedeiro (expressão estável) em locais específicos. Diferentemente dos adenovirus, eles apresentam bem menos imunogenicidade. Porém, devido ao tamanho do genoma destes vírus o tamanho do gene terapêutico usado nestes sistemas é bem limitado 9. Já os vetores adenovirais podem infectar tanto células divisoras quanto não divisoras. Eles apresentam uma expressão transitória, uma vez que os genes transferidos não são integrados no genoma hospedeiro 10. Além do efeito transitório, os sistemas adenovirais também são limitados pela elevada antigenicidade, o que resulta em uma resposta imunogênica potente e de alta citotoxicidade. Vetores não-virais Os sistemas de vetores não virais utilizam uma variedade de tecnologias para transferir o DNA para as células alvo. A principal vantagem sobre os sistemas virais diz respeito à segurança. Nenhum destes mecanismos é capaz de promover replicação do material genético ou de integrá-lo de maneira eficiente no genoma da célula alvo. Assim sendo, estes sistemas promovem apenas uma expressão gênica transitória e bem menos eficiente do que os sistemas virais 11. A forma mais simples de transferência genética sem utilização de vetores virais é com a aplicação de "naked DNA". Este DNA é incorporado pelas células e é expresso de forma transitória. Acredita-se que isto ocorra tanto por um processo de difusão como também mediado por receptores celulares. Apesar de seu efeito transitório e da limitada eficiência em transferir material genético, esta metodologia proporciona o mais alto nível de segurança em transferência genética dentre todos os outros métodos 12. Algumas substâncias podem facilitar a entrada das moléculas de DNA nas células. Isto garante maior eficiência na transferência de genes do que simplesmente a aplicação de "naked DNA". Essas substâncias, ainda em investigação, incluem os lipídeos catiônicos e alguns receptores da superfície celular. Cada um desses agentes parece ter sua eficácia ligada a específicos tipos celulares assim como também à sua toxicidade 11. Além disto, métodos físicos também tem sido desenvolvidos e aplicados com o intuito de facilitar a entrada de DNA nas células alvo. A estimulação por pulsos elétricos intensos e curtos pode modificar a permeabilidade da membrana celular de forma reversível e assim permitir maior entrada de moléculas extra-celulares 13,14. Outro método que está sendo utilizado é a transferência balística 15,16. Com este método, o DNA é misturado com micropartículas de ouro e é transferido para o tecido alvo através de pistolas específicas. Vários investigadores conseguiram inserir DNA no epitélio corneano de coelhos e ratos usando esta tecnologia 17,18. Esses dois últimos métodos requerem equipamentos muito caros e podem causar danos significativos na membrana das células. Aplicações Potenciais Sobrevida do enxerto corneano Os transplantes de córnea são realizados na sua grande maioria sem a pesquisa de compatibilidade de HLA devido ao relativo privilégio imunológico do olho. Apesar disto, a rejeição imunológica continua sendo a principal causa de falência nos transplantes corneanos 19. Sabemos que a rejeição corneana envolve a ativação de resposta imune mediada por células T. Sabemos também do importante papel das citoquinas secretadas por células T e por macófagos nesta resposta imunológica. Assim sendo, a inibição de citoquinas pró-inflamatórias (IL-2, INF-, TNF- ) ou aumento na expressão de citoquinas do subtipo Th2 (IL-4 ou IL-10) passam a ser alvos atrativos em terapia genética para prevenção da rejeição corneana. Rayner et al., usaram adenovírus sem capacidade de replicação para transferir genes codificados para expressar receptores solúveis de TNF em córneas de coelhos cultivadas ex vivo antes do transplante 20. Apenas moderado aumento na sobrevida dos enxertos foi observado neste estudo. Acredita-se que a resposta imunogênica elicitada pelo adenovírus tenha sido responsável por diminuir os efeitos da terapia com anti-tnf-. Como mencionado acima, a IL-10 é um outro alvo na terapia genética contra rejeição corneana. Esta citoquina diminui a expressão de MHC II e de moléculas co- : 3 :

6 REVIEW Junio 2006 estimulatórias nos monócitos, macrófagos, células dendríticas e também inibe a síntese de citoquinas pró-inflamatórias. Em outro estudo, córneas de ovinos foram infectadas com adenovírus codificados para expressar IL-10. Essas córneas demonstraram um aumento significativo no tempo de sobrevida dos enxertos. RNAm de IL-10 pode ser detectado até 21 dias após a deliberação dos genes 21. Modulação da cicatrização corneana A transparência corneana é funda-mental para uma boa visão. Situções como infecções, traumas físicos ou químicos e até mesmo pós-operatório de cirurgias refrativas podem causar opacidades cor-neanas e consequentemente diminuir a acuidade visual dos pacientes. A deposição excessiva de matriz extracelular, a angiogênese e a inflamação são os principais responsáveis pela opacidade corneana. Acredita-se que o papel central na cicatrização corneana, especialmente nos eventos relacionados com a geração de miofibroblastos e deposição de matriz extracelular, esteja ligado ao fator de crescimento tumoral (TGF- ) 22,23. Estes mesmos estudos sugerem que a redução dos níveis de TGF- para as células estromais pode reduzir a formação de miofibroblastos e haze. Sakamoto et al., injetaram adenovírus recombinantes e codificados para expressarem receptores solúveis de TGF-, via intra-muscular, em camundongos, antes de provocar injúria corneana nos mesmos com nitrato de prata 24. O edema corneano, neovascularização, inflamação e deposi-ção de matriz extracelular nestes animais foi marcadamente reduzido quando compa-rado com o grupo controle que recebeu apenas adenovírus expressando LacZ. O papel das metaloproteinases na modulação da cicatrização corneana também está sendo muito estudado como uma outra opção de abordagem em terapia genética para modular as opacidades corneanas cicatriciais. O controle da cicatrização constitui um alvo bastante especial, uma vez que a modulação da cicatrização corneana pode ser necessária por um período curto de tempo, como dias ou semanas. Distrofias corneanas Infelizmente não dispomos de modelos animais apropriados para estudarmos as distrofias corneanas, o que limita o desenvolvimento de novas estratégias de tratamento para estas desordens. Entretanto, novos conceitos sobre a fisiopatologia das distrofias corneanas têm surgido com os avanços alcançados em biologia molecular nos últimos anos. Algumas mutações nos genes BIH3 e Gelsolin já foram identificadas em pacientes com distrofia corneana granular, lattice, avellino e Reis-Bucklers 25,26. Deste modo, a terapia genética pode ser uma arma de grande valia no desenvolvimento de modelos in vivo que simulem as mesmas condições médicas das distrofias corneanas. A partir deste ponto, poderemos investigar a fisiopatologia destas desordens genéticas e desenvolvermos novas abordagens terapêuticas para as mesmas. Conclusão Inúmeros estudos sobre transferência de genes identificam a córnea como uma promessa potencial para aplicação de terapia genética. Estes estudos demonstram que tanto os métodos virais como os não virais podem deliberar genes marcadores ou biologicamente ativos a todas as camadas da córnea. Quando comparados com os métodos não virais, os vetores virais são capazes de deliberar genes com maior eficiência e de expressá-los por maior período de tempo. Porém, estes métodos ainda impõem riscos à saúde dos pacientes. Os adenovírus conseguem transferir genes com muita eficiência, porém com expressividade muito curta. Além disto, são bastante imunogênicos. Os virus adenovirus-associado não são tão patogênicos e possuem baixa imunogenicidade. Entretanto, tem limitações relativas ao tamanho da carga genética que podem transferir. Os retrovírus são preferidos em relação aos outros vetores virais por sua relativa segurança e por sua elevada eficiência em transferir genes. Porém, sua incompetência em infectar células não divisoras limita bastante a sua utilização na córnea. Particularmente, quando estamos diante de patologias estromais e endoteliais. Já os lentivírus, estes mantém a mesma eficiência dos retrovírus em transferir genes, porém com habilidade de infectar células não divisoras. Por esta razão, estes vetores podem se tornar os métodos de escolha para deliberação de genes terapêuticos para os ceratócitos e para as células endoteliais. Mas muitas indagações ainda persistem sobre a segurança do seu uso, uma vez que promovem uma integração não específica no cromossoma das células alvo. Sobre os métodos não virais, estes definitivamente são mais seguros e não patogênicos. Porém, são os menos eficientes em transferir genes. Mesmo assim, ainda podem ser úteis em situações especiais onde se deseja uma expressão gênica transitória como nos processos de cicatrização corneana. Mesmo cientes de todas as ressalvas relativas às limitações dos vetores utilizados hoje em dia, muito progresso já pode ser visto na modificação genética do epitélio, endotélio e ceratócitos com a aplicação destes modelos. Apesar destes notáveis avanços, temos muito a aprimorar nestes sistemas existentes ou mesmo desenvolver novas técnicas de terapia genética antes que estas abordagens sejam colocadas em prática e aplicadas em ensaios clínicos humanos. R E F E R E N C E S : 1.Fabre JW. Prospects for genetic manipulation of donor organs and tissues. Transplantation 2001;71: Marshall E. Gene therapy's growing pains. Science. 1995;269:1050, Felgner PL, Barenholz Y, Behr JP, et al. Nomenclature for synthetic gene delivery systems. Hum Gene Ther. 1997;8: Marshall E. Gene therapy death prompts review of adenovirus vector. Science. 1999;286: Anderson WF. Human gene therapy. Nature. 1998;392: Mulligan RC. The basic science of gene therapy. Science. 1993;260: Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector [comment]. Science. 1996;272: Zufferey R, Nagy D, Mandel RJ, et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol. 1997;15: Kotin RM, Siniscalco M, Samulski RJ, et al. Site-specific integration by adenoassociated virus. Proc Natl Acad Sci USA. 1990;87: Yeh P, Perricaudet M. Advances in adenoviral vectors: from genetic engineering to their biology. FASEB J. 1997;11: Li S, Huang L. Nonviral gene therapy: promises and challenges. Gene Ther. 2000;7: Stechschulte SU, Joussen AM, von Recum HA, et al. Rapid ocular angiogenic control via naked DNA delivery to cornea. Invest Ophthalmol Vis Sci. 2001;42: Mathiesen I. Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther. 1999;6: Harrison RL, Byrne BJ, Tung L. Electroporation-mediated gene transfer in cardiac tissue. FEBS Lett. 1998;435: Johnston SA, Tang DC. Gene gun transfection of animal cells and genetic immunization. Methods Cell Biol. 1994;43: Johnston SA, Tang DC. The use of microparticle injection to introduce genes into animal cells in vitro and in vivo. Genet Eng (N Y). 1993;15: Wang, I.J., Carlson, E.C., Liu, C.Y., et al. Cis-regulatory elements of the mouse Krt1.12 gene. Mol. Vis. 2002;8: Zhang, E.P., Muller, A., Schulte, F., et al. Minimizing side effects of ballistic gene transfer into the murine corneal epithelium. Graefes Arch.Clin. Exp. Ophthalmol. 2002;240: Vail, A., Gore, S.M., Bradley, B.A., et al. Conclusions of the corneal transplant follow up study. Br. J. Ophthalmol. 1997;81: Rayner, S.A., Larkin, D.F., George, A.J. TNF receptor secretion after ex vivo adenoviral gene transfer to cornea and effect on in vivo graft survival. Invest Ophthalmol Vis Sci. 2001;42: Klebe, S., Sykes, P.J., Coster, D.J., et al. Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10. Transplantation. 2001;71: Girard, M.T., Matsubara, M., Fini, M.E. Transforming growth factor-beta and interleukin-1modulate metalloproteinase expression by corneal stromal cells. Invest Ophthalmol Vis Sci. 1991;32: Stramer, B.M., Zieske, J.D., Jung, J.C., et al., Molecular mechanisms controlling the fibrotic repair phenotype in cornea: implications for surgical outcomes. Invest Ophthalmol Vis Sci. 2003;44: Sakamoto, T., Ueno, H., Sonoda, K., et al. Blockade of TGF-beta by in vivo gene transfer of a soluble TGF-beta type II receptor in the muscle inhibits corneal opacification, edema and angiogenesis. Gene Ther. 2000;7: Akama, T.O., Nishida, K., Nakayama, J., et al. Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene. Nat Genet. 2000;26: Afshari, N.A., Mullally, J.E., Afshari, M.A., et al. Survey of patients with granular, lattice, avellino, and Reis-Bucklers corneal dystrophies for mutations in the BIGH3 and gelsolin genes. Arch Ophthalmol. 2001;119: : 4 :

7 Junio 2006 Higiene Palpebral en el Tratamiento de la Blefaritis David Díaz Valle MD, Facultativo Especialista de Area de Oftalmología. Unidad de Superficie e Inflamación Ocular del Hospital Clínico San Carlos (Madrid, España). José Manuel Benítez-Del-Castillo Sánchez, MD, Profesor Titular de Oftalmología (Catedrático habilitado) en la Universidad Complutense de Madrid (España). Jefe de Sección de la Unidad de Superficie e Inflamación Ocular del Hospital Clínico San Carlos (Madrid, España). INTRODUCCION La blefaritis es una enfermedad crónica muy frecuente de la superficie ocular que implica una gran variedad de procesos inflamatorios palpebrales y presenta un amplio espectro de manifestaciones clínicas. Debido a esta alta frecuencia y cronicidad es un motivo muy frecuente de consulta oftalmológica y es, a menudo, frustrante para el paciente y el oftalmólogo. En la actualidad, la blefaritis sigue siendo una enfermedad crónica sin curación. No obstante, con la instauración de un tratamiento adecuado e individualizado puede conseguirse el control y el alivio de los síntomas, así como la prevención de complicaciones asociadas a la blefaritis, como el desarrollo de queratopatías punteadas superficiales, orzuelos, chalación, infiltrados corneales marginales, neovascularización corneal y especialmente cuadros de ojo seco evaporativo por inestabilidad del film lagrimal precorneal. No debemos tampoco obviar la existencia de blefaritis en pacientes que van a ser intervenidos de cataratas, puesto que los gérmenes implicados en las endoftalmitis postoperatoria son los de la flora palpebral del propio paciente, y en estos casos debe instaurarse un tratamiento preoperatorio intensivo para mejorar las condiciones de la superficie palpebral y reducir la colonización bacteriana de la misma. Para realizar una correcta aproximación diagnóstica y terapéutica en esta patología, es esencial clasificar la blefaritis según la localización anatómica de la misma. Clasificación Se han propuesto varias clasificaciones de la blefaritis. Thygesson las dividió en estafilocócicas, seborreicas y mixtas, McCulley propuso una exhaustiva clasificación en 6 grupos clínicos, aunque la clasificación más empleada en la actualidad por su mayor utilidad práctica es la basada en la de Wilhelmus (tabla 1) que diferencia dos subtipos principales según la afectación del margen palpebral anterior que engloba la Tabla I. Clasificación y opciones de tratamiento Localización BLEFARITIS ANTERIOR BLEFARITIS POSTERIOR piel, folículos de las pestañas y músculo orbicular ó del margen palpebral posterior que contiene el tarso, conjuntiva tarsal, así como las glándulas meibomianas y accesorias. Un tercer subtipo menos frecuente en esta clasificación es la blefaritis angular, que afecta la piel y el margen palpebral únicamente en los cantos interno o externo y que se cree asociada a una infección subyacente por Moraxella sp. Clínica Hallazgos Escamas-costras en la base de las pestañas. Telangiectasias en el margen palpebral. Triquiasis. Madarosis. Dilatación y distorsión de los orificios glandulares. Secreciones espesas y turbias difícilmente exprimibles. Figura 1. Blefaritis anterior: Escamo-costras en la base de las pestañas e hiperemia del borde palpebral. Los síntomas de presentación en líneas generales consisten en escozor y picor. Otros síntomas referidos incluyen irritación, hinchazón palpebral y sensación de cuerpo extraño, a menudo más acentuados por las mañanas. Únicamente con los síntomas es difícil diferenciar entre los subtipos de blefaritis anterior ó posterior. Opción principal tratamiento Limpieza palpebral con preparados comerciales o champú infantil diluido. Compresas calientes + masaje. Otras opciones Antibióticos tópicos en casos severos. Lágrimas artificiales si ojo seco asociado. Esteroides tópicos si reacción inmune. Tetraciclinas ó Doxiciclina VO. Lágrimas artificiales Figura 2. Blefaritis posterior: Dilatación de los orificios glandulares y acúmulo de secreción turbia. La exploración biomicroscópica es clave para localizar el área de inflamación. Los signos de afectación del margen anterior son escamas y costras en la base de las pestañas, hiperemia y telangiectasias en margen palpebral, triquiasis y madarosis (fig. 1). La blefaritis posterior, por su parte, es secundaria a alteraciones en la producción y excreción de los lípidos que intervienen en la composición de la película lagrimal; y está, por tanto, asociada a una disfunción de las glándulas de meibomio (DGM). La secreción de estas glándulas debe ser clara y fácilmente exprimible con una ligera presión sobre el margen palpebral. La exploración biomicroscópica en la blefaritis del margen posterior revela unos orificios glandulares dilatados, rellenos de una secreción amarillenta y turbia, difícilmente exprimible (fig. 2). Pueden existir también : 5 :

8 Junio 2006 formas mixtas con hallazgos de blefaritis anterior y posterior asociados (fig. 3). Existen dos enfermedades dermatológicas que pueden asociarse a la blefaritis, como son el acné rosácea y la dermatitis seborreica y deben tenerse en cuenta para establecer un tratamiento integral del paciente, así como un pronóstico en cuanto a su evolución. El acné rosacea es una enfermedad dermatológica crónica asociada a la DGM en la que suele afectarse la cara y la parte superior del tronco. Aparece más frecuentemente en pacientes de años, sobre todo en mujeres. A nivel palpebral, cursa con episodios de exacerbaciones y remisiones y suele manifestarse como una blefaritis posterior, con abundantes telangiectasias palpebrales y engrosamiento y disfunción de las glándulas meibomianas. La dermatitis seborreica, por otra parte, se presenta con un cuadro mixto de blefaritis anterior y posterior, predominando el componente anterior, con escamas grasas y muy adheridas en la base de las pestañas. La inflamación alrededor de los párpados suele tener un aspecto grasoso. Puede existir un eritema con escamas y costras a nivel del cuero cabelludo, frente y cejas. Abordaje Terapéutico Se trata de una enfermedad frustrante para el paciente por la cronicidad de la misma y por la ausencia de un tratamiento curativo. Debe establecerse un algoritmo terapéutico específico y racional, así como conocer exactamente qué enfermedad vamos a tratar, evitando el término inconcluso de "blefaritis". En este sentido, la comunicación con el paciente es clave. El paciente debe comprender la naturaleza de la enfermedad e implicarse en el tratamiento. Debe explicarse que, si bien la curación no es posible, sí lo es el alivio de los síntomas. Asimismo, los pacientes con rosácea deben conocer que en ellos la enfermedad seguirá un curso natural más severo con frecuentes exacerbaciones y remisiones. Opciones de tratamiento En la tabla I se indican las opciones de tratamiento recomendadas para los tipos clínicos de blefaritis más frecuentes. a. Blefaritis anterior El tratamiento de elección es multifactorial, incluyendo higiene palpebral, antibióticos tópicos, lágrimas artificiales y en casos seleccionados esteroides tópicos cuando existe una reacción de hipersensibilidad a- sociada con infiltrados inflamatorios marginales corneales. El elemento básico, en cualquier caso, es la higiene palpebral. Higiene Palpebral Puede realizarse con los preparados comerciales disponibles, o bien empleando un champú infantil de ph neutro que debe diluirse en cuatro partes con agua templada. Para aplicarlo, puede empaparse una gasa en la solución y efectuar una limpieza vigorosa del margen palpebral superior e inferior con los párpados cerrados. También puede emplearse un bastoncillo de oidos empapado en la solución para realizar una expresión mecánica del margen palpebral inferior (fig. 4). En general, se recomienda evitar el uso del bastoncillo en pacientes de edad más avanzada y en pacientes con antecedentes de cirugía intraocular para evitar el riesgo de un excesivo o inadecuado frotamiento sobre la superficie ocular. Posteriormente deben lavarse los párpados con agua caliente para eliminar los restos del producto empleado. Esta operación debe repetirse dos ó tres veces al día. El objetivo del tratamiento es eliminar las escamas y costras irritantes de las pestañas así como eliminar o reducir la carga bacteriana de la superficie palpebral, y mejorar el flujo sanguíneo palpebral. La instilación de lágrimas artificiales sin conservantes inmediatamente después de la limpieza palpebral mejora el disconfort que produce el frotamiento mecánico de la superficie palpebral. Se han realizado estudios comparativos evaluando aspectos clínicos y biomicroscópicos analizando preparados comerciales para el tratamiento de la blefaritis frente al champú infantil diluido sin encontrar diferencias significativas en cuanto a los dos métodos de limpieza palpebral. Otros tratamientos coadyuvantes El empleo de antibióticos tópicos como eritromicina, bacitracina ó acido fusídico con actividad anti-estafilocócica puede ser eficaz durante los periodos de exacerbación y pueden incluso emplearse durante periodos prolongados. Pueden emplearse asimismo cursos cortos de esteroides tópicos en fases de mayor inflamación y cuando se asocian infiltrados marginales corneales que se atribuyen a reacciones de hipersensibilidad local frente a tóxinas estafilocócicas y reconocen, por tanto, un origen inmune. En cualquier caso, y al tratarse de un proceso crónico, los esteroides deben usarse con precaución, periodos de tiempo corto y usar esteroides de escasa penetración y potencia, tipo fluorometolona. Es también muy importante asociar lágrimas artificiales para el tratamiento del ojo seco secundario asociado, frecuentemente de tipo evaporativo, puesto que las formas de blefaritis posterior y mixtas presentan un film lagrimal inestable debido a una inadecuada cantidad y calidad de los lípidos presentes en la lágrima. b. Blefaritis posterior. En este subtipo de blefaritis, la opción terapéutica principal es la aplicación de compresas calientes. Compresas Calientes y Masaje Palpebral Los pacientes deben empapar un paño facial o una gasa en agua caliente y aplicarla sobre los párpados cerrados durante 5-10 minutos. Este tratamiento debe repetirse dos ó tres veces al día, dependiendo de la severidad de los síntomas. El paño no debe estar excesivamente caliente, lo suficiente para ser soportado en las manos sin quemarse y debe ser recalentado si se enfría. Las compresas calientes consiguen derretir las secreciones sebáceas y favorecen el drenaje glandular. Una vez aplicado el calor, debe realizarse un masaje palpebral dirigido a facilitar la expresión del contenido glandular licuado gracias al calor previamente administrado. Las propias compresas pueden emplearse para realizar esta expresión glandular mediante un masaje rotatorio o bien una expresión dirigida hacia el borde libre. Todo este proceso tiene una duración de 5-10 minutos y debe insistirse en establecer una rutina diaria de tratamiento. Es más efectiva realizarla por la mañana puesto que durante el sueño se acumulan abundantes detritus sobre la superficie glandular que de esta forma son eliminados. La expresión manual del contenido de las glándulas de meibomio mejora la composición y el grosor de la capa lipídica del film lagrimal y produce un evidente alivio sintomático. Otros tratamientos La administración de tetraciclinas por vía : 6 :

9 Junio 2006 sistémica ha mostrado un efecto favorable en pacientes con blefaritis posterior y acné rosácea. Esta acción beneficiosa de las tetraciclinas se cree relacionada con la inhibición de las lipasas bacterianas lo cual mejora la calidad de los lípidos producidos por las glándulas de meibomio. Además tiene propiedades anticolagenolíticas y antiinflamatorias que ayudan a eliminar las grasas solidificadas y la queratinización existente en la superficie glandular, favoreciendo el drenaje de las mismas. La doxiciclina tiene un efecto similar y tiene una dosificación más cómoda y es mejor tolerada. Las pacientes en tratamiento con doxiciclina ó tetraciclina no deben quedarse embarazadas, y deben ser avisados de efectos secundarios potenciales como la posibilidad de presentar molestias gastrointestinales, hipersensibilidad solar ó infecciones vaginales micóticas. La dosis habitual es de 200 mg/día de doxiciclina en dos dosis, durante 2 semanas y 100 mg/día de mantenimiento durante varios meses. El efecto favorable tarda aproximadamente un mes en observarse, y los pacientes no deben suspender el tratamiento si tras unos pocos días no observan una mejoría evidente. Deben tratarse los síntomas causados por la sequedad ocular asociada, con lágrimas artificiales e incluso oclusión de los puntos lagrimales en casos más severos. El tipo de lágrima se seleccionará según los severidad de los síntomas y la periodicidad necesaria para el alivio de los mismos, reservándose las lágrimas sin conservantes para ojos secos severos en pacientes que necesitan instilaciones muy frecuentes. Se está investigando si el tratamiento con ácidos grasos omega 6 pudiera ser efectivo en recomponer la composición seborreica normal. Por otra parte, deben considerarse otras anomalías palpebrales asociadas que pueden agravar el problema de la blefaritis, como un pobre reflejo palpebral, hiperlaxitud palpebral, lagoftalmos, queratopatía por exposición y floppy eyelid syndrome. Asimismo, en casos seleccionados y de evolución atípica ó unilaterales no debe olvidarse que pueden existir cuadros que enmascaran blefaritis y que pueden suponer enfermedades potencialmente letales como carcinomas de glándulas sebáceas, carcinomas epidermoides atípicos, carcinomas basocelulares ó incluso formas de lupus eritematoso discoide crónico. En la tabla II se recogen algunos consejos para un mejor tratamiento y seguimiento de los pacientes con blefaritis. Tabla II. Once consejos para un mejor tratamiento de la blefaritis. 1. Buscar signos de acné rosácea. Dilatación y distorsión de los orificios glandulares. Secreciones espesas y turbias difícilmente exprimibles. 2. Considerar tetraciclinas VO en pacientes con acné rosácea o DGM severa. 3. Especificar claramente el tratamiento. Evitar recomendaciones imprecisas como "higiene palpebral" ó "paños calientes". 4. La blefaritis puede asociarse ó producir un ojo seco. 5. Una buena comunicación y la educación del paciente son la clave del tratamiento. 6. Un folleto informativo puede ayudar en pacientes de nuevo diagnóstico. 7. La blefaritis angular es un subtipo poco frecuente. 8. Muchos pacientes presentan formas mixtas anteriores y posteriores. 9. La blefaritis no debe ser ignorada en presencia de otros problemas oculares. 10. Buscar otros problemas palpebrales asociados y tratarlos 11. No olvidar que una blefaritis crónica y refractaria puede enmascarar cuadros más graves Figura 3. Blefaritis Mixta: Hiperemia del margen palpebral y escamas en las pestañas. B I B L I O G R A F Í A En estas formas más severas es recomendable usar Doxiciclina 100 mg/12h durante 15 días y 100 mg/día durante meses. Los efectos secundarios potenciales del fármaco son molestias gastroinetstinales, fotosensibilidad solar e infecciones micóticas vaginales. No usar durante el embarazo. El uso de lágrimas artificiales suele ayudar. El paciente debe comprender la naturaleza de su problema y la ausencia de curación del mismo. Debe además conocer que es extremadamente raro perder visión debido a la blefaritis, así como que existen muchas opciones de tratamiento para mantenerlo confortable y libre de síntomas. En cualquier caso, la comunicación directa y explícita es la mejor. Esta entidad, causada por Moraxella sp, debe ser tratada con antibióticos tópicos e higiene palpebral. Deben asociarse en estos casos, además de otras medidas coadyuvantes, el calor local y la higiene de los párpados según las técnicas descritas con anterioridad. La blefaritis debe ser tratada convenientemente antes de realizar cualquier procedimiento quirúrgico intraocular para minimizar el riesgo de endoftalmitis postoperatorias. Pueden asociarse alteraciones palpebrales como triquiasis, entropión y ectropion. Si la blefaritis es unilateral, refractaria y asocia ulceraciones del borde libre puede realizarse una biopsia para descartar un carcinoma de glándulas sebáceas. Figura 4. Técnica de aplicación delalimpieza palpebral, que debe ser vigorosa con elfin de eliminar todas las costras adheridas y reducir la carga bacteriana de la superficie palpebral. Debe explicarse al paciente y asegurar que seha comprendido puesto que es el elemento principal del tratamiento. 1.Thygeson P. Etiology and treatment of blepharitis: a study in military personnel. Arch Ophthalmol 1946; 36: McCulley JP, Dougherty JM, Deneau DG. Classification of chronic blepharitis. Ophthalmology 1982; 89: Wilhelmus KR. Inflammatory disorders of the eyelid margin and eye lashes. Ophthalmol Clin North America 1992; 5: Driver PJ, Lemp MA. Meibomian gland disfunction. Survey of Ophthalmol 1996; 40: Cady RS, Harrison DA. Management of blepharitis: A step-by-step approach. Comp Ophthalmol Update 2001;2: Harrison DA, Lawlor D. Experiences in treating patients for blepharitis. Arch Ophthalmol 1998;116: Blepharitis and dry eye in the adult. Preferred Practice Pattern. American Academy of Ophthalmology Key JE. A comparative study of eyelid cleaning regimens in chronic blepharitis. CLAO J 1996; 22: Dougherty JM, McCulley JP, Silvany RE, Meyer DR. The role of tetracycline in chronic blepharitis. Inhibition of lipase production in staphylococci. Invest Ophthalmol Vis Sci 1991; 32: Harrison DA. Blepharitis: diagnosis and management tips for happy patients. Review of Ophthalmol 2000;7: : 7 :

10 Junio 2006 Current Role of Anti-VEGF Therapy in the Management of Choroidal Neovascularization J. Fernando Arévalo, MD FACS, Reinaldo A. García, MD, Juan G. Sánchez, MD From the Retina and Vitreous Service, Clinica Oftalmológica Centro Caracas, Caracas, Venezuela. The authors have no proprietary or financial interest in any products or techniques described in this article. Supported in part by the Fundacion Arevalo-Coutinho para la Investigacion en Oftalmología (FACO), Caracas, Venezuela. Correspondence: J. Fernando Arévalo, MD FACS, Clinica Oftalmologica Centro Caracas, Edif. Centro Caracas PH-1, Av. Panteon, San Bernardino. Caracas 1010, Venezuela. Phone: (58-212) Fax: (58-212) address: ABSTRACT The results of preclinical and finished clinical trials revealed the efficacy of antivascular endothelial growth factor (anti- VEGF) therapy as antiangiogenic and antipermeability agents. The safety of single-dose intravitreal injections of different dosages has been established. Macugen has been the first approved anti-vegf drug in the United States since December 2004 for the treatment of neovascular AMD. The results of the ongoing ranibizumab (Lucentis) trials that are currently running will need to compare to that observed with pegaptanib in similarly designed phase trials. In addition, the preliminary data from the bevacizumab SANA study and intravitreal bevacizumab (Avastin) are promising. We are looking forward to the results of the Phase III randomized trials. Probably, the combination of anti-vegf compounds with photodynamic therapy or intravitreal steroids may be the future management of choroidal neovascularization. This article will review the current role of anti-vegf therapy in the management of choroidal neovascularization. INTRODUCTION Exudative age related macular degeneration (AMD) is the most common cause of irreversible severe vision loss in the elderly. 1,2 There is currently no effective therapy for the management of exudative AMD or choroidal neovascularization of other etiologies in most patients. Currently, there are several drugs under investigation with clinical human studies for CNV treatment in AMD. Two of the most important types are the antivascular endothelial growth factor (anti- VEGF) and the angiostatic steroids. This article will discuss the role of intravitreal anti-vegf therapy in the management of choroidal neovascularization. Vascular endothelial growth factor (VEGF) Vascular endothelial growth factor (VEGF) was originally described in highly vascularized tumors where its expression is increased by hypoxia. 3-6 Vascular endothelial growth factor is a homodimeric glycosylated protein that exists in five isoforms of 121, 145, 165, 189, and 206 amino acids and VEGF form a family of related growth factors with structural homology to plateletderived growth factor (PDGF). 7 Vascular endothelial growth factor is an endothelian mitogen, 8 angiogenic protein 5,6,8 and potent vasopermeability factor 9,10 that mediates its effects through the endothelial cell-specific, high-affinity, cell surface transmembrane receptors forms-like tyrosine kinasa (Flt), and fetal liver kinase 1 (Flk-1). 8,11-13 Vascular endothelial growth factor possesses a signal sequence and is secreted from intact cells. 14,15 Is increased by hypoxia in retinal pigment epithelial cells, retinal endothelial cells, retinal pericytes, 13,16,17 and Müller cells. 18 Retinal endothelial cells possess numerous high affinity VEGF receptors, 11,13 however there are other substances with angiogenic activity 19 Anti-Vascular Endothelial Growth Factor (Anti-VEGF) There are two intraocular anti-vegf drugs presently under investigation and one of them has already been approved by the US Food and Drug Administration (FDA). One is the antibody fragment (Lucentis, Ranibizumab; Genentech Inc., San Francisco, CA), and the other is pegylated aptamer (Macugen, Pegaptanib; Eyetech Pharmaceuticals Inc., New York, NY). Ranibizumab is a recombinant antibody that consists of 2 parts: a nonbinding human sequence, which makes it less antigenic in primates, and a high affinity binding epitope derived from the mouse, which serves to bind the antigen. Its molecular weight of 48 kd makes it a much smaller molecule and has been shown to penetrate the internal limiting membrane and access the subretinal space in animal models when injected intravitreously. The anti-vegf pegylated aptamer pegaptanib is a polyethylene glycol (PEG) conjugated oligonucleotide that binds to the major soluble human VEGF isoform, VEGF165, with high specificity and affinity. Pegaptanib is an aptamer composed of 28 nucleotide bases, and was produced and screened for its ability to bind and inactivate VEGF in a manner similar to that of a high affinity antibody directs towards VEGF. Whereas, the recombinant rhufab anti-vegf antibodies (ranibizumab) that are currently available are only partially and are therefore potentially able to elicit a human anti-mouse immune response, aptamers are not immunogenic. Basic Principles of the Potential Action of Anti-VEGF Therapy Anti-VEGF therapy is based on two properties, one is its anti-angiogenic effect, which is well known, and the other is its anti-permeability effect. The scientific basic principles of the potential action of anti- VEGF therapy in choroidal neovascularization is given by the following studies: 1) Kvanta et al 20 reported that surgically removed subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. 2) Lutty et al 21 studied the distribution and relative levels of VEGF in the nondiabetic and preproliferative diabetic human retina and choroid. 3) Tolentino et al 22 showed on the primate retina that VEGF is sufficient to produce many of the vascular abnormalities common to diabetic retinopathy and other ischemic retinopathies. : 8 :

11 Junio ) Adamis et al 23 produced retinal ischemia with laser retinal vein occlusion in 16 eyes of eight animals (Macaca fascicularis). Zero of eight eyes receiving the neutralizing anti- VEGF antibodies developed iris neovascularization and five of eight control antibodytreated eyes developed iris neovascularization. The authors concluded that inhibition of VEGF is a new potential therapeutic strategy for the treatment of ocular neovascularization. Anti-VEGF Therapy Published Studies Ranibizumab, Preclinical studies The phase I and phase II studies sponsored by Genentech evaluated the safety and efficacy of intravitreal injections of an antigen-binding fragment of a recombinant humanized monoclonal antibody directed toward vascular endothelial growth factor (ranibizumab) in a monkey model of CNV. The study concluded that intravitreal ranibizumab injections prevented formation of clinically significant CNV in cynomolgus monkeys and decreased leakage of already formed CNV with no significant toxic effects. The clinical relevance of the study is that it provides the non-clinical proof of principle for ongoing clinical studies of intravitreally-injected ranibizumab in patients with neovascular age-related macular degeneration. 24 Anti-VEGF aptamer, preclinical studies. The cutaneous vascular permeability assay (Miles assay), the corneal angiogenesis assay, the retinopathy of prematurity study and the human tumor xenografs study showed that leakage, angiogenesis in the cornea, retinal neovascularization, and tumor growth can be inhibited with pegaptanib. 25 Then, intravitreal pharmacokinetics of pegaptanib in New Zealand white rabbits showed that the estimated terminal half life was 83 hours, similar to the 94 hour half life observed in rhesus monkeys. 26 At 4 weeks after administration of pegaptanib, drug levels in the vitreous humor remained well above the KD for VEGF suggesting that once monthly dosing in humans is appropriate. In contrast, plasma concentrations were significantly lower and ranged from to µg/ml from day 1 to day 21. Clinical studies and Pegaptanib Clinical Trial Phase 1 The clinical trial phase 1 was a multicenter, open label, dose-escalating study of a single intravitreous injection of pegaptanib in patients with subfoveal CNV secondary to AMD. Phase 1A clinical study showed that single intravitreal doses of the anti-vegf aptamer could be administered safely up to 3 mg/eye. No significant ocular or systemic side effects were noted. At 3 months after intravitreal administration of the pegaptanib, 80% of eyes showed stabilized or improved vision, with 26.7% showing an increase in three or more lines on the ETDRS chart. In phase 1B study, patients were treated with pegaptanib plus photodynamic therapy. Sixty percent of the patients showed an increase of three o more lines on the ETDRS chart, and only 40% of cases needed photodynamic retreatment. 25 Clinical Trial Phase II Phase II study results revealed that 87.5% of patients who received pegaptanib alone given every 6 weeks were well tolerted and stabilized or improved vision 3 months after treatment and that 25% of eyes demonstrated a 3 line or greater improvement in vision on the ETDRS charts. A 60% 3 lines gain at 3 months was noted in patients who received both the anti-vegf aptamer and photodynamic therapy (Fig. 1). 27 Clinical Trial Phase III Showed that pegaptanib sodium with continuous use of 0.3 mg was efficacious in the treatment of neovascular AMD after 1 year regardless of the angiographic subtype, baseline vision or lesion size. 28 The primary end point was the proportion of patients who had lost fewer than 15 letters of visual acuity at 54 weeks.the 1-mg and 3-mg dosages did not exhibit additional benefit. In the combined analysis, benefits for pegaptanib were seen as early as 6 weeks after initiation of therapy and continued through 54 weeks. At week 54, there was a 47% relative difference in mean VA loss between pegaptanib and sham (7.93 vs letters; p < 0.05), and maintained or gained vision (33% vs. 23%; p = 0.003). Severe vision loss occurred in only 10% of patients in the pegaptanib arm compared with 22% of the patients in the sham arm (p < 0.001). A smaller proportion of patients in the pegaptanib arm than in the sham arm had a VA of 20/200 or worse in the study eye at week 54 (38% vs. 56%; p < 0.001). Among the adverse events that occurred, endophthalmitis (in 1.3 percent of patients), traumatic injury to the lens (in 0.7 percent), and retinal detachment (in 0.6% percent) were the most serious. These events were associated with a severe loss of visual acuity in 0.1 percent of patients. VISION study (VEGF Inhibition Study in Ocular Neovascularization) Results of the second year of Macugen (pegaptanib) for the treatment of neovascular AMD showed that over two years, the mean number of treatments for all subjects re-randomized to therapy was 15.7 of a possible 17 injections (92%). Figure 1. (Arevalo et al). A. This patient with subfoveal CNV (A) was treated with both photodynamic therapy (PDT) and three monthly injections of the anti-vascular endothelial growth factor aptamer. The patient's vision improved by 5 Early Treatment of Diabetic Retinopathy Study lines (20/250 to 20/80) with markedly decreased exudation clinically several months after initiating therapy (B). Repeat PDT was not performed at 3 months per the decision of the investigator (Reprinted from Eyetech Study Group. Anti-vascular Endothelial Growth Factor for Subfoveal Choroidal Neovascularization Secondary to Agerelated Macular Degeneration. Phase II Study Results. Ophthalmology 2003;110: with permission from American Academy of Ophthalmology). : 9 :

12 Junio 2006 Patients treated with Macugen 0.3 mg for a second year showed a mean loss of 9.4 letters of vision, compared to a loss of 17.0 letters for sham. Fewer subjects who continued a second year of Macugen 0.3 mg lost 3 lines or more vision: 59% of the 0.3 mg arm (78/133) lost < 15 letters versus 45% (48/107) of subjects receiving sham (P< 0.05). When comparing subjects receiving one vs 2 years of Macugen, the second year of therapy afforded additional protection from vision loss. Sixty seven percent more occurrences of a 3 line loss of vision were noted in patients receiving 1 year of therapy compared to those receiving 2 years of therapy (35 occurrences in the 1-year group versus 21 occurrences in the 2 year group; P 0.05). They concluded that no serious adverse events occurred, and no events directly related to the study drug were identified. The risk of endophthalmitis, with attention to injection technique, was modifiable. There were no reports of endophthalmitis or traumatic cataract occurring within year two. 29,30 Clinical studies and Ranibizumab Clinical Trial Phase I Heier and Puliafito et al 31 reported positive preliminary data from a Phase Ib/II randomized, single-agent study with the investigational anti-vegf product, rhufab V2 (Lucentis, ranibizumab), for patients with the wet form of AMD. Sixty-four patients were enrolled. Patients were treated in one eye every four weeks for four doses (either 300 or 500 micrograms) of rhufab V2 (n=53) or were treated with standard of care (no rhufab V2) (n=11). Three different groups of subjects were enrolled in the study based on disease pattern and prior treatment: minimally classic, predominantly classic, and patients previously treated with photodynamic therapy. Of the 53 patients treated with rhufab V2, 50 patients (94 percent) had stable or improved vision, of which 14 patients (26%) improved 15 letters or more on the ETDRS chart, and 36 patients (68%) had stable vision at day 98. Stable vision is defined as losing or gaining less than 15 letters on the ETDRS chart. On average, patients treated with rhufab V2 gained 9.0 letters at day 98 compared to patients treated with standard of care who lost 4.9 letters. The most common side effects from treatment with rhufab V2 injection were mild transient, reversible inflammation. Heier et al.31 reported their findings at 6 months follow up. Its effect on visual acuity in this trial was similar in magnitude to that observed with pegaptanib in a similarity designed phase II trial. 27 Clinical Trial Phase I/II The FOCUS (RhuFab V2 Ocular Treatment Combining the Use of Visudyne to Evaluate Safety) trial is a Phase I/II randomized, single-masked study evaluating the safety, tolerability and efficacy of Lucentis in combination with PDT in 162 patients with predominantly classic subfoveal wet AMD. In this study, 162 patients with predominantly classic subfoveal wet AMD were randomized 2:1 to receive PDT followed by either 0.5 mg injections of Lucentis or sham injections for 23 months. All patients received initial treatment with PDT but were only retreated at the treating physician's discretion based on the Visudyne package insert. At 12 months, patients treated with Lucentis and PDT gained an average of five letters in visual acuity compared to study entry, while those in the control group lost an average of eight letters. 32 Overall, 90.5% of combination patients in the FOCUS trial lost less than 15 letters of vision, compared with 68% with PDT alone. Twenty-four percent (25/105) of patients treated with Lucentis in combination with PDT versus 5 percent (3/56) of patients treated with PDT alone improved vision by 15 letters or more compared to study entry. At 3 months, only 16% of combination patients required additional PDT compared with 80% in the PDT-alone arm. At month 12, there was an astounding reduction of a mean of only 0.3 additional PDT treatments required in the combination arm to 2.4 PDT treatments in the PDT-arm alone. 32 An analysis of the one-year data showed that there was an increased risk of uveitis in patients treated with Lucentis in combination with PDT compared to patients treated with PDT alone. An amendment to the study protocol was made and then, intravitreal therapy was held for 1 month after PDT was administered. Twelve patients developed severe uveitis when combination treatment occurred within the same month, and no uveitis occurred subsequent to protocol modification. After uveitis, endophthalmitis was the second most common ocular serious adverse event occurring in patients treated with Lucentis. Among non-ocular serious adverse events, the frequency of cerebral vascular events was higher in those treated with Lucentis, while the frequency of myocardial infarctions was higher in the PDT-alone arm. In both cases, the difference between groups was not statistically significant. Severe vision loss (defined as a loss of 30 letters or more) was uncommon (1/105) among those treated with Lucentis and PDT compared with 9 percent (5/56) for those treated with PDT alone. Clinical Trial Phase III MARINA study Minimally classic/occult trial of the Anti- VEGF antibody Ranibizumab (formerly, RhuFab) In the treatment of Neovascular AMD (MARINA) is a Phase III study of 716 patients in the United States with minimally classic or occult wet AMD who were randomized 2:1 to receive intravitreal Lucentis injections or a control regimen. Patients treated with Lucentis were further randomized to receive either a 0.3 mg or 0.5 mg dose of Lucentis once a month for two years. At 12 months, patients treated with Lucentis gained an average of seven letters in visual acuity compared to study entry, while those in the control group lost an average of 10.5 letters. 33,39 Additional key study findings include: 1) Ninety five percent (452/478) of Lucentis-treated patients lost fewer than 15 letters compared to baseline, the primary endpoint of the study, compared with 62 percent (148/238) for the sham injection control group. 2) Twenty five percent (59/238) of patients treated with 0.3 mg of Lucentis and 34 percent (81/240) treated with 0.5 mg of Lucentis improved vision by a gain of 15 letters or more compared to approximately 5 percent (11/238) of patients in the control group. 3) Nearly 40 percent (188/478) of Lucentis-treated patients achieved a visual acuity score of 20/40 or better at 12 months compared to 11 percent (26/238) in the control group. Serious ocular adverse events occurring more frequently in Lucentis-treated patients were uncommon (<1 percent) and included uveitis and endophthalmitis. There were no culture-positive cases of endophthalmitis in the MARINA study. There were 3 cases of culture-negative endophthalmitis, 3 cases : 10 :

13 Junio 2006 of uveitis, 2 retinal tears, 2 with vitreous hemorrhage, and 1 with lens damage. Cerebrovascular accidents occurred, with 1 in the sham group, 1 in the 0.3 mg group, and 3 in the 0.5 mg group. Still, there were no clear statistically significant systemic safety issues with ranibizumab and no observed increase in hypertension. Overall, patients treated with ranibizumab gained 7 letters and the sham group lost 10.5 letters and there seemed to be a trend of improved outcome with the higher-dose group. Based on these results, ranibizumab is expected to be evaluated by the US FDA for the treatment of choroidal neovascularization (CNV) secondary to AMD. Ongoing Phase III Studies Genentech and Novartis Pharma AG are conducting an additional Phase III study of Lucentis, ANCHOR (ANti-VEGF Antibody for the Treatment of Predominantly Classic CHORoidal Neovascularization in AMD). This is a randomized, multi-center, doublemasked, active treatment controlled study comparing two different doses of Lucentis to PDT in 423 patients. Genentech is conducting an additional Phase IIIb study, PIER (A Phase IIIb, Multicenter, Randomized, Double-Masked, Sham Injection-Controlled Study of the Efficacy and Safety of Ranibizumab in Subjects with Subfoveal Choroidal Neovascularization with or without Classic CNV Secondary to Age-Related Macular Degeneration), in 184 patients in the United States with wet AMD. In this trial, Lucentis is administered once per month for the first three doses followed thereafter by doses once every three months for two years. Genentech recently began enrollment in the HORIZON Phase III open-label extension study, which allows eligible patients who have completed participation in certain other Lucentis clinical studies to continue to receive the investigational drug. Systemic therapy and Bevacizumab SANA study (Systemic Avastin for Neovascular AMD) Another method for delivering anti- VEGF therapy to the eye in neovascular AMD patients would be to use systemic therapy rather than an intraocular injection. Currently, a full-length recombinant humanized monoclonal antibody directed against VEGF known as bevacizumab (Avastin, Genentech, Inc.) is approved by the FDA for the treatment of metastatic colorectal cancer. 34 The antiangiogenic properties of bevacizumab have been studied in cancer patients, but never in animal models or humans with CNV. However, fluoresceinconjugated bevacizumab was shown to leak from laser-induced CNV after systemic administration to a cynomolgus monkey. 23 This observation suggested that systemic bevacizumab could leak from CNV in AMD patients and competitively inhibit extracellular VEGF. At the time when Michels et al 35 initiated the open-label prospective clinical study SANA (Systemic Avastin for Neovascular AMD), they proposed that systemic bevacizumab could leak from CNV and bind extracellular VEGF, that inhibition of extracellular VEGF could improve VA outcomes based on the promising results from early-phase studies with intravitreal anti- VEGF drugs, and that bevacizumab could be used for this off-label indication in neovascular AMD patients destined to legally blind for whom no approved therapy existed. Michels et al 35 reported the first nine patients enrolled through 12 weeks of follow-up. One week after the initial bevacizumab infusion, statistically significant changes in VA letter scores and central retinal thickness measurements on OCT were observed in both, the treatment and fellow eye respectively, and these significant changes continued through week 12. By 1 week, both median and mean VA letter scores in the study eyes increased to 62 letters. At week 12, there were 3 eyes (33%) with at least 3 lines of VA improvement and 8 eyes (89%) with at least 1 line of improvement. Recently, Rosenfeld et al 36 reported 14 patients followed for at least 3 months. After this time the median and mean visual acuity letter scores in the study eyes increased by 9 letters and 12 letters, respectively. From 14 eyes, CNV was diagnosed in 12 fellow eyes and median and mean visual acuity increased by 27 letters and 17 letters in these 14 fellow eyes. The median and mean OCT central retinal thickness measurements in the study eyes decreased by 125 and 155 microns, and in the fellow eyes 60 and 81 microns respectively. After 2 or 4 drug infusions during the first month, systemic bevacizumab therapy resulted in durable treatment benefits in 8 of 11 patients through the 6 month visit. Only one of the 11 patients followed for at least 6 months required additional treatment prior to the 6 months visit. Rosenfeld et al believe that these results suggest that bevacizumab may have better durability than similar drugs given intravitreally. 36 Finally, since the first article was published, a total of 18 patients have been followed for at least 24 weeks, and the 24-week results confirm and improve upon the preliminary results observed at 12 weeks. Of the 18 patients, 9 initially received three treatments and 11 received only two treatments, and the majority of patients did not require another treatment through 24 weeks. 37 A major advantage of systemic therapy is the ability to deliver the drug at a therapeutic level directly to the target neovascular tissue in the choroid or retina, the elimination of complications related to intravitreal procedure as infectious endophthalmitis, retinal detachment, and lens injury, the ability to treat both eyes simultaneously in patients with bilateral neovascular disease; and the more predictable outcome in patients who have undergone a vitrectomy or who are aphakic, in which case the half-life of an intravitreally administered drug is probably diminished. 38 However, systemic administration of bevacizumab has been associated with hypertension, delayed wound healing, thromboembolic disease, and spontaneous intestinal perforation with patients with gastrointestinal malignancies. Therefore, the most significant disadvantage of systemic bevacizumab therapy is the possibility of life-threatening adverse events (estimated overall rate of up to 4.4%). Michels et al 35 believe that although the risk of thromboembolic events in elderly cancer patients has been documented, the risk of thromboembolic events in elderly AMD patients receiving intermittent bevacizumab therapy, compared with continual therapy every 2 weeks in cancer patients, is not known. Even if the risk in AMD patients is initially low, as additional treatments are given to our elderly population, the risk of thromboembolic events most likely will increase. The SANA Study group, excluded patients with a history of cerebrovascular accident, transient ischemic attack, myocardial infarction, angina, or any other thromboembolic disease. : 11 :

14 Junio 2006 Intravitreal use of Bevacizumab (Avastin) Positive presentations at retina meetings 39 and the 2 published case reports of VA improvement and decreased retinal thickness 40,41 have led an exponential increase of the use of intravitreal bevacizumab in the past few months. The main force driving intravitreal bevacizumab usage is the high percentage of patients who experience symptomatic relief from active subfoveal CNV and the anatomic changes visualized with OCT (Fig. 2). Because bevacizumab was not created to be given as an intraocular injection, there have been no published animal or human safety data on retinal toxicity. The intraocular safety profile of bevacizumab is unknown in contrast to that of pegaptanib or even ranibizumab. The longer half-life (up to 20 days) of bevacizumab is useful for systemic cancer but may cause safety concerns in the eye. Whether systemic absorption is more likely with a longer halflife drug is unknown. Although an intravitreal injection uses minute fractions of the drug and the systemic absorption is unlikely to be significant, measurement of systemic levels after intravitreal injection has not been done. The clearance of bevacizumab is 100-fold slower than ranibizumab, and the affinity of bevacizumab for VEGF is less than that of ranibizumab. Reasons to use bevacizumab include improved retinal morphology and visual acuity. Indeed, the anecdotal report showed dramatic improvement in OCT appearance and corresponding improvement in visual acuity. 40,41 Other reasons to use it include its low cost and wide availability, with no unexpected toxicity shown to date. Applications of its use include salvage therapy (after lack of efficacy of FDA-approved drugs), lesions that fall outside the indications for approved drugs, patient refusal to use other drugs, and lower cost in uninsured patients. R E F E R E N C E S : 1.Ghafour IM, Allan D, Foulds WS. Common causes of blindness and visual handicap in the west of Scotland. Br J Ophthalmol 1983;67: Hyman L. Epidemiology of eye disease in the elderly. Eye 1987;1: Senger DR, Perruzzi CA, Feder J, Dvorak HF. A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res 1986;46: Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983;219: Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 1992;359: Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992;359: Siemeister G, Schirner M, Reusch P, Barleon B, Marme D, Martiny-Baron G. An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGFstimulated receptor autophosphorylation and proliferation of human endothelial cells. Proc Natl Acad Sci U S A 1998;95: Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 1992;13: Berse B, Brown LF, Van de Water L, Dvorak HF, Senger DR. Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol Biol Cell 1992;3: Connolly DT, Olander JV, Heuvelman D, Nelson R, Monsell R, Siegel N, Haymore BL, Leimgruber R, Feder J. Human vascular permeability factor. Isolation from U937 cells. J Biol Chem 1989;264: Thieme H, Aiello LP, Takagi H, Ferrara N, King GL. Comparative analysis of vascular endothelial growth factor receptors on retinal and aortic vascular endothelial cells. Diabetes 1995;44: de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 1992;255: Simorre-Pinatel V, Guerrin M, Chollet P, Penary M, Clamens S, Malecaze F, Plouet J. Vasculotropin-VEGF stimulates retinal capillary endothelial cells through an autocrine pathway. Invest Ophthalmol Vis Sci 1994;35: Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol. 1991;5: Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem. 1992;267: Aiello LP, Northrup JM, Keyt BA, Takagi H, Iwamoto MA. Hypoxic regulation of vascular endothelial growth factor in retinal cells. Arch Ophthalmol 1995;113: Adamis AP, Shima DT, Yeo KT, Yeo TK, Brown LF, Berse B, D'Amore PA, Folkman J. Synthesis and secretion of vascular permeability factor/vascular endothelial growth factor by human retinal pigment epithelial cells. Biochem Biophys Res Commun 1993;193: Pierce EA, Avery RL, Foley ED, Aiello LP, Smith LE. Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization. Proc Natl Acad Sci U S A 1995;92: Aiello LP, Pierce EA, Foley ED, Takagi H, Chen H, Riddle L, Ferrara N, King GL, Smith LE. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci U S A 1995;92: Kvanta A, Algvere PV, Berglin L, Seregard S. Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. Invest Ophthalmol Vis Sci 1996;37: Lutty GA, McLeod DS, Merges C, Diggs A, Plouet J. Localization of vascular endothelial growth factor in human retina and choroid. Arch Ophthalmol 1996;114: Tolentino MJ, Miller JW, Gragoudas ES, Jakobiec FA, Flynn E, Chatzistefanou K, Ferrara N, Adamis AP. Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate. Ophthalmology 1996;103: Adamis AP, Shima DT, Tolentino MJ, Gragoudas ES, Ferrara N, Folkman J, D'Amore PA, Miller JW. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Arch Ophthalmol 1996;114: Figure 2. (Arevalo et al). A-B) Early and late fluorescein angiogram on a patient with agerelated macular degeneration and juxtafoveal choroidal neovascularization. C-F) Improvement of visual acuity and optical coherence tomography findings at baseline (C), at 1 week (D; from 20/400 to 20/60), 2 weeks (E; 20/40), and 6 weeks (F; 20/40) after intravitreal bevacizumab. 24.Krzystolik MG, Afshari MA, Adamis AP, Gaudreault J, Gragoudas ES, Michaud NA, Li W, Connolly E, O'Neill CA, Miller JW. Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment. Arch Ophthalmol 2002;120: Preclinical and phase 1A clinical evaluation of an anti-vegf pegylated aptamer (EYE001) for the treatment of exudative age-related macular degeneration. Retina 2002;22: Afshari MA, Krzystolik MG, Adamis AP, et al. Therapeutic and prophylactic effects of a recombinant human binding fragment of a monoclonal antibody directed to vascular endothelial growth factor (rhufab VEGF) in a monkey model of laser-induced choroidal neovascularization (ARVO abstract) Invest Ophthalmol Vis Sci 2001; 42:S Eyetech Study Group. Anti-vascular endothelial growth factor therapy for subfoveal choroidal neovascularization secondary to age-related macular degeneration: phase II study results. Ophthalmology 2003;110(5): Gragoudas ES, Adamis AP, Cunningham ET Jr, Feinsod M, Guyer DR; VEGF Inhibition Study in Ocular Neovascularization Clinical Trial Group. Pegaptanib for neovascular age-related macular degeneration. N Engl J Med 2004 ;351(27): D' Amico DJ. Results of the second year of Macugen for the treatment of Neovascular AMD (V.I.S.I.O.N). Program and abstracts of the American Society of Retina Specialists 23rd Annual Meeting; July 16-20, 2005; Montreal, Canada. 30.Mieler WF. The V.I.S.I.O.N study: safety of a second year of Macugen treatment for neovascualr AMD. Program and abstracts of the American Society of Retina Specialists 23rd Annual Meeting; July 16-20, 2005; Montreal, Canada. 31.Heier JS, Sy JR, McCluskey ER; rhufab V2 Study Group. RhuFab V2 in wet AMD- 6 months continued improvement following multiple intravitreal injections (abstract). Invest Ophthalmol Vis Sci 2003;44: Heier JS, FOCUS Study Group. Intravitreal ranibizumab with verteporfin photodynamic therapy for neovascular age-related macular degeneration: year one results. Program and abstracts of the American Society of Retina Specialists 23rd Annual Meeting; July 16-20, 2005; Montreal, Canada. 33.Miller J, Chung CY, Kim RY, MARINA Study Group. Randomized, controlled phase III study of ranibizumab (Lucentis) for minimally classic or occult neovascular agerelated macular degeneration. Program and abstracts of the American Society of Retina Specialists 23rd Annual Meeting; July 16-20, 2005; Montreal, Canada. 34.Adding a humanized antibody to vascular endothelial growth factor (bevacizumab, Avastin) to chemotherapy improves survival in metastatic colorectal cancer. Clin Colorectal Cancer 2003;8: Michels S, Rosenfeld PJ, Puliafito CA, Marcus EN, Venkatraman AS. Systemic bevacizumab (Avastin) therapy for neovascular age-related macular degeneration twelve-week results of an uncontrolled open-label clinical study. Ophthalmology 2005;112(6): Rosenfeld PJ. Systemic bevacizumab (Avastin) therapy for neovascular agerelated macular degeneration (SANA) study: 12 week outcomes. Program and abstracts of the American Society of Retina Specialists 23rd Annual Meeting; July 16-20, 2005; Montreal, Canada. 37.Rosenfeld PJ. Avastin for AMD. Program and abstracts of the American Academy of Ophthalmology 2005 Subspecialty day; Oct 14-15, 2005; Chicago, IL. 38.Andrew A. Moshfeghi. Durability of treatment benefits in the systemic Bevacizumab (Avastin) therapy for neovascualr age-related macular degeneration (SANA) study. Program and abstracts of the American Society of Retina Specialists 23rd Annual Meeting; July 16-20, 2005; Montreal, Canada. 39.Rosenfeld PJ. Systemic bevacizumab (Avastin) therapy for neovascular agerelated macular degeneration (SANA) study: 12 week outcomes. Program and abstracts of the American Society of Retina Specialists 23rd Annual Meeting; July 16-20, 2005; Montreal, Canada. 40.Rosenfeld PJ, Moshfeghi AA, Puliafito CA. Optical coherence tomography findings after an intravitreal injection of bevacizumab (Avastin) for neovascular age-related macular degeneration. Ophthalmic Surg Lasers Imaging 2005;36: Rosenfeld PJ, Fung AE, Puliafito CA. Optical coherence tomography findings after an intravitreal injection of bevacizumab (Avastin) for macular edema from central retinal vein occlusion. Ophthalmic Surg Lasers Imaging 2005;36: : 12 :

15 BASIC INVESTIGATION Junio 2006 Estandarización de una técnica de cultivo de epitelio, endotelio y fibroblastos corneales y evaluación de su crecimiento sobre mallas de colágeno tipo I. Cultivo de epitelio, endotelio y fibroblastos corneales Carlos H. Triana S. MD 1, 3 ; Carlos A. Chiriboga N. MD 1 ; Marta R. Fontanilla D. PhD 1, 2 * Bogotá. D.E. Colombia. 1 Instituto de Biología Molecular - Universidad EL Bosque 2 Departamento de Farmacia, Facultad de Ciencias - Universidad Nacional de Colombia 3 Postgrado de Oftalmología - Universidad EL Bosque *Autor para correspondecia: fontanillamartha@unbosque.edu.com carlotrianas@hotmail.com Transv 9ª Bis No TEL Ext. 291 Si existe interés financiero en el producto del presente trabajo. RESUMEN La córnea es el tejido responsable de la mayoría del poder refractivo del ojo, lo que la convierte en una estructura vital para el adecuado funcionamiento de la visión. Las patologías de la córnea son múltiples y de diferentes etiologías; debido a las características de la cicatrización de este tejido casi todas pueden dañarla, causando alteraciones dramáticas en la refracción del paciente. En estos casos el tratamiento indicado es el transplante de espesor total o parcial. La fuente de córnea son los donantes cadavéricos, lo cual genera riesgo de infección y rechazo. Nuestro grupo ha desarrollado sustitutos de tejido conectivo mucoso y dermis artificiales que en el momento se encuentran en valoración preclínica. En este trabajo nos propusimos aplicar la metodología estandarizada, para establecer cultivos primaros de células de la córnea que luego fueron subcultivados en soportes de colágeno I e incubados. Los resultados indican la obtención de cultivos primarios de células endoteliales, células epiteliales y fibroblastos corneales 5 días post-siembra. Igualmente, que la siembra de fibroblastos en los soportes de colágeno I condujo a su adhesión a las fibras y a la formación de material eosinófilo entre las células. Palabras Clave: Córnea, Cultivo celular, Ingeniería Tisular. ABSTRACT The cornea is responsible for the majority of the eye's refringence, this makes it a vital structure an adequate visual function. Diseases affecting the eye are various and have different causes; however the almost always cause damage to this tissue, which, due to it's scarring characteristics, can have dramatic consequences in the affected individual's refraction. The field of tissue engineering offers us now the possibility to obtain functional tissue substitutes in the laboratory, as has been shown by our group for skin, cartilage and oral mucosa; these substitutes are viable alternatives to grafts and transplants due to the fact that they are not associated with the risks and limitations of these procedures. The aim of this work was to standardize the culture conditions for the three types of corneal tissue and, to seed them in collagen scaffolds. We obtained pure, confluent cultures 5 days post-seeding. Cell morphology allowed primary identification of the cultures. Fibroblasts seeded in collagen scaffolds were seen adherent 14 days post-seeding with very little proliferation and cellular infiltration of the scaffold. Keywords: Cornea, Cell culture, Tissue engineering INTRODUCCIÓN Las patologías de cornea (infecciosas, auto inmunes, trauma, etc), producen deficiencia en la visión y disminución de la calidad de vida. Un porcentaje de estas patologías necesita tratamiento quirúrgico especializado cuando no responden al tratamiento medico establecido o cuando las secuelas son severas. Este tratamiento, genera una alta tasa de rechazo inmunológico 1,2, sumando la escasez de donantes y el costo. Por esto es de gran importancia desarrollar tejidos alternativos que disminuyan estas complicaciones. Nuestro grupo ha podido desarrollar in Vitro sustitutos de mucosa oral, piel y cartílago 3,4,5,6,7 basados en el cultivo de células aisladas de estos tejidos sobre soportes de colágeno. Considerando la similitud de nuestras técnicas con las empleadas para cultivar cornea 8,9,10,11,12 y teniendo en cuenta la importancia que tiene transferir y desarrollar la tecnología para la obtención de tejido corneal artificial 13,14,15,16,17 en este trabajo nos propusimos a estandarizar el aislamiento y cultivo tridimensional de los tres tipos celulares corneales. Materiales y Métodos Establecimiento de cultivos primarios de epitelio, queratocitos y endotelio corneal: En este estudio se utilizaron las córneas provenientes de 6 conejos machos raza Nueva Zelanda provenientes del bioterio central de la Universidad Nacional de Colombia, con un peso promedio de 1.5kg. Los animales fueron sacrificados con una sobredosis intravenosa de pentotalketamina (Eutanex), una vez entraron en paro cardiorrespiratorio se extrajeron las córneas con la mínima cantidad de tejido limbo-escleral. Las córneas fueron transportadas en Solución Salina con Buffer de Fosfatos (PBS, Ph 7.4) suplementada con Penicilina 100 UI/mL y Estreptomicina 100 g/ml. Bajo estereoscopio se retiró todo el tejido limbo-escleral remanente. Siguiendo el protocolo establecido 18, con algunas modificaciones, las córneas fueron suspendidas en una solución de Tripsina 0,25%- EDTA 0,025% (GIBCO BRL) por 10 minutos a 37 C. Luego bajo estereoscopio se identificó la cara endotelial de la córnea y se desprendió la membrana de Descemet unida al endotelio corneal. Tanto la membrana como el endotelio fueron disgregados mecánicamente con ayuda de la pipeta y las células obtenidas fueron sembradas en cajas de cultivo de 25cm 2 (Falcon) con medio DMEM suplementado con 10% de Suero Fetal Bovino (SFB) y vitaminas, Piruvato y Aminoácidos no esenciales al 1% (GIBCO). El tejido corneal remanente fue suspendido en una solución de Dispasa 0,5% (GIBCO) e incubado 1 hora a 37 C y luego toda la noche a 4 C. Una vez transcurrido este tiempo se separó el epitelio, bajo estereoscopio, mediante raspado suave. El tejido así obtenido se disgregó con pipeta y se sembró en cajas de cultivo de 25cm 2 (Falcon) con medio DMEM suplementado de la manera ya descrita. Finalmente, el tejido corneal se sometió a una tercera digestión, con una solución de colagenasa al 0,1% por 30 min. a 37 C. Posteriormente se pasó la suspensión celular a través de un tamiz de nylon (70 µm Falcon Cell Strainer, Becton Dickinson) para obtener fibroblastos disgregados. Los cultivos se mantuvieron a 37C con una atmósfera controlada de 5% de CO2 con cambios de medio cada tercer día. : 13 :

16 BASIC INVESTIGATION Junio 2006 Siembra de fibroblastos cornéales sobre soportes de colágeno tipo I: Las mallas de colágeno tipo I de rata se obtuvieron siguiendo la técnica descrita por Pérez y col 3 con algunas modificaciones. Las colas de rata se preservaron a -20 C hasta el día de su utilización. Luego se descongelaron con etanol al 70%, se despojaron de la piel, se retiraron los tendones y se cortaron en pedazos pequeños. Después estos se sumergieron en ácido acético 0.01M y se mantuvieron a 4 C por 14 días. Una vez transcurrido este tiempo se procedió a centrifugar a g por 15 minutos a temperatura ambiente, y a separar el sobrenadante. Éste fue tratado con NaOH 0.1 N en cantidad suficiente para ajustar el ph a 9, sometido a agitación fuerte y centrifugado a g durante 10 minutos y a temperatura ambiente. El precipitado se disolvió en la mínima cantidad de ácido acético 0.01 N y a dicha solución se le midió la concentración de colágeno por el método de Bradford. Una vez conocida la concentración del colágeno extraído se ajustó a mg/ml. Luego se homogenizó (homogenizador Ultraturrax T-25 Basic S1, IKA WORKS, INC.) y se vertió en cajas de cultivo de 60 mm (Becton, Dickinson Labware, NJ, USA) cubiertas con papel parafinado. Inmediatamente después fueron llevadas a -20 C, para luego ser liofilizadas por 48 horas. Para poder garantizar un ambiente estéril y adecuado para el cultivo, las mallas se esterilizaron con óxido de etileno y se dejaron airear por 24 horas. Los fibroblastos confluentes fueron removidos de la caja de cultivo utilizando una solución de Tripsina 0,25%-EDTA 0,025% por 15 minutos a 37 C y fueron recolectados lavando la caja con 5ml de medio DMEM suplementado. Luego fueron centrifugados a 1000 X g y el pellet celular obtenido se llevó a una concentración de 1 x104 células/ml. Previamente las mallas se colocaron en cajas de 6 pozos (Becton, Dickinson Labware, NJ, USA) con medio DMEM suplementado a 37 C con una atmósfera controlada de 5% de CO2 por 24 horas, pasado este tiempo se sembró 1ml de la suspensión de fibroblastos sobre las mallas y se incubaron bajo las condiciones descritas por 2 horas, antes de agregar 5 ml de medio al pozo. Las mallas así sembradas se incubaron a 37 C con una atmósfera controlada de 5% de CO2 por 21 Figura 1. días con cambios de medio cada tercer o cuarto día. Pasado este tiempo las mallas se fijaron con formaldehído al 10% en PBS y se procesaron en bloques de parafina para su posterior tinción con Hematoxilina / Eosina. Las láminas obtenidas se observaron al microscopio de luz (Nikon LABOPHOT, Japan) para evaluar proliferación celular e infiltración celular a la malla. Los campos microscópicos más significativos fueron fotografiados (Nikon FX-35). RESULTADOS Y DISCUSIÓN Aplicando la metodología descrita anteriormente obtuvimos cultivos de epitelio, endotelio y fibroblastos corneales, confluentes a los 5 días post-aislamiento. La morfología de las células aisladas coincide con la esperada para cada tipo celular (Figura 1). El endotelio corneal muestra células poliédricas, confluentes, con la característica apariencia en "empedrado" de los endotelios, se observan células en estrecha asociación, con un núcleo prominente. En cuanto al epitelio se observan células con morfología epiteloide, algunas de las células demuestran depósitos intracelulares. Los fibroblastos corneales demuestran una morfología ahusada, típica de los fibroblastos en cultivo. La morfología celular confirma su identidad y es coherente con lo reportado en la literatura 18,19. Al sembrar fibroblastos corneales sobre mallas de colágeno tipo I aislado de tendones de cola de rata por 21 días y al Figura 2. procesar las mallas para corte y tinción con Hematoxilina / eosina observamos cúmulos de células adheridos a la malla de colágeno, no se observaron células infiltradas en la malla. (Figura 2). R E F E R E N C E S : 1. Barraquer, I. (2004) Distrofias y Degeneraciones Corneales: Atlas y Texto. Ed Tela. 2. Krupin, T and Kolker, A.E. (2000)Atlas of Complications in Ophthalmic Surgery Elsevier Science Health Science div. 3. Pérez S, Doncel A, Roa C.A, Fontanilla M.R (2001). Estandarización de un método para la elaboración de un análogo de dermis humana. Revista Colombiana de Ciencias Químico- Farmacéuticas, 30: Bello S.A. and Fontanilla M. R (2003) "Evaluation of autologous artificial connective tissues as grafting materials on rabbit oral mucosa lesions". In Abstracts of the 6 th Annual Meeting Tissue Engineering Society International. Orlando, FL, USA,. 5. Balaguera H. (2004) Estandarización de un método de cultivo tridimensional decondrocitos aislados de cartílago hialino. Trabajo de grado - Posgrado de Cirugía Plástica y Reconstructiva - Universidad El Bosque. 6. Posada. M.M. (2004) Estandarización de un método de cultivo primario de Queratinocitos y su cocultivo con fibroblastos en mallas de colágeno tipo I. Trabajo de grado. Departamento de Farmacia, Facultad de Ciencias - Universidad Nacional de Colombia. 7. Abello. S, García. E, Bello. A, López. J.C y Fontanilla M. (2004)Utilización de mallas de colágeno tipo I sembradas con condrocitos autólogos para la reconstrucción de defectos osteocondrales (Estudio experimental en conejos)" en Abstracts 50º Congreso Sociedad Colombiana de Cirugía Ortopédica y Traumatológica. Cartagena, Colombia Mayo de Nishida K. (2003) Tissue Engineering of the Cornea. Cornea 22 (Suppl. 1):S28-S Pellegrini G, Traverso CE, Franzi AT, et al. (1997) Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet.; 349: Sangwan, Virender S. MS; Vemuganti, Geeta K. MD; Iftekhar, Ghazala MSc; Bansal, Aashish K. MS; Rao, Gullapalli N. MD (2003) Use of Autologous Cultured Limbal and Conjunctival Epithelium in a Patient with Severe Bilateral Ocular Surface Disease Induced by Acid Injury: A Case Report of Unique Application. Cornea 22(5): Schwab, Ivan R. M.D., F.A.C.S.; Reyes, Merle M.D., R.N.; Isseroff, R. Rivkah M.D. (2000) Successful Transplantation of Bioengineered Tissue Replacements in Patients with Ocular Surface Disease Cornea 19(4): Amano, Shiro MD; (2003) Transplantation of Cultured Human Corneal Endothelial Cells Volumen 22(7) Suplemento Ilse Claerhout, Hilde Beel, Dirk De Bacquer, and Philippe Kestelyn (2003) Factors Influencing the Decline in Endothelial Cell Density after Corneal Allograft Rejection IOVS; Rieck, Peter W. (2003) Increased Endothelial Survival of Organ-Cultured Corneas Stored in FGF-2- Supplemented Serum-Free Medium IOVS; Lang Roland, Peter I. Song, Franz J. Legat, Robert M. Lavker, Brad Harten,Henner Kalden,Eileen F. Grady, Nigel W. Bunnett, Cheryl A. Armstrong, and John C. Anse (2003) Human Corneal Epithelial Cells Express Functional PAR-1 and PAR-2l IOVS; 16. Ryan David G., Lavinia Taliana, Lijie Sun, Zhi-Gang Wei, Sandra K. Masur, and Robert M. Lavker (2003) Involvement of S100A4 in Stromal Fibroblasts of the Regenerating Cornea IOVS; Yiquin Du, Jing Chen, James L. Funderburgh, Xiuan Zhu, Lingsong Li (2003) Functional Reconstruction of Rabbit Corneal Epithelium by Human Limbal Cells Cultured on Amniotic Membrane Molecular Vision 9: Orwin, Elizabeth J. MS., Allison Hubel, Ph.D. (2000) In Vitro Culture Characteristics of Corneal Epithelial, Endothelial, and Keratocyte Cells in a Native Collagen Matrix Tissue Engineering V.6, No Lanza, R.P., Langer, R and Chick, W. Principles of Tissue Engineering. (1996) R.G Landes Company Austin, Texas, USA. : 14 :

17 Junio 2006 :: Instrucciones a los Autores Visión Pan-América es la publicación oficial de la Asociación Panamericana de Oftalmología. La publicación está particularmente interesada en recibir manuscritos que sean cortas revisiones de materias novedosas de interés para los oftalmólogos miembros de la Asociación. Además de las revisiones, la publicación está interesada en artículos acerca de nuevas técnicas quirúrgicas, nuevas terapias médicas, y casos de correlacion clinico-patológica. Información de Presentación: Los manuscritos deben enviarse electrónicamente al jefe de redacción, Mark J. Mannis, MD a mjmannis@ucdavis.edu o puede enviarse vía el correo a: Mark J. Mannis, MD, Department of Ophthalmology University of California, Davis 4860 Y Street, Suite 2400 Sacramento, CA U.S.A Si se envía el trabajo por correo, este debe ir tanto impreso (a máquina,etc.) y en forma electrónica (CD, etc.). Todas las presentaciones deben ser publicaciones originales que no se hayan publicado en otra parte. Las presentaciones pueden ser escritas en idioma español, inglés o portugués. Todos los trabajos deben tener un resumen en inglés y en español. Formato de Presentación: Los trabajos presentados no deben sobrepasar las 1500 palabras (seis páginas escritas a doble espacio) más las referencias. Las referencias deben ser incluidas como una lista en una página separada al final del manuscrito con referencias citadas codificadas al texto en el orden de aparición. El siguiente formato debe usarse para las referencias: Jones JS, García TL, Perrero M. Retinopatía Diabética en Bolivia. Córnea, 1996; 26 (2): Smith DJ, Caldera MC, Chang N, Ferrer RJ. Managing Trauma Ocular. Hofstra y Publicadores de Kennimore, Londres, Se aceptan figuras de color y deben enviarse en PICT, TIFF o formato de JPEG. El formato de Powerpoint no es aceptable. La página del título debe incluir lo siguiente: (1) el nombre completo de cada autor (es decir, nombre, apellido y la inicial media si usa,) y el grado académico más alto; (2) la ciudad, estado, y país en el que el trabajo se llevó a cabo; (3) el nombre y dirección del autor para recibir pedidos de separata; (4) declaración de los autores si existe o no interés financiero en un producto citado o utilizado en el trabajo. :: Instructions to Authors Vision Pan-América is the official publication of the Pan-American Association of Ophthalmology. The publication is particularly interested in receiving manuscripts that are short state of-the-art review papers that will be of interest to the practicing PAAO member ophthalmologist. In addition to review articles, the publication is interested in articles on new surgical techniques, medical therapies, and case reports that emphasize clinicopathologic correlations. Submission information: Manuscripts should be submitted electronically to the Editor-in- Chief, Mark J. Mannis, MD at mjmannis@ucdavis.edu or can be sent via mail to: Mark J. Mannis, MD, Department of Ophthalmology University of California, Davis 4860 Y Street, Suite 2400 Sacramento, CA U.S.A. All submissions must be provided in electronic form as well as written manuscript form if mailed. All submissions must be original publications that have not been published elsewhere. Submissions can be in Spanish, English, Portuguese or French. All papers should be preceded by an abstract in either English or Spanish. Submission Format: Papers submitted should be no longer than 1500 words (six double spaced type-written pages) plus references. References should be included as a list on a separate page at the end of the manuscript with cited references keyed to the text in the order of appearance. The following format should be used for referenced papers: Jones JS, Garcia TL, Perrero M. Diabetic retinopathy in Bolivia. Cornea, 1996; 26 (2): Smith DJ, Caldera MC, Chang N, Ferrer RJ. Managing Ocular Trauma. Hofstra and Kennimore Publishers, London, Color figures are encouraged and should be submitted in PICT, TIFF or JPEG format. Powerpoint format is not acceptable. The title page should include the following: (1) each author's full name (i.e., first name, middle initial if used, and last name) and highest degree; (2) city, state, and country in which work was carried out; (3) name and address of author to receive reprint requests; (4) statement about the authors' proprietary or financial interest in a product or lack thereof. : 15 :

18 Junio 2006 The Pan-American Ophthalmological Foundation Is Now Accepting Applications For 2007 : 16 : REQUEST FOR INFORMATION (Please print clearly) Last Name(s) Suffix(es)(Sr, Jr, etc) Degree(s)(MD, PhD) First Name(s) Mailing Address Mailing Address (continued) Middle Name(s) City State ZIP Country Telephone I would like to receive information and the application for: Canqui Boyd Scholarship Award David & Julianna Pyott Pan-American Retinal Research Fellowship Gillingham Pan-American Fellowship Award Paul Kayser International Scholar Award Sean B. Murphy Ophthalmic Pathology Scholarship Tim & Judith Sear Scholarship Tyson Travel Scholarship to attend the ARVO meeting Online application forms are available at: Important notes: All applicants must be or become PAAO members. See online application form at: All applicants are required to submit all required documentation at the time of submission of your application form. No applications are carried over from one year to the next. APPLICATION RECEIPT DEADLINE: September 1, 2006 Pan-American Ophthalmological Foundation 1301 South Bowen Road, Suite 365 Arlington, Texas Tel.: (817) / Fax: (817) becas@paao.org Web site: FAX Canqui Boyd Scholarship Award One (1) scholarship in the amount of $4,000 is offered in 2007 to Pan- American members. Candidates must have already been accepted into a training program in Latin America. Funding provided by Canqui Boyd Fund and the PAOF. The applicant should be a general ophthalmologist from Latin America, no more than 2 to 3 years out of his/her residency program. David & Julianna Pyott Pan-American Retinal Research Fellowship This is an opportunity for one (1) fully-qualified ophthalmologist from a Latin American country outside Brazil and Mexico, to spend 2 years at the University of California, Irvine, Department of Ophthalmology as an international retina fellow participating in research and clinical work under the supervision of Dr. Baruch Kuppermann, Professor of Ophthalmology. Applicants should have completed a residency in Ophthalmology, speak fluent conversational English, and be interested in a career in Retina. The value of the Fellowship is U.S. $20,000 per annum. Gillingham Pan-American Fellowship Award Two (2) six-month fellowships in the amount of $10,000 each are offered to qualified Latin American candidates in a variety of sub-specialty interests at various institutions in the United States and Canada. Funding provided by Retina Research Foundation. The applicant should be a general ophthalmologist, no more than 2 to 3 years out of his/her residency program, and from Latin America. The applicant must have been already accepted into an accredited fellowship program in the United States or Canada. The PAAO will not place applicants in institutions. Please include a copy of the acceptance letter with the application form. Paul Kayser International Scholar Program Six (6) two-week scholarships in the amount of $3,000 are offered to US and Canadian candidates for the purpose of introducing exceptional North American ophthalmologists to the best practice and research approaches in Latin America. Funding provided by Retina Research Foundation. The candidate must be a third year resident or a first year fellow. Sean B. Murphy Ophthalmic Pathology Fellowship Award One (1) one-year fellowship is offered in the amount of $10,000 (cdn) at McGill University in Montreal, Canada. The selected fellow will participate in various research projects, Grand Round at various hospitals, and have the opportunity to present at the annual ARVO meeting. Funding is provided by the PAOF and matching funding provided by McGill University. Tim & Judith Sear Scholarship One (1) scholarship in the amount of $4,000 is offered annually to Pan- American members. Candidates must be outstanding students who have presented papers or published during their training period. Candidates must have already been accepted into a training program. Funding provided by Tim & Judith Sear for the advancement of education in ophthalmology throughout the world. The applicant should be a general ophthalmologist from Latin America and no more than 2 to 3 years out of his/her residency program. Tyson ARVO/PAAO Travel Scholarship Up to five (5) travel scholarships are awarded to Latin American candidates yearly, in the amount of $2,500, to attend the ARVO (Association for Research in Vision and Ophthalmology) annual meeting. Funding provided by Retina Research Foundation. The ophthalmology department chairman or national ophthalmological society affiliated with the PAAO must recommend the candidate to the PAAO.

19

20 Preserva la visión alcanzando las menores presiones-objetivo en más pacientes Investigadores de diversos estudios, (AGIS, Shirakashi, Shields) han comprobado que alcanzar y mantener la PIO entre 14 y 15 mmhg reduce la progresión de pérdida del campo visual 1,2,3. Lumigan alcanza la PIO-objetivo de 14/15 mmhg en un mayor número de pacientes: vs. timolol 4 dorzolamida/ vs. vs. latanoprost 6 timolol 5 Porcentaje de Pacientes que alcanzaron la PIO-Objetivo 14 21% 9% 17% 2% 19% 9% Porcentaje de Pacientes que alcanzaron la PIO-Objetivo 15 31% 16% 24% 9% 29% 14% Lumigan (bimatoprost) Forma farmacéutica y presentación. esentación.frascos cuenta-gotas conteniendo 5 ml de solución oftalmológica estéril de bimatoprost a 0,03%. USO ADULTO.Composición. Cada ml contiene: 0,3 mg de bimatoprost. Vehículo: cloreto de sódio, fosfato de sódio hepta-hidratado, ácido cítrico mono-hidratado, ácido clorídrico y/o hidróxido de sódio, cloruro de benzalconio y agua purificada qsp. Indicaciones. LUMIGAN (bimatoprost) es indicado para la reducción de la presión intra-ocular elevada en pacientes con glaucona o hipertensión ocular.contraindicaciones. LUMIGAN (bimatoprost) está contraindicado en pacientes con hipersensibilidad al bimatoprost o cualquier otro componente de la fórmula del producto. Precauciones y Advertencias. Advertencias. Fueron relatados aumento gradual del crescimiento de las pestañas en el largo y espesura, y oscurecimiento de las pestañas (en 22% de los pacientes después 3 meses, y 36% después 6 meses de tratamiento), y, oscurecimiento de los párpados (en 1 a <3% de los pacientes después 3 meses y 3 a 10% de los pacientes después 6 meses de tratamiento). También fue relatado oscurecimiento del íris en 0,2% de los pacientes tratados durante 3 meses y en 1,1% de los pacientes tratados durante 6 meses. Algunas de esas alteraciones pueden ser permanentes. Pacientes que deben recibir el tratamiento de apenas uno de los ojos, deben ser informados a respecto de esas reacciones. Precauciones LUMIGAN (bimatoprost) no fue estudiado en pacientes con insuficiencia renal o hepática y por lo tanto debe ser utilizado con cautela en tales pacientes.las lentes de contacto deben ser retiradas antes de la instilación de LUMIGAN (bimatoprost) y pueden ser recolocadas 15 minutos después. Los pacientes deben ser advertidos de que el producto contiene cloruro de benzalconio, que es absorvido por las lentes hidrofílicas.si más que un medicamento de uso tópico ocular estuviera siendo utilizado, se debe respetar un intervalo de por lo menos 5 minutos entre las aplicaciones.no está previsto que LUMIGAN (bimatoprost) presente influencia sobre la capacidad del paciente conducir vehículos u operar máquinas, sin embargo, así como para cualquier colírio, puede ocurrir visión borrosa transitoria después de la instilación; en estos casos el paciente debe aguardar que la visión se normalice antes de conducir u operar máquinas. Interacciones medicamentosas.considerando que las concentraciones circulantes sistemicas de bimatoprost son extremadamente bajas después múltiplas instilaciones oculares (menos de 0,2 ng/ml), y, que hay varias vías encimáticas envueltas en la biotransformación de bimatoprost, no son previstas interacciones medicamentosas en humanos. No son conocidas incompatibilidades. Reacciones adversas. LUMIGAN (bimatoprost) es bien tolerado, pudiendo causar eventos adversos oculares leves a moderados y no graves.eventos adversos ocurriendo en 10-40% de los pacientes que recibieron doses únicas diarias, durante 3 meses, en orden decreciente de incidencia fueron: hiperenia conjuntival, crecimento de las pestañas y prurito ocular.eventos adversos ocurriendo en aproximadamente 3 a < 10% de los pacientes, en orden decreciente de incidencia, incluyeron: sequedad ocular, ardor ocular, sensación de cuerpo estraño en el ojo, dolor ocular y distúrbios de la visión.eventos adversos ocurriendo en 1 a <3% de los pacientes fueron: cefalea, eritema de los párpados, pigmentación de la piel periocular, irritación ocular, secreción ocular, astenopia, conjuntivitis alérgica, lagrimeo, y fotofobia.en menos de 1% de los pacientes fueron relatadas: inflamación intra-ocular, mencionada como iritis y pigmentación del íris, ceratitis puntiforme superficial, alteración de las pruebas de función hepática e infecciones (principalmente resfriados e infecciones de las vías respiratorias).con tratamientos de 6 meses de duración fueron observados, además de los eventos adversos relatados más arriba, en aproximadamente 1 a <3% de los pacientes, edema conjuntival, blefaritis y astenia. En tratamientos de asociación con betabloqueador, durante 6 meses, además de los eventos de más arriba, fueron observados en aproximadamente 1 a <3% de los pacientes, erosión de la córnea, y empeoramiento de la acuidad visual. En menos de 1% de los pacientes, blefarospasmo, depresión, retracción de los párpados, hemorragia retiniana y vértigo.la frecuencia y gravedad de los eventos adversos fueron relacionados a la dosis, y, en general, ocurrieron cuando la dosis recomendada no fue seguida.posología y Administración.Aplicar una gota en el ojo afectado, una vez al día, a la noche. La dosis no debe exceder a una dosis única diaria, pues fue demostrado que la administración más frecuente puede disminuir el efecto hipotensor sobre la hipertensión ocular.lumigan (bimatoprost) puede ser administrado concomitantemente con otros productos oftálmicos tópicos para reducir la hipertensión intra-ocular, respetándose el intervalo de por lo menos 5 minutos entre la administración de los medicamentos. VENTA BAJO PRESCRIPCIÓN MÉDICA. ESTE PRODUCTO ES UM MEDICAMENTO NUEVO AUNQUE LAS INVESTIGACIONES HAYAN INDICADO EFICACIA Y SEGURIDAD, CUANDO CORRECTAMENTE INDICADO, PUEDEN SURGIR REACCIONES ADVERSAS NO PREVISTAS, AÚN NO DESCRIPTAS O CONOCIDAS, EN CASO DE SOSPECHA DE REACCIÓN ADVERSA, EL MÉDICO RESPONSABLE DEBE SER NOTIFICADO. 1. The AGIS Investigators: The Advanced Glaucoma Intervetion Study - The Relationship Between Control of Intraocular Pressure and Visual Field Deterioration. Am. J. Ophthalmol, 130 (4): , Shirakashi, M. et al: Intraocular Pressure-Dependent Progression of Visual Field Loss in Advanced Primary Open-Angle Glaucoma: A 15-Year Follow-Up. Ophthalmologica, 207: 1-5, Mao, LK; Stewart, WC; Shields, MB: Correlation Between Intraocular Pressure Control and Progressive Glaucomatous Damage in Primary Open-Angle Glaucoma. Am. J. Ophthalmol, 111: 51-55, Higginbotham, EJ et al. One-Year Comparison of Bimatoprost with Timolol in Patients with Glaucoma or Ocular Hypertension. Presented at American Academy Ophthalmology, Nov 11-14, Gandolfi, S et al. Three-Month Comparison of Bimatoprost and Latanoprost in Patients with Glaucoma and Ocular Hypertension. Adv. Ther, 18 (3): , Coleman, AL et al: A 3-Month Comparison of Bimatoprost with Timolol/Dorzolamide in Patients with Glaucoma or Ocular Hypertension. Presented at American Acedemy of Ophthalmol, New Orleans, La, Mejor comodidad posológica: 1 vez al día. No requiere refrigeración. Presentación conteniendo 3 ml.