Terapia Genética nas Doenças Corneanas

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
Junio 2006
MENSAJE DEL PRESIDENTE
PRESIDENT’S MESSAGE
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,
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
demandas de salud del país en el que va a
Enrique Graue
ejercer. Por lo tanto, el disminuir estas difePresidente de la Asociación Panamericana
de Oftalmología.
rencias 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.
T
E
he training in the field of ophthalmology as a specialty has changad with
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.
PAN-AMERICA : 1 :
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
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 acordo 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
U
:2:
PAN-AMERICA
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
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 virais2-4.
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
maligna5,6.
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 líder7,8.
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. Po-
rém, devido ao tamanho do genoma destes
vírus o tamanho do gene terapêutico usado
nestes sistemas é bem limitado9.
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
hospedeiro10. 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 virais11.
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 métodos12.
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
toxicidade11.
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-celulares13,14.
Outro método que está sendo utilizado
é a transferência balística15,16. 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 tecnologia17,18. 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 corneanos19.
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 transplante20. 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 coPAN-AMERICA
:3:
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 genes21.
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 prata24. 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
:4:
PAN-AMERICA
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-Bucklers25,26. 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: 1207-1209.
2.Marshall E. Gene therapy's growing pains. Science. 1995;269:1050,1052-1055.
3.Felgner PL, Barenholz Y, Behr JP, et al. Nomenclature for synthetic gene delivery
systems. Hum Gene Ther. 1997;8:511-512.
4.Marshall E. Gene therapy death prompts review of adenovirus vector. Science.
1999;286:2244-2245.
5.Anderson WF. Human gene therapy. Nature. 1998;392:25-30.
6.Mulligan RC. The basic science of gene therapy. Science. 1993;260:926-932.
7.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;272:263-267.
8.Zufferey R, Nagy D, Mandel RJ, et al. Multiply attenuated lentiviral vector achieves
efficient gene delivery in vivo. Nat Biotechnol. 1997;15:871-875.
9.Kotin RM, Siniscalco M, Samulski RJ, et al. Site-specific integration by adenoassociated virus. Proc Natl Acad Sci USA. 1990;87:2211-2215.
10.Yeh P, Perricaudet M. Advances in adenoviral vectors: from genetic engineering to
their biology. FASEB J. 1997;11:615-623.
11.Li S, Huang L. Nonviral gene therapy: promises and challenges. Gene Ther.
2000;7:31-34.
12.Stechschulte SU, Joussen AM, von Recum HA, et al. Rapid ocular angiogenic control
via naked DNA delivery to cornea. Invest Ophthalmol Vis Sci. 2001;42:1975-1979.
13.Mathiesen I. Electropermeabilization of skeletal muscle enhances gene transfer in
vivo. Gene Ther. 1999;6:508-514.
14.Harrison RL, Byrne BJ, Tung L. Electroporation-mediated gene transfer in cardiac
tissue. FEBS Lett. 1998;435:1-5.
15.Johnston SA, Tang DC. Gene gun transfection of animal cells and genetic
immunization. Methods Cell Biol. 1994;43:353-365.
16.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;15:225-236.
17.Wang, I.J., Carlson, E.C., Liu, C.Y., et al. Cis-regulatory elements of the mouse Krt1.12
gene. Mol. Vis. 2002;8:94-101.
18.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;240:114-119.
19.Vail, A., Gore, S.M., Bradley, B.A., et al. Conclusions of the corneal transplant follow
up study. Br. J. Ophthalmol. 1997;81:631-636.
20.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;42:1568-1573.
21.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;71:1214-1220.
22.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;32:2441-2454.
23.Stramer, B.M., Zieske, J.D., Jung, J.C., et al., 2003. Molecular mechanisms
controlling the fibrotic repair phenotype in cornea: implications for surgical
outcomes. Invest Ophthalmol Vis Sci. 2003;44:4237-4246.
24.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;7:1915-1924.
25.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;26:237-241.
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;119:16-22.
Junio 2006
Higiene Palpebral en el Tratamiento de la Blefaritis
David Díaz Valle MD,
José Manuel Benítez-Del-Castillo Sánchez, MD,
Facultativo Especialista de Area de Oftalmología.
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
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).
Tabla I.
Clasificación y opciones de tratamiento
Localización
Hallazgos
Opción principal
tratamiento
Otras opciones
BLEFARITIS
ANTERIOR
Escamas-costras en la base de las
pestañas. Telangiectasias en el
margen palpebral.
Triquiasis. Madarosis.
Limpieza palpebral con
preparados comerciales o
champú infantil diluido.
Antibióticos tópicos en casos
severos.
Lágrimas artificiales si ojo seco
asociado.
Esteroides tópicos si reacción
inmune.
BLEFARITIS
POSTERIOR
Dilatación y distorsión de los
orificios glandulares. Secreciones
espesas y turbias difícilmente
exprimibles.
Compresas calientes
+ masaje.
Tetraciclinas ó Doxiciclina VO.
Lágrimas artificiales
Figura 1. Blefaritis anterior:
Escamo-costras en la base de las pestañas
e hiperemia del borde palpebral.
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
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.
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
PAN-AMERICA
:5:
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 30-50 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 se:6:
PAN-AMERICA
leccionados esteroides tópicos cuando
existe una reacción de hipersensibilidad asociada 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
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.
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.
3. Especificar claramente el tratamiento.
Evitar recomendaciones imprecisas como "higiene palpebral" ó "paños
calientes".
4. La blefaritis puede asociarse ó producir
un ojo seco.
El uso de lágrimas artificiales suele ayudar.
5. Una buena comunicación y la educación
del paciente son la clave del tratamiento.
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.
6. Un folleto informativo puede ayudar en
pacientes de nuevo diagnóstico.
En cualquier caso, la comunicación directa y explícita es la mejor.
7. La blefaritis angular es un subtipo poco
frecuente.
Esta entidad, causada por Moraxella sp, debe ser tratada con antibióticos
tópicos e higiene palpebral.
8. Muchos pacientes presentan formas
mixtas anteriores y posteriores.
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.
9. La blefaritis no debe ser ignorada en
presencia de otros problemas oculares.
La blefaritis debe ser tratada convenientemente antes de realizar cualquier
procedimiento quirúrgico intraocular para minimizar el riesgo de
endoftalmitis postoperatorias.
10. Buscar otros problemas palpebrales
asociados y tratarlos
Pueden asociarse alteraciones palpebrales como triquiasis, entropión
y ectropion.
11. No olvidar que una blefaritis crónica y
refractaria puede enmascarar cuadros
más graves
Si la blefaritis es unilateral, refractaria y asocia ulceraciones del borde libre
puede realizarse una biopsia para descartar un carcinoma de glándulas
sebáceas.
Figura 3. Blefaritis Mixta:
Hiperemia del margen palpebral y escamas en
las pestañas.
Figura 4.
Técnica de aplicación de la limpieza palpebral,
que debe ser vigorosa con el fin de eliminar
todas las costras adheridas y reducir la carga
bacteriana de la superficie palpebral. Debe
explicarse al paciente y asegurar que se ha
comprendido puesto que es el elemento principal
del tratamiento.
B I B L I O G R A F Í A
1.Thygeson P. Etiology and treatment of blepharitis: a study in military personnel. Arch Ophthalmol 1946; 36:445-447.
2.McCulley JP, Dougherty JM, Deneau DG. Classification of chronic blepharitis. Ophthalmology 1982; 89: 1173-1180.
3.Wilhelmus KR. Inflammatory disorders of the eyelid margin and eye lashes. Ophthalmol Clin North America 1992; 5: 187-194.
4.Driver PJ, Lemp MA. Meibomian gland disfunction. Survey of Ophthalmol 1996; 40: 343-367.
5.Cady RS, Harrison DA. Management of blepharitis: A step-by-step approach. Comp Ophthalmol Update 2001;2: 39-44.
6.Harrison DA, Lawlor D. Experiences in treating patients for blepharitis. Arch Ophthalmol 1998;116: 1133-1134.
7.Blepharitis and dry eye in the adult. Preferred Practice Pattern. American Academy of Ophthalmology 1991.
8.Key JE. A comparative study of eyelid cleaning regimens in chronic blepharitis. CLAO J 1996; 22: 209-212.
9.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: 2970-2975.
10.Harrison DA. Blepharitis: diagnosis and management tips for happy patients. Review of Ophthalmol 2000;7: 61-66.
PAN-AMERICA
:7:
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)
576-8687. Fax: (58-212) 576-8815. E-mail address: [email protected]
ABSTRACT
The results of preclinical and finished
clinical trials revealed the efficacy of antivascular endothelial growth factor (antiVEGF) 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 (antiVEGF) and the angiostatic steroids. This
article will discuss the role of intravitreal anti-VEGF therapy in the management of choroidal neovascularization.
:8:
PAN-AMERICA
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 protein5,6,8 and potent
vasopermeability factor9,10 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 activity19
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 antiVEGF therapy in choroidal neovascularization is given by the following studies:
1) Kvanta et al20 reported that surgically
removed subfoveal fibrovascular membranes in age-related macular degeneration
express vascular endothelial growth factor.
2) Lutty et al21 studied the distribution and
relative levels of VEGF in the nondiabetic
and preproliferative diabetic human retina
and choroid.
3) Tolentino et al22 showed on the primate
retina that VEGF is sufficient to produce
many of the vascular abnormalities common to diabetic retinopathy and other
ischemic retinopathies.
Junio 2006
4) Adamis et al23 produced retinal ischemia
with laser retinal vein occlusion in 16 eyes
of eight animals (Macaca fascicularis). Zero
of eight eyes receiving the neutralizing antiVEGF 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 0.092 to 0.005 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. 15.05 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;110:979-86 with permission from
American Academy of Ophthalmology).
PAN-AMERICA
:9:
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
: 10 :
PAN-AMERICA
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 AntiVEGF 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
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 antiVEGF therapy to the eye in neovascular
AMD patients would be to use systemic
therapy rather than an intraocular injection.
Currently, a full-length recombinant huma-
nized 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 al35 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 antiVEGF 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 al35 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
al36 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 al35 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.
PAN-AMERICA
: 11 :
Junio 2006
Intravitreal use of Bevacizumab
(Avastin)
Positive presentations at retina meetings39 and the 2 published case reports of
VA improvement and decreased retinal
thickness40,41 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.
: 12 :
PAN-AMERICA
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.
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;67:209-13.
2.Hyman L. Epidemiology of eye disease in the elderly. Eye 1987;1:330-41.
3.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;46:5629-32.
4.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;219:983-5.
5.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;359:845-8.
6.Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced
by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992;359:843-5.
7.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;95:4625-9.
8.Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of
the vascular endothelial growth factor family of proteins. Endocr Rev 1992;13:18-32.
9.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;3:211-20.
10.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;264:20017-24.
11.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;44:98-103.
12.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;255:989-91.
13.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;35:3393-400.
14.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;5:1806-14.
15.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;267:26031-7.
16.Aiello LP, Northrup JM, Keyt BA, Takagi H, Iwamoto MA. Hypoxic regulation of
vascular endothelial growth factor in retinal cells. Arch Ophthalmol 1995;113:1538-44.
17.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;193:631-8.
18.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;92:905-9.
19.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;92:10457-61.
20.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;37:1929-34.
21.Lutty GA, McLeod DS, Merges C, Diggs A, Plouet J. Localization of vascular
endothelial growth factor in human retina and choroid. Arch Ophthalmol
1996;114:971-7.
22.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;103:1820-8.
23.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;114:66-71.
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;120:338-46.
25.Preclinical and phase 1A clinical evaluation of an anti-VEGF pegylated aptamer
(EYE001) for the treatment of exudative age-related macular degeneration. Retina
2002;22:143-52.
26.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:S933.
27.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):979-86.
28.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):2805-16.
29.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 AMD6 months continued improvement following multiple intravitreal injections
(abstract). Invest Ophthalmol Vis Sci 2003;44:972.
32.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;8:85- 8.
35.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):1035-47.
36.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;36:331-335.
41.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;36:336-369.
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:
[email protected]
[email protected]
Transv 9ª Bis No 133-25
TEL 6331368 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.
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 inmunológico1,2, 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
cartílago3,4,5,6,7 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 cornea8,9,10,11,12 y teniendo en cuenta la importancia que tiene transferir y desarrollar la tecnología para la obtención de tejido corneal artificial13,14,15,16,17 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 las células obtenidas fueron sembradas en
provenientes de 6 conejos machos raza cajas de cultivo de 25cm2 (Falcon) con
Nueva Zelanda provenientes del bioterio medio DMEM suplementado con 10% de
central de la Universidad Nacional de Suero Fetal Bovino (SFB) y vitaminas,
Colombia, con un peso promedio de 1.5kg. Piruvato y Aminoácidos no esenciales al 1%
Los animales fueron sacrificados con una (GIBCO). El tejido corneal remanente fue
sobredosis intravenosa de pentotal- suspendido en una solución de Dispasa
ketamina (Eutanex), una vez entraron en 0,5% (GIBCO) e incubado 1 hora a 37°C y
paro cardiorrespiratorio se extrajeron las luego toda la noche a 4°C. Una vez transcórneas con la mínima cantidad de tejido currido este tiempo se separó el epitelio,
limbo-escleral. Las córneas fueron trans- bajo estereoscopio, mediante raspado suaportadas en Solución Salina con Buffer de ve. El tejido así obtenido se disgregó con
Fosfatos (PBS, Ph 7.4) suplementada con pipeta y se sembró en cajas de cultivo de
Penicilina 100 UI/mL y Estreptomicina 100 25cm2 (Falcon) con medio DMEM supleg/mL. Bajo estereoscopio se retiró todo el mentado de la manera ya descrita. Finaltejido limbo-escleral remanente. Siguiendo mente, el tejido corneal se sometió a una
el protocolo establecido18, con algunas mo- tercera digestión, con una solución de
dificaciones, las córneas fueron suspen- colagenasa al 0,1% por 30 min. a 37°C.
didas en una solución de Tripsina 0,25%- Posteriormente se pasó la suspensión
EDTA 0,025% (GIBCO BRL) por 10 minutos celular a través de un tamiz de nylon (70
a 37°C. Luego bajo estereoscopio se iden- µm Falcon Cell Strainer, Becton Dickinson)
tificó la cara endotelial de la córnea y se para obtener fibroblastos disgregados. Los
desprendió la membrana de Descemet cultivos se mantuvieron a 37C con una
unida al endotelio corneal. Tanto la mem- atmósfera controlada de 5% de CO2 con
brana como el endotelio fueron disgregados cambios de medio cada tercer día.
mecánicamente con ayuda de la pipeta y
Keywords: Cornea, Cell culture, Tissue engineering
PAN-AMERICA
: 13 :
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 col3 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 12000 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 12000 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 2 - 2.5 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
: 14 :
PAN-AMERICA
Figura 1.
Figura 2.
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 literatura18,19.
Al sembrar fibroblastos corneales sobre
mallas de colágeno tipo I aislado de tendones de cola de rata por 21 días y al
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 QuímicoFarmacéuticas, 30: 9-15.
4. 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 6th
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 2004.
8. Nishida K. (2003) Tissue Engineering of the Cornea. Cornea 22 (Suppl. 1):S28-S34.
9. Pellegrini G, Traverso CE, Franzi AT, et al. (1997) Long-term restoration of damaged
corneal surfaces with autologous cultivated corneal epithelium. Lancet.; 349:990-993.
10. 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):478-481.
11. 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): 421-426.
12. Amano, Shiro MD; (2003) Transplantation of Cultured Human Corneal Endothelial Cells
Volumen 22(7) Suplemento 1.
13. Ilse Claerhout, Hilde Beel, Dirk De Bacquer, and Philippe Kestelyn (2003) Factors Influencing the Decline in Endothelial Cell Density after Corneal Allograft Rejection IOVS; 44.
14. Rieck, Peter W. (2003) Increased Endothelial Survival of Organ-Cultured Corneas Stored
in FGF-2- Supplemented Serum-Free Medium IOVS; 44.
15. 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; 44
17. 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: 635-643
18. 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.4
19. Lanza, R.P., Langer, R and Chick, W. Principles of Tissue Engineering. (1996) R.G Landes
Company Austin, Texas, USA.
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 [email protected] 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 95817
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): 341 - 343.
Smith DJ, Caldera MC, Chang N, Ferrer RJ. Managing Trauma
Ocular. Hofstra y Publicadores de Kennimore, Londres, 1989.
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-inChief, Mark J. Mannis, MD at [email protected] 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 95817
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): 341- 343.
Smith DJ, Caldera MC, Chang N, Ferrer RJ. Managing Ocular
Trauma. Hofstra and Kennimore Publishers, London, 1989.
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.
PAN-AMERICA
: 15 :
Junio 2006
The Pan-American Ophthalmological Foundation
Is Now Accepting Applications For 2007
Canqui Boyd Scholarship Award
REQUEST FOR INFORMATION
(Please print clearly)
Last Name(s)
Suffix(es)(Sr, Jr, etc)
First Name(s)
Middle Name(s)
Degree(s)(MD, PhD)
David & Julianna Pyott Pan-American
Retinal Research Fellowship
Mailing Address
Mailing Address (continued)
City
State
Telephone
FAX
ZIP
Country
Email
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:
www.paao.org/doc/beca_CanquiBoyd_app.doc
www.paao.org/doc/beca_Pyott_app.doc
www.paao.org/doc/beca_Gillingham_app.doc
www.paao.org/doc/beca_Kayser_app.doc
www.paao.org/doc/beca_Sear_app.doc
www.paao.org/doc/2007_tysonARVO_app.doc
Important notes:
All applicants must be or become PAAO members. See online
application form at: www.paao.org/newmember.html
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 76013
Tel.: (817) 275-7553 / Fax: (817) 275-3961
Email: [email protected]
Web site: http://www.paao.org
: 16 :
PAN-AMERICA
One (1) scholarship in the amount of $4,000 is offered in 2007 to PanAmerican 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.
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 PanAmerican 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.
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 visual1,2,3.
Lumigan® alcanza la PIO-objetivo de 14/15 mmHg en un mayor número
de pacientes:
®
vs. timolol 4
®
vs.
dorzolamida/
timolol 5
®
vs. latanoprost 6
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 pr
esentación.
Composición. Cada ml contiene: 0,3 mg de bimatoprost. Vehículo: cloreto de sódio, fosfato de sódio
presentación.
esentación.Frascos cuenta-gotas conteniendo 5 ml de solución oftalmológica estéril de bimatoprost a 0,03%. USO ADULTO.Composición.
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
ecauciones y Adver
tencias. Advertencias. Fueron relatados aumento gradual del crescimiento
Contraindicaciones. LUMIGAN® (bimatoprost) está contraindicado en pacientes con hipersensibilidad al bimatoprost o cualquier otro componente de la fórmula del producto. Pr
Precauciones
Advertencias.
ocular.Contraindicaciones.
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
ecauciones 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
Precauciones
de apenas uno de los ojos, deben ser informados a respecto de esas reacciones. Pr
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.
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.
eacciones 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
No son conocidas incompatibilidades. RReacciones
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,
Posología y Administración.
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
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): 429-40, 2000. 2. 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, 1993. 3. 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, 1991. 4. 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, 2001. 5. Gandolfi, S et al. Three-Month Comparison of Bimatoprost
and Latanoprost in Patients with Glaucoma and Ocular Hypertension. Adv. Ther, 18 (3): 110-121, 2001. 6. 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, 2001.
Mejor comodidad posológica:
1 vez al día.
No requiere refrigeración.
Presentación conteniendo 3 ml.