Upending Wisdom on Angiogenesis Drugs
A new study raises questions about a number of therapies in development for the treatment of age-related macular degeneration. Much of the biomedical research of the last decade has centered on a class of double-stranded RNA molecules called small interfering RNA (siRNA). Today an array of experimental drugs is based on these molecules, including agents designed to treat AMD. The conventional wisdom is that siRNAs work through RNA interference (RNAi), a natural immune system mechanism that inhibits expression of a specific gene.
But a study in Nature1 challenges that belief by identifying a separate and nonspecific mechanism of action for siRNAs. Using mouse models of choroidal neovascularization, the researchers tested a wide range of siRNAs for their ability to suppress CNV. These included 21-nucleotide or longer siRNAs directed at nonmammalian genes, nonexpressed genes, nongenomic sequences, proangiogenesis genes, antiangiogenesis genes and RNAi-incompetent siRNAs.
The investigators discovered that the ability of these nontargeted siRNAs to suppress CNV in mice was comparable to that of the siRNAs designed specifically to target vascular endothelial growth factor-A (VEGFA) or its receptor (VEGFR1) in clinical trials for AMD. Moreover, CNV suppression was not dependent on RNAi in these experiments. Instead, both nontargeted and targeted siRNAs (VEGFA and VEGFR1) suppressed CNV through an entirely new mechanism, by activating a receptor called toll-like receptor 3 (TLR3).
TLR3 is a cell-surface receptor found in multiple types of endothelial cells, including those in the aorta, dermis, lung and umbilical vein. These findings suggest that siRNAs have a class effect that might be harnessed to treat a range of angiogenesis-based disorders. On the downside, the discovery also raises the possibility that siRNAs may have unanticipated—and potentially undesirable—vascular or immune system effects.
These findings have profound implications. According to Jayakrishna Ambati, MD, professor and vice chairman of ophthalmology and visual sciences at the University of Kentucky and senior author of the study, the use of siRNAs or RNA interference has pervaded virtually every area of biomedical research because of its promised specificity.
“An entire industry has emerged around using these molecules as drugs for any number of diseases,” Dr. Ambati said. “By my count, there are now at least five clinical trials under way using siRNAs in various conditions, three of which are for macular degeneration. They all are premised on the belief that siRNAs work by targeting one specific gene and nothing else.”
Finding a new mechanism of action for these compounds calls into question or forces the reevaluation of studies using siRNAs and has implications for the ongoing clinical trials in the eye and elsewhere, Dr. Ambati added. “Not only do the siRNAs behave like a generic class in activating this toll-like receptor, but when TLR3 is activated, they suppress blood vessel growth not only in the eye but in a variety of other tissues and organs as well.”
Additional ramifications of the research are that generic siRNAs may be just as effective as the targeted antiangiogenesis drugs for shutting down blood vessel growth. The researchers also found that 4 to 15 percent of individuals have a mutation in the TLR3 gene that makes them resistant to its antiangiogenesis effects and likely to be unresponsive to that class of drug.
Finally, the discovery of this new, generic mechanism of action for angiogenesis-suppressing drugs may have implications for the AMD agents currently under investigation.
“We found that these drugs also suppress the growth and cause the death of other cells in the eye through this TLR3-initiated mechanism. So if these drugs are approved, we will have to be on the lookout for potential adverse events, perhaps geographic atrophy or other retinal toxicity that could be caused by their generic action,” said Dr. Ambati.
1 Ambati, J. et al. Nature
2008;452:591–597. Published online Wednesday, March 26.
Getting at the Root of Glaucoma
One of the biggest questions in glaucoma is: What pathogenic molecular mechanisms account for the disease? Scientists may be one step closer to finding the answer after discovering that increased expression of secreted frizzled-related protein-1 (sFRP-1, an antagonist of Wnt signaling) in the trabecular meshwork may be responsible for elevated intraocular pressure in some patients with glaucoma.1 Taking this finding one step further, they speculate that restoring Wnt signaling in the trabecular meshwork may prove a viable intervention for treating the condition.
The cross-disciplinary research team included scientists from Alcon Labs, the National Cancer Institute and the University of Iowa. The Alcon investigators, led by Abbott Clark, PhD, vice president of research, used an experimental model of eyes from recently deceased individuals to analyze the effect of sFRP-1.
“Eyes perfused with sFRP-1 solution developed resistance to flow,” said Jeffrey S. Rubin, MD, PhD, a coauthor and senior investigator of cellular and molecular biology at the NCI. “Moreover, sFRP-1 treatment decreased cytosolic beta-catenin in the trabecular meshwork, making the case that the Wnt/beta-catenin signaling pathway was involved in regulating intraocular pressure.”
The researchers then did an intravitreal injection of an adenoviral vector encoding sFRP-1 in mice, which produced an increase in IOP. Five days later, this IOP was significantly reduced by a topical ocular administration of a small molecule that activated the beta-catenin pathway inside the cell, overcoming the block by sFRP-1.
“This result established that an excess of sFRP-1 increased IOP in animals, and this effect was reversed by an agent that increased beta-catenin levels,” Dr. Rubin noted.
“These findings could lead to a new treatment for glaucoma in patients whose disease is due to elevated expression of sFRP-1. However, before this can happen we have to find a way to identify these patients. This is a challenge because sampling the trabecular meshwork is not practical, and we do not now have other ways to predict which patients have abnormal sFRP-1 expression in this tissue. Nonetheless, recognizing the connection between sFRP-1, beta-catenin and glaucoma is a step in the right direction,” he said.
—Lori Baker Schena
1 Wang, W. et al. J Clin Invest
New Progression Index May Guide Therapy
Swedish researchers have reported a new perimetric tool—the glaucoma progression index (GPI) —that they say more accurately predicts the rate of glaucomatous progression than the traditional mean deviation (MD) index and could lead to more targeted care.1 The GPI was designed to meet the need for improved interpretation of series of visual field tests obtained in glaucoma practice.
“The intention with the trend analysis is to identify those having a rapid progression, and to treat these patients more aggressively, with the aim of breaking the rapid trend,” noted Boel Bengtsson, PhD, associate professor of ophthalmology at Lund University in Malmö.
“The new GPI represents a clear improvement as compared with its predecessor—MD,” she said, explaining that MD is more affected by cataract, which can falsely suggest higher rates of progression. GPI software, available from Carl Zeiss Meditec for the Humphrey Field Analyzer/ HFA II-i, is designed to account for several variables affecting treatment decisions: progression rate, current field status and age.
For ease of interpretation, the GPI of a perimetrically normal field is set to 100 percent; a perimetrically blind field is set to 0 percent. A reading of 40 percent, for example, indicates a 60 percent loss of visual field.
Using a GPI-generated graph that depicts a 70-year-old with a 75 percent full field status and annual progression of 3 percent, Dr. Bengtsson demonstrated how the perimetric index works. “By extrapolating the current trend, and assuming that this will not change unless we increase therapy, we can see that this eye will have lost almost 50 percent of its visual field at the age of approximately 78.” Assuming that a 50 percent loss will adversely affect the patient’s daily life, and that the patient will live beyond 78, one might consider increasing therapy, she said.
Dr. Bengtsson plans further validation of the GPI and hopes independent centers will conduct their own studies. The study design does not have to be prospective, she said. “This rate of progression analysis with GPI can very well be performed on retrospective data, preferably clinically collected to better reflect clinical practice.”
In the meantime, she expressed confidence with the instrument based on more than a year of experience with patients and in scientific studies. “We are sure that glaucoma patients are served better by releasing the new method of analysis now, rather than waiting for the results of further analyses, which we hope will be performed soon.”
1 Bengtsson, B. et al. Am J Ophthalmol 2008;145:343–353.
Dr. Bengtsson has a research grant from Carl Zeiss Meditec.
Blue Eyes, Single Lineage
University of Copenhagen researchers have identified a single mutation responsible for blue eye color. Their research suggests that all people with blue eyes have a single common ancestor, according to a study in January’s Human Genetics.1
The researchers examined nuclear DNA and compared the eye color of 162 blue-eyed individuals in countries as diverse as Jordan, Denmark and Turkey to find the mutation. “Originally, we all had brown eyes. But a genetic mutation affecting the OCA2 gene in our chromosomes resulted in the creation of a ‘switch,’ which literally ‘turned off’ the ability to produce brown eyes,” said Hans Eiberg, PhD, associate professor of cellular and molecular medicine at the University of Copenhagen.
Since the article was published, he has examined 800 other blue-eyed individuals with the same results, he added.
“We think the mutation occurred 6,000 to 10,000 years ago, and it probably had a positive effect for people living in the northern part of Europe. The reason is that skin, hair color and eye color are somewhat associated with vitamin D production,” Dr. Eiberg said.
Whether this genetic research has clinical implications remains to be seen. “Our next research project, among others, is to investigate whether people with blue eyes have a greater incidence of cataracts,” Dr. Eiberg said.
1 Eiberg, H. et al. Hum Genet