 All right, good morning, everybody. I think we're just going to get started. I think it just turned to eight just so we can get finished on time as well. It's so nice to be able to give two talks within a four or five day period. And I resisted the urge to talk more and elaborate on our research in the waiver and VEGF. We'll talk a little bit about amblyopia this morning. All right, so just some of the basics about amblyopia. Amblyopia is a unilateral or it's can occur bilaterally as well, a reduction of best corrected vision that cannot be attributed directly to any structural problem within the eye or the posterior visual pathways. It's caused by abnormal visual experience early in life. And it typically affects central vision to a much more of an extent than peripheral vision. And studies in animal models and humans support the concept of critical periods for sensitivity in developing amblyopia. And the critical period, the timing of that critical period and also the sort of the degree to which amblyopia develops depends on the specific type of the abnormal visual experience. So amblyopia is a big problem. It is the most common cause of unilateral vision loss in childhood. It occurs in a significant number of children with estimates ranging up to 5% of children in North America. It's the most common cause of monocular blindness globally and untreated amblyopia is estimated to cause over $7 billion in loss of US gross domestic product annually. The major causes of amblyopia, this is in order of decreasing frequency. So the most frequent cause of strabismus, followed by anisometropia or relatively large refractive errors can cause bilateral amblyopia and stimulus deprivation. So one of the most distressing parts of amblyopia, if we think about it as eye care providers, is that in principle, most amblyopic visual loss, all of this visual loss that we talk about should be treatable or reversible if it can be detected in a timely manner and appropriate interventions can be initiated and completed. So not only is it frequent, but it can cause severe vision loss. And there are many obstacles to successful amblyopia treatment. Compliance is a big factor. We run into this problem all the time in clinic. There are problems with the compliance with patching, spectacle lens compliance. And despite us spending a lot of effort trying to educate parents and people who provide care for these kids about the importance of the disease, its severity, and the length of treatment that may be required, in a lot of situations, it seems like people just don't understand this. And I think that that contributes a lot to this poor compliance and lower success rates in treatment of amblyopia. Amblyopia also has a fairly high recurrence rate. So it's important to continue to watch these kids and initiate therapy. Again, if we see signs of recurrence, this is quite a high rate. 1 in 4 is estimated to show some degree of regression. And unfortunately, this is one of the real big problems that many cases go unidentified. Until its treatments are not as successful, for sure. OK, so we're all familiar with this picture. The visual system has just this remarkable property that corresponding areas between the two eyes are perfectly parallel all the way back from the nerves through the posterior visual pathways. And even in the visual cortex, which is oriented in these really amazing parallel ocular dominance columns. Now, if we look at these ocular dominance columns, at birth, you can see organization into these ocular dominance columns, but they're largely overlapping. And in response to normal development, there's pruning so that these ocular dominance columns become a better defined. They're less overlapping, but there is some overlap that still exists. And this is important for binocular function. So if you look at just a response tracing of individual cells in the primary visual cortex, over on this side, these so-called class number 1 respond strongly to one eye. Class number 7 responds strongly to the other eye. And in between are cells that have progressively more binocular function. Under pathologic situations, for instance, in strabismus, but in this case, the situation, for instance, of a kid that is a cross fixator where there is no preference for one eye, what you see is an absence of these cells that respond to both eyes. So we're losing cells here in the middle. In a situation where, for instance, the left eye is deprived, where you have a situation of stimulus deprivation, for instance, you see that the size of these ocular dominance columns are significantly reduced in the left eye. And so we see a shift of the whole curve towards the right. Now it's thought that generation of these ocular dominance columns initially is based largely on a genetic program and development, and that visual experience leads to the formation of these different patterns during the so-called critical periods. Now an image that is just really striking and reflects these changes. Oh, let's see. We discussed the fact that these are also, this is kind of a direct reflection of changes in function of these cells and the actual signaling of these cells. And we can see that architecture also changes in response to amblyopia. So there are functional and architectural changes. This is just a very striking picture. These pictures are taken from monkeys that were treated with radioactive proline in one eye that traced back to the visual cortex. You can see in a normal monkey, the lighter traces came from the proline reflect the areas of the cortex stimulated by the eye that was injected with a radioactive tracer. You can see that the bright bands and the dark bands are just beautifully evenly distributed across here. And in the situation where the fellow eye was subjected to stimulus deprivation, you can see this gross asymmetry and the width of the bands. OK, so it's obvious from the introduction that I gave that there are still, amblyopia is still a big problem. And there are some problems with our ability to treat it. So there's definitely a need for some new ideas and new treatments for amblyopia. So what is out there? Well, active therapy is the idea that it's important for the amblyopic eye to be actively engaged when we're trying to get the visual cortex to pay attention and rewire in response to this eye. So there's this idea of perceptual learning where specific tasks can be set up. And often these are kind of computer-based activities that specifically and repetitively ask patients to practice specific tasks. There have been some studies that have shown that video games specifically to suggest that video games can help increase the responsiveness of amblyopia therapy. And it's been found that action games tend to do better than non-action games. And there was some discussion at the PDIG meeting at the recent APOS meeting that I was able to attend kind of the PDIG planning meeting there that first-person shooter games were supposed to have the best results. So they were talking about designing, I don't know, something that where you're not shooting people, maybe shooting balloons or something like that, just to keep them really engaged in games. Then there are reports of these therapies that are more stimulatory. Some of these are pretty interesting transcranial direct current stimulation. We had a paper that we discussed in one of the journal clubs on low-level laser therapy where laser light was directed through the Conjuring Tyvens onto the macula. Pleoptics is a device that is kind of like a direct ophthalmoscope. And the objective is to kind of bleach the peripheral retina and then stimulate the macula with light. And all of these have had some reports suggesting that they might enhance responsiveness to amblyopia treatment. I'm going to kind of focus today on what is kind of a holy grail for amblyopia treatment. That's the ability to just take a pill and help with amblyopia treatment. So I'm going to discuss kind of three different groups of pharmacological treatment for amblyopia and what the evidence is that these drugs might have some effectiveness. There's some support for the use of SSRIs. Levodopa and carbidopa has been studied most extensively in the literature. The results seem to be somewhat variable and we'll discuss this some more. It is the subject of an ongoing clinical trial. And in addition to levodopa, other dopaminergic supplements have been studied, specifically this citricoline. And it has, in small studies, shown a favorable result. There's also some studies to suggest that the citricoline esterase inhibitors might be of some use and the use of aerosept is the subject of an ongoing clinical trial. We'll talk about that briefly. All right, so first we'll look at what evidence is there that SSRIs may be useful. And right now this comes from the basic science literature. So before we get into that, we'll just talk kind of at a global level about cortical inhibition during visual development. And there's a concept that before the critical period begins, cortical inhibition levels are too low to allow for the formation of ocular dominance columns. And that during development, inhibition actually has to cross two different thresholds in order to define this critical period. You have to have enough inhibition to allow for the pruning that is required for the formation of the ocular dominance columns. But in order to stabilize cortical circuitry, you cross the second threshold and that makes it so that these ocular dominance columns are kind of set and that is kind of how the critical period ends. Now, one particular neurotransmitter, gamma-amunobutyric acid or GABA, is felt to be a very important inhibitory neurotransmitter in cerebral cortex. And a recent study looked at direct modulation of GABA in the visual cortex. And the way that was accomplished was a mini-pump was implanted directly over the visual cortex of rats. And this mini-pump injected a material called MPA. This is the proper name for it. It inhibits the activity of the enzyme responsible for synthesizing GABA. And so seven days after this mini-pump was implanted, you can see the GABA levels were significantly decreased relative to control. And then they turned it off after seven days. And immediately the levels of GABA came up. So here they were able to really tightly control the level of expression of this neurotransmitter within the visual cortex. So this is just kind of an outline of one of the experiments that was done here in this paper. So this just depicts kind of the critical period for the rat model system. And in this case, they implanted the mini-pump at a stage that is well beyond the critical period. So this is essentially like an experiment in adults. And then they subjected these mice to monocular deprivation. So in a normal situation, here we're looking, this is similar to what we were looking at before with the response of individual cells. And this is kind of the ocular dominant score within the visual cortex. In the normal situation, monocular deprivation in adults doesn't shift ocular dominance because there is no plasticity in adults. And you can see this is just a cumulative curve of ocular dominant scores. It's kind of a summary of these graphs here. In the presence of this gabacynthase inhibitor, so when we decrease gavel levels, we see a shift away from the occluded eye, and that's reproduced here. So this shows that by directly modulating levels of the neurotransmitter, we now induce plasticity in adult mice, mice or rats actually in this study. So there's some evidence that if we can modulate this neurotransmitter, we can change plasticity of the visual cortex. However, there are some problems with direct targeting of GABA-ergic signaling. There's specific concerns over pro-convulsive side effects, and so there's not really a great drug to think about from modulating GABA directly right now. However, indirect modulation of GABA signaling may be possible. So these inhibitory interneurons in the visual cortex that are GABA-ergic signaling are known to be modulated by brainstem pathways that use other neurotransmitters, including noradrenaline, serotonin, and acetylcholine. So we know that we have medications, the SSRIs that already target serotonin. So a study was done to see if we can utilize these neuromodulatory pathways to kind of get at this inhibitory circuitry within the visual cortex. So the experiments that were done were long-term monocular deprivation that span the critical period. And then towards the end of this monocular deprivation, well beyond the critical period, this is essentially in adults, they were treated with an SSRI or with a control and kind of midway between this treatment, this eye was opened and kind of a patching type therapy was initiated. And then when that was completed, they had some measures of visual function. These are actually just lead suturing in order to do the deprivation in the patching. And this long-term, this was considered long-term treatment with an SSRI was four weeks in this model. What was found that if you look at measures of visual acuity, first of all in control mice where you're just treating with a placebo, you see that the deprived eye loses visual acuity and that's just stimulus deprivation, amblyopia. However, treatment with fluoxetine restored this visual acuity. And that was found whether the visual acuity was measured using electrophysiologic mechanisms or using behavioral measures. And they went ahead and looked also specifically at GABA levels within the visual cortex and found that yeah, it looks like modulating these brain stem pathways indeed did change the levels of GABA within the visual cortex. So this mechanism appears to act through this inhibitory neurotransmitter. So this is an exciting initial result because we do have SSRIs that are available, although the use of SSRIs of course is kind of controversial in children and young adults. There are specific concerns about their use, but this is a new area to think about for sure. Okay, let's talk a little bit about what's known about levodopa. So levodopa is a dopamine. It's a precursor to dopamine. It needs to be decarboxylated in order to be converted to dopamine. Dopamine doesn't cross the blood-brain barrier, but levodopa does. And it's used in conjunction with carbidopa because that's a decarboxylase inhibitor that the inhibitor does not cross the blood-brain barrier. So it prevents the drug from being converted to dopamine peripherally but allows dopamine to cross the blood-brain barrier or levodopa to cross the blood-brain barrier and act within the central nervous system. So dopamine was initially postulated to be a candidate neurotransmitter for treatment of amblyopia based mainly on findings that is expressed in rental tissue and that there were alterations in VEP and ERG signaling and Parkinson's disease. This was back in the 70s and 80s. The initial study for the use of levodopa in amblyopia was published in 1990 and it had nine patients with amblyopia that were treated with a combination of levodopa and a different peripheral decarboxylase inhibitor. And then contrast sensitivity and scatoma size were measured shortly after administration of this combination drug. And they found evidence for improvement in contrast sensitivity and scatoma size. And this is one of the figures from that paper just comparing the scatoma size for the nine patients before and after levodopa treatment as compared to placebo. So you can see kind of a, you know, these scatomas are smaller in most of these patients on the levodopa side and really not changed in the placebo side. So since that time there have been several studies looking at levodopa use in amblyopia. Most of the studies have been relatively small and a lot of them have looked at short-term results with levodopa. And regression has been seen commonly. After you stop the medication there's been, in many of these studies, evidence of regression. But enough studies that have a positive result to suggest that there may be something here. So PDIG, this is the pediatric eye disease interest group that is like a, it's a collaborative group within pediatric ophthalmology which is really just a fantastic group for designing and carrying out clinical studies in pediatrics. Has already completed an initial levodopa safety study. And this enrolled 33 children aged eight to less than 18 years old. Inclusion criteria included vision in the range from 2050 to 2400, specific history of strabismus and or anisomatropia. These patients had to be carrying out patching therapy for at least two hours a day at the time of enrollment and have shown no visual improvement, kind of a stable situation in the amblyopic eye at that time. And their vision in the fellow I had to be better than 2025. So this is the way the study was designed. My patients continued the patching that they had to be doing at the time of enrollment and they were split into two groups, high dose and low dose. They continued this dosage for eight weeks and during that time period they had some clinic visits and some phone calls to kind of assess any side effects that might be related to the treatment. Then they tapered the levodopa over one week and then they had their final primary outcome visit and then 10 weeks later they had a final follow-up visit. So what did this study show? This is kind of a large table. We're just gonna kind of focus down here on the results. First of all, these are the columns that would show that they worsened in terms of their vision and there was almost none that worsened. There were several patients that did better. At the four week dose, this is right in the middle of their levodopa treatment. We saw that there was kind of a mild trend towards some improvement and this 95% confidence interval did not cross one. Nine weeks later, this is right after the point that they completed or nine weeks after initiation. So this is right at the point that they completed the taper. The effect was a little bit stronger and 10 weeks afterwards where they were just treating with patching that they'd been off the levodopa for 10 weeks. We see kind of a regression in both groups but still a result with some improvement that were the 95% confidence interval did not cross one. In terms of adverse side effects, these were not considered to be serious. They were headache, cold type symptoms, rash and nausea and vomiting in a few patients and no patients discontinued the medication during the study. So based on this result, the decision was made to proceed with a larger randomized placebo controlled study and that's currently in the recruitment phase. The overall design is very similar to the inclusion criteria that we already mentioned in the safety study except the age range is a little more restricted seven to 13 years. At randomization, patients will be placed in either a group that where treatment consists of patching plus the levodopa-carbidopa combination and they decided to go with the larger, with the higher dose based on the safety study because that was reasonably tolerated. The other group is patching plus placebo. They're gonna be treated for 16 weeks with some phone calls and visits interspersed between there and with a two week taper and that'll be the primary outcome. At that point they'll ask whether or not there was improvement in vision in the amblyopic eye. If not, these patients will stop. If the answer is yes, they'll restart whatever treatment arm they're in for an additional eight weeks with another assessment and in the levodopa group, there'll be an option for the patient to proceed for an additional 13 weeks. In the placebo group, they'll continue with the placebo treatment for eight weeks and then they'll be unmasked and given the option to be inserted back up here into this arm and have the levodopa treatment. See how they do. The enrollment goals are, the goal is to enroll at a two to one ratio with at least 92 patients in the treatment arm and 46 patients in the placebo arm and at the PDIC meeting they were saying that hopefully they'll reach this enrollment goal within about a year or so. All right, so that's kind of where we are as far as levodopa. So what evidence is there to support acetylcholinesterase inhibitors? From the basic science side, there was a recent paper here published a couple years ago to support the possibility of utilizing this pathway. A beautifully designed genome-wide screen was carried out to look simply at factors that were expressed to higher levels in the visual cortex in adulthood relative to the critical period and they used a transcriptome-based approach here. One of the genes that they pulled out is a gene called Lynx1. And here we're looking at protein and mRNA expression levels of the gene within visual cortex and this is just a stain for this Lynx1 protein within the visual cortex. Lynx1 is an endogenous prototoxin that's similar to bungarotoxin and stank venom and it binds to and inhibits acetylcholine receptor. So further experiments were carried out in Lynx1 knockout mice. And this is very similar to the results that we looked at in the RAT model previously where well after the critical period monocular deprivation was initiated. So in wild-type mice, if you look at the white and the gray curve, we see again, you can't shift ocular dominance in adults. But in these Lynx1 knockout mice, you see a shift in ocular dominance. So that shows that genetic disruption of this factor allows for a shift in ocular dominance in adulthood. Again, here's another similar experiment to what we looked at before looking at measures of visual acuity. In this case, they carried out long-term monocular deprivation within the critical period and then looked at visual acuity measurements either in a knockout mice or in response to pharmacologic acetylcholinasterase inhibitor. And what you see, if we look first of all, wild-type mice, of course, is that these mice get amblyopia. They lose vision in response to long-term monocular deprivation. This arm here is just showing that if the mice had the monocular deprivation stop, that is that eye open for one month before they did the visual acuity measurements that they couldn't recover vision in the wild-type mice. However, if we compare this column to the columns in the acetylcholinasterase treatment or in the knockout mice, we see that simply, so in this case, they didn't patch the fellow eye, they just opened up the eye that had been deprived. You can see recovery of vision treatment with either an acetylcholinasterase inhibitor or in the Linx 1 knockout mouse. So these results suggest that targeting acetylcholine neurotransmitter pathway can restore vision in amblyopia in this model. So in these experiments, the acetylcholinasterase inhibitor pharmacological treatment was carried out by performing physo-stigamine injections into the visual cortex. We don't want to do this to kids, but there are acetylcholine inhibitors, acetylcholinasterase inhibitors that are available and one that's used is aerosapt that's available clinically. So currently there's an open label phase one study that is enrolling. Patients greater than eight years of age and this can include adult patients. The patients that are less than age 17 will also receive patching therapy where the adult patients will just receive the aerosept. And the primary outcome will be assessed at 22 weeks with a plan of 12 weeks on aerosept and 10 weeks off. There have been other trials of aerosept in children, mostly in situations like difficult to treat ADHD and there've been also some other psychiatric diseases that where they've had some trials of aerosept. And looking at some of those trials, it wasn't tolerated really well in kids. So there's some question about whether or not how this will really work. It's kind of some mixed results. Yeah, there were quite a few patients that actually ended up stopping the aerosept in those studies but this is, there's some evidence that disrupting this pathway might have some utility. So we'll see. This study is being run through Boston Children's Hospital. All right, so just in conclusion, amblyopia is still an important cause of vision loss and a big problem and there are better approaches. We do need better approaches to treating amblyopia. I think it's true that pharmacologic and potentially genetically targeted treatments do hold promise for treatment for amblyopia in the future but obviously these treatments need to be refined and better targeted. And ultimately, amblyopia is a disease that needs to be attacked with a combinatorial approach that will probably lead to the best outcomes. All right, thank you very much for your attention. I'd be happy to answer any questions or comments. Come on. I mean, it's pretty obvious on some of these anecdotes. Yeah, I think that that's ultimately, yeah and that's been one of the great things about the PDIC group is they've been able to design some nice studies to address those specific issues. How much patching do you need? Do we see improvement with lower levels of patching? Do we see improvement with other types of treatments like atropine and things like that? So there are a lot of options out there and you don't necessarily have to try to push for full-time patching, okay? Oh, sorry, Dr. Warren. Okay, so Lynx1 is a natural inhibitor of acetylcholin receptors. No, I think it's very, very early in terms of the research in that area, but that could be a possibility in thinking about supplementing or treating patients that run into that situation. Can everyone hear me okay? I don't know, is this working? Good, thank you.