 So, good morning everyone, and I'll hopefully not put you asleep, so I'll try to keep you awake and keep you innovated by what I think is a pretty interesting target. And it's being looked at in ophthalmology. So you'll see Rokinae's spoken about or referred to her studies in IOVS. You're also going to see clinical studies being reported out in ophthalmology and AJO. And basically the purpose of my talk is to give you a background. I am a retained consultant and serve as the chief medical officer for Altheos, and I do have a lot of familiarity with this class of compounds. This is not specific to Altheos, it is not meant to be, it's meant to be an overview. So I really wanted to give you an unbiased representation of this target and where it's going in ophthalmology. I'm going to cover Rokinae's pathways, I'm going to talk a little bit about the role of Rokinae's in systemic pathology. In particular, how do we inhibit this pathway in ophthalmology and the potential benefit, as well as how they've been inhibiting this pathway in systemic disease. And sort of keep an open mind, because I think again this target is not only useful for glaucoma, but actually could be applicable to a lot of different diseases that we see in the eye. What I want you to walk away with is an understanding of the role. Some of the slides get pretty complicated, but understand what Rok does, both systemically and ophthalmically. Understand the inhibitors currently in development, and then again just see the potential. And you can purchase, for the researchers here, you can purchase these Rok inhibitors through Sigma, and in Japan there is one oral compound approved, and I'll touch upon that. So what is, what's Rok? Well, it's a row A, which is a member of a row class of small GTPases, and these are similar to Rok, you may have seen, or CDC. And they really regulate an amazing amount of cellular responses, and this is what really enamored me into this target. And I thought, wow, this would be a great compound if we could get into the clinic. It affects cell contraction, cell migration, cell proliferation, gene expression, cellular differentiation, adhesions, and also cell apoptosis. It's present on numerous cell tissues throughout the body, and row A is activated by the exchange of GTP and GDP. Once activated, it works intracellularly and acts on two different kinases, two different enzymes, Rok1 and Rok2. Rok and row A are activated by numerous mediators and proteins. And again, if you think about what activates it, you can understand why it's involved in so many processes. So you've got GTPases activating it, endothelan, which is a potent vasoconstrictor. You've got arachidonic acid, which is involved in inflammation, caspases, which are involved in apoptosis. They all activate the row pathway. Busy slide, but the takeaway is row works on the actinomycin cytoskeleton pathways or structure, I guess you can say. And a lot of it is really just phosphorylation. So you're phosphorylating phosphatases or kinases. You're putting phosphate on, you're taking phosphate off. And what indeed happens is if the MLC, which is myosin light chain, is phosphorylated, it contracts. So row kinase causes phosphorylation of myosin light chains, which then causes contraction. Cellular contraction, contraction anywhere. This is a little bit easier to understand. We have arachidonic acid and we have endothelan. Again row A, GTP is on the cell surface, activates the kinase. The kinase in the active role takes the myosin light chain phosphatase and phosphorylates it. And what you get is contraction. So phosphorylation contraction. Why rock? I had this question actually come up and I thought, hmm, where did the C come from? Well, it's row-associated, coiled, coiled forming protein. Again, there's two, rock one and rock two. Some people believe there's selectivity and specificity, so if you inhibit rock one more than rock two, you're not going to get as much vasodilation. The data's really sketchy on that. How does it work? Well, it's a serine, theranine kinase. It can work through calcium channels to affect contraction, vascular smooth muscle, but it can also again affect the cytoskeleton within the cell that's independent of calcium. The key here is that row kinase regulates vascular tone and it does it two-fold. It affects smooth muscles again through your actomycin contractions, your smooth muscles surround your blood vessels, vasoconstrict, blood vessels clamp down, but it also can directly affect the endothelial cells. So if you remember back to some of your vascular physiology, nitric oxide vasodilates. How do you inhibit vasodilation? You can also inhibit or lower the amounts of nitric oxide. So there's this counterbalance between row kinase activation, the amount of nitric oxide, and also the amount of vascular contraction. And it's a vascular tone. It's sort of a homeostasis that's achieved. So the pictures are sort of the big takeaways. So contraction, actomycin, smooth muscle. Cell junctions. So if you remember back to your cell biology, what affects the cell-to-cell interactions is these tight junctions, these zonular occludins, the desmosomes, and there are actomycin fibers between those cell-to-cell junctions. This is really cool. So cell migration. And if you remember anything from the talk, I think this is really neat. So the actomycin is involved in the cytostructure of the cell and how does the cell move? Well cells move through protrusions. The push-pull. So you've got the rack and the CDC stimulating the formation of those protrusions anteriorly or forward moving. And then you've got row A in the back pulling those protrusions in, and the cells actually migrate. So rock is very involved in those cells migrating from place to place, whether it's a cancer cell, whether it's a fibroblast, or whether it's a lymphocyte. Because of these sort of three top characteristics, rokinase has been looked at in fibrosis, pulmonary hypertension in particular, cancer, tumor cell metastases again because of the cell being mobile and moving around, cardiovascular atherosclerotic disease because of the endothelial function and also the vasoconstriction, chronic and acute inflammation, and ischemic and reperfusion diseases, as well as CNS injury. I took the picture first. So I found this neat picture and I said, I wonder if every organ named in this picture has been shown to be linked to row? And indeed it was. Down to cochlear outer hair cells. So rock and rokinase apparently have something to do with cochlear outer hair cells. Two, there's one approved compound currently, and that's in Japan, it's called fazadel. It's oral and its indication is for cerebral vascular vasospasm after subarachnoid hemorrhage. So basically this compound has shown a benefit in ischemic reperfusion injury. And again, translate that now to the eye and think about all our ischemic reperfusion injuries. Sigma makes a compound 27632, which is a research grade compound, which when we go through some of the data on the research, this was the compound that was used. In Japan, they've actually worked with fazadel. And fazadel was looked at in the United States for myocardial ischemia and also some other ischemic injuries. So in animal models rock or inhibiting rock has actually been shown beneficial in fibrotic diseases in animals. And this is just sort of a list of possible fibrosis that occur or that are induced in animals. And if you inhibit rock through a rock inhibitor, you have some benefit in these animals. In pulmonary disease, it's sort of twofold. So again, it's working on the fibro, the fibro proliferation and the fibrosis, but it's also working on the vascular aspect. And if you inhibit rokinase, you abolish some of the hyperop, hypoxic vasoconstriction. And again, it's this balance that it's a balance between activation and deactivation of row. And the feeling is that rokinase does play a role in the increased basal pulmonary vascular tone in hypoxic pulmonary hypertension. So there's too much rokinase activated. Vascular disease, very similar type of data. If you inhibit the rock pathway, you can alter ischemic events in the heart. You can also inhibit development of coronary artery lesions perhaps in some of these animal models. They did, again, in Japan, use phasodel clinically, and they showed that if you inhibited the rock pathway with phasodel, you actually improved coronary artery spasm in angina patients. Inflammatory disease, well, if you inhibit rock, you can inhibit T-cell proliferation. You can inhibit proliferation of cytokines and mediators, and again inhibit cell migration, and also alter the epithelial endothelial barrier function. So this is when we start to think about the eye, well, what about in the cornea? You've got your epithelial, you've got your endothelial, and there's some interesting papers coming out with rock inhibitors showing different roles in improving cell migration, inhibiting cell migration, improving corneal epithelial defects, causing epithelial defects, so the data is sort of all over there. Rock does affect the epithelial barriers, we don't really understand why or how, and I put this slide in here because if you think about what we're doing in the clinic with these compounds, we're putting this into the eyes of patients with glaucoma, and I think an interesting finding down the road is going to be what's happening to the cornea. Are we having changes in the chemistry? Are we having changes in the epithelial layer? Are we having breakdown of the tight junctions? Are we having changes in permeability of the compound getting into the eye? And again, this is an open area of research. Some more inflammatory, immune-mediated diseases, again, showing that if you inhibit rock, you can have some benefit. CNS, and this again is sort of where it translates to ophthalmology in glaucoma. So the data in CNS has been very, very interesting and encouraging. And if you think about where the drug is approved, it's approved here in ischemic reperfusion injury. So if you do a clue, the MCA in mice, they have found that rock has actually increased two-fold in that ischemic area. And when those mice were pretreated or treated with phasadel, you decrease the ischemic area. Also spinal cord models, they found that if you gave phasadel, again, this work was done in Japan, you improved the outcome in terms of paralysis and hypoalgesia. And they thought it was because of the involvement of astrocytes and granulocyte colony-stimulating factors, so again, growth factors. So somewhere growth factors are involved, various mediators for cytokines. It's a complicated pathway, but it begs the question, again, what is this role? So in ophthalmology, a little bit more closer to home. So it's an active area of research. Rock is present throughout the eye. It's present in the iris, the cornea, the congenitiva, ciliary muscle, retinal ganglion cells. And most importantly, where the interest is, is it's located in the trapecula pathway. It activates fibronectin and extracellular matrix material. So when you think about glaucoma and some of our thoughts on glaucoma with extracellular matrix being upregulated in the trapecula mesh work, trapecula pathway, again, the question is, what is rock inhibitors doing to this pathology? And there's several companies exploring rock eyes currently. They've also looked at rock in the cornea, as I mentioned. So again, cell-cell adhesions, integrity, differentiation of the epithelium, and also endothelium. So if you read into the literature, it's not quite clear what's happening to the endothelium as well. So this is just to remind everybody that the trapecula pathway has the trapecula mesh work, Schlem's canal, episclerol veins. Trapecula mesh work is composed of collagen beams, extracellular matrix cells, and then your canal, Schlem's canal, has the endothelial cells and a lot of the smooth muscle. The smooth muscle is also present in the trapecula mesh work. So you've got smooth muscle and the endothelial cells, so you've got potential targets for the rock kinase inhibitors. So what happens in IOP when IOP is involved with rokinase? Well, again, trapecula mesh work, you've got these beams, you've got actomycin present, rokinase is going to phosphorylate it, it's going to contract those beams, it's going to decrease outflow, it's going to increase the stiffness of the trapecula mesh work. And Vasanth Rao down in Duke has done a lot of this work with David Epstein. In fact, that's where the aricompound came from, and I'll talk about this. So David Epstein was very involved with the aricompound, which is now in the clinic. Rokinase also alters those tight junctions between those cells. So if you've got cells in the trapecula mesh work that are changing their permeability, that could also affect the outflow of the aqueous through those cells. And then lastly, it's the extracellular matrix. So we think that rokinase in the trapecula mesh work, or the trapecula pathway, is working on three parameters. It's causing constriction, it's causing changes in permeability of the cells themselves, and then it's also involved in laying down more of that extracellular matrix. So if you inhibit it, you should reduce IOP, right? You can vasodilate, you can improve permeability if you believe that the tight junctions are affected. And then perhaps you can even change that extracellular matrix that's being laid down. The problem with these compounds is they vasodilate. So what happens when you put them on the eye? They vasodilate, and they cause a lot of hyperemia. The hypereme is not pro-inflammatory, and it's not detrimental, and it's not toxic to the eye, but it is a cosmetic effect that is undesirable. So in development, preclinically, IOP reduction has been shown with these compounds. From a clinical perspective, well, it's kind of neat. It's a novel pathway. To this date, the only compound that works on conventional trapecula pathway outflow is pylocarpine. So we now have the opportunity to have a new compound that works on conventional outflow. Could also be additive. Could be additive to prostaglandins. Could be additive to your aqueous depressants. And it may offer some of these other interesting attributes for glaucoma. Just want to be mindful of time. So preclinical development. So this is the compound that I'm personally involved with. And we have preclinical data that shows that it does lower IOP. Hopefully we're going to be in the clinic in 2012. Like so many of these compounds, and this is not unique to alpheos, but this is a very sort of common finding with all these compounds, is that it does lower IOP and normal tens of rabbits. And depending upon the compound, you can have variable amounts of hyperemia, which is either bulbar conging tiva. There's also some chemosis in edema, which can be seen. And again, that relates to permeability. Because if you're increasing the permeability through those vessels, you may see some edema. All these compounds have looked at themselves compared to zalatan. Again, this is just an example, not unique to alpheos or ATS907. In the monkeys, now keep in mind, too, that a lot of this is species-specific. And certain species don't respond to certain compounds. Rabbids don't respond well to zalatan. Monkeys respond fairly well to zalatan. And across the board, rokinases and monkeys appear to lower IOP better than a prostaglandin. That's kind of the takeaway. So clinical data. Well, what have these compounds done? What have they shown? Ari, again, is probably the leader in terms of development here in the United States, presented their phase II data. And what they did is they looked at their compound, two different dosing regiments, two different concentrations, versus zalatan. And they show that in the clinic, in humans, in a small number of patients, 217 patients, they were actually slightly inferior to zalatan, 0.9 millimeters of mercury. So that would qualify as Dr. Olson referred to as non-inferior. Senju, which is located in Japan, obviously, they published a phase I in healthy individuals. And what I thought was interesting here, and this is why I put it, was that in normal, intensive, healthy individuals, they got a pretty good drop in IOP. They went from 13 downwards of 10 to episclerol venous pressure. So I thought that was interesting in normal tensives. We don't see that a lot with a lot of our compounds, that amount of a drop. Coa is the leading compound in Japan. And again, if you're looking for this data, it definitely will become available in the next year or so. But they reported at RVO a 24-hour nocturnal study, as well as an eight-week study in glaucoma patients. And they had significant hypoeremia. In fact, they had upwards of like 96% of mild to moderate hypoeremia. And they had an IOP reduction of 15 to 22, which is good. I don't know if I would call it great. I think they were a little disappointed. They also looked to be a TID drug rather than a BID drug. So when we think about what's going to be beneficial to our patients, I mean, would a drug like this that's additive to prostaglandin, if you use it three times a day, be helpful to our patients? Perhaps. It would be much better if it was a once a day drug and got a better IOP lowering. Aries currently in the clinic with a combo study, because the big question that comes up is, are these compounds additive to xalitant? So they're currently conducting this study. I think it may read out or report out at AAO, perhaps. But I'm not sure. And they're also doing a 24-hour efficacy study, which is an open-label study. And they're looking at 24-hour IOP control. And I think this is interesting, because, again, these compounds vasodilate. And they vasodilate all the channels on the eye. Episcleryl venous pressure may play a greater role at night, also contributes to IOP. IOP is higher at night. And again, the questions asked is, what is this compound doing at night? This study's being conducted out of Bob Weinraub's sleep lab. COA has two studies ongoing in Japan, phase three. A little unusual for phase three studies. They're calling them phase three, but they're not traditional phase three, as we know them. And again, just looking at IOP endpoints. So this is sort of just where everything stands in terms of compounds. Senju Novartis had a compound. They just continued it, too much hypoeremia. Santin had a compound, too much hypoeremia. And again, COA, ARRI, and then the alfios compound. There's a compound coming out of Denmark as well, but they're very, very early on. They're not even in preclinical yet, I don't think. So beyond IOP, well, what else could these compounds offer us beyond lowering IOP? Well, they could inhibit fibroblasts and macrophage migration. They can alter ICAM and TGF beta. So maybe they can improve inflammation on the ocular surface. Maybe if we use them in our glaucoma patients with ocular surface disease, we may have less ocular surface disease. Maybe they can improve surgical outcomes if you use them in patients who are having trabeculectomies. Well, they vasodilate as well. So again, maybe we can offer a benefit to these patients in the posterior segment. Maybe we can improve optic nerve perfusion or retinal perfusion. And then neuroprotection, actually. I don't like the term neuroprotection because of all the, I don't know, the controversy around the term. It's of interest, but everyone says you can't achieve it and there's no end points. But this compound does seem to have some role in preserving neurons and perhaps retinal ganglion cells. And it also is anti-apoptotic by inhibiting caspase. Some investigators, again, in Japan, did look at the Sigma compound in glaucoma filtering surgery, and this was in IOVS in 2007. And they looked at human tenon fiberblast gel contraction in vitro at several different concentrations and found that there was a decrease in the fiberblast migration, proliferation, and contraction. And then they put this into a rabbit model where they did a pretty straightforward trabeculectomy, actually a full thickness sclerostomy with a conch flap. And they called it a bleb score. I don't know if this is the best way to characterize your data, but the bleb score consisted of vascularization, scarring, and height. And then they also looked at these blebs, histopathologically, and found that, yes, the ones that had been treated with the rokinase inhibitor had less inflammation and less collagen deposition and fiberblasts. So in terms of vasodilation, that would be the other area. So inflammation, vasodilation, and potentially neuroprotective capabilities. Study done with Fazadel, they induced an ischemic optic nerve model in rabbits, and this was accomplished with endothelin. They gave endothelin intravitrally and caused vasoconstriction. They then treated these animals with Fazadel both topically and systemically. And they did show an improvement in blood flow as well as retinal ganglion cell count and visually evoked potential. So you've got some retention of structure and also retention of function by treating this model with these inhibitors. And then lastly, the neuroprotection. These inhibitors, well, rock is involved in astrocyte neuroinflammation. So again, if you inhibit rock, maybe you can affect astrocytes. And we've been speaking and looking at astrocytes in their involvement in glaucoma now for a while. So perhaps we're working through that pathway. The question came up of, well, if it's neuroprotective, where can it be neuroprotective? Where's the site of injury? So there was an elegant study done where they looked at injuries both to the axon, to the optic nerve, as well as to the retinal ganglion cell bodies. And if you treated, again, with a rokinase inhibitor, you had some preservation of the axons. And increased retinal ganglion cell survival. So again, depending upon where that insult is, whether it's in the cell body or the axons, there may be some role for this compound. Or this class of compounds. So in summary, I hope you can walk away with, just saying, hmm, that's an interesting target. And it's an interesting pathway. It has multifactorial mechanisms of action. And if you inhibit this pathway, there may be some benefits, both systemically and ophthalmologically. I think what I'm trying to push for, and as best I can within Altheos and other companies, is just because it doesn't work for IOP, don't give up on this compound. And it may offer other benefits in ophthalmic diseases. So it may have a role beyond glaucoma, and it also may have a role within glaucoma beyond IOP. There's potential here for research. So you can get the compound through research labs, and now that it's in the clinic, there's open INDs. So you actually can get the compound through the companies and look at other diseases in the clinic. It's a relatively, and I don't wanna use the word safe, but it has a good safety profile. So again, there's open opportunities here. So with that, I'm gonna say thank you. I'm gonna ask you if there's any questions, and the world of drug development. So the mice are doing well. Yes, Randy. So there's a discussion for this for a period of time. I don't know if that's gonna be decided. You're right, and from a preclinical development perspective, to get into the clinic, you can only do so much. This does vasodilate, so at high concentrations, you can imagine that it's gonna drop blood pressure, it's gonna give you reflex tachycardia. I'm talking again, oral prean-animals. Yeah, it affects perfusion. So I think it's really gonna come down to how potent is the compound? Where is it actually penetrating into the eye, and how much exposure there is systemically? They don't really know. They don't, yeah, they don't know, and from what I've gathered and understand, from the, again, the in vitro, so when you look at your inhibitory concentrations, apparently that doesn't translate completely to the clinic because it's a kinase, and there's so many other variables and so many other pathways that are affected. So in the cell culture, you could say, okay, we're inhibiting rock one or rock two, say 60%, but that, when you get into a living species, all bets are off. Well, yes, yes, because it actually affects the phosphorylation, so yes. Yes, Paul? No, they haven't, and you know, it's funny because I was just talking to Mimi before, and she said, oh, sorry, that's myself. She actually mentioned ROP, and as I was thinking about this, I'm like, yeah, nobody's looked at that, but it definitely inhibits fibroblasts and fibroblast migration. So it's a good possibility, I would've stopped, sorry about that. Yeah, no, nobody's looked at that. But you're right, I mean, that's a true, it's a great question, Randy, is what is the consequences? And even thinking about patient safety in a first in human study, you try to look at any possible consequence. Yes, Nick? Oh, sorry. Oh, sorry. Phase four. Yep. Yep, exactly. I mean, I think one comfort, but again, every compound's different, is that Fazadel is approved in Japan, and I mean, they're pretty strict, they've got pretty high standards in terms of safety parameters and clinical trials, and their whole development pathway is actually much more complex than ours. They're very in tune to safety, not to say that we're not, but they're even more so. And not too much. You know, it's a relatively well-tolerated drug in an acute setting, that's the other compounding variable. Nick? Mm-hmm. Yep, I saw that paper. So, and maybe a little bit of my personal impressions will come out here, but I'll try to be completely neutral. So what Ari did, if you look at their study and their design, all of these companies are looking at dosing at night, and then looking at the hyperemia scale. Because again, if the hyperemia is limited, so, and it's actually been a question that I've asked people, you know, well, aren't you sort of hiding your safety, if you want to call it safety, but you're hiding that finding in a sense? Maybe that'll make it more acceptable to patients. Some people believe it will, but it's, yeah, it's totally related to the mechanism, and it's tied in, and that was my question actually too, is it tied into efficacy? And it may very well be in this case as well. Yes. So from a glaucoma perspective, or from a systemic perspective? I mean, I can only speak to, you know, basically glaucoma in this case, and no, and I think the question is, people have actually looked upstream, so there's some compounds that, I think it was Merck had at, Paul Kaufman was very involved with a few years ago, and there's tons of papers on this, and it's upstream, so it actually hits more kinase pathways, and there he did a lot of work in monkeys, and there was endothelial disruption. Sure, it worked great at lowered IOP, but hyperemia increased corneal thickness because of the permeability, so you're very right. If we can bring it downstream and be more specific, we can probably eliminate a lot of these side effects, but I don't think that, because this pathway is so relatively new in ophthalmology, and you know, I mean, you could see it dying if it's only looked at for glaucoma, and there's too much hyperemia, then I don't see these compounds moving forward unless somebody gets the drive and the money and says, okay, well, let's look at a different indication. You're welcome. Thank you.