 So our next presenter, we're honored to have Dr. J. Ma. He's the, so he's come from the Department of Physiology. He's a professor and chairman of the Department of Physiology at the University of Oklahoma Health Sciences Center. He did his medical degree in China, and then he did a master's in pharmacology at the Chinese Academy of Medical Sciences in Beijing. And then he went to the University of South Carolina in Charleston to do a PhD in biochemistry. He spent a number of years at the, in South Carolina in the Neurosciences Department and then moved to the University of Oklahoma Health Sciences Center and became a professor in the Department of Medicine and Chronology and Physiology there. And he's here to speak to us about phenolfibrate, the effect on diabetic retinopathy and this target and mechanism of action. Thank you very much for the introduction and the full invitation. It's a pleasure to be here, to meet a lot of old friends and to talk about the science after this. So today I'd like to just talk with most of the four clinicians. I'd like to talk about phenolfibrate. And I went to several places to talk about this. And most endocrinologists are familiar with this drug, but ophthalmologists, a lot of them are not familiar. So just like to give you some background about the phenolfibrate, why we're interested in retinopathy study. And in the late part we have a mechanism study where detailed molecular mechanism probably will not talk too much because you're probably not interested in that. Yeah, I don't need to say too much about the eye structure. Just to show that I have a special vascular structure and the inner retina is supplied by the retina vascular bed here in the inner retina. In outer retina, absolutely no vessel because there's a photoreceptor has to occupy every piece of space because to increase our light sensitivity in the back is a coroid vessel which has no tight junction. And therefore there are two blood-rating barriers. One is the tight junction in the endocelial cell. Our barrier is the RPE, is there is no tight junction here. Here you can consider these capillaries to the coroid as a large vessel. And therefore it is the RPE control what to allow to flow through to the retina to supply the outer retina nutrition. And we studied diabetic renalpathy for many years. And as you know, diabetic renalpathy now is considered chronic inflammatory disease because there's some making some evidence or for example to have a leukocytosis, leukocyte adhere to the endocelium and damages the blood-rating barrier and the cost of vascular leakage. And this is considered the major cause of diabetic macular edema. In the latest stage, of course, you'll see the parasite loss and then new vascularization. And this is the proliferative stage. So what is the phenol fibrate? Phenol fibrate is another new drug. It's a drug for clinical for almost 40 years. And it's a small molecule, PIPA alpha agonist and is originally designed to came to the market for the use of a lower lipid triglyceride. And it's been used, as I said, for hyperlipidemia for almost 40 years. And it's active form is phenol fibric acid. So phenol fibrates the ester form and they get into the blood convert to phenol fibric acid which is active form. It goes through renal excretion and is FDA-approved and is inexpensive. Therefore, we believe this was some problem for the poor patient because 67 cents per day, the patient can, this is the dose we daily used. And later, this has been used for, as I said, many years. Later, we have two large clinical trials. And this is the one associated the phenol fibric with the diabetic renaldoxy. One is called a field study, another is called a study. And I will talk about this as a field study. This is the one published in Lancet, 2007. And the phenol fibrate is a lipid drug, lower lipid drug. So therefore, ABAT sponsored a large clinical trial, 10,000 patients for over five years. All type two diabetes. Because type two are associated with high lipid and obesity. Therefore, they believe this drug may improve the lipid profile and therefore benefit the cardiovascular system. And follow the, so the 5,000 for placebo, another 5,000 for the treatment. But for the primary indicator the cardiovascular protection was not very good. In five years, very disappointing. Almost no protection. But accidentally, the secondary parameter. Define that the eye, the patient in the treatment group has significantly improved edema. And you see the big difference. The laser surgery, you see in five years have a big difference. 32, in total is about 32% decreased progression of diabetic renaldoxy. So this is the first oral drug to my knowledge or ever to show is such a proved efficacy in patients. You know, a lot of drugs in any model works, but in human, it doesn't work. Because in retina disease is hard because the blood retina barrier, a lot of drug you orally take cannot get into retina to sufficient concentration. So in 2009, two years after this, there's another paper published in New England Journal of Medicine. They used about 12,000 patients for four years and about a half use the statin alone. Another half use statin plus phenylphibrate. There's all type two diabetes. They almost the identical result they reported. And 40% improve in retinobacy in these two groups. There are those two papers really cause our attention. So what is why the phenylphibrate is so effective and what's the mechanism, what is the target? That time a lot of people still feel this could be lipid or just off target because this small molecule could be off target effect. And then we look at the PIPA family. There's three members, PIPA alpha, gamma and beta. They all have a similar structure and they're involved in lipid metabolism. And here is the three members and PIPA alpha, PIPA gamma, PIPA beta and the delta. So they're the same. So this is the tissue distribution and they're known function. So first we want to know is this really the lipid effect because a lot of people argue maybe lower lipid penetrated retinobacy. So we choose to use type one diabetes model because type one usually has no hyperlipidemia issue. So we use the type one. And we use the assay, we use the called the vascular probability assay. Basically you inject some marker or tracer into the vessel of the circulation and then you perfuse out from the vessel. Then you dissect retina. See how much the marker means a tracer getting into the retina. The usually the tracer we use is similar side to albimine. Usually the FDTC labeled albimine or Evans blue labeled albimine. So here you can see in the normal and the type one diabetes you have significant increase the vascular probability and after phenolfiberate oral treatment for eight weeks and you see the significant decline. And then we use the mouse model, which is a genetic model. This is STZ induced, which is STZ is a chemical and inject and specifically destroys the beta cell and therefore generate type one diabetes. Akida is a mouse model for diabetes. Originally early people you see people call type two because they have insulin level as normal, but the insulin have a mutation. F4 is not functional. Therefore now people believe this is the type one model and this is genetic model and the pretty reliable easy to use and you guys as you can see have significant increase vascular probability and after phenolfiberate treatment have a decline vascular probability. And another asset we use called a leucostasis asset and basically you flush and use profusion to flush out all the circulating blood cells and as you know more then you stand the vessel and normally in normal kind of non diabetes you see the pretty smooth endocelium there's no cells staying in the cell. It's staying in the vessel and in diabetes you see some leucocytes adhere to the endocelium and therefore after profusion you cannot remove them. They're called the leucostasis and after phenolfiberate treatment those cells apparently decreased. Then you can quantify the inner retina. Retina you flat mound you can see significant decreased inflammation in the retina. And another as I said leucocytes in the parasite drop out and the degeneration of capillary is another pathological change in diabetrinopathy. We also did the so-called parasite quantification in the ghost vessel quantification. Here you see this in diabetes condition you see a lot of the ghost vessel all the cells are die. Only the membrane left they're called ghost capillaries. And also we do the double staining of endocelial marker and the parasite marker you can quantify the ratio. As you can see here in diabetes have the so-called acetyl capillaries such as this have increased and the phenolfiberate oral treatment significantly decreased vascular degeneration. And then you can see the parasite ghost is also decreased. And you see the parasite number is increased back and compared to diabetes. So therefore this we believe there's a phenol five indeed will benefit the type one diabetes. Then next question we ask is this through PIPA alpha or off target? Then we use the PIPA alpha knockout animal. And so here we induce diabetes in wild type and the PIPA alpha knockout. In this mice have no PIPA alpha. Then we applied phenolfiberate. As you can see here in the wild type and you can see the significant improvement of a parasite survival by phenolfiberate from this non-diabetic and diabetic and diabetic with phenolfiberate significantly improved. But in PIPA alpha knockout with diabetes have even more drop of parasite number. And when you use phenolfiberate, there's no rescue. And the same thing is acetyl capillary. You see here and they can decrease by phenolfiberate. Here has no change. So that means this effect on vasculature is dependent on PIPA alpha. Then we isolate the parasite and use primary culture. Isolate the parasite from retina. Then we did a tunnel proctosis assay. And as you can see, we use the palmitate which is oxidant. Similarly, this is a commonly used dresser for diabetes. As you can see here, the increase the cell from wild type of mice and have a significantly rescued by phenolfiberate. But as a cell from the knockout mice have no rescue. That means this parasite protective effect is indeed dependent on PIPA alpha. Here it shows the vasculature probability assay. Again, this is the tracer into the vessel. As you can see here, this is in a normal condition. The knockout in the wild type has no significant difference in vasculature leakage. But in diabetes condition, this is the diabetes wild type and the diabetes knockout. See, the diabetes knockout have significantly higher vasculature leakage compared to non-diabetic wild type mice. And when you apply phenolfiberate treatment, you can see in wild type is a significant decrease but in the knockout animal phenolfiberate has no effect. There's further evidence that phenolfiberate effect is indeed through PIPA alpha rather than off target effect. And then we clone the PIPA alpha into an adenovirus. We use adenovirus infection and we can achieve the same thing. So there is no drug. No phenolfiberate. That means indeed the PIPA alpha is indeed doing the job rather than the chemical has another type target. So next question we ask is, can this therapeutic effect, where is the target? Is it systemic and then affect the eye or is it in the target in the eye? So that means can we achieve this effect by intraocular injection? Then we inject the phenolfiberate into the vitreous and in diabetic animal. And as you can see, we can see the same thing as the oral treatment. That means there's no systemic use of the drug. That means this drug target indeed is in the eye. See this is the so-called OIR model, Oxygen-induced retinobacillus model. This is the ischemia-induced neovascularization model. And as you can see here, one dose injection have significantly decreased neovascularization in this dioxin-induced retinobacillus model. And after injection, you see the VEGF, this is the standing for section of retina and this is the type of standing for nucleus. And you can see for VEGF signal, this is significantly increased VEGF signal. And by the phenolfiberate decrease VEGF signal in the eye. And same thing is the VEGF Western blood in the retina preparation. Then we did the in vitro tube formation assay. You know, endocytic cell and you put it in culture, in certain condition, they have a tendency to form tube-like structure. After you had phenolfiberate, the tube formation is disturbed. And another is migration assay. You scratch in the monolayer of the cell and after 24 hours, the cell migrate toward the acetylapidone and with this phenolfiberate, the migration is slowed down. So now we know this target in the eye. Then we start to turn our eye of the attention to the retina, to the eye. So we start to study, where is PIPA alpha indeed expressed in the retina and is it changed in diabetes condition? Then we collaborated with Dr. Michael Bolton and collected several six patient eyes donor with diabetic retinobacillus, nonproliferative because proliferative too much change. We use nonproliferative diabetic novacy. We got several donors without diabetes. Then we did an immunostaining for PIPA alpha. As you can see here, and the PIPA alpha is widely expressed in almost all the layers of the retina. And in the diabetic donor, you see the significant decrease the standing signal. And the same thing, we use the red diabetic red. You see significant decrease the PIPA alpha signal. Then we did a Western plot compared to all the three members of the family and the PIPA alpha, beta, and the gamma. And each line represents individual red. As you can see here, the diabetic red have a significantly lower, PIPA alpha, but the PIPA gamma is so low. It's not detectable, at least, in use of commercial antibody. And the PIPA gamma is almost no change. And this is a statistic difference. And then we use the other animal models. And Akira is a genetic model. You see also decrease in Akira model. And use the type two. It's a DVDV type two model. You can see here, it's only PIPA alpha changed, but the PIPA gamma is not changed. So that means, that explains why the PIPA gamma agonist is not so effective for retinopathy. But the PIPA alpha agonist phenylphibid is so far, is the only effective ligand. Then we use the knockout animal. You can see the leukostasis. Indeed, after induce the diabetes, indeed without PIPA alpha. And you see more significant, more leukostasis that compare to wild type. Then we inject the PIPA alpha virus. That means the virus express PIPA alpha. This without phenylphibid, just use PIPA alpha. Inject the diabetes in the animal into the eye. You can see the PIPA alpha alone can decrease leukostasis. So that means this PIPA alpha is indeed endogenous protective factor in the eye. So now we go to the mechanism. I don't know how much you're interested. I'll probably just be brief. One thing I'd like to show, this we, our finding in the next talk of today, I will talk about the wind pathway. This is the pathway we identified play a major role in inflammation, angiogenesis, endiabetic synopsis. So I would give you a brief introduction about what is the wind pathway. This is the simplified wind pathway. And the wind pathway, including the core receptor, ARP and the phrasal receptor and the 19 wind ligand. And in the absence of wind ligand, there's two receptors separate. And the beta-catenin, which is a trans-screen factor, is trapped by the kinase complex. Airfoil is constantly phosphorylated. When the beta-catenin is phosphorylated and it becomes unstable, degraded, and therefore cannot get in the nucleus, activity in expression. Upon bending of a wind ligand to the core receptors, those ARP6 become phosphorylated and the recruit actin, therefore dissociate this complex. And beta-catenin become free. It's not phosphorylated. Therefore, beta-catenin accumulate, stabilize, and get into the nucleus, bind it to TCF and activate a number of genes, including several genes associated with diabetoenopathy, VEGF, PDGF, ICAM1, CTGF, TNF alpha. Therefore, we believe this pathway controls so many inflammatory factor and angiogenic factor and the fibrogenic factor. Therefore, it's a promising fact. It's a pathway for diabetoenopathy. So this is our working hypothesis. Since VEGF, PDGF, control the angiogenesis, ICAM1, TNF, alpha control information vascular leakage, CTGF control basement membrane leakage, thickening, and fibrosis. Therefore, if we, I feel if this, we can block this pathway, we can simultaneously block several factors. Theoretically, it should be better than blocking one individual growth factor there. So we, first, we want to know whether this pathway is indeed changed and then you change it by phenolfibrates. Several years ago, we found a wind pathway indeed is activated in several diabetic animal model in human donor eyes with diabetoenopathy. Then we want, here, we show the phenolfibrates that beta-catenin is, we use this as a major and see a beta-catenin is indeed increased in diabetic retinopathy model in retina. And after treatment of phenolfibrates, the beta-catenin is decreased. So it's in the akida. Same thing, in the increase in diabetes and the decrease by phenolfibrates treatment. And this is the so-called reporter mice. Looks pretty ugly. This is the beta-gel standing of the retina. And this, what is this mice, the transgenic and they put the beta-gel like Z reporter mice in reporter gene under the control of a promoter. This promoter is controlled beta-catenin, which is a wind reporter mice. Whenever you see the wind activation, when you stand with the X-gel, you see the blue color. And here, you see in the transgenic animal, when they're non-diabetic, almost no blue color. In diabetes, you see pretty strong blue color. After the phenolfibrates treatment, the blue color is decreased. And of course, in cell culture, we can do the luciferous assay. The similar principle, instead of a beta-gel, the reporter gene is the luciferous. You see the luciferous significantly decreased by phenolfibrates treatment. And then we directly use wind ligand induce the beta-catenin stabilization. The same thing, people half can significantly decrease beta-catenin in this model. So therefore, we believe it's just, indeed, just people half has interaction, functional interaction with wind pathway. So to summarize this part, we found the phenolfibrates therapeutic effect on type one diabetes. So we present, we give this, with this result, we early stage, we send this result in collaboration with field study PI, Dr. Kitch. We contact him, we give him this, we invite him to our university and they will give it all the data. He uses this data to convince Abbott to start. So now the Abbott has approved the clinical trial for type one diabetes. This is because our results convince them that could be work for type one. Therefore they are studying the type one diabetes clinical trial now. And the phenolfibrates inhibit angiogenesis. So this drug target is in the retina. The beneficial effect is people half are dependent and the people half are downregulated in the diabetes. Therefore this downregulation itself contributed to diabetic renalysis. Therefore we found that people half is endogenous anti-inflammatory factor and therefore represent important drug target. So we believe this phenolfibrates as a phenolfibrate, people half agonist has a future in the clinic because a lot of poor patients they cannot afford the arrest in Lucentus and a lot of countries they cannot afford the introvertial injection. So this phenolfibrates is off-pattern drug. There's no pattern, there's a generic drug in 65 cents per day, a patient can, just without any insurance patient can take it. So now routinely this drug has been proved in Australia for routine treatment for diabetic renalysis. The European Union is also applied and is considering phenolfibrates as a drug for the treatment of diabetic renalysis. Yes. Does one focus on diabetic renalysis? Yes, we are working on that. We don't know yet. So there's some model where we're trying to develop. So how does people half this is a more mechanical study. I don't think I want to go too deep. So one thing I want to let you know, we found, so there's no direct in literature. You find the PIPA gamma, not interaction with the wind pathway, but the PIPA alpha zero, there's no nothing. So then how does PIPA alpha, but in our model, indeed we showed you PIPA alpha regular wind pathway, how? So we use, eventually we found this is the VLDLR, the very low density lipoprotein. This one is well known to be regulated by PIPA alpha. So we found that this one also itself is a regulator of a wind pathway. So this is the paper 2007, we found that this, but VLDLR is a regulator of a wind pathway. And here shows a phenolfibrate, the downregulated wind receptor, but the upregulated in the VLDLR in the same cell. Here it shows phenolfibrate upregulated VLDLR by immunostaining the antibody staining. There's a light and banding, both shows the VLDLR increase. This is the VLDLR promoter, is increased by, then how VLDLR interact with the wind pathway and we found that there's a lot of molecular biology study, we did a lot of mutant deletion. And eventually we found that this is the extracellular domain of VLDLR, really dimerized with here, shows that both in the same family, ARP6 is wind receptor, is very essential for the wind activation in the retina. And the both have a large extracellular domain. So now our conclusion, I don't want to go detailed to waste more time. You just want to know, there's two, we identified the dimerized and they regulate each other by the protein level. The dimerized destabilized ARP6, therefore internalized, enhanced internalization ARP6. So that's what we found. This is the mutant we made and we found this alone is the extracellular domain alone can inhibit the wind pathway. And then we did find the degeneration, degradation of ARP6 indeed is enhanced by the VLDLR. And this is the conclusion. They eventually found the immuno-co-presentation that do form the dimer together. So that, this is something we, this part, we have more detailed, today I'm not going to talk in detail. So we just like, we just let you know, VLDLR function as a negative regulator of wind pathway. The wind inhibiting effect is mediated through VLDLR transcription, at least in part through this. Of course, there's some other groups found that there's MPK and then I have a B, yes, we agree, that's probably true. But this is probably, people have multiple regulation target but this VLDLR probably is one of the target. So take-home message is the phenol-5 is an activated PIPA alpha. And the PIPA alpha can down-regulate VLDLR production therefore in the liver and therefore lower the lipid level. And the other side is VLDLR receptor increased and the inhibited wind pathway then inhibited bunch of inflammatory angiogenic factors. And this is the mechanism we believe is responsible for the phenol-fibrate effect on diabetic retinopathy. And the conclusion is that PIPA alpha is promising new drug target for PIPA alpha is for diabetic retinopathy. And the PIPA alpha is a regulator of wind pathway. And then we find that the receptor-receptor, the VLDLR and the RP6 interaction represent a new mechanism for wind pathway regulation. And here are the people working in my lab did contribute to the project. And Yin Chen and King Wang Li, the student did a major contribution. And we got a lot of wind pathway construct with Dr. Ji He from Harvard. And we got collaboration with Tony Kitch from University of Sydney and from Dr. Lyons as well. Thank you. Okay. So a fascinating talk, it's interesting that let's get the light on. We have something like phenol-fibrates, as you say, has been around for a very long period on pre-enix concept. Yes, I just talked to Dr. Kitch. He and I, we approach ADA. Let's see if ADA can use this. ADA said we have to get the FDA approval. We approach FDA. FDA is the standard is if you use clinical trial, you target it on the primary disease. If that doesn't work, that's it. There's no, not that approval. Even your secondary fund is effective for another disease and you have to start a new clinical trial. And therefore if you want the FDA approval, somebody have to invest in another five years for just for diabetic renalysis trial, which is, I think is not a good way to do it. So the politics, I think we're pretty fascinating. The big issue holding it back is the amount of money necessary to get an FDA approval. Yeah, this is another good point. This is of a pattern. That's why nobody want to develop this because there's no protection. So I know several pharmaceutical companies like Genentech, they are developing new people of agonist, and which may be the same. I don't know, maybe the same, yeah. But they just want to, because they have a pattern. And I know Abbott tried to develop phenophobic acid, which is an active form of this. They try to sell that part because they have a pattern on that. And they just have no pattern phenophobic acid, which is caused more. And the final effect could be the same. I don't understand this, yeah. So one of the issues is if you look at this and it sounded like as you were using, and they showed in general, there was a protective effect over time. Yeah. And there have been clinical trials showing that those who are already having diabetic macther or even others that it ameliorates the disease, once it's already in process, there is largely a point. Reverse. We haven't seen that clinical trial yet. Yeah. It seems like there is a... It's mainly for heart, I think. Yeah, I know in Australia, there are routinely you have the diagnosis of diabetes. Routinely you take this, you know, private and that doesn't matter, you have written off. Because also, I didn't talk about the other complication. It's a reduced amputation rate and the reduced nephropathy as well. So therefore it's a broad beneficial effect. So it's anti-information. So the patient should take it routinely, but in this country, I don't know how long it will take to come to the clinic. Thank you very much. Thank you. Thank you. Thank you.