Beyond traditional supportive care in ALF, and I'm going to change slightly the topic later on because hemofiltration for us is a standard of care, we don't call it as beyond traditional supportive measures. These are my disclosures. So remit of the talk, over the next 20 minutes, I'm going to talk to you about what is the rationale for this therapy, why did we think about all this? What are these devices like? We are not intensivists, except one person in the room, I think. What is the existing data to support it, our hypothesis? Then we talk about human hepatocyte transplantation, I'll give you a bit of insight into allogeneic microbe technology, and ASLD insisted that I talk about key points, take-home messages. So the regeneration goes back to mythological times, Prometheus was the first model of human regeneration, or liver regeneration, I would say, not human regeneration. Liver regeneration in humans. You all know the story, that liver was taken by this vulture, but Prometheus didn't die as the story goes. I think he died a terrible death of sepsis or something later on, but that remains to be seen. It's not recorded in the records. Now this slide is important. What you see in this slide is there are two things here, if I could shoot it, yeah. So necrosis and regeneration, there are two things that are happening parallel. The necrosis is the red bit, and these green nodules, what you are seeing is regeneration. Under regeneration, what I've depicted is a diagram of showing how the regeneration happens, because regeneration, the hepatocyte are the only cells, one of the only solid organ cells that can go replication. They stay in one of the binuclear state, and some stimulus has to lead the cells to start replicating. When there's a massive injury, like liver failure, massive hepatectomy, then what you find is that on this slide, what you see is from the cells, from the G0, they have to go to G1 and G2 stage, and then they have to stop, otherwise the liver will keep growing, unless it's stopped by something, and those are the factors that come into place. Why am I giving this slide? Because we talk about the systems that are going to introduce to, they're going to take up all the suck the stuff from their blood circulation, but they're also going to suck out this stuff, which is going to regenerate the liver. Hence, it's extremely important to get this balance right, and how we get that balance right remains to be seen. To introduce the concept, this slide is more relevant to acute and chronic, but I'm using it to introduce what is the problem in acute liver failure is. It's a toxic hypothesis of vicious cycle of auto-intoxication. Initial insult, hepatectomy, or viral hepatitis, or whatever you want to call it, drug, leads to toxin concentration, and what we believe, this is all, and end of all, the liver failure. That is what's killing us, is calling surge response, raised endocrine pressure, et cetera. If you look at this, what it evolves into it, the secondary organ dysfunction. Our previous speakers all talked about it, brain injury. We talked about coagulation failure, immune failure, and renal failure. All these toxic substances that are believed to be accumulating in acute liver failure model lead to the secondary organ dysgenesis, and that is what we see, the phenotype of acute liver failure. So acute liver failure is a syndrome, it's not one disease, it's not one disease where you can characterize because multiple organs get hit, and this continues. So rationale here is, can you do something to block it? Can you take all the bad things out of your blood, and is there any technology exists? If that takes it out, you will be all clean because this cycle will stop. So to do that, we have a lot of systems in our place. So hemofiltration systems, forgive me if you already know about it, but there are two fundamental principles they work around. One is convection method, with the CVVH, there's a counter current mechanism flow, and there's a diffusion method, where this is called as diafiltration or dialysis. Now the difference between the two, for hepatologist is, the system on the top left hand corner, which is CVVH, will only take medium to large size molecules. And for example, the medium size molecules are not ammonia, ammonia is a small size molecule. To take that out, you need to go into the second one on the right side, which is CVVHD, which means dialysis filter, which is a diffusion component to it. If you put them two together, then it becomes CVVHDF or diafiltration. Most of the centers, depending on the personal beliefs of the intensive care and et cetera, they believe on either one of the two or the combination. But you need to have an arm, which is CVVHD, to take out the middle molecules, and the small and middle molecules, which are mainly culprits seen in liver failure, like ammonia, and other aromatic amino acids and middle molecules. The left hand side here is plasma exchange. I'm not going to talk a lot about it, because this has been used, and I will show you some data, but it takes up everything. Then you have to replace all the plasma. So it's like another cleansing system. We tried to classify these things two years back in a review article, and we divided these into artificial liver support and bio-artificial liver support. The word bio means you need to put a component there, which will give the synthetic function to the liver, either a porcine-derived or a human-derived hepatocyte. So that's become bio-artificial. And the other one, which is artificial liver support, are all your cleansing systems. So you can call them cleansing system and biological systems. So these are the several pictures, and they're coming and going. Before we even noticed, depending on where you work, you may have seen all of them, you may have seen one of them, depending on the local setup where you are. And what these devices are, they're a combination of one or the other. So one is on the—I can't—this, sorry—MARS system, which I will come to. The basic system is the biological systems are either putting in porcine cells or human hepatoma cell lines, and the cleansing systems are using various combinations, charcoal filters or other filters, to take out the toxins and replenishing your albumin back, because albumin is expensive stuff. So I'm going to be very negative now for the next three minutes, because I'm going to show you data. I'm not going to show you pediatric studies, and you will say he's biased. I'm a pediatrician, but I'm showing the adult data. The reason is, pediatric studies are run by enthusiasts, small numbers, you can count on leper's fingers. You don't need many. I think if you were to change and believe, you need bigger numbers. So let's look at the data, which is randomized controlled trials, so big numbers, and see what they show. Forty years back, King's College Hospital adult labor unit carried out the study on charcoal hemoperfusion. 1982 to 85, 137 patients randomized into four groups. 75 in grade 3, encephalopathy received 5 or 10 hours of therapy. Survival, 51% versus 50%, so very difficult to say which one worked. 62 in grade 4 encephalopathy received 10 hours of therapy versus standard of care, 39% versus 34%, so here you are, no definite evidence. ELAD study, which device uses hepatoma cell lines, again, has a system of cleansing plus bioartificial support into biological support in it. 203 patients, acute alcoholic hepatitis, 96 received 3 to 5 days of therapy, 107 standard of care, 52% survival in both groups. Now, if you have good statisticians, they can always find ways to make the data more attractive. So if you do a subgroup analysis, then the trend appears there may be some benefit, but for critics like yourself, it's difficult to get convinced. SIRSA-BAL-RCT, which is a porcine-derived system, again, a biological system, 176 patients randomized, 147 acute liver failure, 27 of the primary non-function of the graft. Endpoint was 30-day survival with or without liver transplantation. Look at the range of the treatment they received from 1 to 2.9, 1 to 9, so very varied range of treatment they received to about three courses of treatment per median, mean per three. But survival, 89% versus 80%. Again, I can't convince you that the therapy works over the treatment of the controls. If you do a subgroup analysis, so patients who were non-PNF, mean primary non-function, or known etiology, or unknown etiology, were found there was a benefit that there was, if the known etiology was there, it did better. The MARS, where the principle is that, again, you are replenishing your albumin, which are the big molecules that attach to albumin, and if you take them out, then you can get the cleanser body of that. So this is a very good study. This is a prospective 16 French centers included in the study, 53 patients randomized to MARS and 49 to standard medical care. Six months overall survival, 85% versus 76%. In paracetamol liver failure, however, the survival was a bit better. But the way the treatment was set up, it didn't prove the efficacy of the treatment modality that they were testing. So what do we summarize from these randomized control trials? There are five trials in 718 patients. None delivered a positive survival value. So when you look at the commentaries on these papers, there was a lot of money spent. About half a billion dollars has been spent already. The commentary says there were design flaws. And they may be true, but design flaws are there, which I will explain to you what are the design flaws. We are looking at historic controls. Acute liver failure is not one disease entity. There are multiple etiology. They have their own outcomes. We are comparing with historical data. There are very few randomized control trials because there are not many patients around. Then now we are mixing so many patients with the disease severity. They're walking wounded, what I call them INR of 2.5, but they're running around, okay? But technically they're acute liver failure. Are they really acute liver failure unless they're intensive care? For us, they are not. We are mixing acute and chronic liver failure in these patients, which is a different disease group. And the biggest thing is here in today's era is an artificial event, which is a liver transplantation. It's more artificial now with the availability of living donors because it all depends on the local availability, what is there. So you can transplant a liver or if you wait a few days, maybe patient's liver may regenerate. So to conclude this part of the talk, we use extracorporeal liver assist devices only, hemofiltration. And the reason we use is either for renal dysfunction or for hyper-anemia or metabolic abnormalities. So one of these indications is there, we'll use it, invariably there are more than one indication in one patient, if there are more than grade 2 encephalopathy, there are other abnormalities by then already. But we will not use them for as if any patient walks in. What you have to remember is unlike adults, children you have to put them to general anesthesia, under intensive care, ventilate them to put the big catheter. So you can't just do a CVVH on a three-year-old child in the ward, no you can't. If you do it, tell us how you do it. So let's switch on to now liver regeneration, and what can the cellular therapies offer. So after Prometheus, what has happened? So after Prometheus there was a lot of lull, and nothing happened, till people started using whether do you really need a whole liver to survive? And the concept was, can you get away with a small amount of liver mass, right or a left lobe of the liver, while the native liver regenerates? The concept of auxiliary liver transplantation. So in this slide, what I demonstrate to you in one representative patient, what you find here is at transplantation, or nearly immediate post-transplantation, what you find is that patient's liver, which is this one, has not much activity, everything is donor liver, and the corresponding histology shows massive liver cell necrosis. Fast forward the film, one year, this patient has two livers functioning, this is his own liver, this is the graft, we have stopped the immunosuppression in the meantime, his liver biopsy shows regeneration. So for this patient, liver completely regenerated, and doesn't need more immunosuppression anymore. So our data is, so which is another standard of care if you can do it, everything is in line, we all offer them, all acute liver failure children get offered an auxiliary transplant if everything is available, means the size of the organ given to us is right, right to our surgical colleagues. So there is a 70% native liver survival in this technique. Now this begs the question, are we over-transplanting? And the micro answer will be yes. In the absence of very good prognostic parameters we have, we don't know, so we can't play with somebody's life. We give them benefit of doubt, but if you give this technology, this technique, you don't over-transplant them because after a year they will go back to square one where they started. So the question is, what you have done is you have demonstrated that you can get away with half the liver, patient's native liver regenerates, so Prometheus theory is correct, that patient's liver, human livers do regenerate, and there is a lot of animal data also. So if you look at the large animal model, a lot of animal data is very convincing. So murine models, rat models, monkey models show improvement or survival after hepatocyte transplantation. As a representative slide I'm showing you this large animal experience in a pig model, reversibly immortalized human hepatocytes here, transplanted in these pigs, and what they demonstrated is the pigs who had their own cells, porcine hepatocyte, has 100% survival, while the human hepatocyte also showed a statistically significant survival compared to the control group. So in a very small number, but with a controlled experiment, human hepatocytes which are immortalized and pig hepatocytes showed survival benefit in acute liver failure model in pigs. So can we move on to humans now? So there is enough animal data. If you believe in that, that okay, animal data is there, now the next step is humans. So what was the big block in between? Big block was the GMP isolation of cells, expensive business. So how do you set up? Just to share with you that pain is quite a lot to run these things, this is inside of a GMP unit if you have not been in. How we work is the livers that are not used for transplantation, and you will laugh, what do you mean by that? So these are the surplus livers or segment 4 or reduced grafts or you name it. We get those, we cannulate them, we put collagenase, we mince this, and what you get is a liver cell soup which looks like this. So liver cell soup, you can take it further, cryopreserve it, and put in a cell bank to use it for future when you need it. This is a crude example of saying how the viability is measured by type 1 blue which is a very crude way to test it. Now we come to the next question. So if you are going to use cell therapy for acute liver failure, how are you going to deliver it, and what are the prerequisites? I think we need a synthetic and detoxification function for a few weeks. We need a cell therapy which doesn't need immunosuppression, and we should be able to transplant liver cells in a place which could be safely deposited. We understood that clotting or bleeding complications are very little, but it will take a lot of courage to persuade somebody to put a big catheter in somebody's liver with acute liver failure. So can we meet these three prerequisites? So these three prerequisites would be, we come up with the concept of hepatocytin beads, and beads means can we isolate these cells so that they are protected from the immune system, they could be producing synthetic and detoxification function, and these beads could be deposited safely into the peritoneal cavity of the humans. So briefly the methodology is very simple. We isolate the cells, we go to this expensive bubble maker, this is about $50,000, and this bubble maker produces these cells into salmon eggs, and one salmon egg has got about 400 cells in the hepatocytes. Just to crudely cut everything short, which is 20 years in development, what we got is to prove to you cell function and viability that these beads, the cells they have, they have mitochondrial activity, they have albumin synthesis, they have urea production, and they have a factor VII production. So what we wanted to produce is a synthetic and detoxification model, which we did. Then the question is, how do you know these bead cells inside the beads are dead or alive? So 3D confocal microscopy shows that these cells are equally distributed, the red ones are the dead ones, and the green ones are alive ones. So went on after regulatory, it takes a lot of time, this was the cartoons that you can keep the toxins away, that means toxins means ammonia, etc., if you take a surrogate marker. The waste product is urea, can we measure it out, and immune cells could be kept out. So we had to do a lot of immune function testing on these before we could demonstrate that the immune cells, these things do not activate the immune system. The procedure is very, very simple. So anybody, medical school graduate background should be able to do it. And what it does is, we have to glorify it a little bit, otherwise people won't like it. So do the ultrasound, make a big fuss around it, but it's very simple. So the idea is that one day you can put in a drone and send it anywhere you want, thaw the cells and put them back. These are the cells before transplanting the beads, and this is after. So this slide precedes, you know, before the outcome. The protocol is to take the beads out when the patient goes home, if you survive, or at the time of transplant, when the transplant operation happens. The point here is a learning point, because these beads don't look very different from the other ones, but they have been in the human body for several months. So what it doesn't, hasn't created is immune reaction, and the function has been preserved. So we did eight babies, various diagnosis, outcomers. They all were listed for liver transplantation, means fulfill the criteria for transplantation, and if the transplantation came, we have to give it. So 50% native liver survival. I can talk to you more about these patients if you want to know at the time of discussion. The point is that it was safe, efficacy, we could argue. Then the question came, the journal paper we sent this, tell us how did it work? Was it a magic? So they're human cells, and to demonstrate human proteins in the human blood is very difficult. So what we have to do is we set up experiments in the lab looking at putting human cells into the rat model and showing human proteins in the rat blood. So that was the nearest we could go. So this slide shows that in the rat model, we are using two different technologies here with the mesenchymal stromal cells, shows that human albumin we could demonstrate in the rat. So that means there was a transfer of these proteins from the rat peritoneum into the rat blood. Similarly, alpha-1 antitrypsin was also demonstrable. Now while we were doing it, we were having a struggling with getting good quality cells, so we looked at beneficial effect of mesenchymal stromal cells. And what we found is that if you co-culture human hepatocytes with MSCs, they improve their viability, they improve their functionality, and they improve their attachment, which is extremely important for us. And these are several mechanisms on this side, anti-inflammatory, immunomodulatory, oxidative stress reduction, and tissue regeneration. We did demonstrate in our lab that you can demonstrate it, and we published it subsequently when we cultured the cells in the acute liver failure sera. And what we showed is that the mesenchymal stromal cell co-culture was a better way to do it, rather than just isolated hepatocytes. While we were doing it, a group from China published their paper looking at intraportal transplantation of human bone marrow-derived mesenchymal stromal cells. And they have this deglactosamine model of liver failure in pigs, and what they demonstrated is if they were transplanting the pigs into the portal venous system, their survival was higher, while the ones who didn't get the interportal vein, they didn't survive. So our next clinical trial, so we have gone on to the next phase, now setting up a clinical trial. Using this technology, we identified a special molecule in our alginate which has a better function than the other, and that's intractable properties and pending at the moment, and this will be the clinical trial with the power calculation to prove the efficacy or otherwise of this treatment protocol. What we're all expecting to me to talk about is stem cells, and I have no idea about stem cells, what they do. So what is happening in stem cells is broadly is people are working on IPS technology, people are working on embryonal cell technology, and people like me, what we are waiting is bring us a bucket of cells which look like, walk like, talk like, and eat like liver cells, we will use them. Currently the IPS or ES-derived cells are not doing all this. Once they do it, I think the whole thing will change. I'm sure it will happen one day, but at this point in time, it is not there yet. So the key points, ladies and gentlemen, hemofiltration for us is a standard of care. Liver assist devices hasn't delivered what they set out to be. They haven't avoided transplantation or eliminated liver transplantation. Hepatocyte, again, like liver assist devices, maybe they're in the similar stage, and stem cell-derived hepatocytes are still, we are eagerly awaiting their arrival, which are of clinical use. And I take this opportunity to thank my group in the lab, and particularly the people who give us the money, and the organ donors, and National Health Service blood and transplant. Thank you very much.

liver failure treatment

hemofiltration

hepatocyte transplantation

liver regeneration

extracorporeal liver assist devices

cell therapies