Knock-in mice aid Alzheimer’s treatment
Knock-in mice aid Alzheimer’s treatment
Behind the Science – Ozgene podcast
How far are we from effectively treating or curing Alzheimer’s? In this podcast episode, we chat with Prof. John Mamo from Curtin University about a groundbreaking study that has discovered a possible cause of Alzheimer’s disease with new prevention and treatment possibilities.
- Becoming a scientist (00:21)
- Early research – from Physiology to Alzheimer’s (04:18)
- What is Alzheimer’s disease? (08:26)
- What causes Altzheimer’s disease? (09:31)
- Alzheimer’s, capillary vessels and amyloid (13:50)
- Unique mouse model producing human amyloid in the liver (21:43)
- Research process and experiments (24:24)
- Clinical trial for potential Alzheimer’s treatment (30:00)
- Further preclinical Alzheimer’s studies with mice (32:15)
- Potential Alzheimer’s drug, Probucol (34:32)
- Future of Alzheimer’s or dementia treatment (38:22)
- Alzheimer’s research opportunities (40:58)
- Will there be a cure for Alzheimer’s? (44:10)
- Alzheimer’s clinical trial details (45:16)
Becoming a scientist (00:21-04:18)
Welcome to the inaugural episode of the Ozgene podcast. I’m one of the hosts coaching and joining me is my co-host, Maarit.
And today the guest for the show, we have the director of the Curtin Health, Innovation and Research Institute, John Mamo. Welcome to the show.
Well, thanks very much and I didn’t realise I was so privileged to be launching your inaugural podcast. So great to be here.
Happy to have you on the show.
OK, well why don’t we get straight into it then? So, John you’ve been a scientist obviously a number of years, but how did it all start for you? When did you first realise that you want to become a scientist?
Well, I’d have to say it probably was back in the primary school days, as it turned out I have really high affinity for animals and volunteered my services to the RSPCA. So as to sit there and mend birds with broken wings and so forth. Just had this real interest in terms of the health and well-being of animals back then, and really wanted to be a vet. But I was born and raised in South Australia and at the time no veterinary courses available. So I thought, well, what am I going to do? I was too young, didn’t want to leave home and decided I might go into the science arena, but focus on Physiology. I remember bringing some crazy experimental books home from the library and trying a few things at home like yeah, that’s where it just grew from, essentially.
Wow, now I’m curious. So, what were some of those things you tried at home then?
Probably a little bit embarrassed to say a few of them, but let’s go with the more mundane things about you know using lemons to get light bulbs to glow and I did have some mentorship through my mum and she was just sort of this natural physiologist and used to fix chickens and things like that. There was an avian plague going down the street here many years ago and you know, just follow her tails and do things like that. More often than not it was pretty successful. So yeah, just one thing led to another and did a little bit of work in the farming area. Domestic animal production systems and always had a real eye for animal health and well-being and their welfare, so I guess that’s where that curiosity has come from.
So, sounds like your mum played a bit of a role in you becoming a scientist, but was there anyone else who sort of inspired you along the way?
I just give another shout out to Mom actually because she was a post war immigrant with my dad came from Malta and unfortunately never had the opportunity to have an education that I had, but she was this natural physiologist. A really wise woman and that, I think, was important to me. And I lost my dad when I was very young, so she was a very big influence in my life.
I think the major or contemporary people that have impacted, was opportunities I’ve had to spend some time with wonderful Australian Nobel Prize laureates but had the opportunity to host Peter Doherty for a day in WA, and we spent the whole day together looking at the science and chatting about science. And, you know, I sort of asked him how he got through the crunch times, and he said, it’s really quite simple. Dance to the beat of your own drum. If you’ve got a research line of inquiry and you if really believe in it, just follow through with that.
And the other person that has had a big impact on me by way of research thinking, has been Barry Marshall and Robin Warren. Of course, they won the Nobel Prize in Medicine Physiology for their pioneering studies in Helicobacter pylori and its links with ulcers. Yet the two never had any NHMRC grants that were supporting the line of research inquiry. But again, just total commitment, total belief. Our total persistence, you know diligence and you know. Western Australian born and bred. I mean it’s just exemplary.
I come across Barry periodically and he came to a retreat at the institute just last week and gave an inspiring talk to our early career researchers. So just to see people who you are so good at their craft, and you know I can sort of consider them as the sort of lifelong Olympians they just keep at it, and they just keep making the team. They have this extraordinary success, and then they give themselves to others. You know, they’re keen to be mentors to others, so they’re the people that have really inspired me.
Early research – from Physiology to Alzheimer’s (04:18-08:26)
That’s a really fascinating story, especially coming across all of these people and how they’ve influenced your research thinking. So I wonder, coming from Physiology and talking to these people, particularly as you said, the Nobel laureates, how did you go from Physiology into more sort of the field of Alzheimer’s disease research? Specifically, how did you find yourself in that position or what was it that you chased?
Yeah, OK, so it’s. It’s a bit of a journey. My earlier research interests were principally in the cardiovascular arena, and I had some difficulty fully accepting the dogma of the day, which is that heart disease is simply a reflection of disturbances in metabolism. Cholesterol is associated with low-density lipoprotein and the reason I say that is because nowadays about 80% of people who end up in a cardiac ward probably have pretty much normal cholesterol levels, so there’s another part of the jigsaw puzzle that’s not clear there. And I had invested my formative years of research looking at how lipoproteins these are the fat carrying articles that are made by the body to transport fats in blood, how lipoproteins which are made by the small intestine might be involved in heart disease ideology? And small intestine basically is responsible for packaging the dietary fats that you ingest, and the dogma of the day was that the particles made by the small intestine don’t directly contribute cholesterol to the arterial wall and to sort of summarise 15 – 20 years of research in a minute or two, we basically provided equivocal evidence.
We developed the first methods to look at lipoprotein cholesterol penetration and retention in the arterial vessels, and sure enough there was plenty of these particles that were coming from the small intestine, so you know that led to some other studies by other organisations, including pharmaceutical companies, to develop drugs to influence that, and the journey into our Alzheimer’s disease on that foundation and some epidemiology studies. So there’s some great studies, the Rotterdam study is one and they showed that, interestingly, there was a really strong association for risk for Alzheimer’s disease in people that were consuming diets that were enriched in the types of fats that normally we associate with cardiovascular disease. And there was a lot of uncertainty in terms of what’s the trigger for Alzheimer’s? Where’s this platforming? And I came up with this concept that might there be a relationship in terms of what’s happening outside of the brain with the trigger for the disease. Because when you look at the pathophysiology of Alzheimer’s, it’s really complex and I don’t think it had been completely disentangled properly, so that’s where that journey started.
Roughly, what year has that been because the gut brain axis is quite a new angle of research, that’s very much in its infancy, so I was just wondering if you could kind of contextualise when you were thinking that maybe Alzheimer’s cause lies outside of the CNS?
Yeah, it was it. It probably started about 15 years ago or so and we weren’t focusing on anything to do with the microbiome, which is a really hot topic nowadays. And that’s probably a different line of Interplay there, but what I wanted to explore with the early career team that I had at the time, and many of them are still with me. As it turns out, what I wanted to explore is the possibility that there might be an interplay between the metabolism effects as they transported through the blood with these lipoproteins, and where there might be an interplay with the axis where blood is essentially making itself available to brain, because the brain needs lots of blood as energy. We had some ideas that disturbances in lipid metabolism might be compromising the vasculature of the brain and serving as a risk factor for the disease.
So, with that in mind, we’ve come from your journey, took you through cardiovascular disease, and then specifically in lipid and fat metabolism, then moved on to this concept where there’s a link between lipid metabolism or lipid diets, and potentially Alzheimer’s disease. I wonder if you could give us some context on what Alzheimer’s actually is for viewers who might not be so familiar with neurodegeneration.
What is Alzheimer’s disease? (08:26-09:31)
Sure, often people who don’t have scientific backgrounds kind of coin Alzheimer’s and dementia as one and the same thing. It’s not quite right. Dementia is sort of a motherhood descriptor which is characterising people who are suffering from memory impairment, and Alzheimer’s is the most common form for dementia. Probably about 70 to 80% of people who get dementia have Alzheimer’s as that sort of pathway to reaching that state. So, internationally in terms of wanting to reduce the global burden of disease, focus has been in Alzheimer’s, because that’s theoretically we’re going to get most impact. That’s not to say that other forms of dementia through other pathways aren’t just as important, and certainly for the person experiencing the disease critically important. But we can learn from the various versions of Alzheimer’s, and again, you know when you think of cardiovascular disease, some people have high cholesterol, some have high blood pressure, some have other lifestyle factors. They smoke or they’re sedentary. Or they have diabetes. All risk factors for cardiovascular, and here we’re talking about Alzheimer’s, that there might be something specific that’s causing a particular type of the dementia phenotype. So Alzheimer’s is basically the main form of memory loss.
What causes Altzheimer’s disease? (09:31-13:50)
So with Alzheimer’s disease itself as you said, there’s a couple of different forms. As I also understand that there’s multiple different hypotheses about what brings about Alzheimer’s. Specifically, I was wondering if you could comment on is Alzheimer’s disease an accelerated ageing process, or is there a genetic cause and age seems to be a risk factor or kind of concomitant to genetic cause.
It’s probably a bit of both. I mean, population studies clearly show that age is certainly associated with Alzheimer’s disease risk. But having said that, that doesn’t tell us a lot about what might be the potential pathways for getting you there, and this is something we really need to identify, because if we’re going to come up with strategies which will prevent risk for developing the disease or slow the progression of it, we need to know what we’re dealing with. Are we lowering cholesterol? Are we lowering high blood pressure? There is the analogy once again. I think of the many thousands of studies that are being done over the decades, I think in some ways we’ve been a little bit confounded in the context that the focus has been on pathophysiology. So you know 110 years ago, Alois Alzheimer made these very elegant illustrations of what’s occurring in Alzheimer’s subjects brain, and the focus has been on the material that’s developing in there, and because the brain is considered essentially an isolated organ from the rest of the body and the focus has been that disease is occurring within the brain itself, and I think I would say, you know, if I had to guess that probably 80% of the research for the last two or three decades has been focusing on what’s occurring within the brain that might be accelerating the cause or triggering this process. And then we’ve got people who study dementia and they’re looking at the other main form, which is vascular-based dementias.
And when people talk about vascular based dementia, they’re often talking about people experiencing stroke, for example, but we know in subjects that have Alzheimer’s, they too have almost without exception, disturbance in the vascular architecture that’s occurring. So then the question is, what’s the horse? What’s the cart? And there’s certainly very hot debate of whether Alzheimer’s pathology that we commonly consider that see plaque, those protein aggregates, forming in the brain. Is that the first thing that happens or is that a consequence and there’s the vascular aberrations occurring beforehand. The studies we’ve got from clinical evidence, is that vascular disturbances occurred decades before, so I think there’s a bit of a shift in people in this field that certainly that there’s acceptance that vascular biology is a big part of the player. But I think there’s a shift that this may be more associated with cause or amplification of that decline process?
It’s interesting to have seen that shift, Alois Alzheimer was very reasonable in the deduction that you have these plagues, and this was what must be causing the disease. But without further research, we would have never known that that’s actually scar tissue, which is where the field is leaning to at the moment, as opposed to the wound or the injury or the injury process. I think it’s a very challenging jigsaw to fit together, particularly when you start as you said, Cart and Horse. Which one comes first? Yeah, and one of the challenges in molecular biology, just very broadly speaking, as we talked about just before we started, was the concept that every time you look at anything in molecular biology, you kind of ignore time. You take a very narrow slice in time to look at something like this. And when you look at it, how do you know which is progressing which form?
Yep, so very true. I think you know, in Alzheimer’s, there’s been nearly 400 clinical trials, and all of those trials are focused on essentially 3 different opportunities. One is to either prevent the accumulation of the material occurring, one is to support communication between brain cell types. And a fairly recent announcement is using antibody systems to try and chisel away the plague. To date there’s been nothing that’s shown remarkable efficacy and I think if this was a disease that was occurring principally within the brain, we may have been seeing better results. I guess the counter argument could be that, well, you’re already getting into the people at very late stage of the disease, so maybe the expectations are too high. But we certainly haven’t had the same degree of interrogation from the vascular biology side, and I think that’s deserving of consideration.
Alzheimer’s, capillary vessels and amyloid (13:50-21:43)
Do you think that’s mainly the biggest gap in our understanding of Alzheimer’s, or what in your mind would be that?
Yeah, that that is exactly right, but I want to drill it down a little bit more because I alluded to the fact that the brain is this energy demanding organ, it receives about 20% of cardiac volumes. So in humans that’s about 1000 litres of blood per day. Each passage of blood through the brain, you have about 50% of the glucose that’s removed. Even if you were just sort of sitting sedentary and thinking of nothing, so it’s a very high demand organ. And you’ve got to appreciate that the arteries and then the small arteries will branch out to this microvascular network of vessels called capillaries and the audience who don’t have scientific backgrounds, these are microscopic. You can’t see them, and the human brain probably has a 25-meter surface area of capillary vessels. So that’s quite extraordinary, and the reason for that is essentially every brain cell needs to touch the blood it needs to get the oxygen and needs to get the nutrients for that purpose. But when you think of the number of studies that have focused on the, on what controls or regulates the integrity of those capillary vessels, these are called the blood brain barrier, there are positive studies with respect to their fundamental importance, and this is not only true to Alzheimer’s, but any of the neurodegenerative diseases whether it’s multiple sclerosis, it’s relevant to conditions such as epilepsy, it could be schizophrenia or it could be mood and behaviour.
And the capillary vessels, in some ways they haven’t been investigated because one of their functional properties is that lining them are cells which are really tightly stuck together and ordinarily they will only allow the glucose and the oxygen to penetrate into the brain. And that’s why we say the brain is an isolated organ. But we’ve known for decades that under certain conditions, the integrity of those vessels can become compromised, and I guess our early hypothesis was that maybe in Alzheimer’s there’s something that’s compromising the integrity of the capillary vessels. And then we’re getting some kind of insult occurring’s, which would involve a neurovascular inflammation, and it’s that trigger that insult that’s occurring, that might be key to whether you’re getting that toxic plaque forming. So I would like to see, you know, much greater emphasis and understanding of the microvasculature in the human brain. Because we’re living longer. Because of better public health environments that we live in and because of medical interventions, so neurological disorders or diseases are going to be an increasing burden globally, requiring huge care, support from family and loved ones, and from the health care system. So I think the gaps the gap is in understanding microvasculature in the brain and I’d love to see a lot more vascular biology studies with that focus.
So how’s the research you and your team are doing? How is that filling some of that gap?
OK, so we about 15 years ago we thought we had an idea what might be potentially a trigger for insult for those capillary vessels.
So the plaque material which characterises Alzheimer’s disease is enriched in a protein called amyloid. Lots of tissues in the body can make amyloid for various reasons, but we were aware that the organs and tissues of the body that make those lipoproteins the fat-carrying particles A2 can make amyloid. And we were the first group to show that in the small intestine where you absorb dietary fats, when the lipoprotein particle is made, amyloid production is made with it, and we think it’s normal function is to direct where that fat particle goes. Does it go to muscle, or does it go to the liver? It’s kind of part of its ordinary physiological function. But what we found is this is in mouse models that when you fed mice particularly with diets that were rich in saturated fat, the amount of amyloid being produced with these lipoproteins was increased enormously.
How did they become disturbed? They essentially began to leak and the particles which were transporting the fat and the amyloid in blood were moving from blood into brain. That’s not supposed to happen, because the capillaries are supposed to prevent that from happening, and once it penetrates into the brain, it essentially gets retained or stuck on substances which normally anchor the cells together, it’s like the glue between the cells and when that occurs that then triggers inflammation. You don’t know what’s occurring, you can’t feel it, you don’t have a headache. But if that persists then you have a risk of damaging the brain cells through an oxidative stress pathway. It’s very similar again to cardiovascular disease, lipoprotein cholesterol getting stuck in the vessel wall. Similar pathway, but this time I’m not talking about the fat. I’m talking about the amyloid. We think the amyloid is the thing that’s particularly disturbing the capillary vessel. Now if that’s true, that raises the hypothesis that we developed then, could the plaque material that forms in Alzheimer’s disease be coming from outside of the brain into the brain delivery pathway? And if that’s true then we have opportunities for both prevention and treatment.
That’s a really interesting chain of information to actually piece together, and it’s a very subversive hypothesis this idea that you know it’s the amyloid plaques, the brain is an isolated organ. Of course, it can’t be isolated, it needs oxygen. It needs nutrient, waste transfer and it needs the support from the rest of the organs, and I think it’s really interesting to make that jump and put that together, especially as amyloid is produced elsewhere in the body, not just in the brain. As I know standard there’s also multiple different amyloid species that get produced depending on how it’s processed from its precursor protein. Do you think the different amyloid species have different impacts on this vascular junction?
I think they do, but we haven’t studied that in great detail yet. When we first did these fat feeding studies in mice, these are wild type mice, genetically unmanipulated and it would have been great if we were able to demonstrate plaque formation in those mice, but one of the challenges in that model is that whilst mice make amyloid very similar to humans, it’s just a little bit different. And its characteristics don’t lend itself to forming that aggregate material, so you can never prove that the plague is forming. You can never prove the disease is caused by this pathway.
And the other thing that was available to us when we were thinking about how do we extend this was that there were commercial strains of mice that had been genetically engineered by others, and they were essentially humanised. So these genetically engineered mice were producing human amyloid, and as you’d expect, great, you’d see lots of plaque formation. But these mice had expression of amyloid that was exaggerated everywhere, but particularly in the brain. So whilst pathologically they were interesting, they weren’t particularly useful for our hypothesis, which was to explore whether there’s a blood to brain pathway for disease ideology.
So I think where we were all challenged back then and when you say with amyloid isoforms within the human context, there certainly are different ones. We talk about an isoform called beta-amyloid 1-42 versus beta-amyloid 1-40 and they do have differential characteristics in terms of what’s potentially more toxic or what might have a different effect on the vascular architecture. But we haven’t delineated how that interplays with the lipoprotein metabolism as yet.
Unique mouse model producing human amyloid in the liver (21:43-24:24)
So speaking further to your research, you’ve recently published a paper in PLoS one. How does that paper fit into the grand historian? And how do you intend to move forward with your research with that paper? What was the question that you were asking?
Yeah, well, we were really interested in trying to get some stronger evidence of whether we were onto something here. And I put my hand on my heart and said, look, you know maybe the approaches to, if it was at all possible, genetically engineer mice so that they were producing human amyloid in one of these organs outside of the body, either the liver where you make lipoproteins or the small intestine. But I know nothing about genetic engineering of mice. Absolutely nothing but fortuitously, as it turns out – I’m at Curtin University and just literally, across the road is a great organisation called Ozgene. They’re Australia biggest and strongest provider of genetically engineered mice and provide mice internationally. So I was not shy in coming forward and I went over to Ozgene.
I managed to score an appointment with the CEO and I said, “Look, Frank, I know nothing about this, but can you make me a mouse that can produce human amyloid only in the liver.”
And he said “Yeah, no problems we can do that for you, John”.
He wanted to tell me how they’re going to do it. But gee whiz, it was like double Dutch to me. It’s like, “Yep, that’s great. Frank, that’s great. But when can we get them?”
They successfully did that for us. So we had these knock-in mice that were unique. No one else had made them. They’re producing human amyloid only in the liver, and we also chose a genetic play which wouldn’t make it supraphysiological so it’s just completely off the Richter scale and everything is going to develop the disease process. It’s a really subtle kind of level of expression, and when you measure the amyloid in blood, it’s pretty similar to what we’re seeing with humans, so that’s encouraging, and we essentially got these mice and aged them and just monitored what was happening with age, and we looked at a whole bundle of different things. We looked at that microvasculature integrity. We looked at leakage from blood into brain. We looked at inflammation. We looked at what’s happening with the brain cells and cut the long story short, as the hypothesis predicted. Yep, we get capillary disturbances. We’re getting the lipoprotein moving into the brain and we’re getting inflammation. And we’re getting brain cell death and the mice developed cognitive impairment. So it’s really consistent. We don’t super age the mice, so they haven’t yet formed that hardcore plug, and that’s very telling. As far as I’m concerned. because it’s saying, look, we’re seeing a really strong neurodegenerative phenotype before we’re getting the plaque. That to me it’s telling me what’s the horse and what’s the cart.
Research process and experiments (24:24-30:00)
Yeah, I remember I was reading through the paper and as you said, you’ve got quite a few different experiments that you performed from RT-qPCR to all the way through behavioural studies. Even though it was just the one, the passive shock test. One of the things that I wanted to know that’s kind of in between the lines of the paper, especially as an early career researcher myself, was how did these results start coming in?
I know that with a lot of research projects, particularly with papers, the results don’t come in necessarily the order that you want, and then you kind of have to start piercing this paper together or start piercing the story together. How did you go about doing that and was it as easy as it looks?
No, not at all, you have to be very patient. I can tell you that much. So you know our paper looked at mice that were 6, 12 and 18 months of age and with a lot of the microscopic measures that we describe in there. When you do those measures that have to be done all at once, you have to batch process them, because if you do them on different days you can get that can be a confounder with the data, so we had to wait for a lot of the studies to collect everything at 18 months of age, so that was a bit of a challenge for us.
Where I thought this was particularly interesting is when we did this brain imaging. So there’s a technique called positron emission tomography or PET scanning. Essentially, you inject the mice with a tracer or an isotope which tags the amount of amyloid that’s abundant in brain. And what excited me about this is that we generated the mice, we have them all ready and prepared, and then we took them to our neuroradiology colleagues and a shout out to them, but they were blind to treatment, so they had no idea whether the mice were control mice (genetically unmanipulated), or whether they were our genetically differentiated mice. So, they did their wizardry and did some pretty comprehensive and complex analysis and then they gave us the results and then we unblinded it. And sure enough, we saw this really marked difference between the mice that we had genetically manipulated and we could easily show that there was an age dependent accumulation of amyloid load in the brain in these genetically engineered mice, which got greater at 6, 12 and 18 months of age. And as you took sequential slices essentially little brain, it was there.
When we saw that data for the first time, I’ve got two pivotal colleagues in the team – shout out to Ryusuke Takechi and Virginie Lam, HMRC fellows in the group who did their PhD with me. We basically looked at each other and thought we might be onto something here. So that was the first real insight.
What a wonderful moment! After working together for all of those years to be able to see that data together as a team.
Ah, it was extraordinary. We had NHMRC funding for the precursor for this study and we actually won the Marshall and Warren award for what they called the most innovative and potentially transformative research. So someone liked us back then, but it was pretty hard to get funding thereafter. But it was a long journey. It was probably I would say maybe five or six years working with Ozgene by the time we commissioned the mice, had them available to us and then grew them out. They (Ozgene) have been an instrumental part of that collaborative framework, so they haven’t just been a service provider for us, they have been part of the scientific mouse behind that as well.
Well, thank you for all the kind words and we’re very grateful to be a part of that process.
What really struck me on reading this paper was that here we have this amyloid protein that’s a primary suspect for a long time, being produced outside of the brain, more or less in access to normal physiological levels in the liver – that was the genetic modification. Then you said about and demonstrated that over three to two year period, give or take that you’re seeing the symptoms, these very early molecular and slightly larger psychological impacts very early on at, as you said, physiologically, relevant levels that must have been quite rewarding to go through the hypothesis and see all those results together, especially knowing that these mice are, you know, you get them and you start your study 18 months later, you’re going to actually have the data and just trusting in your hypothesis. Did you feed those mice a high-fat diet as you said in some of your earlier work?
Yeah, we do have some studies where we have fed mice with the saturated fatty acids and it was interesting that it didn’t add much to it. So just having the exaggerated production of the amyloid at fairly physiologically relevant levels was in itself enough to cause this sort of neurodegenerative pathway, and that’s an important finding because there’s a huge body of evidence, and we know that saturated fatty acids or the fatty acid moiety can be some way cytotoxic. You know it causes oxidative stress in the mitochondria and endoplasmic reticulum and that’s not a good thing, but it didn’t have a substantial value to add to that proposal. Now that’s not to say, that in humans said we were translating to humans who were producing too much lipoprotein amyloid as a consequence of their dietary behaviour. That stopping intake wouldn’t be, you know, a good thing to do, or reducing intake wouldn’t be a good thing to do. So in this particular strain, no, it didn’t add much to that.
Clinical trial for potential Alzheimer’s treatment (30:00-32:20)
So you’ve now had this great result. This significant, some even call it ground breaking paper come out, and so what’s next now for you and your team, in light of everything you’ve found?
Well essentially, we’ve got two very major lines of inquiry from the studies we’ve done in the preclinical or the animal models that has led us fortuitously to be able to launch a clinical trial. And that’s a trial with a difference. Again, using the mouse models without spending too much time on it.
We identified an old historic drug which had some really interesting metabolic properties on that lipoprotein amyloid cascade. It’s a drug called Probucol. And we found that it suppressed the synthesis and secretion of the lipoprotein amyloid, and it protected the capillary vessels, and because it was a drug that was used in the clinical context, we know lots about it. But we took that proposition to the medical research future fund and said, look, it is an old drug. This is a pathway we think might be a really big risk factor for Alzheimer’s. There’s nothing on the horizon that we think has got efficacy and we’d like to explore. And they gave us some funding for it. So we just launched the trial in Perth just a couple of months ago in Western Australia. But we are trying to leverage the grant funding because it’s limited to see if we can twin it in other states or cities and whether that’s in Australia or internationally, doesn’t matter. We haven’t had a lot of pharma interest in it. The reason for that is because the patency position is really weak on it because there’s too much prior art, so you know it’s fair enough if they’re going to invest lots of money to investigate potential efficacy of a drug, they need to have some return on that investment, and you know until we have perhaps a new analogue.
I think the bigger imperative is we’ve got a drug. We know it’s generally well-tolerated and it’s safe. We’ve got to test it and there’s been no other studies that have investigated this potential pathway for reducing risk or progression. So what we want to see there is stabilisation of memory or cognitive performance, so that’s the clinical side.
Further preclinical Alzheimer’s studies with mice (32:15-34:32)
But I’m equally excited, if not more so, with the preclinical because we’ve got lots of work to do, if this lipoprotein pathway is part of that. Hoping our research will stimulate others to follow suit. But again, we’ve reached out to Ozgene and we said, look, there’s another really important genetic consideration that I think we have to think about in exploring this further. There’s a gene called APOE, and in humans there’s three forms of this ApoE, E2, E3 and E4. You get one gene from mum and one from dad. If you have one copy of the E4 or two copies of the E4, you’re progressively in much higher risk for developing Alzheimer’s disease.
Now, ApoE, is very much involved in the metabolism of the lipoproteins that carry the amyloid, and I don’t really understand why that 30-40 years ago, when they discovered there was a risk factor, why didn’t everyone immediately go to looking at lipoprotein metabolism? Because ApoE is always with the lipoprotein, but they didn’t.
And there’s been lots of other studies in different contexts. So, we think that there’s something unusual about the E4 versus the E3 in terms of what is it doing with the lipoprotein amyloid? Is it keeping it longer? Is it making the insult to the capillaries worse? Is it sticking to the cells for a longer period of time, is it promoting inflammation? So we’ve reached out to Ozgene and come January, we’re going to start getting our first trickle of mice, which are going to be exploring the potential into play with the ApoE gene. And that I think it’s going to be really telling for us.
Of course, we’ve got a whole bucket of potential candidate compounds, which historically have been used to modulate lipid and lipoprotein metabolism, and we’ve got a priority list and we want to kind of explore how some of them might or may not be effective, so it’s not just about looking at lipid lowering drugs. It’s about looking at regulating the metabolism of the amyloid associated with the lipoprotein, and it’s a big point of difference. So big preclinical studies are planned ahead of us.
Potential Alzheimer’s drug, Probucol (34:32-38:22)
Sounds great and yeah, really wonderful for us to be involved in all of this research with you. I’m just actually going back to that drug you mentioned, Probucol, right? And well, you mentioned it was an old drug. So what context has that actually been used previously?
So Probucol was a cholesterol lowering drug and it happens to be the most potent antioxidant that’s ever been identified. And again, about 30 years ago there was a big hypothesis, that at the core of heart disease is this oxidative stress phenomenon. And in animal models, Probucol really slowed down attenuated that atherosclerotic cascade that leads to cardiovascular disease. But it would lower cholesterol by about 20 – 25% in most people. And at the time the new generation statins were coming into the market and they were much more potent cholesterol lowering drugs.
And Probucol also reduced another form of cholesterol, called HDL cholesterol, and we know that HDL cholesterol is a good thing for heart disease. It kind of protects it and the way it protects it is by stripping cholesterol out of the artery vessels. So people thought, oh, that’s not very good, Probucol is lowering HDL cholesterol, but then some years later they found the reason it lowered HDL cholesterol because it pushed it out faster, so it was actually a good thing. But anyway again, patency was out of market and there were these new drugs available. And then Probucol has got a couple of other properties which led us to that. I knew from my early days in cardio, that Probucol would accelerate the clearance of the lipoprotein amyloid from blood. So I thought that’s great. We can reduce exposure. Capillaries should be protected.
We didn’t know at that time that Probucol profoundly suppresses the production of the amyloid, so you’re not putting it out into blood, so that’s a really good thing. And I mentioned that it was an antioxidant, but it’s also a really potent modulator of a group of enzymes called heme oxygenases, and they’re pivotal to inflammation.
So here we have a drug that basically suppresses the amount of amyloid in blood – tick – it suppresses inflammation – tick – and it’s an antioxidant– tick – and it’s lipophilic. So the way it’s transported in blood has to be part of the lipoprotein moiety. So you essentially, even if some of that lipoprotein amyloid gets into the brain, you’re essentially delivering the bullet to sort of knock it out, notionally speaking.
It’s the perfect kind of trifecta for a drug candidate. Yeah, and you’ve also got delivery or targeting already built into it. I wonder has it been utilised in a clinical trial for Alzheimer’s disease?
No, so we’re the first with them. It’s really interesting. We’ve had some conversations with some other Pharmaceutical industry players. And they always come back to us and say, well, what’s your singular side? What is your singular side of point of control? And then can you produce an analogue or something similar to control that one thing. They don’t want to know about the trifecta as you put it. They want to know is that the lipoprotein secretion is at the inflammation, or is it the antioxidant?
And I say, well, the sum of the parts is probably what gives this thing more bang than potentially other things that we might be considering. Why wouldn’t you want to look at it holistically, but they want the mechanism, and I say no. I want physiological effect before I want to focus on the mechanism. So that’s been a bit of a realisation for me, beause commercialisation hasn’t historically been a big driver for us. But having said that, we are certainly trying to invest a limited amount of human resources in exploring. Are there ways that we can improve the efficiency or the efficacy of the Probucol? Can we increase availability? Can we do something smart with it? And we’ve got some interesting preliminary data down that road, but it’s early days yet.
Future of Alzheimer’s or dementia treatment (38:22-40:58)
I recall quite a few years ago now I was at a round table Q&A for neurodegeneration and one of the questions that was posed was what do you think is the future of Alzheimer’s or dementia treatment looks like? Unfortunately, I can’t recall the speaker, but they suggested that the future of Alzheimer’s disease treatment is not treatment, rather it’s prevention. You will be identified at some point to meet a certain number of risk categories, and then there’s a drug that you will take for the rest of your life, and they suggested that that was the primary reason why a lot of the clinical trials for Alzheimer’s treatments have failed. As we’ve outlined in this conversation today is that it’s kind of too late once somebody has the phenotype or has those symptoms and they undergo Alzheimer’s disease and its progression or other forms of dementia. The treatment is more or less too late at that point, you have to come in quite early. Do you think that your Probucol treatment would fall into that category, this idea that it’s kind of a lifelong supplementation once you hit a particular set of risk factors or likelihood?
Well, I think for the for the drug itself. It would be wonderful if we could at least slow cognitive decline. So I want to flatten the slope. Now that’s the new term isn’t it with COVID? I want to flatten the slope a little bit because you know most people who get Alzheimer’s disease are sort of in their later years of life. And you know, if you can even give them five years better quality of life, that’s a big thing. And it has a big impact on the family. And there’s other biological issues that come into consideration that might lead to, you know, people passing. When we do a straw poll survey of people, older age Australians and ask, what worries you most about your health and wellbeing and it always comes back at about 85%, they always say, it’s dementia. As many people who can live or face a terminal illness, cancer or heart disease, and I’m not by any means saying, you know, that’s less important disease, but people still being robbed or stripped of their dignity when their memory is going to be failing, and they know it’s coming. So my hope with any pharmacological treatment, because, as you say, the disease has already been there for 20 or 30 years is to stabilise, to stop the rot, so to speak, that would be extraordinary if we can have something that does that.
Alzheimer’s research opportunities (40:58-44:10)
By way of prevention, I think we’ve opened up a Pandora’s box. Potentially we’ve got 50 years of cardio knowledge. Is having a Mediterranean diet effective for reducing risk for Alzheimer’s? We know that people who have diabetes have a higher risk for developing dementia. We don’t know exactly whether it’s an Alzheimer’s dementia or not, but it’s certainly a vascular-based dementia and people who have diabetes or poorly controlled diabetes tend to produce more of the lipoproteins that we have identified at transporting the amyloid. So there’s ranges of drugs and interventions and things that we can do at better understanding of genetics. What’s the interplay with genetics which might give us some great opportunities by way of prevention? So I’m really quite excited, but it’s huge amount of opportunity for research to do in that space.
You know, I think of Winston Churchill and Ronald Reagan. Both smoke, drank, lived pretty long lives, I know Winston Churchill was pretty sedentary. I’m not sure about Ronald Reagan. He probably rode a few horses. Churchill got stroke. That’s what killed him in the end. But Reagan got Alzheimer’s disease. So, here’s 2 blokes who lived pretty long, why the difference? Why did one get it and not the other?
There’s lots of opportunity, the one area that I do have a little bit of concern about that we haven’t resolved yet is we’ve put forward proposition that we may have identified a risk factor, right? So if you want to assess if any intervention, whether it’s lifestyle or drug, is affording any protection you need, you need to be able to accurately quantitate and measure the risk factor. I’m talking about lipoprotein amyloid. And there’s quite a few methods that have been published and shown, and they measure something in blood. But I can tell you now, I’m not confident that any of those measures are accurate. The reason I say that is when the amyloid is part of the lipoprotein moiety, this fat-rich complex, the presence of the lipids profoundly interferes with its detection. And you normally have two types of detection techniques, one of them is an antibody technique, well it completely compromises that consideration. And the second way is an analytical technique where you put it through basically a piece of equipment which puts it through a filter, and you get the measure. They immunocapture it beforehand and I can tell you we’ve done tracer studies where we deliberately label the amyloid so we can see where it’s going and you just can’t separate it from the fat.
And these classical methods that have been published for decades. You know, this is how you separate fat, lipids or fats from protein, and the protein goes here. Well, it doesn’t. The amyloid follows the fat, and then it’s a real complicating factor for us. So, at the moment we and others measure amyloid in blood, but we can’t tell you exactly, you know, are we measuring what’s with the lipoprotein as such, is that is what we’re measuring a good surrogate marker? So, we need to sort that out because if we’re going to investigate this lipoprotein pathway for Alzheimer’s, until we get that sorted, it’s going to be challenging.
Will there be a cure for Alzheimer’s? (44:10-45:16)
So you talk about sort of preventing Alzheimer’s and then potentially slowing the disease once you’ve got it. But do you think we will ever truly have a cure for it? And what could that potentially look like?
Well, you know, expecting a cure for a disease that normally appears in someone who’s probably at the earliest in their late 60s. There are some people that get it earlier or 70s or 80s, is a big ask. There were some earlier conversation about what occurs with natural ageing and we know that with natural ageing you do get capillary dysfunction and you do get changes in the brain architecture, which you know is not like a healthy young brain. So, I think to say we’re going to have a cure is probably not really in realistic terms, something that we should be expecting. But a significant slowing down of cognitive loss would be a tremendous outcome. I think all the opportunities got to be around prevention.
Alzheimer’s clinical trial details (45:16-46:09)
Thank you so much John for your time today and all your great insights into Alzheimer’s and your research. And we hope that your clinical trials go well, and obviously benefit all of us in the future since we’re all getting old.
Well, thank you so much and maybe we can have a plug for the clinical trial. We’ve got a web address, it’s www.piastudy.com.au gives you a little bit of information, hopefully to trial, we can open up at least in Australia if not abroad. Also, many thanks to Ozgene, they’ve been really a great partner and working with us to achieve the sort of strains of mice that we need to delineate what’s occurring in this really complex disorder. So we’re very grateful for their professional services.
Thank you, John. Thanks very much for your time.