#255 ‒ Latest therapeutics in CVD, APOE's role in Alzheimer's disease and CVD, familial hypercholesterolemia, and more | John Kastelein, M.D., Ph.D.

30:00I want to come back to this distinction when we start to talk about therapy in as much as it changes either the initial steps we take or the expected number of steps we take therapeutically. But before we do, I want to put a bow on a few other things. Can you formally state again the Dutch lipid clinic criteria? And then let me know if that sort of differs from others, because I want to make sure that, because there are different criteria for this, correct? And I think one could argue, maybe you're biased, but I might share your bias, that the Dutch criteria might be the most rigorous. Is that?

30:36There is the Simon Broome criteria, there is the WHO criteria and the Dutch lipid clinic. Now, the Dutch Lipid Clinic criteria were put together by one of my co-workers, Peter Landsberg. And this set of criteria was externally and internally validated with mutations and huge numbers. And every time you do a comparison between the Dutch and the rest, the Dutch are winning in terms of their power to predict FH. So it's not unlike Max Verstappen winning the most races also being Dutch, right? Just to clarify that. I don't know how much of a fan you are of that. I think Max Verstappen is actually a totally brilliant guy and a fantastic athlete.

31:20This is just something that you get points. So if you have a first degree relative with known premature coronary disease, you have a score of one. Or if you have a first degree relative with high LDL, you get one. Then if you have children or you have this xantometer, you get I know this is an audio, but it's a long list. And what's interesting is that if you have a mutation, you actually get eight. And a diagnosis of definite FH is above eight. So you have definite FH, probable FH, possible FH, and unlikely FH, which is great because you can divide your patient population into these categories.

32:03And as you're saying, it has a therapeutic consequence. Because if someone has definite FH, we treat from the age of six. That's in our national guidelines. And then we start treating immediately, just to give you a feel for that. let's just make sure people understand what was just said there, right? If we can, through this very rigorous diagnostic criteria that just for the record was not developed by Max Verstappen, if we can establish that a person has FH with such a high degree of certainty that we would call it definite, we would be treating an individual as young as six years of age, which means our certainty with which this person's life is at risk in as early as the third decade of their life, i.e.

32:54in their 20s, we're so sure of that, that we would do something that honestly, I think a lot of people who don't, especially if you don't really understand the pathophysiology of lipid metabolism, would think that's absolutely insane. Now, I obviously share your view, which is that's absolutely not insane. That's the only way that person's going to go on to live a long life. But I would imagine that that's a very difficult discussion to have with the parents. Well, sometimes. Yeah, yeah. I was just about to say, it would depend on what they've already experienced. Absolutely. It's all basically determined by family history.

33:28I have seen in the pediatric lipid clinic, especially in the first five to 10 years, mothers with no father anymore. some mothers in their 30s their husband was deceased she came with three children and then one child had a total cholesterol of 10 which is like 400 or something the middle child had normal cholesterol and the youngest child had a cholesterol of 300 or something and so that mother really wants her child treated yeah she knows that two of her three children inherited the genes that killed her husband at 30. Exactly. Totally unannounced. The first heart attack was the last. Terrible.

34:15One more question, actually, John, before we get to treatment. What is your best guess as to two things? One, the fraction of people with FH who do not go on to develop premature ASCVD. So we'll call this the fraction of people with FH that seem immune to the phenotype. And then of course, the more interesting question is, what would be some plausible explanations you would offer for that? So let's start with the easier question if it's knowable. We estimate based on our long-term follow-up of that large Dutch cohort that about 5 % of people escape any disease symptoms at all. So that includes coronary artery, calcium scoring, CT angios, zentalasmata, zentomas, arcus, anything.

35:12They seem completely immune to the elevated LDL cholesterol. Now I have to say that the vast majority of those are women. So that's the first thing. In men, it is extremely rare to have that. So the majority are women. And very often we discover those women when we go into the family. A child is referred, total cholesterol 380. There's no thyroid problem, no renal problem. Then we go to the father and the mother and the uncles. And then we find an aunt who's 76, has never been treated. and we measure cholesterol and she also has an LDL of 250. And then we do mutations because in Holland, the mutation screening is free.

35:58The government pays for that. So we do mutations in every one that comes to our clinic, basically free. And so then we find a mutation in her too. And there are some explanations. Very frequently, those women have very high HDL cholesterol. So perhaps some people have such an efficient reverse cholesterol transport system, that they can take care of the deposited LDL in the macrophage, on the tendons, everywhere. It's almost like the reverse cholesterol transport back to the liver is so fast that you can dump anything on them, doesn't matter. So in other words, they have something protective. Yes, they absolutely have something protective.

36:42And so they are almost never smokers. So if you're a man and you smoke and you have FH, it's a death sentence. If you're not treated in the old days or people that are not discovered right now, then really that interaction between one pack or two packs a day with LDL is the worst in terms of heart disease risk. So the women are very rarely smoking. They also almost never have diabetes. So they are thin, active women, have high HDLs and don't smoke. But we have desperately tried, we even had grants and everything to understand genetically if something may be with these people. We and no one, I think, on the planet has ever found a real good biological reason as to why some people are so resistant against LDL cholesterol.

37:36And I assume the sample size is not that large, but has anybody looked at monozygotic twins, both with FH, to see if there are differences in progression as a function of various lifestyle factors that might give us a sense of how much of that is driven by divergence in behavior. It's very interesting, by the way. There is one of the largest monozygotic twin cohorts is in Amsterdam at the other university. Doret Boomsma, a wonderful researcher, but a phage is just too rare to have enough of those monozygotic twins to make any conclusion. By the way, I'll just add something based on the discussion with Dan Rader that you referenced, that is, the HDL cholesterol story is a pretty complicated story.

38:21It's a far more complicated story than the LDL cholesterol story. The LDL cholesterol story is actually relatively straightforward. We don't know when we measure an elevated HDL cholesterol if it is a biomarker of something good that is happening, i.e. HDLs delipidating foam cells, macrophages, things like that, or if it's a bad thing. I went really down the rabbit hole three years ago on the literature of people with elevated HDL cholesterol who were developing premature ASCVD. So it posited that these people with elevated HDLC, that was actually a biomarker for very dysfunctional HDLs. Yeah, those are people with SRB1 mutations.

39:04Exactly. We found one family and published that in New England. Very interesting. These people had very high HDL, but they had premature coronary disease. But remember, the woman I'm describing is a nice, thin, elderly grandmother who's very active, jumping up and down, doesn't smoke, and her high HDL is definitely not a sign of something dysfunctional. My point being is this is such a complicated, for lack of a more productive word, it's a multifaceted problem for which there might just be in these 5 % of people, again, mostly healthy women, who seem immune to the phenotype, there's an alignment of the stars where enough other things are working in their favor that it's offsetting this damage.

39:53So let's now talk about treatment. And I want to talk about it through the lens of, are there any differences in how we treat people by sex, by age, and by level of disease at time of diagnosis. So whether we're dealing with primary prevention, and that's a term, I don't know if you would agree with me on this, but I describe primary prevention as treatment when there is not a single discernible sign of disease. Whereas I would call secondary prevention, if you have a calcium score of five, we're already in secondary prevention, even though you haven't had an event. Or if there are abnormalities on a CTA, of course.

40:34That's right. No, I completely agree with you. Primary prevention is rare. That's exactly right. So secondary prevention is, yeah, your CTA might be zero calcium score, but there's a soft plaque well inside the artery, zero luminal obstruction, doesn't matter. You have ASCVD that can be documented by the naked eye effectively, not even microscopically. So we're in the secondary prevention world. So maybe walk us through primary versus secondary prevention, men versus women, children versus adults, and take that in any order that you like as far as how you think about treatment. Let's start with the child.

41:09So the child comes to us at the age of six years is the first time a child understands what they eat. And in heterozygous FH, it's very important to start with a very healthy lifestyle early on. So we give them extensive anti-smoking training and we give them extensive dietary counseling for all the good, healthy choices and also tell them sports, physical exercise. So there's a large, okay, but that never detracts from the fact that we will start with a statin at the age of six. Before you get to that, can you give some insight or color around what type of dietary advice? Are you recommending saturated fat restriction?

41:54Because obviously saturated fats, I think in FH, you probably have a greater likelihood of exacerbating the condition. Is it true that people with FH are more sensitive to dietary saturated fat? Or does it just seem that way anecdotally to me? Yeah, that's, I think, anecdotally. The problem is that we all know that these lifestyle measures are not going to cure FH. They are just supportive, which is very different than for the general population, of course, but heterozygous FH is driven by the LDL and how long a patient is exposed to that LDL. So the earlier we can intervene, the better. Actually, it's pretty clear that if you start intervening early in LDL, you probably add 15 to 20 years to that life versus doing nothing on the medication front.

42:45So you need to start treating the child. And of course, you start with a statin. Do you have a preference for statin, John? We used to have a preference for prevastatin because it was kind of seen as the mildest and we were only allowed in the beginning to use prevastatin because we had done a two-year trial where we showed that carotid IMT in these children in a randomized trial of prevastatin versus placebo, there was a generation of progression. So we showed already in kids between eight and 18 years of age, that was a Yama paper, actually that we could stop the progression of athero when we treated them with pravastatin.

43:26And this was way back. And now there are guidelines for the American College of Pediatrics. There are European guidelines. And in some countries, they start at the age of eight. Other countries, it's six, but it's somewhere between six and eight. and then you have statin and I don't think there's much of a preference anymore for a certain statin actually some people have a preference for rosuver statin because you can dose it at two and a half milligram to start with which is a tiny pill for a kid and then you add azinamide because you don't go to goals like you do in adults but you try at least to have an LDL below 130 so 130 is kind of the number I mean there's no it's very interesting because there's no intervention evidence for this at all, like in adults.

44:12You must have seen the Fourier-Olay results that came just out two weeks ago, where even LDLs below 20 are better than below 55, are better than below 70. We don't have those data for kids. But I was going to say, I was actually kind of surprised to hear that you would have such a modest goal of 130, given that kids without FH have LDL cholesterol levels of 20 and 30. In other words, we know - That's fighting conservatism. Yes, yes, yes. No, I understand. I just want to make sure - I don't agree. I personally don't agree with the 130, like probably you don't agree, because you and I know what a healthy LDL is for your endothelium, and it's much lower than 130.

44:56But this is always a fight, where there are people that say, yeah, maybe we've done a lot of research. We've looked at growth, mental state, at learning ability. We've looked at maturation, hormones, menarche, puberty development, and we've never found any negative effect in kids of statins, but there are conservative people. And so we have more modest goals, but I think it's very diverse. There are pediatricians who treat kids more aggressively. I guess my point is it's so ironic because we have the natural experiment right in front of us, which is kids are born with an LDL cholesterol of 10 milligrams per deciliter.

45:39And as they go through puberty, it starts to go up. But during the most important periods of development, i.e., when their brain is developing, that's doing so in an environment where cholesterol is virtually undetectable. Absolutely. The first year your brain grows by far the fastest and you have basically no LDL left to go anywhere. So it's completely scientifically agree with you. And by the way, John, has the thinking changed with PCSK9 inhibitors? Because I guess you could make the argument, well, statins are impairing cholesterol synthesis and maybe in a developing child, that could be problematic.

46:23But a PCSK9 inhibitor has no bearing on cholesterol synthesis. It's simply amplifying LDL clearance. So does that change the thinking at all? Well, it changed my thinking, and I agree with you. But there are other indications, all preclinical, so pretty useless in my opinion, that in the embryonic stage, PCSK9 has a role in brain development. And there are some Mendelian randomization studies that pick up an increased signal for Alzheimer's disease with low PCSK9, while all the trials actually have never shown any effect on cognition. But of course, those arguments are again used by people that are more conservative.

47:09Listen, we have done all the trials because we have so many kids with heterozygous FH for the files of both alurochimab, evalochimab. We've done all the trials in children for heterozygous FH, also for simvastatin plus azitamide, all the statins alone. We've done Prava, we've done rosuverstatin, we've done simvastatin, and then simvastatin-azitamide, and then the PCS-canine monoclonals. And so we've done all that. And the kits are, It's so interesting. Kids are not statin intolerant. Meaning you don't see kids developing myalgias and statin-related muscle symptoms. No. Because in adults, we see that about 5 % of the time.

47:54You're saying you don't see that in kids? No, because they don't write the inserts. They don't read the inserts. Yeah, exactly. Sorry, they don't read the inserts. But do you think it's also because you're disproportionately using prevastatin, which is milder, or do you think it's true even in the presence of rosuvastatin? No, it's also true in the presence of atorvastatin and rosuvastatin. Yeah. And listen, it's very interesting because we have now a follow-up. And I think that was a New England paper. We have, of course, the kids that were 8 are now 18. Then you follow up. And the kids that were 18 are now 28.

48:32So we have compared these kids with the generation above them at that age. And none of these kids actually got a heart attack or angina or anything. And the generation above them, who didn't get any treatment, of course, because there were no statins or nothing, their early mortality, early coronary disease was rampant. So I think we've already shown, although it's not a real randomized controlled clinical trial, but it's the only way you can do this. You can't randomize a kid. Of course, you can only do observational data. Is that treating from an early age actually protects the kids against premature death and premature coronary disease.

49:16Okay. So I want to move on to another topic. But before I do, I guess the last thing I want to contrast this with is the adult, middle-aged or otherwise, who shows up either with or without disease. How aggressive do you go there? As aggressive as the guidelines tell us to go for non-FH patients. So the kids are transferred from the pediatric lipid clinic to the adult lipid clinic when they're 18, and then we immediately start with PCSK9 monoclonals and pretty soon with Inclisiran and Azidamide. And we really strive for the lowest LDL possible for that individual. And in the homozygotes, we do high-dose statin, Azidamide, Evalocumab, and now Evinacumab, the Regeneron NH-PTL3 monoclonal antibodies.

50:11So those four things constitute the state-of-the-art therapy for homozygous FH. it gets a lot of patients to relatively normal LDL levels. And that is a miracle for homozygous FH. In heterozygous FH, there are about 10 % of patients that even with triple therapy, we can't get them to a reasonable LDL. So we call these now severe heterozygous FH. They probably have more than one mutation. They probably have another mutation somewhere else that makes it a more severe phenotype. But that, we officially don't know that. Meaning these patients on PCSK9 inhibitor plus statin plus ezetimibe, you can't get below 70 milligrams per deciliter?

51:03Oh, no, we don't get them below 100 or below 120. So some of them are really, they have nasty opposition against your therapy. And it's linked, by the way, to the starting LDL. So we say that if your starting LDL is above 300, you have severe heterozygous fh, and that's about 10 % of the overall heterozygous fh population. Of course, when you start at 300 and you have a statin that takes off 45%, then another new baseline, 15 to 20, and then you can calculate that it's not that easy to get below 100. Is apheresis even a viable option? Is it easy to pull out? I know it reasonably works for LP little a, but does it work for the majority of the APOB-bearing lipoproteins?

51:52It does. We have an apheresis center in Amsterdam because we've sometimes diagnosed homozygous FH after six months in kids of six months that had a very severe homozygous FH. And then we've had two LDL apheresis kits. But I can tell you one thing, evinacumab is the golden rescue for these children. It's still only used in adults, but I hope that we will be able to use that quadruple therapy rapidly in kids also, because it obviates the need for the alapheresis in many instances. What's the frequency that it needs to be given? The drug is dosed, how frequently? It's intravenous dosing, I think, once a week.

52:38Wow. But they are working on a subcutaneous formulation of it because, of course, intravenous, but these kids are used to something. Yeah. I mean, these kids don't have veins. I mean, you're basically putting PICC lines in kids to give them treatment or something. Yeah. Wow. So there is a small but vocal cadre of people out there who kind of refuse to believe that LDL is causally related to ASCVD. or another sub-variant of this group who believe that it's only causally related to ASCVD in the context of metabolic illness, but if you're metabolically healthy, then LDL is not problematic. Based on your knowledge and experience with FH, which spans the spectrum of metabolically healthy to unhealthy people, how likely do you think that is?

53:32These patients that you mentioned before are always used as the stick to beat people like me, because they say if LDL is truly causal, it's impossible that some people don't get heart disease while having FH. I try to explain that most genetic diseases are modified by other environmental and genetic factors, but someone, these cholesterolcritics.org. It's a website of a strange bunch of people that absolutely don't want to accept that LDL cholesterol is bad. But my experience with FH, my entire career, I've seen these families. I've worked 100-hour weeks for years to find all these people. And we were able to find the largest cohort on the planet of heterozygous FH.

54:23And these stories are all heartbreaking. So for me, it is so simple because these people have one thing, one, a mutation in a single gene that doesn't do anything else than raise LDL cholesterol. And they drop dead, you know, when they're like 25. So for me, I don't even listen to it anymore. Yeah. I mean, it's the sort of Mendelian randomization also makes that clear because it doesn't just include FH. It goes all the way down. And my response to people when they point out the observation that there are some people with high LDL cholesterol or high ApoB who don't have coronary artery disease is there are also people who smoke their whole lives and don't get lung cancer.

55:05And by the way, there are people who never smoke who do get lung cancer. Neither of those facts remotely diminishes the causal case for smoking and lung cancer. I couldn't agree more, Peter. Absolutely. And I find it also because, of course, the FH argument was always the scientific argument of proving that LDL cholesterol, if it's elevated, sits at the core of atherogenesis. And then, of course, when it became clear that there were a few people among these families who could actually live without a problem, they used that as an argument. But I completely agree that smoking is a very good example, but there are many more.

55:46So let's pivot now to a class of drugs that I discussed at length with Dan Rader, the CTEP inhibitors, because you're very involved in not just the latest of these, but potentially the first one that really appears to have a shot at working. So let's back up for a second. And for folks who either don't remember the podcast with Dan or didn't hear it, let's provide an explanation of what CTEP is and maybe a little bit of the sorted past of the CTEP inhibitors and basically what is prologue to where you are now? The prologue unfortunately is very long, Peter, and it's one of the best examples of how big pharma can make big mistakes.

56:37So the CTP protein was discovered by Phil Barter, the Australian KOL that's now retired, but he discovered this protein. And I think he actually discovered it in rabbits. And why did he look in rabbits? Because if you give a rabbit egg yolk or cholesterol, that rabbit gets atherosclerosis. if you do the same diet to a mouse or to a rat or a hamster for that matter, they don't get athero. And so it was extremely interesting what made a rabbit different from other rodents. And that is CTP. So a rabbit has CTP. So all strategies to lower CTP were tested in that New Zealand white rabbits, like siRNA, gene therapy, small molecules, antibodies against CETP.

57:33And in all instances, you could cure the athero with lowering of CETP activity. At the same time, it became obvious that in Japan, there are many people with a mutation in CETP, so they have very low CETP. And initially, the first report said these people live longer than we do and they are free of coronary disease. So then loss of function of CTP became a longevity gene. It is still by many people called a longevity gene because if you don't have it, you live longer, you have less Alzheimer, you have less diabetes, and in general, you are just simply more healthy. Sort of like PCSK9 loss of function as well, also a longevity gene.

58:20Yes, you don't need that. You don't need PCSK9. The theory is that we all went through the evolutionary funnel about 10 ,000 years ago during the last ice age. There were very few humans alive, especially in the north, and they went through the evolutionary funnel where everything was directed against being thrifty. people that could absorb the last calorie out of a mammoth were favored. And actually, we now think that those genes that we selected during that evolutionary funnel are now bad for us. Like we don't need PCSK9. We don't need CETP. We probably also don't need NGPTL3. And so all of these genes were meant to conserve energy and to conserve cholesterol.

59:09And so what CTP does is a very simple thing is it grabs a cholesterol ester molecule from HDL, from an HDL particle. CTP sits on the HDL particle like a little cap. It's a curved protein that sits on top of a sphere and it has an opening and it sucks a cholesterol molecule out of HDL. then that particle collides with an LDL particle and there's a little tunnel and it spits the cholesterol ester molecule straight into LDL. So the consequence of that is that HDL cholesterol goes down and LDL cholesterol goes up which in the days of very active LDL receptor activity was a great idea because then all the cholesterol went back to the liver and a cholesterol molecule is very expensive to make.

1:00:05It costs 27 ATPs. In a nasty environment like the Ice Age, you want to conserve that molecule. And the best way of conserving a cholesterol is by sending it back to the liver and then the liver can decide what to do with it. Put it in VLDL or in bile or it can do a lot of things with it. But in a situation where our LDL receptors are not that active anymore, adding cholesterol to LDL is not a very good idea. and all the Mendelian randomization studies have shown that people with high activity of CTP have more heart disease, more heart failure, more kidney disease, more diabetes, more Alzheimer, blah, blah, blah, the whole works.

1:00:51And so Pfizer was the first to say, ah, atorvastatin's patent is going to expire within a few years. Let's have another atorvastatin. So we make a CTAP inhibitor called torsetrapib I was involved in all these large trials. So I was on the steering committee of the Eversetrapib trial on all of these trials, except revealed. That was done by Oxford. But Pfizer did a phase two. And in the phase two, after four weeks, blood pressure went up a little bit. And then comes the big pharma mistake. Why do we care about a little blood pressure increase if HDL goes up by 70 %? They didn't understand why the blood pressure went up and they moved the drug into phase three.

1:01:38That is a fundamental mistake. If you don't understand a side effect in phase two, you don't move a drug into phase three until you've understood it. But listen, it was my fault also. I was on the executive committee of the outcome trial with Tercetrapib and we all thought, oh my God, this is going to be the next fantastic wave of drugs in cardiovascular disease. But that drug killed more people than it saved. I remember in September of 2006, exactly the street I was standing on in the financial district of San Francisco when the trial was announced that it was being halted. And I was so, I mean, to quote planes, trains, and automobiles, if I had woken up with my head sewn in the carpet, I would have been less surprised than that outcome because I I2 was so optimistic based on the HDL cholesterol increase, even though it was clear this trial was kind of a stupid land grab from an IP perspective on the part of Pfizer.

1:02:44You had to combine it with the Torvastatin and all that kind of stuff. Putting that aside, I thought this is going to be a world beater and it turned out to be an enormous failure. Then everybody was fired except the basic scientists who got the assignment to understand what happened. And it was so interesting because they took the drug and they infused it into a rat. Now, a rat does not have CTP, but the blood pressure went up in 10 minutes. Poof! So that told everyone that this is a drug that has an off-target effect. Fortunately, it has nothing to do with CTP. But the drug actually raced straight into your adrenals, where it promoted aldosterone production, cortisol production.

1:03:30It raced into your endothelial cells, where it promoted endothelin-1, which is like an angiotensin-2, terrible vasoconstrictor. And all of that led to water and sodium retention, low potassium, high blood pressure, because these were secondary prevention patients. And you don't want a drug like that in a secondary prevention patient. So that was the most unfortunate beginning of a new story in our field. The wrong drug. You know, it's so funny. This was not too long after another epic failure, but one that I would argue resulted in a drug being removed that shouldn't have been removed, but instead required a little bit more work to determine who the susceptible individuals were.

1:04:15And of course, that's the drug Vioxx. I don't know if it had the same name in Europe, but truly a remarkable COX-2 inhibitor in a league of its own makes Celebrex look like drinking water in terms of its impotence. And there's no question that there were a subset of patients in whom Vioxx slightly raised blood pressure and led to a small increase in events. But I think Merck, I think it was Merck that was the company that made Vioxx. Again, I don't want to overspeak, but I think their arrogance and refusal to act in a timely manner resulted in just the loss of a drug that I think to this day we'd be better off with than without.

1:04:54There's just something about big pharma where they are often deserving of the reputation they have. Not always, but often they are their own worst enemies. Yeah. This was a prime example of how can you make such a, I mean, I'm now a drug developer myself, so, with Michael Davidson, whom I greatly admire. The idea that we would push a drug in phase three while not understanding a side effect in phase two is just incomprehensible. And then what's interesting, and not many people realize that, Peter, then came Roche. Yes, and this is what? This was dulcetrapib. Dulcetrapib, yeah. Dulcetrapib. That drug was extremely important for all the science because that drug only raised HDL.

1:05:42It was a very weak CTP inhibitor. It only raised HDL by 30%. And there was no effect on the Kaplan-Meier curve in the cardiovascular outcome style whatsoever. And what people didn't realize, that drug was the end of the HDL hypothesis. Because there was no effect on LDL, no effect on FOB, no effect on non-HDL. but there was a 35 % raising of HDL cholesterol, and that did not translate into one less heart attack or stroke. So that ended the HDL hypothesis in a way. Yeah, and I think we would only go on afterwards to see two Mendelian randomizations. One looking at genes that raised HDL cholesterol, one looking at genes that lowered HDL cholesterol, neither were found to be causally linked to ASCBD.

1:06:33So when I hear people tell me that their high HDL cholesterol is protecting them from coronary artery disease in the presence of high LDL cholesterol, I have to restrain myself in the context of CTEP inhibitor failure number 1, 2, 3, 4, 5, 6, 7, and Mendelian randomizations that, as you said, completely fly in the face of this hypothetical belief system. But what is extremely interesting is that in those days, we didn't understand that you really need to lower LDL cholesterol with a CTP inhibitor to see an effect on ACVD. So Merck then actually was the third. They got a drug called Anacetrapib.

1:07:18And by that time, people weren't sure anymore whether they had to power an outcome trial on the basis of the HDL cholesterol increase or the LDL. And Merck said to Oxford, just make the trial large enough. And they did a 30 ,000 patient trial, which is, I think, until now, still the largest cardiovascular outcome trial ever. 30 ,000 patients. But they overestimated the LDL lowering. The drug only lowered LDL by 17%. The baseline LDL in that trial was 60 milligram per deciliter. so the absolute LDL was 11 milligram per deciliter that predicts if you put it on the matter line that predicted a nine percent reduction in mace and that's exactly what they got so that trial validated that CTP inhibition only lowers heart attacks by virtue of its LDL lowering and that it answers to the same law as statins, azitamide, and PCSK9 monoclonals.

1:08:26It is on the same regression line. So then Michael and I understood that what we needed to find was a CTAP inhibitor that didn't have the off-target effect of torsetrapib, that was way more potent than dalsetrapib, and lowered LDL robustly so we could repeat the anacetrapib trial, but then with a much bigger effect size. And we found that drug at Mitsubishi. Before we talk about Obisetribib, I want to kind of highlight two things you said. The first is you have literally provided the most lucid evolutionary explanation for our species-wide transition to the preservation of ApoB. I have to be honest with you, I had never heard it explained the way you did, and it makes so much more sense than any other kind of teleologic or evolutionary explanation.

1:09:24So I just want to make sure I heard it correctly because I'm going to use it often. I'm going to be the most boring guy at the parties now because I'm going to use this story to explain it. It's called the thrifty gene hypothesis, is. And it's more often used to explain, for example, that people in Asia get type 2 diabetes at a much lower BMI than weak occasions because they've gone through much more famine when the rice failed, huge famine in the Far East. Yeah. And I've always been familiar with those arguments as they pertain to diabetes and obesity. I just had never taken the additional leap of, hey, I know it takes 27 ATP to make a molecule of cholesterol, but I never made the additional leap, which was think about how expensive that is.

1:10:19And in an environment that is so resource constraint, which up until 150 years ago, we were. So we spent hundreds of millions of years in an environment where preservation of resources was the second priority only after reproduction, that of course we would be so effective at LDL clearance, and therefore, of course, we would want to be in the business of shuttling as much cholesterol from HDL into LDL via RCT because we knew it was going to a good place. Back to the liver, It would be circulated as bile. We would ultimately recirculate that pool. We could make more hormones. We could digest more food stuff.

1:11:07Yeah, it all makes sense. And then lo and behold, of about an effect of like 150 years ago, it was not such a premium on that now. And we've got more than enough cholesterol to spare. And that thing, that gene that we worked for millions of years to preserve now is biting us in the ass, just like the same genes are around adiposity and insulin resistant. And there's a very interesting additional piece of evidence is that if you make a mouse look like a human heterozygous FH and you infect that mouse with bacteria and compare the results of infection with bacteria in the non-FH mouse, the FH mouse is better resistant against bacterial infection.

1:11:50And so the theory is, and we've seen that in our pedigrees is that FH in 1860 was not such a severe disease as it's right now. And maybe there is so much FH because again, in the ice age, having high LDL might have been an advantage as a protection against bacterial infection. These two things converge. Yeah. And think about how amazing that is in terms of a parallel to APOE4. Exactly. Like this is the exact same story as APOE4, which basically was the only APOE isoform we had until what, 200 ,000 years ago? Yeah, something like that, I think. Yeah. And it offered remarkable protection against infections.

1:12:38And of course, it's only today, A, with our longer life, but B, I would argue with all of the insults that come with our longer life, that APOE4 is such a predisposing factor to both cardiovascular and neurodegenerative risk factor. Okay. So first off, that was an amazing explanation of the story. So that was actually also, I think, a fantastic prologue for those who missed the discussion with Dan. So let's recap where we are with Obacibitrib, right? That's the current one? Yes. So it is how potent? I guess you've done things to - Yeah. I don't, listen, I am still a scientist and I don't want to sound like a salesman, but we were lucky finding this drug in Mitsubishi.

1:13:23Yeah. So tell us more what that means. So help folks understand how do you find a drug? How is drug discovery done? How did this thing come about? So I am a consultant to many biotech companies and sometimes companies ask me to look at the pipeline and see if there's anything good or bad in it. So I was invited by Mitsubishi to look at a number of things. And I saw the phase one data of this compound that was still called TA8995. And I saw that at one milligram, that drug lowered LDL by 27%. Now, remember, docetripib was used at 600 milligram and it didn't do anything on LDL. So I thought, holy moly, this drug is very potent.

1:14:09And then I looked at the CTP inhibition at 10 milligram, which is a dose we're now using, there's a 97 % inhibition of CTP. So it's simply in that sense, and surprisingly potent CTAP inhibitor. And that translates into an LDL lowering of about 50 % on top of high intensity statins, and an HDL increase of 165%. Now, you have to control yourself when you see those numbers, because you know that the HDL cholesterol increase is not going to do anything for heart attacks and strokes. But Michael and I are not only working on this drug to develop it, we're also scientists. And we wanted to understand all the genetic and epidemiology and Mendelian randomization data.

1:15:00And so we've gone far beyond heart disease with this drug. We're looking at Alzheimer's, age-related macular degeneration, septicemia, and diabetes. Because if you really inhibit CTP, you not only stop the transfer from cholesterol from HDL to LDL, but you completely change lipoprotein metabolism and you force the liver to produce more apoA1. And you and I can, I think, agree on the fact that apoA1 is a fantastic molecule because it is the molecule that ABCA1, the cholesterol pump on the cell membrane, recognizes and actually exports cholesterol to. So that was a first. And then we also know that if you produce lots of EpoA1, you produce lots of pre-beta HDL particles, the small HDL particles, and these particles suck cholesterol out of peripheral tissues.

1:16:06Now, in endothelial cells and macrophages, that's probably good. But what's much more interesting is that they suck cholesterol out of the beta islet cells in your pancreas. and you undoubtedly know that with life if you're a type 2 diabetic more and more of these cells die till the point that you become insulin dependent that is because of the lipotoxicity of that cell that cell takes cholesterol can't export it the sterols are oxidized they become pro-inflammatory toxic the cells go into apoptosis and they die so that is all and this is proven now for all four CTP inhibitors. So all four CTP inhibitors in their outcome trial had less diabetes in the treatment arm than in the placebo arm.

1:16:55And we've published that in a meta-analysis about half a year ago. Just to be sure I understand, John, is that because you think regardless of which of the CTEP inhibitors we saw, we were seeing more heterotypic exchange between the ApoBs and the Okay, so let's explain for people. Tell them the difference between homotypic exchange and heterotypic exchange, which is pretty easy to define, but more importantly, clinically, where these are occurring and why these are leading to the outcomes that we're about to get into. So the sequence is probably this. You stop the transfer of a cholesterol molecule going from HDL to LDL.

1:17:40That will make HDL higher by definition and LDL lower. Now, Dan has done stable isotope turnover studies and have shown that the reason for the lower LDL is that they are sweeped up by the liver. So these LDLs actually get 50 % lower, which is a huge drop because the liver upregulates and what can the liver upregulate? Their LDL receptors. At the same time, these large HDLs take EPOE on board. And as you know, EPOE is also a ligand for the LDL receptor. So the large HDL particles are also cleared by the liver. And the liver does that because it produces large amounts of EPOA1, which kind of disturbs the balance.

1:18:39So there is a new equilibrium between removal of lipoproteins by the liver and production of small HDL particles, but that only happens if you almost completely knock out CTP. So you have to really inhibit it by about 90%, which is very close to the homozygous patients in Japan. They also have half LDL and about a triple HDL and every other thing that I just described. So it's for me, and I think also for Tom Dayspin and people that really know lipids, this is extremely interesting because it is complex. It is complex, but until now, it is extremely exciting because there are two groups in the world.

1:19:25So this is the diabetes part, you know, where you suck cholesterol out of the beta cell. Then there is the septicemia part that I never really knew about. There are two groups in the world, one in Vancouver and one in Leiden, that have shown that if you're born with a loss of function variant in CTP, you are much better protected against septicemia. And not a little bit, but like mortality, big effect. Actually, there are multiple presentations this year of these two groups. And they, of course, want our drug to test it in septicemia. Let me make sure I understand something. And I want to go back and ask a question about FH to bring it back to that.

1:20:03Is there any evidence that FH patients untreated have a lower mortality due to septicemia? It's not great science, but there are some indications, but most of that work is done preclinically. So it's not as straightforward as, look, if you have high peripheral cholesterol, you have more precursor to make corticosteroids and therefore support immune function in the time of sepsis. It's much more complicated than that. No, it's way more complicated. And it's most likely has to do with the scavenging function of lipoproteins. So if your CTP is low, when you get septicemia, your HDL stays high. And it's very likely that the HDL particle functions as a sink for endotoxins and everything else.

1:21:01And so having a high HDL and a constant high HDL during septicemia is a very good thing. And I, on a recent podcast, shared that I used to witness the opposite in the ICU, which was a drop in HDL cholesterol. Yes, you always witnessed that. Yeah, and that would actually be a poor prognostic indicator. Not necessarily poor, it's just that's the normal indication. Yes, if your HDL drops like to nothing, that's a poor prognostic indicator. You are protected against that drop if your CTP activity is low. because it's like having a CTP inhibitor on board, which stops removing cholesterol from HDL. So again, this is a function issue that is not just a blind phenotype issue.

1:21:49The sort of paint-by-numbers approach to this problem is high cholesterol good because it's more precursor. No, no. It's CTP inhibition good because you have a bigger sink to dispose of toxic waste. Absolutely. That's it. So that is the septicemia part and the diabetes part. Now, the diabetes part is proven in a meta-analysis. For example, the Roche drug, and that's so interesting, the Roche drug only raises HDL. Doesn't lower LDL? No, zero. And so its protective effect against diabetes can only be connected biologically with the HDL, of course. And so it's now thought that if you raise HDL with CTP inhibition, you protect the pranqueous against apoptosis.

1:22:41It's an effect of about 16 to 20 % in the new onset type 2 diabetes between placebo and active treatment arm. So it's not like a tiny effect. It's almost as big as the negative effect of statins on diabetes, which is also at the highest dose, 15 % or so. So let's just make sure, again, we bring it back to this idea, because am I correct in saying that obocetrapibs, the first CTEP inhibitor that impacts both heterotypic and homotypic exchange? That is very hard to say, Peter, because there is not much work done with the older CTP inhibitors. Dan Rader has done a huge amount of work with the Merck and the cetrapib.

1:23:28He has, for example, shown that HDL particles from anacetrapid-treated patients have more cholesterol efflux from macrophages. He has shown that SRB1 is upregulated in the liver by CTP inhibition with that drug. So he has done a lot of that work with the other drugs. Not so much. Not so much. Okay. This is pretty exciting. A sort of student of this world would have to be forgiven for having a little bit of anxiety and fear about being too optimistic here, right? This is one of those things where we've been burned on every one of these, right? There's a track record of four or five consecutive failures, some more epic than others, perhaps none more epic than Pfizer's.

1:24:20and yet let's just say like if you were a wall street analyst trying to get your arms around this you'd have a really hard time getting excited based on how many times you've been burned but we're not stock analysts and we're really just trying to come at this through the lens of science what would you say is the greatest risk that this does not pan out so let's explain what has what we know so far. You've completed phase one. So there's no toxicities. And phase two. We've also completed phase two. And you had four phase two trials? We have one, two, three, four phase two trials. Yes. And we are now fully in phase three.

1:24:59And in fact, all our phase three trials will be fully randomized this year. So we have gone, we've pulled every plug we could. We've started the outcome trial at the same time as our lipid trials, Because we, of course, realize that what we need to show is robust LDL lowering, robust non-HDL lowering, robust APO-B lowering. And then we need to show safety. So blood should be no blood pressure effects, nothing, good safety, good tolerability. It's a tiny pill, a 10 milligram pill. And then, of course, at the end of the day, we also need in the future to show outcomes in a cardiovascular outcomes trial, of course.

1:25:39But I have to say that until now, Peter, this drug is well tolerated. We haven't seen any side effects in phase two. And it lowers LDL by half, which makes it just as potent as the injectables. But then in a 10 milligram pill, when we first saw these LDL results on top of high intensity statins, I was kind of amazed at this. Yeah, so I don't want to get too far ahead of ourselves, but I'll just assert something here, which is if this pans out to be as good in large phase three studies as it has been in phase two, the only thing that would stand in the way of this displacing every statin on the planet is cost because you'd have equal or better lipid lowering efficacy.

1:26:29And instead of having a small increase in the risk of type two diabetes, you would be patently reducing that risk. We're going to talk about the brain in a moment as well. There may even be some benefits there, but let's put that aside for the moment. So there's a lot riding on this. It's cheap, Peter. It's very cheap to make. Well, I was going to say, given that it's not an injectable monoclonal antibody, this - Yeah, it's cheap to make. And yeah, it's actually very cheap to make. I think this is public knowledge. It will cost$36 per year to make this drug at the time when you have lots of patients.

1:27:04So what's called peak sales. So the drug is cheap to make and that will allow us to price it reasonably and ethically. And that is something that Michael and I have always wanted, is to have a drug that lowers LDL, that you can use on top of statin or in a fixed dose combination with azitamide, that robustly lowers LDL and ApoB, has little or no side effects, and is easy to put in your pillbox because the vast majority of my clinic in Amsterdam, they have pillboxes and they are a bit afraid of a needle and they want an extra pill to get their LDL down. So let's talk about the three trials. Just to be clear, the three phase three trials, you have Broadway, Brooklyn, and Prevail, right?

1:27:53So Broadway is 2 ,400 patients one year. it's looking at drug - ASCVD patients. ASO, these are high risk. So this is secondary prevention in people who are on their existing maximally tolerated lipid-lowering therapy and you're going placebo versus drug. It's not open label, correct? Yep. No, no, no. It's placebo-controlled, two-to-one randomization. And is the belief that this trial in 52 weeks with 2 ,400 patients is significant enough to see a difference in MACE? Or is this not powered for MACE and it's just a biomarker study? No, it's not powered for MACE. But of course, every phase three trial, whether you've alirocumab, avalocumab, incliceran, or even bampidoic acid, we are adjudicating events.

1:28:43We expect about 120 events in its entirety. And of course, we hope to see a trend. That's what you hope. Yeah, so in other words, rather than 80-40 between your two-to-one randomization, there may be a significant difference, although you're not really powered to it unless the difference is significant. We are not allowed to do statistics. The FDA does not allow to do statistics, but this is just descriptive. And exactly like Inglisiran, Inglisiran had about a 30 % difference in events, if I remember correctly. And that at least gives you an indication that it's moving in the right direction. So everyone hopes for that, of course.

1:29:23Okay. So then you've got Brooklyn, which is a very small trial, but it's also - Yes, exactly. That's in heterozygous FH, 300 patients, same thing. Same thing. And then prevails the big one, right? So that's where you've got 9 ,000 patients. And are these people with existing ASCVD as well? So it's also secondary prevention. Yes, it's hardcore ASCVD. And we've even entered a lot of risk-enhancing factors into this trial. Because we understand that the higher the risk, the easier it is to show a benefit. And we also strive for a baseline LDL of around 100. Because if your baseline LDL is around 100 and your LDL lowering is 50%, then your absolute difference is 50 milligram per deciliter.

1:30:16If you plot that on the CTT metoregression line, you have a 27 % MACE reduction, which of course you can't do because there will be people on off drug. There will be people who are going to take a PCSK9 monoclonal, but at least we are very sure that it's going to be more than 20 % MACE reduction. And aside from not having coronary artery disease, what are the exclusions for Prevail? If your LDL is below 55 milligram per deciliter. So we have adapted Prevail to the new American College of Cardiology guidelines. So one of the things that I've talked about, I think, on this podcast is that when Fourier and Odyssey were launched, especially Fourier, I was personally quite skeptical.

1:31:08Not because I didn't believe PCSK9 inhibitors would work. I really believed PCSK9 inhibitors were going to be a home run. I believed that they were going to be incredibly safe. So all these things that ended up being true, I actually believed, but I thought the trial would fail because the patients were coming in. This was also secondary prevention, so a very similar patient population to prevail. These people have an average LDL cholesterol of something like 70 milligrams per deciliter on the way in. So you're taking people who are heavily drugged to LDL cholesterol of 70 milligrams per deciliter, randomizing them to PCSK9 inhibitor versus placebo.

1:31:45My thought was you can't do that study long enough to see a difference. Well, I turned out to be completely wrong, right? That study was halted at something like 3.2 years. But do you worry about that risk here, which is you've got patients that are so heavily medicated, Yes, you're going to exclude them if they're down at 55. But look, you're going to have a lot of people at 70 milligrams per deciliter. Yeah, but it's interesting is that we have already randomized a very robust number because we expect to be ready before the end of the year with 9 ,000. So you can imagine that we already have a substantial amount of people.

1:32:20And baseline LDL is still exactly where we want it, around 100, because we have actually kind of promoted the inclusion of high LDL patients into our trial, in our discussions with the sites and everything. And what was another very big mistake of Fourier was that it was not 3.2, but it was 2.3 years. Oh, gosh. I got my numbers mixed up and it becomes even more. Yeah, it doesn't matter. It was 2.3 and that was too short. That was too short. So we've said after our last patient goes in, we at least want to have a two and a half year follow-up. So that determines that your trial, because of course, everyone before that already has follow-up and then you add another.

1:33:11So we think that we'll have a median follow-up of three and a half to four years, and that's really long enough to see the full effect of lipid lowering. Is Prevail being run in Europe and North America? Yes, absolutely. Canada, North America, South Africa, Europe, Eastern Europe, Western Europe. Any other side effects show up in phase two? Obviously, you're not seeing insulin resistance. You're not seeing any muscle soreness, any of the typical statin-related side effects. Any GI side effects, anything? Or is this truly a - Zero. I hate to sound like a salesman. And we have a paper in the Nature Medicine Lancet, and we are submitting our ROSE2, which is our fixed dose combination with azitomib to another very good journal.

1:33:58And so you look at the tables. There are no side effects. We have not identified a single side effect related to study drug until now. Now, of course, that will change because in phase three, the numbers are bigger. But in phase one and phase two, we have seven phase one studies and four phase two studies. There's nothing. By the way, which was also true for the Lilly C-Tap inhibitor, the Rose C-Tap inhibitor, and the Merck C-Tap inhibitor. There were none of these three had side effects. 60 ,000 patients in outcome trials. So the drugs, if you discount the Pfizer misery, the rest of them was very safe.

1:34:38Yeah. So in that sense, from a side effect profile, we're really moving into a world where between I mean, bempendoic acid, ezetimibe, potentially obisetripib, and PCSK9 inhibitors, you're talking about a class of drugs that don't have side effects. It's really the statins, which obviously do have side effects, although in relatively fewer people than is generally perceived, that kind of give the overall class a bit of a bad name in terms of side effects. Yeah, there's still, you know, books out there like the cholesterol myth and statins are toxic. And it's interesting because in people that really need it, like severe heterocyclic FH, you see a lot less statin intolerance than in people in primary prevention that just have it as a lifestyle drug.

1:35:30So there is a large psychological component to all of this, undoubtedly. So do we see any benefit on LP little a reduction? We do. How much? 56%. Oh, so more than with a PCSK9 inhibitor then? Yes. Okay. So say more about that. So again, Dan Rader, you know, he's my lifesaver. So Dan did a stable isotope study with anesetropib. and found out that that CTP inhibitor inhibited the synthesis of APO little a. Now, how that is possible, I have no idea at all because I cannot connect an intracellular synthesis of a protein to a drug that sits on an HDL particle in your circulation. So there must be a link that we still don't understand.

1:36:32So he, but he, that was a stable isotope study published in a good journal. He's going to do all of that again for us. So we are going to do a stable isotope study with Dan for EPOB containing lipoproteins. And we're going to look at EPO little a, but in our ROSE trial, which was, you know, nature medicine publication, LP little a went down by 56 % of the 10 milligram dose and 43 on the five milligram dose. What's interesting is that other CTP inhibitors lower LpA by about 20%, but they are about one-third of our efficacy. So it feels like the LpA lowering is in conjunction with the LDL lowering.

1:37:20But again, sorry, Peter, I can't explain this. I can't. No, no, it's amazing. And of course, for those listening with elevated LP little a, which of course is hands down the most common genetic finding that leads to premature ASCVD. I mean, if we know that there are a few thousand people that are, you know, listening to this who have FH or some trait, there's tens of thousands listening who have elevated LP little a. As impressive as a 50 % reduction is, we don't yet know if that's clinically enough to reduce outcomes. And that's where I still think I have not had Sam Tamikas on the show, but remains to be seen if the ASO inhibitors will be the lifeline there.

1:38:02So I think that we'll have to put a TBD pin in that. I want to go back to something else that we didn't talk about, but I want to just remind the audience of the Mendelian randomization. So we go back to the observation, which is that CTEP activity is largely genetic. And we've already talked about the fact that there are some people who basically are CTEP hypo-functioning individuals. These people tend to live a very long life. They have less heart disease. They have less Alzheimer's disease. They have less diabetes. Less heart failure and less renal disease also. Oh, I didn't know about the renal disease.

1:38:45Okay. So we basically say, look, in addition to APOE2 as a longevity gene, FOXO, APOC3 hypofunctioning, we can now add hypofunctioning CTEP to the list of things like hypofunctioning PCSK9. When you go and look at the MR, the Mendelian randomization, it makes it very clear that for every one microgram per milliliter decrease in a genetically determined CTEP concentration, we're going to see about a 0.1 millimole per liter reduction in LDL-C, about the same reduction in triglyceride, this enormous 2 plus nanomole per liter reduction in LP little a, the 0.2 to 0.25 millimole per liter increase in HCL cholesterol, et cetera, et cetera.

1:39:33But one of the things I never realized was you're also seeing a reduction in blood pressure, about 0.2 millimeters per mercury, which again doesn't sound like much until you realize that this is just normalized to one microgram per milliliter. You also see a 0.1, roughly, millimole per mole change in hemoglobin A1c. In other words, this is a potent antihypertensive agent as well. What in the heck explains that? Yes. So there's absolutely no explanation for that. It was, by the way, interesting because those data on blood pressure were already known at the day of torsatropeb. Which should have been a red flag to these guys, right?

1:40:16Exactly, which should have been a double red flag to these guys that if you inhibit CTP, the blood pressure increase has to come from something else. But no, no one understands the blood pressure at all. Because CTP is made by the Kupfer cells in the liver and it gets synthesized excreted. It sits on the back of an HDL particle. One in 10 HDL particles have a CTP protein on their backs. And the half-life is actually the half-life of an HDL particle, which is about a week. So it's a very simple protein. It sits in your circulation or an HDL, does what it needs to do. And how on earth it can have anything to do with blood pressure, I would surely not know.

1:41:04The relation with brain lipid metabolism, there's much more known about that now than, let's say, two years ago. So there we're making great strides in understanding how loss of function of CTP influences brain cholesterol metabolism. That's fantastic science. So I know I promised you that I wouldn't keep you up too late in the evening in Amsterdam, but I just don't see how we can end this podcast now without going down that rabbit hole a little bit. Would you grant our listeners a little bit more of your precious time that you've been generous in sharing? Okay. Thank you, John. So if you look at late onset sporadic Alzheimer, which is the vast majority of Alzheimer patients, 65 % have an EPOE4 molecule on board.

1:41:53They're either E4, E4, or E3, E4, or E2, E4. So they have an E4. If you have E4, E4, your risk for Alzheimer's is 16 times higher than when you have E3, E3. And if you have E3, E4, your risk is about four and a half times higher. We now begin to understand why carriership of EpoE4 is so bad for the brain. EpoE4 is an insufficient molecule to get cholesterol out of cells in the brain that have too much or bring cholesterol to cells in the brain that have too little. So it fails on both accounts. It is not a good acceptor of cholesterol and it's a bad bringer of cholesterol. And so if you are E4 and those cholesterol abnormalities accumulate over life, You actually get sterile accumulation in neurons.

1:42:51And if you have sterols in a cell too long, they get oxidized. I mean, everything oxidizes in us. We rust like anything else. If we are exposed to oxygen, we get oxysterols. Oxysterols are what kills cells. It gives a pro-inflammatory signal. It drives cells into apoptosis. It is the worst thing that you can have. Now, an oxysterol is not much of a problem if you have a functioning particle in your brain that sucks the sterols out of the cell and then converts it to 24-hydroxycholesterol, and that gets through the blood-brain barrier to your liver, up into bile, and it's gone. that whole normal process where the brain needs a lot of cholesterol for myelinization, building synapses, building these sprouts.

1:43:43And at the same time, if they have too much, they want to get rid of it fast in order not to get is all wrong in an ApoE4 carrier. So what is the protein that can help here? It's only one protein, ApoA1. EpoA1 can take over all these functions of EpoE4. And how do you get EpoA1 in the brain? By raising it substantially in the circulation. Because unlike EpoE, EpoA1 can get through the blood-brain barrier because it's small enough and there's very likely a specific receptor that actually pushes it through brain cells. and which drugs do raise EpoA1 by far the most CETP inhibitors. So that is the connection.

1:44:35So these large HDLs, they acquire EpoE because they get larger and they lose their EpoA1. And we think, and we have preclinical already some evidence for that, but we're doing a trial in humans where we tap CSF to look at EpoA1 that if your EpoE1 concentration goes up in the circulation enough, you'll push it into your brain. And once it's in the brain, it takes over the function of this dysfunctional EpoE4. It's a fantastic story, Peter. The nomenclature makes this complicated. So I guess let's make sure people understand what we're talking about. It's always a problem when the gene and the protein have the exact same name, right?

1:45:19At least with LP little a, we have the LPA gene and then LP little a, the lipoprotein. But here, it's the same name. We denote it differently, right? We use all caps versus not and italics and all that. But when we talk about the APOE4 gene having these three isoforms, two, three, and four, you're going to get six different combinations of them. But what you're talking about is the protein. You're talking about the thing that is made by the gene. Again, what's just another remarkable insight into the complexity of biology is the protein ApoE, which is not designated 2, 3, or 4. It's just the protein ApoE.

1:45:57The one that is made, that is transcribed and translated by the E4 isoform, I believe it only differs in one amino acid from the wild type. It's a very subtle difference, right? It is. It is an arginine for a glutamine, I think, or something at amino acid 152 or 118. I can never remember. I always thought it was 127. So yeah, one of us is wrong, but yeah, whatever. Yeah. Well, I'm sure I'm wrong. It's only one amino acid. And that changes the three-dimensional conformation of that protein completely and makes it basically a lousy cholesterol acceptor and transporter. If you carry an E4, you have like a list this long of things that go wrong in your brain.

1:46:44It's pro-inflammatory. It is insufficient in lipoprotein metabolism. It is no longer a chaperone for beta amyloid. It's like a very long list. And it's terrible for people that have E4. I think one of these Hollywood actors actually knows that he is an E4 carrier. He's that athlete. Chris Hemsworth. Yeah, he disclosed this during the Limitless series, which I think was really valuable for a lot of people to see, to bring a lot of awareness to this. And by the way, I've made the point many times that it is very valuable for someone like Chris to know that he has APOE4 because the earlier you take steps to prevent the exacerbating risk factors, the better your odds.

1:47:33I mean, the one thing that's important to point out, you opened this discussion by explaining the risk factors. I look at data that suggests it's a little less than 16 and fourfold. I think it's sort of maybe closer to 10-fold and two-fold, but we don't need to worry about that. There's no question it's high risk, but it's important to note it's not deterministic. No, it's not deterministic. And in fact, it's far less penetrant than FH is. Yes, it is. There are lots of people walking around with APOE4 that are not getting Alzheimer's disease. And as you pointed out, a third of people with Alzheimer's disease don't have ApoE4.

1:48:04So everything we're talking about here is fair game to everybody. What you said that's interesting that I didn't realize until today was ApoA1 can traverse the blood-brain barrier. And therefore, if you have a therapy that raises ApoA1, you can potentially offset the damage of a defective ApoE in response to APOE4. Do we have a sense for many of the preclinical work that's been done, or even the early clinical work that's been done, what the magnitude of that can look like? Where I'm going with this, of course, is how could we begin to quantify the potential benefit of this? Is this something where, as you probably know, Mike Davidson also very involved in the clotho space, right?

1:48:55Also very involved in looking at the observation that those with clotho KLVS variants seem to have almost complete protection from their APOE4 gene. It's a remarkable finding. Yeah. So there is one large Mendelian randomization study in E4 carriers and which gene can protect you, loss of function of CTP. So we already have mandatin randomization data in humans that low CTP protects an APOE4 carrier against Alzheimer's. But what you just asked me is, can we have an effect size here? We are doing a proof of concept trial where we tap cerebrospinal fluid and we look at, I mean, like 50 biomarkers, because what we hope is that EPOA1 goes up in the brain.

1:49:51That the consequence of that is that the cells are going to normally synthesize cholesterol. So the desmosterol and lactosterol levels should go up because cholesterol synthesis is normalized again. At the same time, 24-hydroxycholesterol should also go up because cells are getting rid of cholesterol in a normal fashion, and the inflammation biomarker should go down because you basically substitute for this dysfunctional E4. So in order to understand the effect size, we need to be able to make a story where we say dysfunctional E4 replaced by A1, normal cholesterol synthesis is on again. normal cholesterol removal is on again.

1:50:39Inflammation goes down. And of course, this proof of concept trial is only six months. So there won't be much in Alzheimer biomarkers. But if we can show that we improve lipoprotein metabolism in the brain, it is a first step into a fascinating journey, I would say. Will you be measuring desmostrol and lithosterol in any of these trials or in all of them? Yes. Yes. No, in the Alzheimer trial, we do. Which is prevail is the one where you're going to... No, no. The Alzheimer trial has no name. The Alzheimer trial is a trial in... Oh, it's a fourth trial. It's a fourth trial. It's a fourth trial.

1:51:14Yes, it's a fourth trial. But we have no name for it because it's in a single center in Amsterdam and it's a large Alzheimer's center where we have basically mild cognitive impairment with the diagnosis of Alzheimer's. So early Alzheimer's, we give them our drug, and we serially measure both blood and CSF by spinal taps. I got it. But you will not be in Prevail, for example. Will you be looking at lithosterol, desmostrol levels? We have a lot of spare tubes. So that is definitely something that, you know, we have quite a wish list of things where we want to look at. Yeah. Well, inquiring knuckleheads like me want to know.

1:51:55So let's close out our discussion, John, with an explanation of the role of APOE in cardiovascular disease. Because APOE gets a lot of attention for what it's doing in the brain. We just had a pretty brief but insightful discussion on that. But I think people are less clear on the relationship of APOE in the heart. So what can we say about atherosclerosis and APOE? Again, EPOE4 might have been wonderful during the ice age, but now it's bad because it's associated with higher LDL. It's associated with a more pro-inflammatory state, and it's associated with more heart disease. Now, if people have a hard time believing how it's possible that when you're in E4 that you have higher LDL, The explanation is that EPOE, of course, sits on VLDL and on VLDL remnants.

1:52:51And EPOE4 actually is a better ligand for the LDL receptor. It's an amazing story, the EPOE story. Better ligand than E3E2. And so, especially the chylomicron remnants and the VLDL, so IDL races into your liver, and that will downregulate the LDL receptor, and therefore LDL goes up. Sorry, just to make sure folks understand that, because it's a bit counterintuitive, right? This is a little bit of a paradox. It is totally counterintuitive. If you have APOE4 and it's a higher affinity ligand for the LDL receptor, it should mean that APOE4-generated proteins lead to more rapid clearance of LDL. But if I understand you correctly - No, not LDL.

1:53:39There's no APOE on LDL. Yes. Okay. Sorry. Sorry. Yeah. It's all on the remnants and on VLDL. I see. And that's the point. So because it's the remnants and the VLDL, those readily get attracted, downregulate the LDL receptor. So there's less LDL receptor to get rid of LDL. Is that the chain of events? That's the chain of events. But there's much more to E4 than just a little lipoprotein metabolism. There's, enough association with other things that you don't want. I mean, this chronic pro-inflammatory state that people like Paul Ritker, you know, has made his life's work of is also associated with an E4 carriership.

1:54:25And does the association with APOE4 in terms of risk vanish once you normalize for APOB, or is there still residual risk based on these other factors such as inflammation that exist and persist once you've normalized for APO3 versus APO4 in the context of the same LDL-C slash APOB? If you ask Alan Snyderman, he'll say that if you control for APOB, everything falls away. And that it's the number of, you can also look at NMR, LDL particles, but that APOB is really what drives all our statistics in cardiovascular disease. It's very interesting. For example, you've seen the bampoic acid data. So the clear outcomes trial was on the line of absolute LDL lowering versus MACE reduction.

1:55:21So where is the room or the space for the CRP lowering effect if you're on the line? And I really admire Alan Niemann. He has been stoic in his emphasis on FOB for so long. And finally, he gets this kind of vindication now where most people would agree that non-HDL and EPO-B are a better prognostic marker and a better measure of therapy than LDL cholesterol by the Friedewald formula. But if you ask me personally, Peter, I think that EPO-E4 has a few properties that you cannot completely knock out statistically or biologically with EPO-B lowering. Yeah, I don't think that's a big stretch. And it might be that Alan's view and that view are not completely at odds, right?

1:56:09It could be that those other effects are small enough that on a clinical level, you would need really large sample sizes and lifetime exposure metrics to see the difference. It also could be that there are other amplifying features. In other words, if you take two healthy people, one E4, one E3, with the same ApoB, the risk is relatively similar. But if you take two unhealthy people, type 2 diabetes, NAFLD, profound insulin resistance, one E4, one E3 with the same ApoB, it could be that those other factors create more of a gap between those people on a Kaplan-Meier curve. I think that that's very reasonable.

1:56:55And I tend in that same direction. I mean, I think for me, part of the takeaway here is just that I'm trying hard not to be excited, I think that, as I said before, when you're once bitten, twice shy, and I've been a very vocal critic of these trials, meaning the last 15 years of CTEP inhibitors. And I would say that perhaps I've been too harsh and I've been mostly a critic of the HDL hypothesis and I've relied on the CTEP story along with the Mendelian randomization as my rationale for that. And in reality, I think that that has created a little bit of a blind spot in my eye towards a more pure biologic understanding.

1:57:42And of course, I'm the last person who should have this blind spot because part of my interest is in longevity. And it's clear to me that CTEP is a longevity gene, meaning the hypofunctioning variant is as much a longevity gene, if not more, than the hypofunctioning PCSK9. So it's with the tincture of embarrassment that this is sort of my mea culpa to say, I have been too down on CTEP inhibitors, and I'm very hopeful that Obisetrabib not only redeems the field, but also gives me something to be excited about clinically in my practice. Thank you very much for that. And to be quite frank, I was much like you for a long time.

1:58:20And it is, you know, you have to imagine the kind of turnaround you have to make in your head. I was a strong believer in the HDL hypothesis until the Roche trial. Then I had to turn around away from HDL back to LDL and FOB and then understand the blood pressure effect, understand the mistakes in the trials, try to find a drug that didn't have all that baggage. My first New England paper was in 1987. What we did is we measured CTP activity in a coronary angiography trial. In those days, we only had coronary angiography with pravastatin. And it was, if you had high CTP activity, you had the worst progression of coronary disease over that two-year trial.

1:59:05So that taught me that CTP was bad. And since that day, I'd wanted to find an inhibitor that was something that I could work with. It's such a beautiful story. You've articulated it very well, John. And I do hope that at some point you find the time to write kind of a clinical philosophical paper about science, which is we lost our way, we as the field, meaning I'm just observing the field, but the field lost its way in this drug based on the wrong biomarker. We were using HDLC as a biomarker because we didn't have a better biomarker for CTEP inhibition. And so the initial insight in the biology was right, but by having the wrong biomarker and failing to understand the mechanism of that, 15 years and I don't know how many billions of dollars, but it's probably approaching 10 billion, if not more dollars, were wasted.

2:00:10And tragically, by the way, it should be noted, some lives were lost. We don't want to lose sight of the fact that this resulted in lives lost during these clinical trials. And that's the price we do pay as a society to advance knowledge. I don't want to suggest that you can never have a loss of life in a clinical trial, but it's just a sobering story. But if you would forget about everything that happened before, what we want nowadays is strong Mandelian randomization evidence. we want lots of phase one and phase two trials to show that there are no some bizarre side effects and then you gently go into a phase three with a dsmb and everything and you don't do a phase two where you see your blood pressure affecting you say oh what the hell and you know that would not be possible today anymore fortunately and that's a good thing yeah yes well john i'm really glad that Tom reached out to you to ask if you'd be willing to be on this podcast.

2:01:09I'm grateful that you were able to accommodate my time today. And thank you again for the work you're doing. And I'm very excited to follow this story as will the other listeners today. We'll have data on Alzheimer in the summer. Really thrilled to understand what's happening in the brain. Yeah. Fantastic. Okay. Thank you so much, John. Have a great evening. Have a great weekend, Peter. Thank you for listening to this week's episode of The Drive. If you're interested in diving deeper into any topics we discuss, we've created a membership program that allows us to bring you more in-depth exclusive content without relying on paid ads.

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2:04:10Thank you.