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The Long Saga of Apo-A1 Milano

It’s been six years since I titled a post “Remember Apo-A1 Milano?” If you go back even further, I wrote about the topic on this blog back in 2003 (!); scroll down to the November 11 post on that page. You can see from that one that the saga goes well back into the 1990s, but it’s really even older than that. New developments this week make the whole story wel worth examining, because it tells us a lot about both the good and bad sides of using human genetic markers to advance new therapeutics.

Here’s a brief summary. The name refers to a mutated form of the Apo-A1 lipoprotein, first identified by researchers at the University of Milan, when a patient presented from a village in northern Italy (Limone sul Garda). This person had blood chemistry that could only be described as alarming: very low HDL and elevated triglycerides. One would normally think that someone with that profile was at great cardiovascular risk, but the patient showed no signs of atherosclerosis or any other problem of the sort. It turned out that the patient had a mutated form of lipoprotein A-1, a major constituent of HDL particles. It’s a single amino acid replacement – arginine for cysteine at position 173 of the protein. Careful work with blood testing of all the residents and genealogy established that all the people with this mutation (just over 3% of the village, so maybe three dozen people in all) descend from a single person, Giovanni Pomarelli, who was born with it in the 1700s.  Further study of the population in Limone showed that the mutation did seem to have an unexpected cardioprotective effect – how else to explain the unfavorable blood profiles with the lack of the usual problems?

Well, good. What we have here is one of nature’s random shots that tells us something very interesting about cardiology and human lipid handling. A great deal of work has gone into trying to figure out what it is about the mutation that makes it different – as far as I know, current thought is that it’s both the loss of the arginine and the addition of the cysteine contribute. But the next step was to try to figure out how to turn these insights into a therapy for the large number of people in this world who are not descendants of Giovanni Pomarelli. As is so often the case, that was where things started to get really tricky. Gain-of-function mutations like this are rare, and they present a real problem for drug development. Put simply, we’re a lot better at messing things up. We can make inhibitors and antagonists, ligands that block interactions and so on, so if you have a beneficial loss-of-function mutation, we can often come up with some way to mimic that. Might be a small molecule, might be a big ol’ antibody, but one way or another, we can often take some good cracks at blocking whatever needs to be blocked.

But a mutant protein that’s actually working better poses a problem. How do you recapitulate that with a small molecule? In this case, it’s hard to see a way to do it – the only thing that seems workable is to find a way to get that new protein into a patient. Out-and-out gene therapy in mice, using a viral vector, looks impressive: forcing the mice to make the new protein that way, along with a low-cholesterol diet, led to “rapid and significant regression” of atherosclerosis, but gene therapy in humans is still in its early days, and no one is going to line up to be the first for this one. To go even further, eventually we’re going to have the nerve (and the knowledge that lets us get that nerve!) to mutate the human germ line itself, and for all I know, everyone a hundred years from now will be born with something like this mutant lipoprotein. But we’re definitely not there yet. So the brute-force approach, which is available right now, is to just give the mutant protein itself as an injection, let it circulate around in the blood and do its thing as if the body were producing it. That gives you a mixed situation that’s not found in nature – a handful of people have only Apo-A1-Milano, the rest of us have none – but it seems plausible.

And it certainly seemed plausible in animal models, that’s for sure. Infusion in both mice and rabbits seemed to reduce atheroma lesions in their blood vessels. Safety trials were done in humans with recombinant protein, and in 2003, a study funded by Esperion Therapeutics was published. Patients got five weekly treatments, and the results were positive – but were they positive enough? Esperion couldn’t produce enough of the protein for a longer trial, as I understand it, and what they saw was an increase in atheroma volume in the control group (+0.14%) and a decrease in the treatment group (-1.06%). That certainly seemed real, but you would have to have more to declare victory: a longer trial in more patients, long enough to determine real cardiovascular outcomes. Esperion could not deliver that, but they were bought up by Pfizer (for an impressive $1.25 billion dollars) around the time that work was published, and Pfizer can deliver that sort of big, expensive effort.

That did not work out. Not even a little bit. Five years later, the whole thing was a wipeout – Pfizer spun out what was left of Esperion and sold Apo-A1 Milano to The Medicines Company for $10 million, a whacking loss of greater than 99% on that deal. The Medicines Company are turnaround specialists – their thing is to buy what they feel are undervalued drug programs from larger companies and try to get them to market in some form. They’re smaller, so they don’t need multibillion dollar payouts to be successful (although I’m sure that they wouldn’t be averse).

Cerenis Therapeutics also took up the challenge. They’re a French company founded by a former research head at Esperion, and they went into the clinic with their own variation on Apo-A1 Milano HDL, but reported disappointing results. “Disappointing” is hardly even the right word: no change at all in atheroma volume between treatment groups and controls. Instead of a dose/response, they got what looks like random noise, a complete bomb. That first link has some interesting comments from cardiologists about these results – they were reluctant to think that this whole approach was in trouble, but kept holding out for better trials.

Meanwhile, the Medicines Company trial got underway, but back in August came some disquieting news. An independent committee was examining the data, and the optimistic case said that they might find a strong enough response to stop the trial early. But they recommended that it continue as planned. And now a later look at the numbers have led the company to completely stop development. That leaves the whole Apo-A1 Milano story in disarray; to the best of my knowledge, no one is is working on it, and after these results, you’d have to have a pretty compelling reason to get back in.

So what’s going on? You can come up with several explanations, not all of which exclude each other:

  1. The original cardioprotective data from Italy weren’t correct. As far as I know, though, these still stand up, so the human mutation probably is still considered to be beneficial. But there are some data from both these human patients and the animal studies that don’t quite fit the story (see here).
  2. Having the mutation protects you, for reasons yet to be figured out, but infusion of the protein itself doesn’t. That would seem to fit the facts, but it’s hard to see how that can be true, given the animal data. Either there’s something wrong with those numbers, or the split between humans and the animal models is very wide, or. . .?
  3. The human trials just weren’t done the right way. That was (as mentioned above) one explanation that was offered after the Cerenis failure, but it’s getting pretty beaten-up-looking by now. You’d have thought that there would be at least a weak signal from one of these trials, but nothing.

Back when Pfizer bought up Esperion, there were mutterings that it was all done just to take them off the market so that Lipitor could have the field all to itself. But it doesn’t look as if Apo-A1-Milano, as a drug, is able to do anyone any good at all. No one could have believed this outcome back in the early 2000s, but the numbers are the numbers, and (once again) we don’t know as much about human lipid handling and the cardiovascular system as we think we do.

Update: John Carroll of notes that CSL Behring just announced results of a trial giving extra regular plasma-derived Apo-A1 protein to patients at cardiovascular risk. After four infusions, they seem to be OK on toxicology/safety, but seem to have found no difference in efficacy in the treatment group. If they’re going to see anything, it’s going to have to be in a big, long Phase III, apparently. . .

13 comments on “The Long Saga of Apo-A1 Milano”

  1. mallam says:

    Those with the natural mutation have had it from birth, and all the Apo A1 is mutated, not just some of it. When Melano is infused, there would seem to be a problem in that the native Apo-A1 is still present, and can function normally. So the obvious question becomes how much Melano is needed to successfully compete in decreasing the normal function of native Apo-A1? Can any amount of Melano be successful at all in the presence of normally functioning native protein? With the clinical results, the answers become more and more obvious, and it’s not clear to me why this is necessarily such a surprise….the questions and situation were obvious from the get-go and the outcome was reasonably binary.

  2. Vaudaux says:

    Those with the mutation are heterozygous, so you would expect that half their ApoA1 is mutated and the other half is wild-type.

    1. David Borhani says:

      Data show that Milano carriers express only ~25% normal Apo-AI. Weisgraber et al., J. Biol. Chem. (1983) 258:2508.

  3. luysii says:

    The history of two proteins ‘known’ to cause disease — Huntingtin and SOD1 is relevant. Over 10,000 papers have been published on each, and we still don’t know why the 150 or so known mutations in SOD1 cause disease — more aggregation prone? production of a new effect? loss of an old effect? All are reasonable possibilities. Theories abound but we don’t know. The same applies to Huntingtin, we’re not sure why it is so needed by a few brain neurons when it is present in every cell outside the brain, which act nearly normally despite containing the same mutation.

  4. bk says:

    Another twist to this story involved the fraudulent trading of Esperion stock by Durus hedge fund (Scott Sacane) in 2003. They accumulated large positions without reporting to SEC. Googling around, one can find this story in a hedge fund operations text book. Always wondered if Durus sold it shares prior to Pfizer buyout.

  5. z says:

    Maybe a good target for Crispr?

    1. Barry says:

      yes, CRISPR has people dusting off a lot of gene-therapy dreams from twenty years ago.

  6. Paula Lario says:

    One has to wonder if the success in the animal model is due to a different MOA (housekeeping and/or immune response?) and that the formulated dimeric protein is not functional in the same way as the endogenously expressed protein. I have no background with this work or this protein, but a quick glance at the structure Apo A1 sends red flags as to its conformational complexity and stability.

  7. Gunter says:

    The melano story has the problem that associations arent necessarily causal. As a thought experiment hypothesise that this mutation actually kills off people who are vulnerable to heart disease before they have a chance to reproduce? Then some other factor is responsible for the low cardiovascular disease incidence among those who survived and giving the mutated apoA1 could even have the opposite result.

    1. loupgarous says:

      I like the thought experiment hypothesis per se, but for it to work you’d have to consider genetically lethal mutations that attack those at enhanced risk for dying later in life from heart attack. Such an animal would be the polar opposite of apo-a1-Milano.

      That opposite case’s more likely – like the familial hypercholesterolemia which pops up in Quebec, which wasn’t close to genetically lethal until the carriers moved to affluent, agricultural North America from places in Europe where over the centuries, rich food’s been too expensive to overeat. So in Quebec, home of poutine, that gene actually gets a chance to do its stuff, making kids really morbidly obese, and in extreme cases not passing that gene on.

  8. Trumpistan says:

    Derek loew

  9. Bertrand says:

    In fact there were further development after Cerenis phase II failure (the link you provide).
    After that, there was another post-hoc analysis at SAHMRI, Sthepen J. Nicholls, who is also the investigator of the MILANO-PILOT study from The Medicine Company. The new analysis found that on patients with high baseline PAV (>30%), the p-value would be ‘statistically significant’, even if I don’t like to use this misleading expression (p-value 0.038 on N=69 / 58 pbo vs 3mg arm). Another intermediate study demonstrated that 3mg was the optimal dose (LOCATION). You can find these data here:
    So Cerenis has another try on this sub-population, also doubling or so the number of infusions, hoping for a clearer signal (NCT02484378).
    Enrollment has ended, results in Q1 2017. They also test the same HDL mimetics in a genetic HDL deficiency called Familial Primary Hypo-Alphalipoproteinemia.
    So we’ll know more soon enough, but far sure you can be very skeptic after both CSL and MCDO data presented at AHA last week.

  10. Maurice jung says:

    i have been looking into this since it was first reported on 60 minutes back in the 80s i think it was. i have read studies that the wild APO a1 WAS BETTER THAN THE MUTATED VERSION AS YOU CAN NOT PATENT A NATURAL GENE ( OR AT LEAST I THINK SO)
    So where are there any clinical trials for either the natural or mutated gene being done. I believe Cedar Sinai had done some test ( about 10 years) and found very good result in removing the plaque. Do you have any updates on that here or say something overseas?? What type of info can you furnish me, and what type of test can/need be done to be a candidate. would fresh cell therapy also be a candidate in this filed or would the clinics that provide that be able to do this as well????

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