Q&A

There are a lot of functions and properties and benefits of senescent cells that you went over in this presentation, at least in some cases. Is senescent really the right word? And how is it defined, is it just that they stop replicating and beyond that there are a lot of different possibilities of what they are doing – both good and bad?

• The definition of senescent cells is basically entry of a cell into essentially a cell cycle arrest, that’s irreversible unless there are further mutations or things that happen to the cell. It’s a cell fate, like replication, differentiation, apoptosis or necrosis. It’s the fifth cell fate. So any cell can become senescent. They can become senescent in response to damage signals, repeated replication, problems with replication, with intracellular organelles, in response to infectious pathogens, drugs, radiation, or other kinds of damaging insults. Senescence is basically defined as essentially irreversible loss of capacity to divide. It takes 10 days to 6 weeks for a cell to become fully senescent, so it is a slower process than other cell fates. Some but not all senescent cells can spread senescence and interfere with stem and progenitor cell functions, damage tissues and can produce a range of signals that detract, activate, and anchor immune cells. So this is a cell fate, a necessary process you don’t wanna interfere with. But if they become senescent and they persist and reach a threshold above which they spread faster than they are cleared out by the immune system, they start to take off and systemic problems can develop. There are probably two types of senescent cells at least, the nature of senescent cells depends on the nature of the starting cell type, what induced the senescence and how long the cell has been senescent. But 30-70% (so called deleterious senescent cells) produce damaging factors. And the rest do not produce damaging factors (so called helper senescent cells) – they are important for wound healing and so forth.

You said that “mitochondrial DNA is released from senescent cells” – that sounds like a serious problem. What’s going on here? Is it purely maladaptive or is there some good reason for it? It looks like a serious failure inside the cell system.

• One of the changes in senescent cells is that there is a really complex transcription factor cascade that enforces senescence, where literally thousands of genes expression gets changed. Mitochondrial dysfunction is a hallmark of senescence. Senescent cells are resistant to dying but they have a so-called “partial Warburg shift”, much like cancer cells, they depend on sugars rather than fatty acids. There’s a lot of turnover of mitochondrial DNA and changes in mitochondrial dynamics, so they release mitochondrial DNA and a lot of other non-coding nucleotides as well, like microRNAs that affect a lot of tissues as a great distance. Mitochondrial DNA is recognized as DAMP – danger associated molecular pattern protein – so other cells when they recognize that cells nearby are breaking down, they become senescent cells. So this is how senescence can spread even at a great distance. We’re working with some of the space agencies on space radiation research, we’ve got cells going up in 18 months on the Axiom flight.

Based on what you said about senescent cells and the Warburg effect – doesn’t that suggest that a really low carbohydrate diet might be senolytic?

• Not necessarily senolytics as such, but there are metabolic ways of getting at senescent cells and you do find that diet might impact senescent cell generation as it does with cancers – like caloric restriction which delays senescent cells accumulation and simultaneously improves healthspan. But we don’t know whether it’s the caloric restriction itself or carbohydrate restriction, so more work needs to be done. But these diets and lifestyle interventions are very hard to introduce in trials, especially in sick elderly people, compared to small molecule pills.

What the difference is between sensecent and terminal differentiation and how do senolytics achieve specificity?

• Terminal differentiation means that the cell has acquired a specialized cell fate and stops dividing. Senescent is in essence a terminally differentiated state, but some other terminally differentiated cells like neurons or cardiac myocytes can acquire senescence like state. That’s why it’s really hard to define senescence. It’s beginning to look like non-replicating cells can acquire a senescent like state as well, so the definition is tricky and there’s a debate and emerging view that terminally differentiated cells can acquire what looks like a senescence related fate.

Will you be doing mid-term interim analysis in the trials that are at half-way point?

• Some of them yes, most of them no, because of data safety and monitoring boards (these are FDA regulated trials).

Quercetin & Fisetin aren’t as strongly regulated as drugs so actual amounts of active ingredients vary from labeled amounts, sometimes a lot. (Companies like ConsumerLab make a business of testing these kinds of things.) How do you source for the trials & what do you do to ensure accurate dosing?

• We had to get a full IND for Fisetin. The minute you use a natural product to treat a disease, it is considered a drug. We had to register with the FDA as a drug. It took 2 years and 450 pages. We had to set up something called “Good Manufacturing Practices” manufacturing. We had to do stability testing, look for degradation products, test in multiple species, do safety and tolerability, we had to do all of that just like it’s a brand new drug before we were allowed to do clinical trials. We contract to a GMP manufacturer and then the Mayo Clinic mass spec facility monitors their product and we have to furnish to the FDA every couple of months safety and stability data.

Wound healing is important, so we don’t want to prevent cells from becoming senescent cells. In the case of wounds, we think most SnCs get cleaned up after. Do we know yet if that cleanup is less good with aging (maybe due to immunosenescence)? I.e., would we expect higher SnC burden in animals with higher rates of wounds?

• Some senescent cells produce platelet growth factor which helps wound healing. Those are the senescent cells that are not pro-inflammatory. Senolytics do not target those. Senolytics target 30-70% of cells that have SASP – senescence associated secretory phenotype – and are not active in wound healing. So we do not see the inhibition of wound healing with senolytics.

Are you saying that all the types of senescent cells that are involved in wound healing do not have SASP? Or just some of them?

• The SASP may be necessary early on to clear away dead tissue. But later on, it’s been shown in the animal models, if you clear cells (some of which are senescent) by targeting p16 expressing cells, you delay wound healing. But if you add back platelet growth factor, you restore wound healing. It turns out it’s the senescent cells that don’t have a SASP that produce that. So you do need them early on to help clear up the wound, you wouldn’t want to give these drugs right away, but in a chronic wound they may help.

So as we undergo immunosenescence and maybe the cleanup is imperfect, would we expect that animals that get higher rates of idiosyncratic repeated wounds will end up with a higher senescent cell burden?

• We’re not sure about that, but we see that pattern in other kinds of scenarios. There is what we call “stairway effect” – you have something that causes senescent cell burden, and then you get a bit better and you get back to baseline. Above a certain threshold, senescent cells start impeding immune system function and their own clearance.

You said monthly hit-and-run dosing is as good as continuous. Would you expect annual dosing to be effective?

• It depends on the rate of senescent cell formation, so it depends on the condition. If you blast a mouse with a one-time burst (like radiation), you just need to give a couple doses in the animal’s entire life to alleviate dysfunction. If you’ve got continuous high fat feeding, new senescent cells are generated at a particular rate, so it depends on the stimulus. It depends on the cell type that’s becoming senescent and a lot of other things. We’re only just beginning to get a handle on that. One of the things we use the blood test for is to try to figure out when senescent cells are coming back. We try to miniaturize that and make it as fast as possible so we can do in some of these trials intermittent blood testing like every few days and determine when there is a takeoff of senescent cells again so we can dose at that point.

How much is the senescence of several layers of cells in the liver an adaptive response to the aggressive environment and how much could senolytics interfere with this response?

• You don’t see much senescent cells in the liver in younger healthier individuals. You see them in conditions like primary sclerosing cholangitis, where they are very abundant and where they occur in one particular part of the liver. There is a clinical trial envisaged in that, so that will be coming along. In the case of hepatic steatosis where there is fat tissue that gets into the liver in the context of obesity and diabetes, there are different kinds of cells in the liver than in sclerosing cholangitis, so it’s a different set of cells in a different part of the liver that are affected. But you tend not to see many that we are able to detect in healthier individuals in the liver.

Are there any known negative interactions between the human immune system and senolytic therapy?

• It depends on the drug, there are probably a well over a 100 drugs now. Some drugs are very toxic, things like Noviclax for example. It is not an approved drug, it is being investigated, and it does have a lot of effects on the immune system. It mainly affects platelets but can cause unpredictable decreases in the white count, especially neutrophils. And after a while, there seems fairly severe neutropenia. While other senolytics don’t seem to have a big effect on immune cell numbers.

How much did you end up paying for that whole GMP for Fisetin work?
 (CMC and the other IND-enabling studies)

• Quite a lot, don’t know the exact numbers, we negotiated with different manufacturers, so it depends on the particular study. One thing we’re finding about Fisetin is that it’s pretty stable in our hands, we see that when we do mass spec on it. We were worried it would break down easily. In the right storage conditions, it is remarkably stable.

Will that data be available for that whole process with Fisetin – purification, stability testing, and all that kind of stuff?

• Yes, it will be, when we will be publishing some of the trials. We are developing a website about that particular trial with NIH, which should be coming online in the next week or so. It won’t have everything at the beginning, but eventually it will. Everything not considered business confidential will be available.

Do you have the clinical blood tests & expanded biochemistry tests from patients before and after administration of senolytics? Did you try any of the biological aging clocks trained on this data (e.g. Young.AI)?

• Yes, that’s all being done. But we don’t know yet, the data are just starting to come in. There is a group at Yale interested in the data, epigenetics processing is done at University of Minnesota, and then Morgan Levine and others at Yale are looking at the results. The data will eventually be public, because these are publicly funded trials.

You made a decision to go with and study systemic administration. Oral dosing, because of the signaling aspect, etc. Given the number of studies, any plans to try any local administration in any trials? (Unity’s failure could be due to locality, but could also be the wrong molecule.) (Unity’s failure could also be a poor choice of indication. Other indications with higher and maybe more temporary SnC burden may be better for local administration.)

• We find there is a spread of senescence in model organisms and based on early data in humans as well. Because of the spread of senescence throughout the organism, we’re concerned that local accumulation will be a harbinger of systemic problems, therefore systemic is the way. Also because of the problem of local administration and the many things it can mean when there is damaged tissue and a lot of blood supply to that tissue. And local administration spreads in the end systemically as well, so you could basically be giving at least intramuscular injection if not intravenous injection, when trying to administer something locally to a damaged tissue. Skin and inhaled agents are something different and might be worth looking into.

I suspect it doesn’t make sense to do local when you’re looking at the very very frail which those trials seem to be, but as you start to look into trials for younger healthier people possibly there are conditions where there’s a very local high senescence cell burden that hasn’t spread out too much yet?

• We’ve got some stuff emerging from Jon Hopkins that in fact in some younger people with local accumulation of senescent cells we see that they’re everywhere if we look.

What can the group do to help with your work? What would be helpful?

• We’ve got a lot of help from the government and foundations. What’s very important for people is to get the message out to not take the drugs in an uncontrolled way, because we don’t know whether these drugs are safe and effective. I give it maybe a 50:50 chance. We’re doing the trials because we don’t know if they’re going to work. As a physician I am very concerned with the Hippocratic oath and the first principle – “first, do no harm”. It’s a very fundamental process we are playing with, so we’ll have to see what happens and prefer conservative approach at this point. But we’re trying to move quickly because we feel it’s important to know one way or the other, but I worry.