848 replies to this topic

[recon]

The researcher said 3 cources of 30mg each would be enough.

3x30mg per lifetime?
If true and indeed effective, that's probably the best rejuvenation approach to date.
Hope you are right.

I think it means 3x30mg to effectively eliminate senescent cells. Senescence will still occur after and will accumulate as usual. Then another triple-course will be required.

[meatsauce]

Im going to get the first part of the order after the new year. 

[extendcel]

Are there still extra amounts for the group buy? What is the purity? 85%?

[Nate-2004]

I'm sure the holidays have slowed all this down, what's the news/status on FOXO4 DRI? 

 

Also has anyone else besides our n=1 blogger managed to get their hands on some? If so what have you experienced? 

 

The more I read on SENS research progress the more I realize how much focus we need on senescent cell clearance. Inflammation is one of the key targets in regenerative and rejuvenation research.

 

I may be at the point where I can join in on this group guy in the next month but I want to make sure this is even more effective than the Dasatinib.

 

Also anyone able to get their hands on Navitoclax or any HSP90 inhibitors?

Edited by Nate-2004, 29 December 2017 - 02:00 PM.

[meatsauce]

They are shipping the first part of it next week!

[extendcel]

You're getting it from a US company? What's strange is that this chinese company I'm in contact with is giving the same timeline.

[Hrub]

Hello  Guys. I am following this topic already for some time.

I am from the Netherlands and became interested because of news last march from Erasmus University.

To my surprise this FOXO4.dri stuff that is used there seems to be made in a company PepScan which to my surprise is 2 miles 

from my home.

If I can be of any help ... but I am not authorized to get this stuff as it is only authorized to be used in labs.

 

 

 

 

 

 

 

[meatsauce]

Hello  Guys. I am following this topic already for some time.

I am from the Netherlands and became interested because of news last march from Erasmus University.

To my surprise this FOXO4.dri stuff that is used there seems to be made in a company PepScan which to my surprise is 2 miles 

from my home.

If I can be of any help ... but I am not authorized to get this stuff as it is only authorized to be used in labs.

Yea can you sneak in and get us all some?

[RenewYou]

Hello FOXO4 peeps

 

I have read your entire 7 pages of this thread as well as studied FOXO4 as much as is available

 

I'd love to have the opportunity to be part of the next group purchase of FOXO4

 

Please let me know what I"m to do to become a part of the next group purchase as I'm as eager as you all to get started 

 

Cheers & Happy New Year

[jdlancaster519]

Hi all,

 

I was wondering if you group buyers have come together to select a protocol you will be following? Also, are many of you going to be taking objective tests... perhaps blood tests or anything that could be objectively put together? It's a risk to experiment on yourselves, and I appreciate that you are for all our benefit. Will you be reporting your results? It would be great if we could get some data that could analyzed after enough people tried it. 

[smithx]

This makes a lot of sense. I wonder if Longecity would subsidize any part of the costs of some set of standard blood tests, for anyone who can't afford them.

On the other hand, this stuff is pretty expensive, so most participants can probably afford to take a before and after test.

For US users, this one is under $100 and seems good. I've used them with success before, and the tests are done by Quest, which is probably the better of the major labs in the USA:
https://www.directla...US/Default.aspx

Hi all,
 
I was wondering if you group buyers have come together to select a protocol you will be following? Also, are many of you going to be taking objective tests... perhaps blood tests or anything that could be objectively put together? It's a risk to experiment on yourselves, and I appreciate that you are for all our benefit. Will you be reporting your results? It would be great if we could get some data that could analyzed after enough people tried it.

[The Capybara]

Quick question.....
Since the "L" amino acids are so much less expensive than the "D" type, is there any evidence to show that this FOXO4-DRI peptide sequence in "L" form wouldn't be just as effective, even if it might need to be taken in a larger dose or a few more times due to the possibility of faster enzymatic degradation?

[jdlancaster519]

The wrong form, l as opposed to d or vice versa, would not have the same effect. The shape of a protein and the charges are key to how they interact with receptors,other proteins, and molecules. The best example of why not to do this are the thalidamide babies. The orientation in space of a molecule or protein greatly changes its effects. A protein with the same polypeptides can fold or be oriented different ways with different interactions.

[The Capybara]

It absolutely would be the correct fit. In fact it should be a better fit on FOXO4. You'd have to reverse the amino acid sequence. Reread the original paper. The reason they used the "D" amino acids was to avoid enzymatic degradation, likely at a slight cost to binding avidity. That's the point of the "DRI" --> D-Retro-Inverso
So again I need to ask, why not use the reversed "L" amino acid sequence at, perhaps, a higher dose to compensate for the possible higher degradation, at a possibly much cheaper price?

[chris1299]

The reason they used the "D" amino acids was to avoid enzymatic degradation, likely at a slight cost to binding avidity. That's the point of the "DRI" --> D-Retro-Inverso

I'm pretty sure they used the D version so that it would interfere with naturally occurring FOXO4 that binds to p53, allowing p53-triggered apoptosis to occur. The L version wouldn't interfere, since that just is natural FOXO4.

Edited by chris1299, 04 January 2018 - 10:19 PM.

[The Capybara]

Chris1299: Don't speculate, just read the paper so that you understand it.
I'm not riding you, but you're wasting time trying to debate about something that's in the original peer reviewed article.

You are absolutely mistaken on several issues.

Edited by The Capybara, 04 January 2018 - 11:05 PM.

[jdlancaster519]

Edit: added a short version

 

In short: "Neither the same peptide in L-isoform (Figure 3C) nor an unrelated DRI-peptide based on a distinct Forkhead protein, FOXM1 (Kruiswijk et al., 2016), affected senescent cell viability (Figure 3D)."

 

In long: 

 

 I stated that enantiomers interact differently and nothing you stated disagrees with that. You said that the L-form of this protein should be a better fit. What does it bind to better, to what degree? Please quote and reference for this specific protein (Not proteins in general). 

To which paper are you referring as "original?"  If it is a matter of simply reading a paper then you should do so and make a statement with references so that we may all learn from you. If you are referring to  Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging, Baar et al. 2017:

 

"Research on peptide chemistry has shown that protein domains containing natural L-peptides can sometimes be mimicked by using D-amino acids in a retro-reversed sequence (Guichard et al., 1994). Modification of peptides to such a D-retro inverso (DRI)-isoform can render peptides new chemical properties, which may improve their potency in vitro and in vivo (Borsello et al., 2003)."

 

What you should take from that is that the L and D forms do not have the same chemical properties and the D form may be more potent. You should also take that DRI form don't always mimick the effects of their L form bretheren. 

 

"Several DRI-modified peptides have been shown to be well tolerated and therapeutically effective in clinical trials. These include a double-blinded, randomized, placebo controlled Phase IIb trial (Beydoun et al., 2015; Deloche et al., 2014; Suckfuell et al., 2014) and a Phase I trial for systemic treatment of solid tumors (Warso et al., 2013), together showing there is precedence for DRI peptides in clinical therapy. This provided the rationale for designing the FOXO4 peptide in a DRI conformation, henceforth named FOXO4-DRI."

 

What you should take from this is at that the authors of the paper used this form because it was well tolerated (low side effects/toxicity) and effective in clinical trials.

 

"Incubation of senescent and control IMR90 with increasing concentrations of FOXO4-DRI showed FOXO4-DRI to potently and selectively (11.73-fold difference) reduce the viability of senescent versus control IMR90 (Figure 3A) and other normal cells (Figure S3A). Real-time cell density measurements revealed the effect to occur as soon as 24–36 hr after administration (Figure 3B). Neither the same peptide in L-isoform (Figure 3C) nor an unrelated DRI-peptide based on a distinct Forkhead protein, FOXM1 (Kruiswijk et al., 2016), affected senescent cell viability (Figure 3D). These results show that FOXO4-DRI can target senescent cells, and they highlight the importance of the DRI-modification for its potency."

 

What you should take from this is that the L form of the protein doesn't work well. You can see this in Figure 3C. Increasing the amount of the L form of the protein (or any drug or protein) doesn't necessarily increase on-target effects. Also, you are likely to increase off-target effects (toxicity/side effects) with no scientific evidence that the on target effects you desire will come from a higher dose. The authors explicitly state that the DRI-modification is important.

 

I will not argue that the L form of the protein in high doses may or may not have the desired effects, but the authors of the paper believe the D form is the most safe and effective. In high enough doses the L form will kill senescent cells... but is it due to toxicity....? how will it affect normal cells....? How will it affect other metabolic pathways? There is no scientific evidence to back the using of a high dose of the L form of the protein. If you take it at a high dose, you may be able to save a few bucks... but at what cost?

 

 

 

 

 

Edited by jdlancaster519, 04 January 2018 - 11:53 PM.

[The Capybara]

You know, I just showed up to ask a question:

"Quick question.....
Since the "L" amino acids are so much less expensive than the "D" type, is there any evidence to show that this FOXO4-DRI peptide sequence in "L" form wouldn't be just as effective, even if it might need to be taken in a larger dose or a few more times due to the possibility of faster enzymatic degradation?"

I think that's a good question to ask.

I don't know why I'm defending any position.

"Research on peptide chemistry has shown that protein domains containing natural L-peptides can sometimes be mimicked by using D-amino acids in a retro-reversed sequence (Guichard et al., 1994)."

Yes! So the question is that if D-amino acids in a retro-reversed sequence can mimick an L-amino acid sequence, then why is everyone dismissing the Levo form of FOXO4-DRI, which is likely already out there with the cell entry sequence at a much cheaper price than a peptide sequenced from the Dextro amino acids? It may even be affordable and just as effective and likely not patented.

"Modification of peptides to such a D-retro inverso (DRI)-isoform can render peptides new chemical properties, which may improve their potency in vitro and in vivo (Borsello et al., 2003)."

Yes! That's likely because of their resistance to enzymes and differences in their steric hinderance(s). The point is that if the DRI form may work, the Levo form will almost certainly work and likely have greater avidity since the intrinsic FOXO4 protein is of the Levo form.

Again, please read the first paragraph. Is there anyone that knows of any reason why the Levo form wouldn't be an effective and far less expensive option to FOXO4-DRI?
That's all I'm asking.

[meatsauce]

The first portion of the order has been shipped out so ill have some soon. I might get a sample from another company that says they can make it quicker and test it against the current batch. That way we could get a second buy going for those who missed the first round. 

[chris1299]

You know, I just showed up to ask a question:

"Quick question.....
Since the "L" amino acids are so much less expensive than the "D" type, is there any evidence to show that this FOXO4-DRI peptide sequence in "L" form wouldn't be just as effective, even if it might need to be taken in a larger dose or a few more times due to the possibility of faster enzymatic degradation?"

"Neither the same peptide in L-isoform (Figure 3C) nor an unrelated DRI-peptide based on a distinct Forkhead protein, FOXM1 (Kruiswijk et al., 2016), affected senescent cell viability (Figure 3D). These results show that FOXO4-DRI can target senescent cells, and they highlight the importance of the DRI-modification for its potency."

I have read the paper, and I'm quite sure I'm not wrong about this. Why would introducing additional FOXO4 interfere with naturally occurring FOXO4?

To me it sounds like you fundamentally don't understand the apoptosis mechanism at work here. FOXO4 (the L version) prevents p53-triggered apoptosis in senescent cells. The researchers were looking for a way to prevent this activity of FOXO4, and seem to have been successful. Adding more FOXO4 would absolutely not reduce senescent cell viability--probably the opposite.

Edited by chris1299, 05 January 2018 - 03:12 AM.

[Vantika]

TL;DR: It is my considered opinion that FOXO4-DRI is in all probability a safer, more effective, and *more cost-effective* senolytic than its L-isoform cousin, FOXO4-L, despite the ability to synthesize FOXO4-L at somewhat reduced prices.

 

 

I think that's a good question to ask.

I don't know why I'm defending any position.

 

Hey, The Capybara.

 

It's a fair enough question to ask. I certainly don't mind you asking it and welcome the debate. We're all seeking the truth here, whatever that may be. Along those lines, please interpret my tone in this response charitably, with all of my remarks meant only as friendly debate, not an attack of any kind.

 

Having said that, I should say that I think jdlancaster519 answered the question pretty well. Maybe it would be worth reconsidering his arguments while taking an even closer look at the de Keizer paper.

 

The point is that if the DRI form may work, the Levo form will almost certainly work and likely have greater avidity since the intrinsic FOXO4 protein is of the Levo form. [emphasis added]

 

This claim -- that the L-isoform "almost certainly" will work -- is, in my opinion, speculative and purely theoretical, with what available empirical evidence we actually have indicating that FOXO4-L (the term coined and used by the de Keizer paper's authors for this variant) will not work effectively, even at high doses.

 

Take a look at Figure 3D (relatively easy to interpret).  Note that FOXO4-L had very little effect on viability of the senescent cells tested, even at concentrations as high as 25 uM.

 

Now take a close look at Figure 3C (mostly the same data, though requires a close reading).  Specifically, take note of the FOXO-L curve interpolation for senescent cell clearance (the red line). Now, mentally put that curve through a low-pass filter to smooth out the noise of the random measurement errors at each tested concentration.

 

Notice that FOXO4-L's (relatively meager) effectiveness at killing senescent cells plateaus at around 25 uM concentration, with no observed improvement in senescent cell clearance at the higher tested concentrations of 37.5 uM or 50 uM.

 

[I]s there any evidence to show that this FOXO4-DRI peptide sequence in "L" form wouldn't be just as effective...?

 

The apparent trend (from what limited data we actually have) indicates that FOXO4-L does not become more effective at higher concentrations (beyond 25 uM), contrary to your speculation that it should.

 

Granted, it would be nice if we had test data at even higher concentrations, but if that data exists it's not presented in the paper (unless I missed something). Given the data we do have, I would argue the preponderance of the evidence indicates that FOXO4-L probably will not become meaningfully or competitively effective at higher doses.

 

...even if it [FOXO4-L] might need to be taken in a larger dose or a few more times due to the possibility of faster enzymatic degradation?

 

Now, furthermore, I would argue that even if I'm wrong about FOXO4-L's dose-effectiveness plateau, it still would likely not be cost-effective to use it.

 

Let's assume for the sake of argument that I'm wrong, that dose-effectiveness does not plateau, and, hypothetically, that FOXO4-L does suddenly start becoming effective at, say, 100 uM (off the right of Figure 3C, into terra incognita).  Let's assume senescent cell viability under that concentration (100 uM) suddenly plunges to about 20%, which would be roughly equivalent to using FOXO4-DRI at a 10 uM [sic] concentration.

 

So you would then need ten times as much.  Are you still saving money at this point, buying ten times the amount of FOXO4-L vs one tenth the amount of FOXO4-DRI?

 

Next we need to take into consideration enzymatic degradation, autophagy, lysosome activity, etc. as you yourself have already astutely pointed out.

 

De Keizer et al have already carefully measured the persistence of FOXO4-DRI in cellular cytoplasm: "Using antibody against HIV-TAT, we observed FOXO4-DRI to ... remain detectable for at least 72 hr (Figure 2J). ... [T]his indicates FOXO4-DRI effectively enters senescent cells at high intracellular concentrations, which remain abundant and stable over a prolonged period of time," [emphasis added].

 

We have no data on the persistence of the FOXO4-L form inside of cells (none is published, if any even exists, as far as I'm aware).  

 

From eyeballing Figure 2J, FOXO4-DRI's half-life seems to be somewhere in the neighborhood of 12-16 hours in cytoplasm (a better estimate could likely be done if someone actually bothered to precisely measure the fraction of pink vs black area in the Figure 2J images).  And de Keizer et al have already empirically established a working treatment protocol for FOXO4-DRI, using doses spaced 48-hours apart with this intracellular pharmacokinetic information in mind, presumably keeping intracellular peptide concentrations within therapeutic levels throughout the duration of each dosing cycle.

 

How would one go about estimating FOXO4-L's half-life in cytoplasm without any data?  How are you proposing to keep concentrations within the therapeutic range (which, I might add, in the absence of our purely hypothetical 100 uM assumption above, we also don't know for FOXO4-L)?

 

Do you just err on the side of dosing higher and/or more often, to increase the likelihood of staying in the therapeutic range?  How can you ever be sure that you are inside of this range?

 

And, now, in light of these considerations, how much more FOXO4-L do you need?  Let's say you (optimistically, IMO) estimate the FOXO4-L intracellular half-life to be half that of FOXO4-DRI.  So you dose twice as often to compensate for this.  You now need, in total, twenty times as much FOXO4-L as you would FOXO4-DRI for the same effect.  Are you still saving money?

 

And all this theorizing still ignores safety issues.  We don't know what effect such high doses of FOXO4-L would have on non-senescent cells, since those concentrations (> 50 uM) have never been tested even in vitro (or if they have, the data hasn't been published).  We have no measurement of FOXO4-L's Selectivity Index (see de Keizer paper) at those doses (in contrast to FOXO4-DRI's excellent Selectivity Indices at lower doses).  And we have no in vivo data of any kind for FOXO4-L.

 

 

The Capybara said:

[FOXO4-L] absolutely would be the correct fit.

...

[T]he Levo form will ... likely have greater avidity since the intrinsic FOXO4 protein is of the Levo form.

 

 

I'm not sure exactly what you mean by "correct fit."

 

First of all, let's clarify that FOXO4-DRI is not the D-retro-inverso form of the entire FOXO4 protein, but rather only of a fragment of it that is involved in binding to p53, using a subsequence of the full FOXO4 protein that is conserved between mice and humans.  The portion of naturally occurring FOXO4 that binds to DNA-SCARS at DDR sites in senescent cells is omitted from FOXO4-DRI (and FOXO4-L, for that matter) by design.

 

FOXO4-DRI does not bind to FOXO4, if that is what you were suggesting (forgive me if I've misinterpreted you; I'm not sure what you meant). Rather, it competes with FOXO4 for binding to p53.

 

If instead you were suggesting that FOXO4-L might be a better "fit" for the naturally occurring, full FOXO4 protein than FOXO4-DRI is, in the sense that FOXO4-L folds into a shape more closely matching that of the same corresponding region of the naturally occurring, full FOXO4 protein, I'd contend that that's speculation.  You may be right, but there's no way to know with certainty exactly how a protein or peptide folds or what three-dimensional shape it will ultimately assume without actually performing X-ray crystallography on it, which, AFAIK, has not been done for either FOXO4-DRI or FOXO4-L.  Consider also that an exogenous HIV-TAT sequence has been appended to each.

 

Regardless, even if you are right and FOXO4-L does more closely match the same region of the full FOXO4 than does FOXO4-DRI, I don't see this necessarily having any bearing on the relative therapeutic effectiveness of FOXO4-L vs FOXO4-DRI.  It's entirely possible that FOXO4-DRI, via its unnatural D-retro-inverso form has an even higher binding affinity for p53.

 

And indeed this seems to be the case, with FOXO4-DRI out-competing naturally occurring FOXO4 for binding to p53.  De Keizer et al write: "Stepwise addition of the FOXO4-DRI peptide to this complex caused the CSPs of FOXO4 to be reverted back to the unbound state, indicating FOXO4-DRI competes with FOXO4 for p53 binding in a dose-dependent manner and doing so with higher affinity (Figure 2I)," [emphasis added].

 

This freeing of sequestered p53 from FOXO4 attached to DNA-SCARS inside the nucleus, making p53 available for its role in the intrinsic apoptotic pathway is how FOXO4-DRI works. FOXO4-DRI apparently doesn't attach to these DDR/DNA-SCARS sites (which FOXO4-L might, for all we know), and it also apparently attaches to a region of p53 (in a way and with a binding affinity) that doesn't interfere with p53's role in apoptosis.  Either of these considerations could also cause issues with the FOXO4-L form, in addition to its binding affinity and pharmacokinetic issues discussed above.

 

---

 

Ultimately, unless and until new empirical evidence emerges overturning that already available, I'm afraid I have to disagree with your conjecture on this.

 

Anyway, I appreciate the discussion, The Capybara. Thanks for posting.

 

 

[jdlancaster519]

Thank you Vantika, you write things in a much more understandible way than I do. Capybara- I appreciate your search for a cheaper solution, but I care about you and everyone else. The data supports the D form and I'd like everyone to try the d form and report their results. The authors of the report said the L form was not effective. I'd appreciate all the data from everyone that tries the FOX-04 DRI protein... I am a biologist, but only have a few few peer reviewed publications.... mostly on nematode-bacteria symbiosis and CBRNE. I'm just trying to survive and save my loved ones like everyone else.

[recon]

The first portion of the order has been shipped out so ill have some soon. I might get a sample from another company that says they can make it quicker and test it against the current batch. That way we could get a second buy going for those who missed the first round.

That’s a good move. I’m sure many will be willing to participate the next run if the first one is proven successful.

[meatsauce]

We are finally getting the first portion of the buy. We will see how they do on time with the rest of the order. I am also working on getting a sample from another company who says they can do it quicker.  We can open up another group buy now if you guys want to or wait for results from the first. 

[Valijon]

Id like to see results from the first group buy before commiting myself to anything.

[Rocket]

We are finally getting the first portion of the buy. We will see how they do on time with the rest of the order. I am also working on getting a sample from another company who says they can do it quicker. We can open up another group buy now if you guys want to or wait for results from the first.

Hope I made it into the first group buy. If it matters i will do pre and post blood tests. The only things I will be on is megadosing liposomal resveratrol, cjc and bpc for the next 5 weeks.

[smithx]

Hope I made it into the first group buy. If it matters i will do pre and post blood tests. The only things I will be on is megadosing liposomal resveratrol, cjc and bpc for the next 5 weeks.

I know it's off topic, but what are those (aside from the resveratrol) and why are you megadosing them?

[meatsauce]

 

Hope I made it into the first group buy. If it matters i will do pre and post blood tests. The only things I will be on is megadosing liposomal resveratrol, cjc and bpc for the next 5 weeks.

I know it's off topic, but what are those (aside from the resveratrol) and why are you megadosing them?

 

cjc 1295 is a peptide that causes a the pituitary gland to constantly release growth hormone which will cause an increase in IGF1. BPC - 157 is another peptide that is produced in the stomach that was found to have systemic healing effects when injected. 

[recon]

We are finally getting the first portion of the buy. We will see how they do on time with the rest of the order. I am also working on getting a sample from another company who says they can do it quicker. We can open up another group buy now if you guys want to or wait for results from the first.

Hope I made it into the first group buy. If it matters i will do pre and post blood tests. The only things I will be on is megadosing liposomal resveratrol, cjc and bpc for the next 5 weeks.

Shouldn’t one reduce IGF-1 levels for longevity? There’s a BBC documentary, and an accompanying online article, that explores the use of fasting to extend lifespan and one bio marker they used for the efficacy of the fasting was the IGF-1 levels that they tried so hard to reduce.

[helix66]

We are finally getting the first portion of the buy. We will see how they do on time with the rest of the order. I am also working on getting a sample from another company who says they can do it quicker.  We can open up another group buy now if you guys want to or wait for results from the first. 

Hi meatsauce, I would be interested in getting in on the next group buy now rather than wait. My logic being that members will probably have started posting anecdotal reports to the forum buy the time the second order is received.

 

On sourcing a different supplier I would rather stick to your current supplier, as it seems they're doing a fair amount of due-diligence. I'd rather not roll the dice on a new supplier.

 

Finally although I earlier requested 200mg in a PM, I would like to increase that to 300mg.

 

Thanks for all your efforts so far.

 

cheers.