848 replies to this topic

[sthira]

Long-time senescence researcher and blogger Dr. Dominick Burton:

...

(Burton's revamped senescence blog:
https://cellsen.wixs.../senescentcell/

This is a good share, thanks. Burton evidently understands the value of writing about the complexities of senescence in a plain, straightforward voice that's publicly accessible.

[Moondancer]

If we split the costs, I too would be willing to invest in third party testing, as among others Smith suggested. Frankly I think this is important. 

 

[Zed]

 

 

 

 

Stealth as in "reasonable deniability" . You dont want to mislabel something (eg call it "childs doll" )  as that could be a red flag. However you can use vague or tangential references .."ie Beauty Product "  or as is normally put on the numerous vials of RC's and peps I have received -"MS samples"  - whatever that is.

 

I have received literally over a dozen peps in vials posted in plain US express post envelopes  wrapped in bubble warp plastic - no sweat (US $13.50) - 10 working days with online tracking . Being in an envelope may even help being waved through.

 

I suggested Fed Ex Box (3-4 days) as I thought you needed an ice pack or something -if not express envelope is fine.

 

A friend getting similar RC's - boldly marked "Research Chemical - Not for Human Consumption" had her package opened by the Customs - and a letter to that effect inserted but all good - no warnings, questions whatever. I guess its the luck of the draw - if its a chemical that comes up on their radar then they will throw the book at you - otherwise not.

PS -I generally make a small animal sacrifice to Mercury the messenger of the Gods every time I order a package - seems to work..

 

 

 

Thanks for your feedback Zed. Did the packages listed as MS Samples usually have a $ cost of packaged items listed? And if yes, what was the ballpark price?

 

Also what animal do you suggest I sacrifice... Would a cockroach do?

 

 

Recent package pic attached. Marked "MS Samples" - a couple of vials wrapped in bubble wrap and popped into the envelope. All safe and sound. Cost marked 1.50 Actual cost $150 ..could have been a typo..again "reasonable deniability".

 

Given its the TGA I would think a cockroach would be most fitting.
 

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[Nate-2004]

 

Long-time senescence researcher and blogger Dr. Dominick Burton:

...

(Burton's revamped senescence blog:
https://cellsen.wixs.../senescentcell/

This is a good share, thanks. Burton evidently understands the value of writing about the complexities of senescence in a plain, straightforward voice that's publicly accessible.

 

 

These were all such good explanations about not only what senescence really is in terms of cells but good explanations as to the causes. This should be posted somewhere else.

[Krell]

I am interested in the group buy, including third party testing.

[TaiChiKid]

I can do and have previously offered to do GC/MS testing of samples, but we need a standard to compare them to. That's why I proposed going with Pepscan, which produced the peptide used in the study, or at least getting a sample from them which we can then compare to everything else.

 

Smithx, how much would you charge to do GC?MS testing, and how much of a sample do you need?  Do you have enough from the amount that you are ordering?  It would be nice to get an indication about purity and so forth.
 

[smithx]

I wouldn't charge anything for GC/MS analysis, however what we do need is:

- A sample from Pepscan to serve as the Standard which we compare other samples to. They did the synthesis of the compound used in the published study, so presumably they would provide the same compound to us
- A sample to analyze
- Each sample should be at least 10mg

I would also appreciate getting some of either sample for my own use. I have not so far ordered any.

I can do and have previously offered to do GC/MS testing of samples, but we need a standard to compare them to. That's why I proposed going with Pepscan, which produced the peptide used in the study, or at least getting a sample from them which we can then compare to everything else.

 
Smithx, how much would you charge to do GC?MS testing, and how much of a sample do you need?  Do you have enough from the amount that you are ordering?  It would be nice to get an indication about purity and so forth.

[malbecman]

For a peptide you will want LCMS testing...it wont go thru a GC column (its not volatile).   Also, keep in mind that LCMS is not the best way to test purity.  It will confirm identity but some compounds dont ionize

in an LCMS (give no signal) or give a disproportionate signal to their amount.

 

  For purity, ideally, you want to run LC with a UV detector set at a wavelength to detect everything like 220nm.  

 

Source; I am a PhD chemist.

[meatsauce]

I have another US company that will test it for free. 

 

[meatsauce]

The rest of the first order is ready to go I pm'd everyone in the US about if they will be home the next couple days for delivery so please get back to me. Also everyone outside of the us please PM and tell me what is your preferred carrier for delivery. 

[meatsauce]

Sorry there is some confusion out there about the order groups. What is completely finished now is the first order that took place months ago. The one that people paid for last month is ongoing. I wish everything would be quicker but this one shouldn't be too long. 

[extendcel]

No updates on any noticeable effects?

[meatsauce]

I need Reed Hearon to PM me I don't have your address.

[meatsauce]

No updates on any noticeable effects?

 

I still think I'm the only one who has tried it. 

 

The other people who have received the first batch still haven't started it I think. One go them had to stop after a small test dose because of an injury. Senescent cells are involved in healing injuries so taking this with a severe enough injury can result in it not being healed properly. 

[Invicta Immortalem]

Again:

 

No updates on any noticeable effects / from no one / until now (except @Darren Moore)?

Edited by Invicta Immortalem, 02 March 2018 - 01:06 PM.

[Vantika]

Again:

 

No updates on any noticeable effects / from no one / until now (except @Darren Moore)?

 

See: http://www.longecity...ndpost&p=840454

[Moondancer]

Is perhaps known already when the second batch will be ready and shipped? Thanks!

[Rocket]

Again:

No updates on any noticeable effects / from no one / until now (except @Darren Moore)?

Sounds like no one except a 32 year old has tried it. And I don't know why anyone that young would get any results at all since there are likely hardly any senescent cells to cause issues. When I was 32 I was healthier than when I was 22 simply from diet and exercise. If I was 32 there would be no way I would administer cancer medicine in hopes of slowing down aging. At 32 most of the aging you have is really poor health from poor diet, alcohol, tobacco, and poor sleep habits and by then your bodies production of hgh drops and you are more prone to sports injuries and slower injury healing but that is not due to senescence. I doubt anyone under 40 would notice positive effects from this therapy.

[TaiChiKid]

Now that I have received my FOXO order, does anyone have any thoughts on simultaneously taking three senolytics:  FOXO4, D&Q, Rapamycin.  My theory would be that the three taken simultaneously would catch more snescent cells...  Any thoughts?

[Vantika]

Now that I have received my FOXO order, does anyone have any thoughts on simultaneously taking three senolytics:  FOXO4, D&Q, Rapamycin.  My theory would be that the three taken simultaneously would catch more snescent cells...  Any thoughts?

 

DO NOT combine FOXO4-DRI with Rapamycin.  If you do, you will almost certainly be undermining the senolytic effect of FOXO4-DRI.

 

Rapamycin is not a senolytic, but a "senomorphic".  It doesn't kill senescent cells, but only temporarily reduces/suppresses their secretions[1].

 

In the de Keizer paper[2] they tested FOXO4-DRI in combination with various compounds that either enhance or mitigate SASP.  Compounds enhancing SASP (IL-1alpha, Lipopolysaccharides (LPS)) increased FOXO4-DRI's potency against senescent cells.  Compounds suppressing SASP (IL1 receptor antagonist, cortisol) reduced FOXO4-DRI's potency against senescent cells (killing proportionately fewer at any given dose, thereby reducing FOXO4-DRI's various Selectivity Indices).

 

See Figures 6H and 6I of the de Keizer paper[2].  You really should look at Figure 6I.  I implore you to do so.

 

They write:

 

"[W]e wondered how FOXO4-DRI would function under such high-SASP conditions. In vitro experiments showed FOXO4-DRI to be more potent against senescent cells in which SASP was transiently boosted by recombinant IL1a/b or lipopolysaccharide (LPS), whereas an IL1 receptor antagonist or the general anti-inflammatory drug cortisol reduced its potency (Figures 6H and 6I). Thus, FOXO4-DRI actually is most effective against senescent cells expressing high levels of SASP and could as such be particularly effective against loss of renal function."

→ source (external link)

 

Relevant signal transduction pathways:

• LPS --> TLR4 --> NF-kB --> {IL-1alpha <--> NF-kb feedback loop}    (LPS agonizes TLR4 receptor, which upregulates NF-kB, which in turn fuels (adds feedback to) the IL-1alpha/NF-kB positive feedback loop that underlies most of the inflammatory secretions of SASP[1].)
• Cortisol --| [IL-1alpha --> {IL-1alpha <--> NF-kb feedback loop}]    (Cortisol antagonizes IL-1a receptor, which downregulates/blocks the SASP IL-1alpha/NF-kB positive feedback loop[1][3].  See "Figure 2" in [3].)

If you were to combine FOXO4-DRI with Rapamycin, you would get:

• Rapamycin --| [mTORC1 --> {IL-1alpha <--> NF-kb feedback loop}]    (Rapamycin inhibits mTORC1, which is a necessary intermediary in the same SASP feedback loop by enabling translation of IL-1alpha[1][3].  See "Figure 2" in [3].)

From [1]:

 

"We propose that rapamycin selectively targets the SASP-initiating cytokine IL1A through translational inhibition, thereby suppressing establishment of the signalling  cascade that activates NF-kB and the transcription of many genes encoding SASP factors (Supplementary Fig. 7)."

→ source (external link)

 

The effect would be very similar to using cortisol: comparatively very few senescent cells would die (see the solid red line in Figure 6I of the de Keizer paper[2]), and you would effectively be wasting your FOXO4-DRI.

 

I think someone even found some unpublished data from de Keizer et al and posted it earlier in this very thread, where his group had specifically tested Rapamycin in combination with FOXO4-DRI and confirmed it to have the above expected Nerf-ing effect, rendering the FOXO4-DRI largely impotent.

 

Similarly, any anti-inflammatory drugs/compounds/supplements you may be taking likely downregulate NF-kB (thereby suppressing SASP), directly or indirectly, and thus would have similar counterproductive effects in combination with FOXO4-DRI.  These must be washed out before beginning your FOXO4-DRI session.  That might include Quercetin, which you mentioned explicitly.  Even though it is a senolytic in itself, we don't know that its mechanism of action is synergistic with FOXO4-DRI's.

 

---

 

Also, I think you mentioned earlier in the thread that you are Han Chinese.  This greatly increases your risk of having an adverse reaction to Allopurinol, if you were thinking of using it for hyperuricemia prophylaxis.  Febuxostat is an alternative.

 

Actually, I see now that you have already sequenced your DNA.  That ought to tell you whether you have the high-risk allele[4].

 

 

References:

 

[1] MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation.  DOI: 10.1038/ncb3195

Pubmed: https://www.ncbi.nlm...pubmed/26147250

Sci-hub: http://sci-hub.tw/10.1038/ncb3195

 

[2] Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.  DOI: 10.1016/j.cell.2017.02.031 

Pubmed: https://www.ncbi.nlm...pubmed/28340339

Sci-hub: http://sci-hub.tw/10...ll.2017.02.031 

 

[3] Therapeutic interventions for aging: the case of cellular senescence. DOI: 10.1016/j.drudis.2017.01.004

Pubmed: https://www.ncbi.nlm...pubmed/28111332

Sci-hub: http://sci-hub.tw/10...dis.2017.01.004

 

[4] https://en.wikipedia...harmacogenetics

[poonja]

Thank you for the valuable information.  How long a period would you recommend to wait after ceasing raps use before beginning a course of foxo use and how long before beginning raps use again?  Again, thank you or taking the time to share this valuable information.

[Nate-2004]

My guess is that most people will not see any significant results for at least 60 days. I don't even know how people are dosing or if they're dosing properly or following the recommendation of inducing more inflammation during treatment or what.

[tintinet]

My guess is that most people will not see any significant results for at least 60 days. I don't even know how people are dosing or if they're dosing properly or following the recommendation of inducing more inflammation during treatment or what.

 

 

Just received it. Not started dosing yet. Ideas about optimal dose, schedule, etc.?

[meatsauce]

The protocol that was followed in the study with rat to human conversion is 30mg day 1, 30 mg day 3, 30 mg day 5.  The dose is supposed to depend on your weight. For those who got 100mg if you want to use it all you can dose. 35mg day 1, 35 mg day 3, 30mg day 5. 

[Vantika]

Thank you for the valuable information.  How long a period would you recommend to wait after ceasing raps use before beginning a course of foxo use and how long before beginning raps use again?  Again, thank you or taking the time to share this valuable information.

 

From Judy Campisi's paper[1] (slightly edited for formatting):

 

Sustained effect of rapamycin on the secretory phenotype:

 

If rapamycin suppresses the SASP by preventing establishment of the IL1A--NF-kB feedback loop, transient treatment with rapamycin might suppress the SASP for an interval after removal of the drug.

 

In culture, a single exposure of HCA2 cells to rapamycin for only 24 h immediately after ionizing radiation suppressed IL6 secretion by ~80% for 7 days thereafter (Fig. 7a), similar to the suppression seen after continuous treatment for 7 days (Fig. 1a).

 

Although it is possible that the lingering effect of rapamycin after removal from the medium was due to intracellular retention, cell-associated rapamycin declined by ~80% and >90% 2 and 6 days, respectively, after removal (Fig. 7b). Interestingly, the MTOR pathway remained partly suppressed on the basis of S6 phosphorylation 2 and to a lesser extent 6 days after rapamycin removal (Fig. 7c).

 

Eventually, however, the loop was re-established. Suppression of IL6 secretion and SA-beta-gal activity slowly recovered after rapamycin withdrawal, reaching the level of untreated cells ~3 weeks after withdrawal (Fig. 7d,e).  [emphasis added]

→ source (external link)

 

Other targets for suppressing SASP don't seem to have nearly as long-lasting an effect as inhibition of mTORC1 does[2].  So as for non-mTOR-inhibiting SASP suppressors (many anti-inflammatories, often via (indirect) NF-kB downregulation), my guess is that a one week washout period would probably be good enough.  Others have speculated in this thread[3] that a 1-2 week period would work, based on typical surgical pre-op instructions.

 

Of course, if the compound itself (or any of its active metabolites) has a very long half-life, that changes things.  You would need to wait until its serum and intracellular concentrations reach zero, then start the clock on full reestablishment of SASP.   Use your own judgment.  YMMV.

 

 

References:

 

[1] MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation.  DOI: 10.1038/ncb3195

Pubmed: https://www.ncbi.nlm...pubmed/26147250

Sci-hub: http://sci-hub.tw/10.1038/ncb3195

 

[2] Prolonging Life Span? - Judith Campisi - Rejuvenation Biotechnology 2016.  Time-index: 12:00.

Youtube:

 

[3] http://www.longecity...ndpost&p=840139

[Vantika]

How long a period would you recommend to wait ... before beginning raps [rapamycin] use again?

 

FOXO4-DRI persists inside of cells' nuclei (possibly still being at therapeutic levels) for as long as 72 hours after dosing.

 

From the de Keizer paper[1]:

 

Using an antibody against HIV-TAT, we observed FOXO4-DRI to be taken up as soon as 2-4 hr after administration and to remain detectable for at least 72 hr (Figure 2J). Given that the affinity of antibodies is generally low, this indicates FOXO4-DRI effectively enters senescent cells at high intracellular concentrations, which remain abundant and stable over a prolonged period of time.

→ source (external link)

 

Accordingly, I would wait at least 96 hours after the last dose before resuming any anti-inflammatories or other potential SASP-suppressors.  Waiting a full week might be better, if you really want to be sure to get your money's worth.

 

 

References:

 

[1] Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.  DOI: 10.1016/j.cell.2017.02.031 

Pubmed: https://www.ncbi.nlm...pubmed/28340339

Sci-hub: http://sci-hub.la/10...ell.2017.02.031

 

Edit: typo in sci-hub link.

 

Edited by Vantika, 03 March 2018 - 10:39 PM.

[poonja]

Interesting article in Fight Aging Newsletter regarding protocol for testing Foxo4-dri.  Would link but do not know how. Looking forward to my n-1 testing.

[Nate-2004]

It's an incredible guide that I would definitely be using if I could afford to do this.

[PWAIN]

Just taken my first 5mg dose. Only observation so far is that the injection site hurts which is probably a reaction between the FOXO and surrounding tissue.

 

Will keep observing and reporting. Tomorrow I will take 10mg together with D&Q and again the day after. After a week, I will start using Rapamycin again. Hopefully I get a clean out of some senescent cells and some noticeable health effects. Is anyone else taking it yet?

[poonja]

Are you doing subq or IM.