When I realized that this quote is the exact rationale behind your entire experiment - and I think it would make a great quote in the paper you are writing… because it so perfectly explains why your approach is likely the best approach we have today to increase levels of rapamycin in our bodies, without significant side effects:
Analysis of rapamycin levels in blood suggests that daily i.p. injection of 8 mg/kg yields blood levels of around 1800 ng/mL one hour after injection and 45 ng/mL 24 hours after injection (Johnson et al., 2013c). For comparison, blood levels of 3-4 ng/mL rapamycin were measured following dietary delivery at 14 ppm in the same mouse strain (Zhang et al., 2014) and studies from the ITP have reported between 9-16 ng/mL at this dose and 23-80 ng/mL in animals receiving the 42 ppm rapamycin diet (Miller et al., 2011). Thus, daily i.p. injection of 8 mg/kg rapamycin, which appears to be well tolerated in wild type mice (Johnson et al., 2013c), yields circulating levels of the drug that are at least 20-fold higher than the highest concentration that has been carefully tested for effects on normative aging.
All good! May have to up my dose, not having any side effects💪
The sting of the injections seems to be lessening somewhat as time passes.
Same as last update, only gastro remodelling is noticeable change. I am tracking my sleep and still observing to draw more meaningful conclusion as to impact of my protocol. If anything, sleep “seems” a bit more fragmented than pre protocol.
Although early weeks it seemed I might be trending downward in weight, no real change vs pre rapamycin, my weight is stable…even at this super high dose. I “might” be compensating some re calories for the amplified hunger signal during the day (recall I am OMAD, fast all day until dinner). The rapamycin has definitely changed the continuous hunger signal I feel during the day…it feels almost like I have a stomach bug sharp bite signal, that sort of sensation, but of course I feel normal. This is rapamycin.
Doing a full blood panel in 2 days, with deeper immuno markers added, along with the usual suspects. Will of course be including Sirolimus trough and will take another dose immediately post trough draw, and will be adding a few other Sirolimus time/concentration time stamp draws such as 6hrs and 24 hrs to help fill in some data points on my first curve “guesstimate”. Recall first panel, I had trough, 1, 2, and 3 hr data points. This panel will also be a snapshot proxy of the practical issue of stability of my Rapamycin source powder dissolved in DSMO and kept in freezer between doses, with subsequent freeze/thaw cycles.
After dose this week, I will have taken 17 weekly 15 mg IM/5.8 mg IN doses.
Memory/cognitive is my #1 longevity focus, and of course anectodal, but seems very sharp on protocol. I have been travelling recently and have very vivid recall of places/moments. Certainly no diminution, but my memory was quite good going into protocol, so difficult/too early to tease out any signal. I will be doing another online memory/cognitive test near future to add to my first done several months ago.
All is well, continued weekly dosing protocol. Feeling great, normal routine.
I definitely feel different re gastro oral vs current IM + IN, but not any better that I can quantify, but not comparing apples to apples (more below*). The fact I don’t feel any worse on therapeutic dose levels (compared to pre rapamycin), as compared to many members reporting some ill effects on FAR lower dose oral tablets is part of the apparent novelty of this protocol. This is very intriguing given the chronic side effects and drop out rates with therapeutic ORAL dosed rapamycin.
*My “oral” rapamycin experimentation earlier this year was with raw rapamycin powder (not commercial tablets most everyone here takes), which we now know delivers very low bioavailability (was not getting a Sirolimus blood signal even at high dose) unless one takes (maybe) mega+ doses. So I skipped the entire low dose commercial tablet experimentation and jumped right into therapeutic dosing with this IM+IN protocol. Having raw powder was a major enabling factor, combined with my explorative risk taking demeanour.
Attached compilation of labs I am tracking. My last panel was Nov 9, 22. I started this protocol Jul 6, 22, so it’s been just over 5 months of weekly dosing.
I added some immunological markers to my last blood panel Nov 9, 22.
The most interesting change was a significant drop in the weekly trough Sirolimus level, from 11.1 ng/mL Aug 3, 22 to 3.9 ng/mL Nov 9, 22. I am not 100% sure the cause of this drop. It might be the repeated freeze/thaw and possible contamination cycles of the weekly syringe filling. Recall I produce a batch of 10mL of DSMO with 300 mg raw powder, for a 30 mg/mL concentrate. I store this in the fridge, and then every week, I open the glass vial, draw out 0.5mL (15mg) and inject. I then close the vial and return back to fridge.
Commensurate with the drop in trough Sirolimus, the typical dysregulated markers came back down some (TG, Remnant Cholesterol, hbA1c, FBG). Even the high dose Aug 3, 22 markers didn’t cause me much concern to abandon the protocol. As a leading rapamycin oncologist told me “that’s how you know it’s working”. Is this perturbation part of the dysregulation dynamic of lifespan extension? Tinkering with mTOR2 and insulin-glucose pathway? TOR2 is poorly understood, considered “bad”, but many of the longevity studies utilizing a variety of molecules (non Rapamycin, ACA, 17bEstradiol) keep reporting TOR2 activation as associated with lifespan extension, even when not much TOR1 downstream signalling markers impacted.
I did have a recent Cardiologist consult last week coincidentally (my CAC changed from 4.5 yrs ago, 26 > 61…chronic endurance exercise increases CAC over time, yet delivers all cause mortality reduction: the “runners paradox”), but he’s not concerned given my exercise/profile phenotype. No discussion of pharma intervention (even with apparent elevated LDL) or changes to lifestyle. He was comforted that I had an Lpa (genetic risk marker) test done years ago, and was super low. He did order some followup ultrasounds of my carotid/femoral arteries just to further baseline for any CVD.
My iron is in a bit of flux, combination of pausing 8 week regular blood donations but ongoing dsyregulation from high dose rapamycin. Suffice to say, I am still likely very much clinically iron deficient anemic, but it dosen’t seem to be impacting my daily running/exercise or tiredness! Hormesis adaptation from chronically lower cellular iron stores, the body has become much better at retaining and using available iron? I continue to eat foods containing iron, so I might be building back cellular reserves, will need to do a full iron panel again next labs. Still paused on blood donations pending trending.
As a means of continuous improvement, avoiding contamination and freeze/thaw cycles, and to reduce the amount of DMSO per injection, I implemented a few changes batch/protocol #2:
I prepared a new batch of 300mg raw rapamycin powder (long term deep freezer storage) in only 8ml of DSMO, for a 37.5mg/mL concentrate. I could then only need to inject 0.4mL to deliver 15mg IM of rapamycin (a reduction from 0.5 mL/injection). I sonicated this solution for 20 mins at room temperature. Solution looks crystal clear to the eye.
From the batch in 1, I immediately I preloaded the full batch (20 doses) into individual 0.4mL/15 mg dose syringes. I then froze the entire batch of syringes in the deep freezer (vs fridge). DSMO solidifies at 18C, well above normal fridge temperature, but perhaps deep freeze is superior. Each weekly dose would thereafter entail simply removing a single syringe from the deep freezer, allowing a few minutes to defrost, and then injecting. This would eliminate possible contamination and freeze/thaw cycles.
I initiated this new protocol on Nov 15, 22. I instantly noted a return to the pretty intense stinging on the initial foray dose delivery, which dissipated quite quickly (few mins). The trending of the last doses of previous concentrate batch #1 protocol, prior to my Nov 9, 22 labs, the stinging was much less than the very initial syringes injected from the very first concentrate, suggestive of some type of rapamycin degradation. This confirmed with much lower trough Nov 9, 22 labs.
Repeating something previously reported, that gastro/stool is the very most noticeable change with high dose rapamycin. Consistently, for example, 2 days after my last dose this week, I had 3 bowel movements by 2pm, producing about 25-30 pieces of small, soft, tanned colored stool (similar to the small, higher number stool pattern of the mice in seminal rapamycin/longevity paper by MK).
This stool pattern is completely foreign to me in my pre high rapamycin dosing life. Prior to high dose rapamycin, I typically experienced varying level of constipation, and typically a single, large, harder, dark stool. So clearly my stool and micribiome is undergoing some massive alteration with the high dosing rapamycin, but yet NOT DIARRHETIC (commonly reported with higher levels of oral tablet dosing). I feel no diarrhetic type symptoms, it’s completely a stool output phenomena.
I have to draw another blood panel in a few weeks to confirm trough Sirolimus levels, but between the intense injection stinging and the stool pattern, it would seem (hoped) I have returned to much higher AUC levels of blood Sirolimus. My injection stinging and stool repeat weekly pattern may be the confirmatory signal.
The Intranasal administration continues unchanged. At some point in the future, I will do a washout of IM, and just assess blood Sirolimus with just IN. I am currently doing both for the objective of maximizing translation of systemic and brain delivery of rapamycin (trying to extend lifespan whilst running an experiment!), without having to resort to even higher peripheral injection doses to help increase BBB passage. IN as previously reported, is focused on enhanced brain delivery.
It was speculated by MK that this gut microbiome remodelling “could” be a central pathway to longevity extension, perhaps via immuno modulation, cancer/bacteria mitigation. We know factually rapamycin is BOTH an anti-microbial and immune modulator? Smoking gun? I reached out to MK, not heard back.
Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice
In this seminal paper by MK, the significant change in high dose rapamycin stool prompted the authors to do a full micribiome sequencing/species (metagenomic) analysis.
"We hypothesized that changes in the microbiome may underlie this phenotype (high dose rapamycin) and therefore analyzed the fecal microbiome for each of the cohorts used in this study by deep-sequencing of bacterial 16S rRNA. Rapamycin treatment induced a significant change in the composition of fecal microbiome. Among the most notable changes in fecal bacterial DNA content seen in the global microbiome analysis was a significant increase in prevalence of segmented filamentous bacteria (SFB, Candidatus Arthromitus sp.) in the rapamycin treated animals. SFB are intestinal Gram-positive bacteria with a segmented and filamentous morphology, and are not normally present at high levels in aged mice . The SFB genome lacks a majority of virulence factors and SFB are not invasive; however, their tight adhesion to the intestinal epithelial cell induces differentiation of host immune cells. To the best of our knowledge, this represents the FIRST pharmacological intervention to increase SFB in ANY animal. It will be of interest to determine whether these and other effects of rapamycin on the microbiome are shared across species and play any causal role in the beneficial or detrimental effects of this drug"
This is a truly profound finding, under reported and under studied in longevity studies…lost in the mTOR hysteria.
What are these SFB and are they “associated” with longevity phenotype?
“SFB are notable for their ability to modulate adaptive host immune responses, including induction of regulatory Th17 T cell Peyer’s patch maturation, and gut IgA production. A recent study from the laboratory of Joyce Wu at the University of Arizona has revealed a novel innate immune function”
We know rapamycin is a potent immune modulator, but how mediated…via gut SFB?
Genome Sequence of Segmented Filamentous Bacteria Present in the Human Intestine
“SFB are physically attached to the host’s intestinal epithelium and affect several functions related to the immune system, among them IgA production and T-cell maturation”
Presence of Segmented Filamentous Bacteria in Human Children and Its Potential Role in the Modulation of Human Gut Immunity
"We previously reported that the colonization of SFB in humans mainly occurs by 36 months of age, and is difficult to be detected afterward. Collectively, these data suggest that SFB is a rare member of microbiota, and may play an important role in the development of human gut immunity.
Wait, SFB don’t exist in aged mice or humans…but yet they are produced in LONG LIVED mice on rapamycin? A human translation lifespan correlation and therapeutic opportunity?
In another recent paper, some additional profound findings relating to HUMAN cancer and fungal/bacteria:
Cancer-microbe associations have been explored for centuries, but cancer-associated fungi have rarely been examined. Here, we comprehensively characterize the cancer mycobiome within 17,401 patient tissue, blood, and plasma samples across 35 cancer types in four independent cohorts. Clinically focused assessments suggested prognostic and diagnostic capacities of the tissue and plasma mycobiomes, even in stage I cancers, and synergistic predictive performance with bacteriomes. Recent studies found metabolically active, immunoreactive, intracellular, and cancer type-specific communities of bacteria and viruses in tumor tissues. Fungi are detected by multiple staining methods in human tumors. Different cancer types exhibit cancer-type-specific mycobiomes. Intratumoral mycobiome-bacteriome-immunome interactions. Statistical and machine learning analyses demonstrate cancer-type-specific mycobiomes. We observed strong positive correlations between fungal and bacterial diversities, abundances, and co-occurrences across several cancer types, suggesting tumor microenvironments (TMEs) may be non-competitive spaces for multi-domain microbial colonization, which we term a ‘‘permissive’’ phenotype. This differs from the gut, especially under anti-cancer or antibiotic therapies, where fungal and bacterial populations alternate and compete over shared resources—an ‘‘antagonistic’’ phenotype"
A connection between fungi/bacteria/cancer and anti-microbial rapamycin…mediated by the gut-microbiome?? Wait, what about mTOR…isn’t that THE causal pathway to cancer mitigation, lifespan extension in mice?
I wrote to this author and asked him if possibly rapamycin and its longevity benefits could be mediated by this cancer/fungi/bacteria connection…he wrote “we’ve not studied this, but so many others have asked me the same question”.
“The intestine is a critical target organ for improving the health of aged animals and
humans. Therefore, we speculate that the gut microbiota could be a new anti-aging target.”
What about Acarbose and it’s longevity mechanism to longevity enhancement? Does it have anything to do with glucose lowering or mTOR…or maybe all to do with the gut microbiome remodelling pathways? Are we blinded by TOR theories, missing the fundamental mechanistic underpinnings?
“Acarbose reproducibly modulated the composition of the microbiota and increased the concentration of SCFAs in mice, especially the abundance of propionate or butyrate. There was a correlation between fecal SCFAs and lifespan in mice, suggesting a role of the gut microbiota in the longevity-enhancing properties of acarbose. In another study, acarbose increased the abundance of Bifidobacterium and Lactobacillus, whereas the abundance of Bacteroides was decreased at the genus level. Consistent with a study in mice, acarbose treatment increased the abundance of Bifidobacterium, Eubacterium, and Lactobacillus and decreased the abundance of Bacteroides in T2D patients.106 Similarly, the gut microbiota Bifidobacterium longum and Enterococcus faecalis were increased significantly after 4 weeks of acar-bose treatment in T2D patients”
I take a daily tablespoon of green banana leaf power to help with SCFA production.
From above anti-aging paper: “A novel theoretical basis for improving our understanding of the mechanisms by which acarbose extends the life span of mice has been suggested. This review linked inflammation, mitochondria, and telomeres with the GUT MICROBIOTA, illustrating individual mechanisms involved in acarbose associated life span extension. Acarbose improves the immune system, inflammatory response, and mitochondrial function by affecting the gut microbiota”
No mTOR connection central to Acarbose lifespan mediation? I reached out to a researcher at the ITP group re Acarbose/micribiome/longevity, not heard back.
We know from the ITP, the combination of rapamycin + acarbose was synergistic in further lifespan improvement in mice? Are they both working via the gut microbiome remodelling? Two fundamentally different mechanistic molecules yet both delivering lifespan extension? But yet both must be mediated to cancer in some way…mice only die of cancer. Microbiome ties the two…could it really be that mechanistically simple?
It is becoming more and more readily appreciated/investigated that the gut microbiome is a CENTRAL mediator of systemic health. By extension, longevity? Are the SFB that MK found in his study associated with longevity? Causal?
I plan on sending out my stool sample shortly for whole gene sequencing metagenomics, and will look to see how my micribiome changed with this high dose rapamycin protocol, and if it’s associated with the species/shift found in long lived mice studies. I’d have to do a washout to get a no rapamycin microbiome baseline.
Is this gut/stool remodelling I am experiencing a “youthing” effect? If you’re not experiencing this massive gut remodelling, can you be moving the needle with rapamycin?
I plan to add Acarbose to my intervention soon*, once I’ve further steadied my rapamycin protocol AND baselined my gut rapamycin mediated micriobiome. Will then repeat all labs and gut microbiome analysis.
*Most likely starting with lower than typical human or mouse translation dosing, for reasons of not wanting to tip into chronic hypoglycemia (I’m already close to hypoglycemia on keto/OMAD). Btw, if you look at the ITP Acarbose study, the mice were fed 1000ppm w/w in their diet. Average human in the world consumes 1.85kg/day. So equivalent w/w for human translation would be 1.85 x 1000 = 1850 mg ACARBOSE/day. Using HED metrics: 1000ppm w/w diet = 166mg/kg mouse body weight (using: a dose in food of 42 ppm is equivalent to a dose of 7 mg/kg body weight per day for a 30 g mouse eating 5 g of chow per day.) Divide by 12.3 for HED mice to human, thus, 166/12.3 x 75 kg human = 1012 mg per day. Most people take at most 300 mg/day acarbose. So once again, like most all of the ITP studies, mice are taking much higher than human doses.
Does the mouse diet in the Acarbose ITP study (same exact diet as Rapamycin) have any bearing on the longevity effects re HUMAN TRANSLATION??
65/22/12 carb/fat/protein calorie macros. So a pretty high carb diet, but not too far western diet macros?
The food stuffs: "Ground wheat, ground corn, wheat middlings, ground oats, fish meal, dehulled soybean meal, corn gluten meal, dehydrated alfalfa meal”. Variety of carbohydrates.
Does it matter what MICE eat vs what HUMANS eat for rapamycin/acarbose remodelling and longevity translation? I am strict keto, don’t eat carbs (although I am getting plenty resistant starches and prebiotics), but yet experiencing this massive gut/stool remodelling on high dose rapamycin with NO change in my diet pre/post rapamycin?? Does diet matter IF you’re taking a proven lifespan (rodent) extending small molecule intervention? Do these billions year old microbes rule?
So many unanswered questions, too incredibly complex to tease out for human translation.
If I can make a high dose rapamycin/gut/stool/bacteria remodel connection…might be a compelling outcome for further human translation exploration of my protocol.
@MAC, I’m with you on the microbiome being likely having a significant influence on healthspan/lifespan, as I’ve also noticed significant changes in my stools, whilst my biological age as per Ageing.AI has been dropping significantly.
Also, I recently started Acarbose and I used a CGM for the first two weeks just to make sure I wasn’t tipping into hypoglycemia, and have to say, the spikes were lower but the troughs stayed the same for me, so no issues at all.
Keep up the trailblazing work as I think we are all definitely staying tuned!!!
Firstly, the Acarbose hasn’t changed anything as far as I can tell, but only been on it for 3 weeks.
The stool changes took place over the last 1 to 3 months, from rock solid dark (almost black) pebbles to large soft brown (very clear colour difference) very fat sausage shapes.
3 months ago was the step up from 6mg oral Sirolimus fortnightly to 8mg oral fortnightly (and have since moved to 10mg). However, I must add, in a concerted effort to get on top of my high LDL-c, Amla Powder and Psyllium Husk was added into the daily intake (along with Ezetimibe about 6 weeks ago).
Obviously therefore, we can’t conclude that this was down to the Sirolimus, and given the Husk is 90% fibre this has probably had the biggest effect. (Amla powder quantity I don’t think is anywhere big enough to have a noticeable effect and I’m not aware that Ezetimibe would do anything here either and that came in after the changes were noticeably taking place). Diet otherwise completely unchanged.
That said, my latest blood test results came back yesterday with me now right in the middle of the normal range for LDL-c (down from double the top of the normal range - so that triple combo has worked extremely well for this n=1) so from today I’m going to leave out the Psyllium Husk and Amla powder for a couple of weeks and we’ll see what happens.
I will report back to you.
I think I can already report back having dropped the Psyllium Husk first, the stool is almost back to the rock solid dark pebble shapes - the colour remains a lighter brown though.
I guess the Rapa hasn’t really had much of an effect if any for me after all.
MAC, If you have concern about your CAC I’m going to show a link to Patrick Theut’s story about his very high CAC and his approach to fixing it. It’s on a website where he sells vitamins and I am not promoting his vitamins, but I believe his story is likely true. I’ve heard him talk about it, but I don’t have a link to his talk. Click on the “My Story” link if you have any interest.
We’re all here to push out ACM (all cause mortality). One of cancer, neurodegenerative, CVD or a metabolic dysfunction related to one of these pathways is going to get us one day. From the volume of papers I’ve read on CAC rising with chronic endurance exercise (the latter of which is hugely inversely correlated to ACM), the lack of any diffusivity in my CAC, the still relatively very low CAC, given my fitness/diet/insulin sensitivity/family history phenotype, CVD is the least of my mortality pathway concerns at this point in time. However, I am under the care of a cardiologist, and I do NOT assume anything, we are actively investigating this pathway further (additional investigative testing forthcoming).
Just a single reference on rising CAC with chronic endurance exercise:
