#1

From the Dudley Lamming Lab, a pre-pub paper. This is a very high dose example, so not directly applicable to our dosing levels in our longevity efforts.

Purpose: Genetic deletion of mTOR has protected against post-traumatic osteoarthritis (OA) in male mice, however, effects of pharmacological mTOR-inhibition are equivocal and have not been tested in aging models nor in female subjects. Therefore, the goal of this study was to determine if mTOR-inhibition by rapamycin can modify OA pathology in aging non-human primates and female mice.

Methods: Common marmosets were administered oral rapamycin (1mg/kg/day) or vehicle starting near mid-life until death. Five-month-old, female C57BL/6J mice were treated with vehicle or rapamycin (IP, 2mg/kg, 3x/week) for 8-weeks following non-invasive ACL rupture. Knee OA pathology was assessed via microCT and histology. Phosphorylation of mTORC1 (p-RPS6S235/36) and mTORC2 (p-AktS473, p-NDRG1T638, p-PKCαT348) substrates were evaluated via western blot in articular cartilage, meniscus, and/or infrapatellar fat pad. ATDC5 cells were cultured with rapamycin to determine time and dose effects on mTORC1/2 signaling.

Results: In marmosets, rapamycin did not impact age-related radiographic OA severity or cartilage pathology but increased medial meniscus calcification and lowered lateral tibia subchondral thickness, particularly in females. In female mice, rapamycin worsened ACLR-induced meniscus calcification and cartilage pathology. In marmoset and mouse joint tissues, rapamycin inhibited mTORC1 and increased p-AktS473 but not p-NDRG1T638 or p-PKCαT348. This mTOR signaling pattern was replicated in ATDC5 cells during exposure to low concentrations of rapamycin.

Conclusions: Rapamycin attenuated mTORC1 signaling with feedback activation of AktS473 in articular cartilage, meniscus, and/or infrapatellar fat pad and was accompanied by deleterious effects on meniscus calcification and/or cartilage pathology in female mice and common marmosets. marmosets.

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#2

Thanks for keeping us informed sir.

#3

I don’t agree that these are “very high dose example[s], so not directly applicable to our dosing levels in our longevity efforts.” As previously noted, 1 mg/kg is what Salmon used in the marmoset lifespan study, and they used that dose after a dose-finding study found that lower doses were insufficient to lower mTORC1 signaling (and even 1 mg/kg wasn’t enough for the chubbier animals).

The 2mg/kg, 3x weekly in mice is harder to suss out, and they unfortunately don’t seem to have reported on trough levels or any other PK clue, but in Kaeberlein’s late-life multi-month lifespan study, i.p. injections of 8 mg/kg daily led to serum drug levels similar to 378 ppm in food, which is 27x what was used in the first ITP (14 ppm). This is 1/4 of that dose given 3/7 as often, so the cumulative exposure might be something shy of 3x the ITP dose — high, but not stratospheric.

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#4

1mg/kg/day is a very high dose (for humans). My weight is 50 kg, so it’s 50 mg/day/every day. I can hardly tolerate 1 mg/day/every day.

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#5

Sure: you never simply apply a mg/kg dose from a smaller species to humans — you apply one or another conversion factor. As discussed in my linked post, the dose in the marmoset study (1 mg/kg) led to daily trough levels similar to those of transplant patients.

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#6

What’s the HED of 1 mg/kg in marmosets?

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#7

By the simple FDA formula, 1 mg/kg marmoset / (37/6) = 0.162 mg/kg human, so 11 mg/day for a 70 kg adult.

However, this assumes a 60 kg human and a 350 g marmoset. If we start with a 70 kg human and a 430 g marmoset (Supplemental Table S3), we get

HED = 1 mg/kg x (.4301 kg/70 kg)[exponent]0.33 = 0.183 mg/kg, or 12.8 mg.

But again, these are all rules of thumb for estimating a starting dose. We have the actual blood trough levels for the marmosets.

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#8

Confused here. If we are talking about 11 mg/day in humans, it’s a mega dose, and I’m not aware that any transplant patient would be prescribed such dose. I’m prescribed 1mg/day. I did develop the first stage of osteopenia (detected at 68). It’s stable and doesn’t progress and even improved a little (monitoring for 2 years now).

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#9

10mg per day is usually only done in Cancer patients, and the side effects are pretty horrendous…

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#10

We’re not.

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#11

Agree with @RapamycinCurious. When blood levels are already available, there’s no need for inaccurate formulas to convert doses across species/protocols. 5.2ng/mL trough in marmosets vs. 5-15ng/mL target trough in transplant patients who take 2-5mg/day, so we are talking a HED of 2mg/day.

Also, this is around the same blood concentration that’s the minimum for life extension in mice and is unfortunately higher than what the longevity community takes and what is safe IMO.

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#12

I would think this adds to the argument that chronic inhibition of mTOR is to be avoided. We should, therefore, estimate a trough level that is the maximum and ideally a period of time (proportion?) for which to maintain levels below this trough. That will inform dosing frequency. 5ng/ml is arguably too high, but gives a figure to judge higher intermittent dosing.

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#13

@DrFraser @Olafurpall any thoughts

#14

Is this correct? I don’t know anything about that, but per ChatGPT:

  • “Even if trough levels appear similar, differences in absorption, distribution, metabolism, and elimination between marmosets and humans can affect how the drug works. The 5.2 ng/mL trough in marmosets might not perfectly equate to the same pharmacological effect in humans.”
  • “The serum level is only a snapshot of what is circulating, but rapamycin exerts its effects inside cells (for example, by inhibiting mTOR in target tissues). Differences in tissue penetration, cellular uptake, and local drug accumulation mean that the same serum trough might not translate to similar intracellular concentrations. For instance, marmosets may have a different distribution pattern than humans, so a 5.2 ng/mL trough in marmosets might not activate (or inhibit) mTOR to the same extent as a 5–15 ng/mL trough in humans.”
  • “The terminal half‐life of rapamycin differs across species. For example, in humans, rapamycin’s half‐life might be longer than in marmosets. Thus, even if a trough level is matched, the duration and fluctuations of drug exposure over time may differ, influencing both efficacy and side effect profiles.”
  • “In transplant patients, the target trough range is broad (5–15 ng/mL), with effective doses ranging from 2 to 5 mg/day. If the marmoset level of 5.2 ng/mL is at the lower end of that range, a corresponding human dose might be closer to 2 mg/day—but this depends on how the drug’s efficacy and safety translate between species.”
  • “Thus, a more comprehensive assessment—including pharmacodynamic endpoints and full PK profiling—is needed to accurately determine the human equivalent dose (HED) rather than relying solely on serum levels.”

Still, the serum level estimate might be better than the rule-of-thumb FDA conversion. So we can say that the HED is most likely between 2 mg/day and 13 mg/day for a 70 kg human.

bioRxiv is down at the moment so I can’t read the paper: what are the serum levels, peak and trough?

#15

I agree with @John_Hemming, the side effect and benefit profiles are different with continuous mTORC1/C2 inhibition as opposed to cyclic use with primarily mTORC1 being inhibited.

My experience on taking care of individuals who are in their 70’s who have been on Rapamycin for many years, is that I’m not seeing Osteoarthritis as a listed diagnosis on many of them - certainly less than community average by a long way.

I’d expect both muscle loss and bone loss with continuous rapamycin, so the findings aren’t a surprise.

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#16

Yes, the standard for a researcher extrapolating doses from preclinical data is to compare blood levels, unless you’ve also measured the levels in your target tissue or have a PD/functional assay.

ChatGPT is basically saying: yes 2mg/day all else equal but there might be other factors. We don’t know if there are meaningful PD and tissue distribution differences across species, but blood levels are the best proxy. There’s no reason to fall back to crude scaling formulas when you already have the blood levels, which take into account formulation, administration, absorption, and metabolism differences. On the other hand, I disagree with ChatGPT’s reasoning about half-life, because we’re already comparing trough vs. trough on continuous dosing. The best guess is 2mg/day but it could be higher or lower.

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#17

Unfortunately, the takeaway from the paper may be that low or intermittent doses are also risky. The negative outcomes presented here are attributed to mTORC2 activation (not inhibition) as a feedback mechanism when mTORC1 is inhibited. This wouldn’t be possible at higher continuous doses like in transplant patients or the more effective mouse doses for life extension, where mTORC2 would also be inhibited.

#18

I’m not sure we know this is mTORC2 activation? The way these studies are dosed would lead to inhibition of mTORC2.

I probably have a healthy population, but I’d say that in my patient’s on Rapamycin, the observations would be the opposite in regard to osteoarthritis and rapamycin use.

If I suddenly see a whole group get afflicted unexpectedly with Osteoarthritis I’d take note of this, but this hasn’t been the case. I have many patients who are in their 70’s or older who have been on Rapamycin for 5+ years without Osteoarthritis being on their list of diagnoses or issues they are symptomatic from.

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#19

We should check the full paper because the website is down, but the abstract attributes it to activation of Akt, the mTORC2 substrate. I think at 1mg/kg/day, the other paper showed the lowest marmoset in the 1s ng/mL at trough (with 5.2ng/mL trough being the average). For the mice, it’s 3x/week which matters given the shorter half-life in mice, as opposed to continuous in the ITP studies via diet. So, these don’t seem to be continuous, full-suppression type doses to me.

#20

That 5.2 ng/mL trough is almost certainly going to inhibit mTORC2 as my patients essentially have a trough of 0 or close to it, and have 30% of the time above 3.0 ng/mL. So I would say having a trough of 5.2 ng/mL is continuous use and continuous mTORC1 and at least partial mTORC2 inhibition rather than activation. Unless they have proof mTORC2 was activated for the duration I’d doubt that claim - and even then, why not attribute it to continuous mTORC1 inhibition? Seems a bit random - but need to look at the paper and see if they have a reason for such a claim? Anyway, it’s a very different dosing scheme than I utilize.