Rapamycin and the Issue of Getting Through the Blood Brain Barrier

[RapAdmin]

Over the past year or so I’ve head many times that Blagosklonny takes a higher dose of rapamycin (20mg+) every two weeks because (he’s suggested) it helps increase the passage of the rapamycin through the blood brain barrier (and therefore increased mTOR inhibition in the brain), and I’ve asked him what research he based this on, but never got a response.

Today I just found what may be the information / conjecture that this belief is based on. Here is the quote and paper below:

The focus of this research study is in part to see the effect of rapamycin on the mouse model of Leigh Syndrome (LS), which has a significant impact on the brain, as specified below:

Leigh Syndrome (LS) is a severe mitochondrial disease that occurs in about 1:40,000 newborns and is associated with retarded growth, muscular deficits including myopathy and dyspnea, lactic acidosis, and a characteristic progressive necrotizing encephalopathy of the vestibular nuclei, cerebellum, and olfactory bulb (Budde et al., 2002, 2003; Anderson et al., 2008). Ndufs4 encodes a subunit of Complex I of the mitochondrial electron transport chain; mutations in the NDUFS4 gene cause LS in humans (Budde et al., 2000, 2003; Darin et al., 2001; Anderson et al., 2008; Quintana et al., 2010), and the Ndufs4 knockout (KO) mouse is a murine model of LS

conjectures on the rapamycin / Blood Brain Barrier issue that is widely discussed with regard to higher doses…

High-dose injection provided the greatest benefit to survival in the [mice]… the bolus provided by injection provides an enhanced benefit over steady dietary delivery in the mitochondrial disease model, perhaps by overcoming blood-brain barrier. Daily IP injection of rapamycin at 8 mg/kg alters neural activity associated with aging (Yang et al., 2012) and robustly reduces whole brain levels of phospho-s6, an indicator of mTOR activity

…

Effective inhibition of mTOR in the brain may be necessary for the full benefits of rapamycin in both Ndufs4KO mice and in normative aging.

Source: Dose-dependent effects of mTOR inhibition on weight and mitochondrial disease in mice

[MAC]

These are mutant transgenic mice.

We know from this paper in HUMANS back in 2008, that even 2mg/day passes the BBB, and the higher the dose, the higher the mTOR suppression in the brain.

A weekly dose of 10mg, you’re still at approx 2mg after a week (half life calculator)

Antitumor activity of rapamycin in a Phase I trial for patients with...

[MAC]

“Daily IP injection of Rapamycin at 8 mg/kg alters neural activity associated with aging”

Human equivalent 60kg person, 40mg/DAY.

Dr B at 20mg every 2 weeks, nowhere near.

We just don’t have a lot of good human data…

[RapAdmin]

Thanks for posting. Its interesting how much variation there is in terms of personal dose responses in terms of rapamycin / sirolimus levels in the brain tumors. Huge variations…

(A) Rapamycin concentrations in tumor tissue (filled squares) and peripheral blood (empty circles) grouped by rapamycin dose cohorts (2 mg, 5 mg, or 10 mg per os daily). Intratumoral rapamycin concentration for patient 11 could not be determined due to insufficient frozen tumor material. The last preoperative dose of rapamycin was given on the day of craniotomy and peripheral blood was collected within 24 h of surgery.

[desertshores]

It would seem to me that because of the molecular weight of rapamycin, very little passes the blood-brain barrier. Maybe someone smarter than me can calculate the theoretical amount that would cross the BBB based on rapamycin’s molecular weight.
Your thoughts?

Rapamycin has a molecular weight of 914.17

“Generally, only lipid soluble (lipophilic) molecules with a low molecular weight (under 400–600 Da) cross the blood-brain barrier Possible strategies to cross the blood-brain barrier. Possible strategies to cross the blood–brain barrier | Italian Journal of Pediatrics | Full Text
Note” 1 Da = 1 g/mol.

“Blood-brain barrier permeation decreases 100-fold when the size of the drug is increased from an MW of 300 Da, which corresponds to a surface area of 50 square angstroms, to an MW of 450 Da, which corresponds to a surface area of 100 square angstroms.”

Drug transport across the blood–brain barrier - PMC.

Rapamycin = 95 HPLC, powder 53123-88-9

Rapamycin | CAS 53123-88-9

[MAC]

Notwithstanding general rules surrounding molecular weight, structure, lipophilicity, etc, at the end of the day, all that matters is signal in target tissue.

And for that, we have an abundance of papers in mice, rats, other larger animals, and humans (above), that Rapamycin readily crosses the BBB.

The bigger question…is their a dose/concentration response, what target tissues matter, and does it actually manifest in superior human anti-aging.

[desertshores]

Sorry, I am still skeptical that suddenly, poof, a molecule of a molecular weight that has been proven repeatedly in scientific literature proven not to easily cross the BBB suddenly finds it very permeable. We humans are not mice rats or any other mammal. Can you supply a definitive study on humans that proves your point? If so, I stand corrected.

[MAC]

Yes, I posted above, relevant Figure 2.

Antitumor Activity of Rapamycin in a Phase I Trial for Patients with...

Brain tissue was sourced by “salvage surgical resection” by craniotomy.

As an aside, in this small cohort taking 2, 5, or 10 mg orally:

“Of particular importance, there were no perioperative bleeding complications. Five of 15 patients had grade 3 adverse events (hypokalemia, hypercholesterolemia, and cytopenias) during postoperative rapamycin treatment, which were managed with supportive care and did not require treatment discontinuation”

[RapAdmin]

Perhaps easier to blow up the text and include another image for evidence. From the paper:

Rapamycin Crosses the Blood–Brain Barrier and Blocks mTOR in Tumor Tissue

**

**

(A) Rapamycin concentrations in tumor tissue (filled squares) and peripheral blood (empty circles) grouped by rapamycin dose cohorts (2 mg, 5 mg, or 10 mg per os daily). Intratumoral rapamycin concentration for patient 11 could not be determined due to insufficient frozen tumor material. The last preoperative dose of rapamycin was given on the day of craniotomy and peripheral blood was collected within 24 h of surgery.

(B) Quantification of mTOR activity in tumor tissue by immunohistochemistry. The cartoon to the left depicts the S6 kinase 1 branch of the mTOR signaling pathway resulting in phosphorylation of S6 ribosomal protein at serine 235/236 and serine 240/244. The panel to the right shows a comparison between immunoblotting (top) and IHC (bottom) for the determination of S6 phosphorylation in tumor tissue from rapamycin patients 1, 2, and 3. The fold change in serine 235/236 phosphorylation between S2 and S1 for patients 1, 2, and 3 were 0.45, 1.01, and 0.45, respectively (see Figure S2A).

(C) Changes in S6 phosphorylation between S2 and S1 (y-axis: ratio of S6 phosphorylation in S2 sample to S6 phosphorylation in S1 sample) for all patients for whom matched S1 and S2 samples were available (14/15 rapamycin patients and 9/9 patients who did not receive rapamycin). S6 phosphorylation was determined by IHC using phosphosite-specific antibodies against serine 235/236 (left) and serine 240/244 (right). Please see Figures S1 and S2 for details regarding IHC scoring method and results for individual tumors. p-values for the difference in mean S2/S1 ratios for each group were determined using the Kruskal Wallace test.

and Figure 3. in the Supplementary materials below - click on the image to see larger image:

Figure S3: S6 Phosphorylation at Ser 240/244 in Matched S1/S2 Tumor Tissue Pairs IHC-based quantification of S6 phosphorylation at Ser 240/244 in (A) matched S1/S2 tumor samples from 14 patients in the rapamycin clinical trial cohort and (B) matched S1/S2 tumor samples from nine glioblastoma patients who did not receive rapamycin prior to S2

[MAC]

Thank you RapAdmin.

So I’d really like to know more about Dr B’s “BBB” rationale, other than just trying to get a higher peak pulse in the brain at higher dose, yet avoiding side effects?

My gut says you need to constantly signal mTOR reduction in the cellular target to get max effect, akin to constant CR in long lived CR mice? But what is autophagy signal minimum?

Maybe 20mg every 2 weeks, is sufficiently near 2mg/day??

Maybe it’s a loading dose issue?

Understanding Steady State Pharmacokinetics

Maybe someone could model 20mg every 2 weeks vs 2mg/day for “fun”.

[desertshores]

From the article:
“Because rapamycin is a macrolide natural product whose size could prevent distribution across the blood–brain barrier, we measured rapamycin concentrations by mass spectrometry in an aliquot of tumor tissue obtained at S2. Rapamycin was detected in 14 of 14 tumors”

"Alternative explanations include variations in penetration of the blood–brain barrier or tumor hydrostatic pressure among patients.

Yes, I don’t dispute that rapamycin crosses the blood-brain barrier, but this article has a very small sample size and really doesn’t address the issue, of whether or not rapamycin crosses the BBB in large percentages or small percentages.

A sample size of 15 is not very compelling.

Because of the ambiguity I and others favor a high-dose pulse regimen.

[RapAdmin]

@desertshores

Yes, I don’t dispute that rapamycin crosses the blood-brain barrier, but this article has a very small sample size and really doesn’t address the issue, of whether or not rapamycin crosses the BBB in large percentages or small percentages.

Yes - that is a fair statement. If anyone finds any papers that address these issues, or with larger sample sizes, please post.

@desertshores I’m not arguing one way or another regarding your dosing strategy, just trying to learn what the state of knowledge is for this issue at hand. Your strategy may be entirely optimal - I don’t know. As with so many things, we don’t seem to yet have the rigorous data that we’d like.

[MAC]

This is sufficiently large cohort to demonstrate clearly that Rapamycin fundamentally does cross the human BBB. It’s been shown in countless mice and rat models, but few human studies…getting a craniotomy isn’t much fun unless you have few choices as a recurrent cancer patient.

The amount crossing matters less per se than mTOR suppression in target cells (out of our control other than dosing). Of course, for those Pharma companies looking to improve permeability, then yes, they would be studying rapalogs to further enhance.

The study showed directly that mTOR suppression was correlated with Rapamycin dosing, so perhaps simply passive diffusion.

I am with you, inclined to take higher doses until I see see some glucose/lipid dysregulation. The oncologist said to me “that’s when you know it’s working”.

From a human perspective, we are flying blind!

[desertshores]

Well, fortunately, or unfortunately, I am old, so I am willing to take more chances than I would recommend to a younger person.
I have watched enough Dr. Brad Stanfield videos on Youtube(LOL) to think he wouldn’t lend too much credence to this article. Also, there is not much actual data available at this stage.
I wish you and all the others and all the others here taking this journey the best. May, we all meet on the other side of 100.

[MAC]

This is all I’ve found so far on Rapamycin/BBB and HUMANS.

Humans don’t readily volunteer for craniotomies.

The fact that there are so many studies showing Rapamycin passage into the brain of mice/rats, also is supportive.

I’ve calendarized our utopian meetup.

[MAC]

Here’s a paper (2014) reviewing various pharmacological approaches to treating recurrent glioblastoma.

Molecularly targeted therapies for recurrent glioblastoma: current and future targets (UCSF)

nihms-820562.pdf

“Sirolimus—Given its large natural structure, there was initial concern that sirolimus might not be able to traverse the BBB; however, sufficient levels of sirolimus can be found in brain tumors when
administered at therapeutic doses (17)”

Reference 17 is the paper on Glioblastoma posted above from 2008.

[Joseph]

Craniotomie is not required.

I have posted this before;

PET scan

"....the upper limits of rapamycin?...The more important question?

I post this in another thread on this forum In my opinion the best way to measure would be; Non-invasive measurement of mTORC1 signaling with 89Zr-transferrin The real issue would be cost, all is off the shelf equipment, Not covered by insurance A PET scan as in the published would cost around $3,000. per scan

[RapAdmin]

@MAC great find - thanks for posting. From that paper you added, the 2014 (Molecularly targeted therapies for recurrent glioblastoma: current and future targets (UCSF)), I found this paper particularly interesting with regard to the rapalog “Everolimus” - which people here are starting to take instead of (or in addition to) rapamycin. It seems that Everolimus might be particularly good at passing through the Blood/Brain Barrier (BBB).

Everolimus — Everolimus is a specific mTOR inhibitor with lipophilic properties that allow it to readily traverse the BBB. The use of everolimus with bevacizumab as a combined modality therapy for newly diagnosed glioblastoma has shown some efficacy; the PFS compared favorably to previous reports of standard radiation and temozolomide therapy.(47).

The referenced paper (47) is this paper:

Phase II study of concurrent radiation therapy, temozolomide, and bevacizumab followed by bevacizumab/everolimus as first-line treatment for patients with glioblastoma.

Full Paper here: https://www.hematologyandoncology.net/files/2013/05/ho0412_hainsworth1.pdf

Note: For people interested in learning more about the rapalog (acts like rapamycin) drug called everolimus - see this discussion thread: Everolimus instead of Sirolimus / Rapamycin? Anyone else trying?

In it, it states:

Conclusions: The use of bevacizumab and everolimus as part of first-line combined modality therapy for glioblastoma was feasible and efficacious.

Additionally, from the paper:

Four weeks after the completion of radiation therapy, patients began oral everolimus 10 mg daily, and continued bevacizumab every 2 weeks; therapy continued until tumor progression or unacceptable toxicity.

The dose of everolimus was modified on the basis of either hematologic or nonhematologic toxicity. Everolimus was interrupted if grade 3 or 4 toxicity occurred, and was held until the toxicity improved to grade 1 or less. At that time, dosing was resumed with a 1-level dose reduction (5 mg orally once daily). Patients were monitored for development of hyperlipidemia or hyperglycemia; if these events occurred, appropriate medical management was initiated.
…
Patients with objective response or stable disease began treatment with concurrent bevacizumab and everolimus. Patients were reevaluated at 8-week intervals with MRI scans until tumor progression was documented.

Interestingly, researchers have been testing Everolimus with many other cancer drugs, where they are finding that not only does Everolimus cross the blood brain barrier well, but it also helps other cancer drugs enter the brain and target tumors better (it seems). Here are some examples:

The research team at the Institute of Cancer Research, London, led by Professor Chris Jones, found that a drug called everolimus could enhance vandetanib’s capacity to pass through the blood-brain barrier in order to treat the cancer.

Combining the two drugs increased the amount of vandetanib in the brains of mice with DIPG by 56 per cent. The researchers also found that the treatment extended survival in mice by 14 per cent compared with those receiving a control treatment.

Source: Scientists identify new drug combination for children with incurable brain cancer

And:

Our results show that the efficacy of dasatinib treatment of PDGFRα-driven HGG was enhanced with everolimus and suggest a promising route for improving targeted therapy for this patient population.

Source: Everolimus improves the efficacy of dasatinib in PDGFRα-driven glioma

[MAC]

They are quite similar pharmacologically. Not nearly as many longevity papers in mice, as with Rapamycin.

Thoughts?

Differentiating the mTOR inhibitors everolimus and sirolimus in the treatment...

Sirolimus and everolimus in kidney transplantation

https://sci-hub.se/https://doi.org/10.1016/j.drudis.2015.05.006

[RapAdmin]

I think Everolimus bears a closer look - perhaps as something we alternate with rapamycin, or perhaps dose simultaneously with rapamycin, or perhaps as a replacement for it.

There won’t be many mice studies or longevity studies (if any) because Everolimus is so close to rapamycin in terms of molecular structure (and also from a functional standpoint its almost identical), and because it has only recently gone off-patent (so no motivation by drug company to test in anti-aging, and too expensive for academic labs to have used in their trials/research).

Here is what Matt Kaeberlein has said about it:

Detailed discussion on Everolimus here: Everolimus instead of Sirolimus / Rapamycin? Anyone else trying?